Keywords:

In summary, in this thesis we show the value of C-arm CBCT in assessing CRCLM response to chemoembolization and enlarge the scope of intra-arterial therapy to suicide gene therapies which appear promising.

Title : Study of antitumor effects of hepatic intra-arterial therapy (chemoembolization and suicide gene therapy). Role of the Intraprocedural imaging: preclinical and clinical evaluation.

Abstract :

Colorectal cancer liver metastases (CRCLM) are a leading cause of death. They occur in 50 -75% of patients during the course of the disease. Intra-arterial treatments for CRCLM have led to continuous progress; but these treatments still face significant limitations regarding tolerance and results. We explored two different avenues in trying to improve the results of intra-arterial treatment. The first one consists in optimizing the intraprocedural imaging technique aiming at anticipating the tumoral response.

The second one consists in developing a completely new approach based on so called "gene directed enzyme prodrug therapy" (GEPD).

As a 1

st part, we explored the intra-procedural response to chemo-embolization based on various approach using C-arm CBCT. We first demonstrated the feasibility of tumor segmentation with semi-automatic software on MRI image and then apply the same method a VX2 liver tumor model. We found a strong correlation of tumor size between Carm CBCT and CT scan (rho = 0.993, p < .001) but also between CBCT and histopathology (rho = 0.996, p < .001). In addition, we found a minimal bias (0.3 cm 3 ), and high precision (0.674) when measuring tumors with C-arm CBCT. Since the C-arm CBCT precision was demonstrated, the next work was focused on the quantification of tumor enhancement before and after trans-arterial chemo-embolization (TACE); as a surrogate biomarker of tumor response with the goal to predict tumor response at an earlier stage. Dual-phase C-arm CBCT was first used, showing significant neuroendocrine tumor enhancement decrease after embolic injection accounting for a noticeable micro-embolic effect at the tumor capillary bed level. In another study, Dual-phase C-arm CBCT failed to demonstrate its utility in CRCLM detection because of a lack of accuracy (Sp.58% Se. 51%). We then orientated our search on intraprocedural evaluation of the parenchymal blood volume (PBV). In this work we showed that initial PBV was strongly correlated to tumor shrinkage after chemo-embolization (rho=-0.8587; p=0.00001). Moreover the increase of 1 ml/1000ml in the blood volume of the tumor before TACE leaded to a decrease of 0.54mm in the diameter change of the tumor. Finally, patients with a 1% decrease in PBV after DEBIRI had a 10% lower likelihood of achieving disease control (OR=0.9, 95% confidence interval CI: [0.81 -1]; p=0.0493). In conclusion of this 1 st part, we anticipate the need for a dedicated clinical study that would evaluate prospectively the tumoral outcome based on PBV, with possible triage of responding/non responding tumors PBV toward alternative liver directed technique.

As the 2 nd part, thanks to a strong collaboration with INSERM U 1147 team we developed a new intra-arterial treatment based on a suicide gene therapy consisting of an engineered gene coding for a mutated cytochrome responsible of the metabolism of a prodrug. This new approach uses transduced mesenchymal stem cells (MSCs) as carrier of the suicide gene. Our work consisted in in-vivo experiments in rabbit VX2 liver tumor and showed significant tumor volume difference between control and test groups associated to tumor necrosis significantly greater for rabbits receiving the GDEPT (78% of total tumor surface) compared with control animals (22% of total tumor surface (p=0.006). These findings deserve further confirmations, which are undergoing (refine the strategy, document toxicities…).

Titre:

Etude des effets antitumoraux des traitements intra-artériels hépatiques (chimio-embolisation et thérapie par gène suicide). Rôle en imagerie per-opératoire, évaluation préclinique et clinique.

Résumé:

Chez les patients atteints de cancers colorectaux, les métastases hépatiques (CRCLM) sont une des principales causes de décès. Elles surviennent chez 50-75% des patients au cours de la maladie. Les traitements intraartériels pour CRCLM ont conduit à des progrès continus; mais ces traitements restent limités par leur tolérance et leur efficacité. Nous avons exploré deux voies différentes pour essayer d'améliorer les résultats des traitements intra-artériels. La première consiste à optimiser la technique d'imagerie per-opératoire, visant à anticiper la réponse tumorale. La seconde consiste à développer une nouvelle approche basée sur une thérapie par un gène suicide par voie intra-artérielle.

En 1

ère partie, nous avons exploré la réponse à la chimio-embolisation (TACE) suivant diverses approches en utilisant le Cone Beam CT (CBCT). Nous avons démontré d'abord la faisabilité de la segmentation tumorale avec un logiciel semi-automatique en IRM puis appliqué la même méthode à un modèle de tumeur VX2 du foie de lapin. Nous avons montré une forte corrélation de la taille de la tumeur entre le CBCT et Scanner (rho = 0,993, p <.001), et entre le CBCT et l'analyse anatomopathologique (rho = 0,996, p <.001). Le CBCT a un biais minimal (0,3 cm3), et une haute précision (0,674) dans la mesure des tumeurs. Puis, nous avons axé notre travail sur la quantification du rehaussement tumoral avant et après TACE en tant que biomarqueur de substitution de la réponse tumorale. Le CBCT double phase a été utilisé en premier, montrant qu'une importante diminution de l'atténuation des tumeurs neuroendocrines est quantifiable après injection de particules emboligènes et qu'un effet micro-embolique notable était observable. En revanche dans une autre étude, le CBCT double phase n'a pas démontré son utilité dans la détection de CRCLM par un manque de sensibilité et spécificité (Se.58% Sp. 51%). Nous avons alors orienté notre recherche sur l'évaluation intra-procédurale de la perfusion tumorale. Dans ce travail, la valeur initiale du volume sanguin tissulaire (PBV) était fortement corrélée à la réponse tumorale après TACE (rho = -0,8587; p = 0,00001). Nous prouvons que l'augmentation de 1 ml / 1000 ml du volume sanguin de la tumeur avant TACE conduit à une diminution de 0.54 mm du diamètre de la tumeur sur l'imagerie scanner à 2 mois. De plus, les patients présentant une diminution de 1% en PBV après DEBIRI ont une probabilité de 10% d'observer une réponse tumorale (OR = 0,9, 95% CI intervalle: [0,81 à 1]; p = 0,0493). En conclusion de cette 1 ère partie, une étude prospective plus large permettrait d'évaluer prospectivement la réponse tumorale basée sur PBV, permettant de sélectionner les répondeurs et de proposer aux non répondeurs un traitement local alternatif.

Dans la 2

nde partie, grâce à une collaboration étroite avec l'équipe INSERM U 1147, nous avons mis au point un nouveau traitement intra-artériel basé sur une thérapie par gène suicide, fondé sur l'injection dans l'artère hépatique de cellules souches mésenchymateuses transduites par le gène mutant codant pour le cytochrome CYP2B6TM. Nos travaux in vivo sur la tumeur hépatique VX2 du lapin montrent une différence significative du volume de la tumeur après traitement entre les groupes de contrôle et test. La nécrose de la tumeur était significativement plus élevée chez les lapins recevant la thérapie par gène suicide (78% de la surface totale de la tumeur) par rapport aux animaux témoins (22% de la surface totale de la tumeur p = 0,006). Ces résultats nécessitent d'autres expérimentations qui sont en cours (documentation plus précise de la toxicité, des doses optimales…). En résumé, dans cette thèse, nous montrons la valeur du CBCT dans l'évaluation de la réponse des CRCLM à TACE et nous avons élargi le champ des traitements intra-artériels une nouvelle thérapie par gène suicide qui semble prometteuse.

Mots clés:

Métastases hépatiques de cancer colorectal Traitement intra-artériel hépatiques Chimio-embolisation Cone Beam Computed Tomography Thérapie par gène suicide

A Maxime, André, Jean-François et Marie-Thérèse Pellerin

Médecins

"Exercez-vous à voir large, net et simple, et allez tout droit, paisiblement, sans vous inquiéter de ce qui se dit." Pierre Teilhard de Chardin S.J.

Remerciements

ien u'une th se soit un tra ail personnel, je souhaite ici rendre hommage et exprimer ma profonde gratitude tous ceux ui, de pr s ou de loin, ont contribué sa r alisation et son aboutissement.

TABLE OF FIGURES AND TABLES

Figure 1: A proposed algorithm for selection of systemic treatment and integration with surgical resection in patients with metastatic colorectal carcinoma, based on available clinical and molecular data. 5FU, 5fluorouracil;CAPOX,capecitabine/oxaliplatin;FOLFIRI,FOLFOX,WT, Flow diagram illustrating the effect of intraregional differences in blood flow describe by Dedrick RL. et. al.. (42).C is the drug concentration in the blood (μg/ml); V is the volume of distribution (ml); Q is the arterial blood flow rate (ml/min); I is the infusion rate of the drug μg/min); and the subscripts b, t and h refers, respectively, to the infused remainder of the body, the tumor and the healthy tissue.........................................

Figure 4:

Illustration of the measurement methods of the three response criteria. The gray area within the nodules represents the enhanced viable lesions, and the white area represents non enhanced or iodized oilretaining lesions. A, the maximum diameter of the entire tumor before treatment (pretreatment); A′, the maximum diameter of the entire tumor after treatment (post treatment); A″, the maximum diameter of the enhanced area of the tumor after treatment; B, the diameter perpendicular to A; and B″, the diameter perpendicular to A″ ...............................................................................................................................................

Figure 6a, b, c :

MDCT at the portal phase (Figure 6a), dualphase C-arm CBCT: eAP (Figure 6b), and dualphase Carm CBCT: IAP (Figure 6c) in a53 years old woman with widespread liver invasion by CRCLM. Figure 6a show 6 tumor involving segment II, III, IV, V and VI appeared as hypodens lesion surrounded by liver parenchyma enhanced. The eAP (Figure 6b) show three tumors with moderate peripheral enhancement associated to minimal streak artifact from catheter or contrast media (white arrow head). All the three lesions were classify as uncertain by readers. Figure 6c ................................................................................................. Figure 8 a,b,c,d : Bland and Altman plot for reader 1 at the early arterial phase (8a) and the late arterial phase (8b). Bland and Altman plot for reader 2 at the early arterial phase (8c) and the late arterial phase (8d). .......

Figure 11:

Flow chart shows the acquisition protocol used to generate the parenchymal blood volume images of the liver using an Artis Zee Q System (Siemens). The patient is told to hold his breath shortly before the first rotation (mask run) starts [START_REF] Ferlay | Estimates of cancer incidence and mortality in Europe in 2008[END_REF]. Right after finishing the mask run, automatically triggered contrast injection is started [START_REF] Ferlay | Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008[END_REF] to ensure a constant contrast flow in the liver parenchyma. The C-arm rotates back and immediately starts the second rotation (fill run) [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF]. The overall scanning time is of about 19 s. ........................................... . GDEPT protocol. ..................................................................................................................... Figure 28: the Figure 28a shows representative tumor histology section stained with hematoxilin-eosin-saffron of the control rabbit (group A) (original magnification ×2.5). Multiple VX2 nodules can be seen with large viable (vT) and limited necrotic (nT) tumor tissue surrounded by fibrotic (F) linear area. The Figure 28b shows Representative histology section stained with haematein-eosin-saffron of a tumor from group C2 (original magnification × 2.5) [START_REF] Rosen | Colorectal cancer screening disparities related to obesity and gender[END_REF]:1043-52 (12). ........................................................................................... Figure 33: Conventional chemotherapy and Targeted therapeutic agents. Abbreviations: EGFR, epidermal growth factor receptor; IgG1,immunoglobulin G1;VEGF,vascular endothelial growth factor. ........................ Figure 34: Experimental plan .............................................................................................................................. Figure 35: Animal daily chart. ............................................................................................................................. .............................................................................................................. ......................................................................................................................................................... CRCLM cTACE. nr: non reported,* in months. ................................................. Table 18: DEBIRI relevant publications ............................................................................................................... Table 19: Y90 studies results ...............................................................................................................................

INTRODUCTION

Colorectal cancer is one of the most commonly diagnosed cancers in Europe and worldwide [START_REF] Ferlay | Estimates of cancer incidence and mortality in Europe in 2008[END_REF][START_REF] Ferlay | Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008[END_REF]. Colorectal cancer liver metastases (CRCLM) are a leading cause of death, their occurrence accounts for around 50 -75% of patients during the course of the disease [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF].

Since many years, intra-arterial treatments for colorectal liver metastases (CRCLM)

have led to significant progress; but these treatments still face significant limitations regarding tolerance and results. Further progress could be obtained by better understanding of the intra-procedural imaging features of hepatic metastases and their response to intraarterial treatment.

In addition, while the variety of drugs/device available for intra-arterial injection is slowly growing, there are still significant limitations in the quality and variability of clinical results and variability of response, to such an extent that, so far, IA treatment is not yet a standard treatment.

In this Thesis we will present and discuss our research works on the various methods to explore the peri-procedural response to chemo-embolization treatment and especially the potential role for C-arm CBCT to detect and characterize colorectal cancer liver metastasis.

Also we will thoroughly study, as this is a parallel research topic for our team, the issue of a new intra-arterial treatment the so called "gene directed enzyme prodrug therapy" (GEPD).

Three chapters compose this Thesis.

The first chapter describes the state of art of CRCLM care. Currently available intraarterial approaches are surveyed with rationale, concept, preclinical evidence, embolic materials used, and current clinical results.

The second chapter, entitled "Evidence and pitfalls: value of C-arm CBTC in detecting colorectal liver metastases" presents the research problem and reports our results to assess the C-arm CBCT accuracy for detection and measurement of tumors and the inter observer variability. Experiments were first performed using the rabbit VX2 model of liver tumor and, then, the same methodology was applied to human CRCLM clinical cases.

After the demonstration of the accuracy of C-arm CBCT we worked on the quantification of tumor enhancement before and after trans-arterial chemo-embolization (TACE); aiming at identifying where embolic agent of embolization are located, in order to observe the impact on the blood flow modification before and after TACE, and to correlate to the short-term results. Two approaches were evaluated. The first study focused on tumor attenuation measurement with C-arm CBCT before and after embolization procedure based on clinical cases. The second measured blood volume in-patients receiving chemoembolization using Irinotecan drug eluting beads.

The third chapter reports our ongoing research on a completely different concept of intra-arterial treatment of liver malignancies: the suicide gene therapy consisting of an engineered gene coding for a mutated cytochrome responsible of the metabolism of a prodrug. This new approach uses transduced mesenchymal stem cells (MSCs) as carrier of the suicide gene. Herein are presented the in-vitro and in-vivo results of in a preclinical animal model, e.g., rabbit VX2 liver tumor. Sadly, the majority of patients are diagnosed with advanced CRC disease at presentation.

Although most CRC-related deaths are preventable through screening colonoscopy, it is estimated that less than 50% of eligible patients are screened for CRC [START_REF] Baron | Recommended intervals between screening and surveillance colonoscopies[END_REF]. In the United

States, Black Americans have a higher incidence of CRC compared to Whites, with the disease developing at a younger age and at a higher tumor stage; often, they develop proximal colon cancer. Although not exclusive, left sided and rectal cancer is more common in Whites. Men and women are equally affected with a proximal predominance in women and a distal predominance occurs in men. In developing countries, the incidence rate of CRC in non-Caucasians is low, although it is steadily increasing [START_REF] Rosen | Colorectal cancer screening disparities related to obesity and gender[END_REF].

The spread of primary CRC can occur by lymphatic and hematogenous dissemination, as well as by contiguous and trans-peritoneal routes. The first site of hematogenous dissemination is usually the liver, followed by the lungs and bones. At CRC diagnosis, 20-25% of patients exhibit metastatic disease (stage IV), in which synchronous liver metastases from colorectal cancer (mCRC) are present in 15-25% of cases and metastases are confined to the liver in 70-80% of these cases [START_REF] Adam | The oncosurgery approach to managing liver metastases from colorectal cancer: a multidisciplinary international consensus[END_REF]. The prognosis for patients with untreated liver mCRC is poor; in the Burgundy registry which stands up as a paradigm at the French level, fewer than 30% of patients with untreated disease were alive after 1 year and fewer than 5% survived 5 years after diagnosis [START_REF] Manfredi | Epidemiology and management of liver metastases from colorectal cancer[END_REF]. Data from this fully documented registry also showed that 5-year survival rates were shorter with synchronous than with metachronous liver mCRC (3.3% vs 6.1%, respectively) [START_REF] Manfredi | Epidemiology and management of liver metastases from colorectal cancer[END_REF][START_REF] Conrad | In patients with colorectal liver metastases, can we still rely on number to define treatment and outcome?[END_REF]. The reported percentage of synchronous liver mCRC is increasing compared with metachronous metastases, probably due to improved imaging techniques leading to earlier diagnosis [START_REF] Conrad | In patients with colorectal liver metastases, can we still rely on number to define treatment and outcome?[END_REF].

Genetic and biomarkers

Carcinogenetic mechanisms identified in CRC (11)

The clinical behavior of a colorectal cancer results from genetic interactions at many levels. The current challenges are to understand the molecular basis of individual susceptibility to colorectal cancer and to determine factors that initiate the development of the tumor, drive its progression, and determine its responsiveness or resistance to antitumor agents [START_REF] Perkins | Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer[END_REF].

So far three mechanisms of colon carcinogenesis have been sequentially found out and proposed: i) chromosomal instability (CIN) that concerns about 80% of sporadic CRC as characterized by aneuploidy, chromosomal gains or losses associated with mutations of specific genes (TP53, APC, KRAS & PI3K), ii) genetic instability (15%of CRC) as mainly defined by microsatellite instability (MSI) as well as altered Miss Match Repair (MMR) machinery and

iii) epigenetic modification leading to the CIMP phenotype (CpG Island Methylation) [START_REF] Perkins | Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer[END_REF].

Main altered signaling pathways in CRC (12, 13)

Deregulation of 3 main signaling pathways hallmark CRC, namely:

-The APC pathway resulting from a constitutional alteration of the APC gene, a tumor suppressor gene, the deregulation of which leads to the familial adenomatous polyposis. The APC gene inactivation, as observed in 60-80% of colonic cancers with CIN phenotype, induces an acti ation of the cell cycle through β-catenin interaction.

-The activation of Epidermal Growth Factor Receptor (EGFR), a member of the tyrosine kinase receptors family, triggers an intracellular signaling pathway through RAS / MAPK (mitogen-activated protein kinase), and PI3K (phosphatidylinositol inositol 3 kinase) /

AKT according to a cascade process from the cell membrane to the nucleus, resulting in abnormal cellular proliferation. Mutations of genes such as RAS (N,K & H types), B-RAF as well as PTEN hallmark the deregulation of these intracellular signaling pathways and can be advantageously used as prognostic factors (14, EGFR-KRAS pathway as an example).

-TGF-β and TP53 proteins that negati ely control the cell cycle are frequently inactivated and represent also critical pathways for CRC.

Recent knowledge of these pathways deregulation enable-proposing prediction of anti-EGFR therapies in mCRC and improved treatments.

A molecular presentation of main features and altered signaling pathways is developed in the Annex I.

Treatments of metastatic colorectal cancer (15)

Although the prognosis for patients with metastatic disease without specific treatment remains limited, over the last two decades, the deciphering of signaling pathways described above has led to multiple new medical treatment options of metastatic colorectal cancer (mCRC). As a result, median survival, from approximately 6 months in the 1990s, has increased to nearly 30 months in the latest large randomized study [START_REF] Venook | CALGB/SWOG 80405: Phase III trial of irinotecan/5-FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) with bevacizumab (BV) or cetuximab (CET) for patients (pts) with KRAS wildtype (wt) untreated metastatic adenocarcinoma of the colon or rectum (MCRC)[END_REF][START_REF] Scheithauer | Randomised comparison of combination chemotherapy plus supportive care with supportive care alone in patients with metastatic colorectal cancer[END_REF]. Such an improvement in survival has been driven by the sequential deployment of a variety of seminal chemotherapy combined with the so-called targeted therapeutic agents. This latter class of agents includes monoclonal antibodies directed to various growth factors and their enzymatic machineries: vascular endothelial growth factor (VEGF) (bevacizumab) and epidermal growth factor receptor (EGFR) (cetuximab and panitumumab), aflibercept, a recombinant fusion protein also directed against VEGF, as well as regorafenib, an active inhibitor of multiple tyrosine kinases. Finally, a small but substantial minority of patients with isolated sites of metastases may potentially be curable with surgery and liver-directed therapies. The availability of multiple therapeutic agents for the treatment of mCRC therefore requires a strategic approach to maximize patient benefit, in terms of both life expectancy and quality of life. When choosing the initial treatment, the first step is to evaluate whether the patient is potentially curable by a surgical resection of metastases, either at the time of diagnosis or after conversion therapy. This approach will guide the choice and timing of chemotherapy. Treatments with the potential highest response rates and the greatest potential to downsize metastasis are the most appropriate for potentially curable patients. If the patient does not seem curable, treatment regimens that offer the longest progression-free survival (PFS) and overall survival (OS) and that maintain quality of life as long as possible are to be preferred. More detailed informations are available in Annex II.

Algorithm of treatment

The Figure 1 summarizes the patient algorithm that combines available clinical and molecular data, leading to selection of systemic treatment and integration with surgical resection in patients with mCRC [START_REF] Mi | Integrating systemic and surgical approaches to treating metastatic colorectal cancer[END_REF].

Liver surgery for mCRC

Out of the patients diagnosed with colon and rectal cancer, 50% will develop disseminated disease and a large proportion of metastases will be liver metastases (CRLM) [START_REF] Tzeng | Colorectal liver metastases[END_REF]. Unresectable disease is the norm in these cases but, among the estimated 20% who are able to achieve complete resection, there is an associated overall 5-year survival from 50% to 60%, which is related to a hazard ratio of 1.3 for disease recurrence [START_REF] Tzeng | Colorectal liver metastases[END_REF]. In fact, liver resection is the sole treatment associated with long-term survival in patients with liver mCRC [START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases[END_REF][START_REF] Stangl | Factors influencing the natural history of colorectal liver metastases[END_REF].

One must keep in mind that the surgical management of CRLM is complex. The rationale for hepatic resection is based on the fact that patients are cured by surgery at a rate similar to that for some primary non-metastatic malignancies. The goal of surgery is to obtain a complete tumor resection with safe margins (R0) and, although non-anatomic wedge resections are sometimes sufficient, wider segmental or lobar resections may provide a better chance of surgical success. Overall, the rate of operative resectability is high and a small minority of patients undergoes non-therapeutic laparotomy. Predictors of poor survival include size and number of tumors, short disease-free interval, node-positive primary tumor, positive margins, and the occurrence of extra hepatic disease.

However, these factors do not always exclude patients from resection given the possibility of long-term survival, even in the presence of poor prognostic factors. Indeed, overall mortality after hepatic resection for liver mCRC is less than 1%, likely resulting from improved operative technique and patient selection [START_REF] Tzeng | Colorectal liver metastases[END_REF]. Systemic chemotherapy has improved survival of patients with unresectable liver mCRC, but to date randomized trials have not shown a significant improvement in outcomes with modern regimens given in the perioperative setting [START_REF] Nordlinger | Perioperative chemotherapy with FOLFOX4 and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC Intergroup trial 40983): a randomised controlled trial[END_REF]. Response to neo-adjuvant therapy is not considered as prognostic factor as even patients with progression who remain resectable can experience long-term survival after resection.

In this setting, it is interesting to stress that, for patients with unresectable CRLM, conversion to complete resectability can be obtained using a combination of systemic and Hepatic Arterial Injection (HAI) therapeutic approach [START_REF] Goere | Prolonged survival of initially unresectable hepatic colorectal cancer patients treated with hepatic arterial infusion of oxaliplatin followed by radical surgery of metastases[END_REF].

Systemic therapies for metastatic colorectal cancer

In patients with unresectable CRC that represent most cases, systemic treatment focuses on tumor control. Occasionally the treatment may be symptoms directed (palliative)

and not with a curative aim. The treatment goals are to increase life expectancy while maintaining quality of life for as long as possible. In this context, the model of distinct lines of chemotherapy is being abandoned in favor of a continuum-of-care approach, similar to that taken in other chronic illnesses [START_REF] Goldberg | The continuum of care: a paradigm for the management of metastatic colorectal cancer[END_REF].

The 3 active conventional chemotherapeutic agents for mCRC are fluoropyrimidines (including intravenous 5-fluorouracil or its oral prodrug equivalent, capecitabine), irinotecan, and oxaliplatin. Patients clearly benefit from access to all active agents [START_REF] Goldberg | The continuum of care: a paradigm for the management of metastatic colorectal cancer[END_REF][START_REF] Grothey | Overall survival of patients with advanced colorectal cancer correlates with availability of fluorouracil, irinotecan, and oxaliplatin regardless of whether doublet or single-agent therapy is used first line[END_REF]. A variety of targeted therapeutic agents are also available, which are incorporated into conventional chemotherapy regimens at various time points across the continuum of care.

First-line Treatment: the Chemotherapy Backbone

Data from head-to-head comparisons suggest that outcomes with first-line Oxaliplatin, 5-Fluorouracil, and Leucovorin (FOLFOX) and combination of Irinotecan, 5-Fluorouracil, and Leucovorin (FOLFIRI) are similar [START_REF] Colucci | Phase III randomized trial of FOLFIRI versus FOLFOX4 in the treatment of advanced colorectal cancer: a multicenter study of the Gruppo Oncologico Dell'Italia Meridionale[END_REF]. The choice between initial FOLFIRI and FOLFOX should be based on expected treatment-related toxicity in the context of coexisting co-morbidities for any given patient.

Anti-Vascular Endothelial Growth Factor Agents

Bevacizumab, an anti-VEGF monoclonal antibody, has been shown to be beneficial for mCRC patients in combination with first-line fluoropyrimidine-, oxaliplatin-and irinotecan [START_REF] Hurwitz | Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer[END_REF][START_REF] Kabbinavar | Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer[END_REF][START_REF] Vincenzi | Bevacizumab in association with de Gramont 5-fluorouracil/folinic acid in patients with oxaliplatin-, irinotecan-, and cetuximab-refractory colorectal cancer: a single-center phase 2 trial[END_REF].

Aflibercept, a recombinant fusion protein that binds to VEGF-A with higher affinity than does bevacizumab also constitutes an option of treatment; however, so far, its challenge versus bevacizumab is unknown and, thus, is currently tested in clinical trials.

Anti-Epidermal Growth Factor Receptor Agents

Anti-EGFR agents can improve outcomes in mCRC disease, both as single agents and in combination regimens but, in all cases, they are used pending biomarkers analysis (KRAS status) that optimizes EGFR inhibitor to be used.

Dual antibody therapy targeting both VEGF and EGFR has been tested and found to lead to worsened outcomes and, therefore, has been not recommended [START_REF] Tol | Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer[END_REF][START_REF] Hecht | A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer[END_REF].

Noteworthy, so far the Regorafenib, that targets a variety of kinases implicated in both angiogenic and tumor growth-promoting pathways, shows a modest improvement of survival in mCRC patients treated by standard systemic regimens (conventional and targeted therapies); thus, this agent is strictly reserved for refractory cases [START_REF] Grothey | Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial[END_REF].

Maintenance regimens

Approximately 75% of patients discontinue first-line chemotherapy in trials for reasons other than progressive disease; this evidence addresses the question of whether to considering maintenance chemotherapy or taking chemotherapy break. The OPTIMOX and CAIRO-3 trials had been performed to deal with this issue but did not conclude definitively [START_REF] Simkens | Maintenance treatment with capecitabine and bevacizumab in metastatic colorectal cancer (CAIRO3): a phase 3 randomised controlled trial of the Dutch Colorectal Cancer Group[END_REF]. Thus, decisions regarding maintenance therapy versus treatment breaks must also take into account patient preferences and cost [START_REF] Simkens | Maintenance treatment with capecitabine and bevacizumab in metastatic colorectal cancer (CAIRO3): a phase 3 randomised controlled trial of the Dutch Colorectal Cancer Group[END_REF].

Potentially curable advanced colorectal cancer

The liver is the first site of hematogenous dissemination in most patients with colorectal cancer. Approximately 10% of patients can live past 5 years even with metastatic disease. The definition of resectable metastatic disease is evolving, and there is not an accepted standard yet: even bilobar metastases and extra-hepatic disease are no longer considered contra-indications. Decision-making in this setting requires multidisciplinary collaboration: a practical approach requires that patients should be medically fit for surgery, existing liver disease should be resectable with adequate liver remnant, and extra-hepatic disease should be controlled. Surgical resection is the preferred treatment in patients with oligo-metastatic disease primarily in the liver, with 5-year survival rates of approximately from 50% to 60% in patients with favorable prognostic features [START_REF] Rees | Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients[END_REF]. Approximately onefifth of such patients survive beyond 10 years in some series, and this applies also to non-hepatic disease in some series [START_REF] Tomlinson | Actual 10-year survival after resection of colorectal liver metastases defines cure[END_REF][START_REF] Chau | A VELOUR post hoc subset analysis: prognostic groups and treatment outcomes in patients with metastatic colorectal cancer treated with aflibercept and FOLFIRI[END_REF]. When the primary tumor site is controlled and the metastatic disease is limited to lungs without extra-pulmonary location (except for resectable or resected hepatic lesion), resection of isolated pulmonary metastases can increase survival rates up to 40% at 5 years [START_REF] Chau | A VELOUR post hoc subset analysis: prognostic groups and treatment outcomes in patients with metastatic colorectal cancer treated with aflibercept and FOLFIRI[END_REF]. Patients with initially unresectable liver metastases may become eligible for resection if the response to chemotherapy is sufficient.

This approach has been termed conversion therapy to distinguish it from neo-adjuvant therapy. Conversion therapy allows 12% to 33% of initially unresectable or borderline resectable metastases to become eligible for metastasectomy [START_REF] Masi | Long-term outcome of initially unresectable metastatic colorectal cancer patients treated with 5fluorouracil/leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) followed by radical surgery of metastases[END_REF]. The five-year survival average rate is 30% to 35%, which is substantially better than expected with chemotherapy alone.

Generally, a regimen leading to a likelihood of objective response is preferred to that expected with chemotherapy alone, but so far no guideline has been proposed. For instance the triplet chemotherapy regimen FOLFOXIRI plus bevacizumab has been shown to better response rates and PFS than FOLFIRI plus bevacizumab according to a recent phase III TRIBE trial [START_REF] Masi | Long-term outcome of initially unresectable metastatic colorectal cancer patients treated with 5fluorouracil/leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) followed by radical surgery of metastases[END_REF]. With regards to wild type K-RAS patient treated by a combination of anti-EGFR inhibitor with either irinotecan-based or oxaliplatin-based regimens, the improvement of resection rates remains modest. [START_REF] Adam | Patients with initially unresectable colorectal liver metastases: is there a possibility of cure?[END_REF].

Liver-Directed Treatments

Several other regional therapies, including local tumor ablation, regional hepatic intra-arterial chemotherapy or chemo-embolization, and radio-embolization, stereotaxic body radiation therapy, had been proposed as options for liver-isolated mCRC patients who are not candidate for surgery [START_REF] Goere | Prolonged survival of initially unresectable hepatic colorectal cancer patients treated with hepatic arterial infusion of oxaliplatin followed by radical surgery of metastases[END_REF][START_REF] Fiorentini | Intra-arterial infusion of irinotecan-loaded drug-eluting beads (DEBIRI) versus intravenous therapy (FOLFIRI) for hepatic metastases from colorectal cancer: final results of a phase III study[END_REF]. These therapies are often incorporated with initial hepatic resection or used as alternatives in patients who present not medically fit enough to undergo surgical resection. Although these methods can provide excellent local control, so far, long-term survival outcomes have not studied in depth [START_REF] Pellerin | Intra-arterial treatment of liver metastases from colorectal carcinoma[END_REF]. Intra-arterial liver directed treatment clinical outcomes are presented in Annex III.

Hepatic Intra-arterial Treatment (HIAT) for colorectal cancer liver metastasis

When metastatic disease is still confined to the liver, hepatic intra-arterial treatment is a complementary approach for loco-regional disease control. Furthermore, even when the disease has spread beyond the liver, (e.g. small lung deposits) coexisting with large hepatic metastases, the patient's life is immediately threatened by metastatic progression in the liver much more than by other metastatic location. Thus effective loco-regional hepatic therapy could still prolong survival. The following features are indeed very attractive: i) high therapeutic agent exposure to the tumor (enable of overcoming drug resistance mechanisms); ii) targeted delivery of the therapeutic agent while both extra-hepatic tissues and healthy hepatocytes are relatively protected. Moreover, arising microscopic liver metastases, newly so-called "missing" metastases could be treated in the same manner. A summary regarding clinical outcomes of patients treated by intra-arterial liver is presented in Annex III.

Concept and rationale for hepatic intra-arterial treatment

Intra-arterial (IA) therapy is defined as the local delivery of any active agent such as a cytotoxic pharmacological or a radio-nucleotide agent, combined or not to the occlusion of tumor arterial network by embolic material. This embolic material can be bland or carry the therapeutic agent itself.

Optimization of this approach will need to consider the respective role of several key points including the metastases characteristics, the liver vascular input, and the pharmacokinetic advantage of a drug when locally delivered.

Tumor arterial feeders

Metastases basically originate during passage of tumor cells from the primitive colon tumor through the portal system resulting in the seeding into the liver. Once initiated, metastases derive exclusively their own vascularization from the arterial network, therefore supporting the rationale that using the IA route will exhibit a high potential to reach the tumor cells through its natural vascularization [START_REF] Dezso | Development of arterial blood supply in experimental liver metastases[END_REF].

Liver vascularization allows safe occlusion of the arterial input

Two distinct vascular input systems constitute the liver vascularization. The portal system is involved in 75% of the liver vascular input. It provides all nutrients from the digestive tract, and directly vascularizes hepatocytes. The hepatic artery derives from aorta and leads the 25% remaining of the blood supply. Thus, the ratio between the two vascular inputs supports the safety of arterial occlusion without liver function damage as long as portal flow is not compromised [START_REF] Liapi | Transcatheter arterial chemoembolization: current technique and future promise[END_REF]. The Figure 2 shows the liver and tumor vascular in-put. described the arterial drugs PK with the intention to homogenize results [START_REF] Eckman | A critical evaluation of the principles governing the advantages of intra-arterial infusions[END_REF]. The concept of pharmacokinetic advantage of IA infusion was engineered. The PK advantage of intra-arterial chemotherapy is calculated by the comparison of drug delivery to the liver following intraarterial and intravenous (IV) injection. The entire advantage of regional delivery must be accrued on first pass through the liver. Once the drug has entered the systemic circulation, it behaves as if it is injected IV. The overall selectivity for regional administration (Rd) can be expressed as the ratio of the steady state drug concentrations in the liver (C Liver) for IA and IV injection divided by the ratio of systemic concentrations (C systemic) for the two routes of administration (IA and IV), as expressed in the following equation 1:

( ) ( )
In this model, it is assumed that the drug is infused at a constant rate, into an artery until steady state is achieved. The concentration is given as the area under the concentration time curve (AUC). However liver drug concentration is difficult to determine without direct tissue chromatography examination. To overcome this difficulty, alternative formulae for assessing regional advantage have been derived. The pharmacokinetic advantage (Rd) of IA administration will be determined by the factors that govern Fickian diffusion. For arterial infusions this is determined by i) the hepatic arterial flow rate (Q), ii) the rate of drug elimination from the body or total body clearance (CL tb), and iii) the extraction ratio of the drug during first pass through the liver (E), as proposed by equation 2:

( )

It is readily shown that where CLtb is the total body clearance obtained during intravenous infusion, Q is the blood flow rate to the region, and E is the fraction of drug that is extracted during a single pass through the region while (1 -E) is the fraction that has not been extracted.

Thus, the ideal cytotoxic drug for intra-arterial hepatic therapy should have the following characteristics: i) high total body elimination rates (CL tb), ii) high hepatic extraction rates (E) and iii) low hepatic perfusion rates (Q).

Hepatic extraction of cytotoxic agent and tumor blood flow are the two crucial factors that can modify the liver drug pharmacokinetic.

Hepatic extraction ratios for cytotoxic drugs have been determined experimentally and are summarized in Table 1.

)
Blood flow is the major parameter able to modify the pharmacokinetic advantage of a drug. However, so far the difference of drug concentration existing in healthy liver versus tumor has not been firmly established. It seems logical that increased blood flow to a site within a liver region should increase drug delivery to that site. Drug upload is increased, but the mean time drug download is increased too. Thus, the pharmacokinetic advantage is not necessarily obtained.

To evaluate local effect, it is theoretically possible to separate the tumor and the healthy tissue in 2 separate compartments. Tumor blood flow can be enhanced by the infusion of selective vasoconstrictors (angiotensin II), thus maintaining or even doubling the blood flow to liver metastases. This experimental condition is valid for a low-extraction drug.

If the drug is highly extracted on a single pass through the tumor but not through the normal tissue, then redistribution of blood flow could lead to greater tumor exposure.

It is also possible to increase the arterial pharmacokinetic advantage of a drug by reducing the blood flow. This supports the use of intravascular occlusion of the hepatic artery as a combination with local drug delivery, and is clinically known as chemo- Dedrick RL. et. al.. (42).C is the drug concentration in the blood (μg/ml); V is the volume of distribution (ml); Q is the arterial blood flow rate (ml/min); I is the infusion rate of the drug μg/min); and the subscripts b, t and h refers, respectively, to the infused remainder of the body, the tumor and the healthy tissue.

The Flow diagram presented in Figure 3, shows the effect of intraregional differences in blood flow (Q). The drug concentration entering the region is equal to the arterial blood concentration (Cb), plus the increment in concentration l/Q, caused by the infusion. This concentration Cb + I/Q, is the same both for tumor and healthy tissue. If neither tumor nor healthy tissue removes a significant amount of drug, then the pharmacokinetic advantage is identical for both and is also independent of the individual values of intraregional blood flows, Qt and Qh. The pharmacokinetic advantage calculated from equation 2 depends only on total regional blood flow (Q), and total body clearance (Cl).

Trans Arterial Chemo-embolization (TACE)

Chemo-embolization was first described in the 1980', as a combination of an increase in local drug concentration with local ischemia by means of embolization [START_REF] Kato | Clinical observations on chemoembolization for genitourinary cancer. I. Chemoembolization of kidney cancer with microencapsulated anticancer drug (author's transl)][END_REF]. The injection of embolic material in an artery generates arterial occlusion. Depending on the embolic material size, the local hemorheologic conditions will be changed downstream to the embolization point. In case of truncular occlusion, this will induce downstream blood pressure decrease, whereas in case of distal occlusion the embolic material is responsible of ischemia and necrosis [START_REF] De Luis | In vivo evaluation of a new embolic spherical particle (HepaSphere) in a kidney animal model[END_REF][START_REF] Khankan | Embolic effects of superabsorbent polymer microspheres in rabbit renal model: comparison with tris-acryl gelatin microspheres and polyvinyl alcohol[END_REF][START_REF] Laurent | Arterial distribution of calibrated tris-acryl gelatin and polyvinyl alcohol embolization microspheres in sheep uterus[END_REF]. In most of the current available treatment, the embolic material is generally packed within a common vector carrying the therapeutic agent.

Currently, two classes of material are used: Lipiodol® and drug eluting beads.

Lipiodol® (Lipiodol® Guerbet, Roissy-France)

Lipiodol®, an ethiodized oily iodinate contrast media, has noticeable properties: i) high viscosity allowing temporary vessel occlusion, ii) lipophily responsible of specific affinity for primary and secondary hepatic tumors. Lipiodol® is prepared in a water/oil manner as an emulsion with chemotherapy encapsulated in micro droplets (30 μm). When injected to the tumor vascular bed, droplets create occlusion thus increasing the contact time between the tumor and the chemotherapy (50).

Drug eluting beads

Drug eluting beads are biocompatible microspheres that are calibrated, nonresorbable, and loadable. These microspheres elicit drug release properties in the arterial tumor network. Microspheres loading operate by electrochemical binding according to a reversible ionic exchange between a positive radical of the drug and the negative sulfoxide radical contained in the bead. Doxorubicin and irinotecan are the only drugs commercially approved for clinical use in this situation. Beads release chemotherapy through a concentration gradient from vascular bed network to the tumor [START_REF] Willis | The interface of medical devices and pharmaceuticals: Part I[END_REF][START_REF] Willis | The interface of medical devices and pharmaceuticals: Part II[END_REF].

Lipiodol® vs drug eluting beads: advantages and drawbacks

Microspheres elicit several advantages: i) calibration of sphere ensuring standardization and reproducibility, ii) hyper-selectivity of the microspheres-chemotherapy complex, iii) control of chemotherapy pharmacokinetics, iv) lasting with a weak systemic passage [START_REF] Lewis | DC bead: in vitro characterization of a drug-delivery device for transarterial chemoembolization[END_REF][START_REF] Varela | Chemoembolization of hepatocellular carcinoma with drug eluting beads: efficacy and doxorubicin pharmacokinetics[END_REF]. On both pharmacodynamic and pharmacokinetic points of view, this technique enables a highly significant and lasting chemo-toxicity towards hepatic tumor cells [START_REF] Namur | Embolization of hepatocellular carcinoma with drug-eluting beads: doxorubicin tissue concentration and distribution in patient liver explants[END_REF][START_REF] Namur | Safety and efficacy compared between irinotecan-loaded microspheres HepaSphere and DC bead in a model of VX2 liver metastases in the rabbit[END_REF][START_REF] Namur | Drug-eluting beads for liver embolization: concentration of doxorubicin in tissue and in beads in a pig model[END_REF]. The major limit of drug eluting material is that only a few drugs can be loaded because of the need of particular electrochemical properties. Up to now, only anthracyclin and irinotecan can be used, despite they are not the best drugs to treat liver cancer.

The iscous li uid nature of Lipiodol® allows embolic ad antage. A "front door/backdoor" embolization is performed by droplets translocation from the arterial side to the portal side through periportal space [START_REF] Kan | Ethiodized oil emulsions in hepatic microcirculation: in vivo microscopy in animal models[END_REF]. It has been demonstrated that super selective TACE using Lipiodol® allows consistent delivery of the Lipiodol®-drug combination into HCC lesions including the whole periportal space [START_REF] Kan | Ethiodized oil emulsions in hepatic microcirculation: in vivo microscopy in animal models[END_REF]. In contrast to drug eluting particles, Lipiodol® is a polyvalent drug carrier agent and many different drugs can be prepared as emulsion with Lipiodol® [START_REF] Idee | Use of Lipiodol as a drug-delivery system for transcatheter arterial chemoembolization of hepatocellular carcinoma: a review[END_REF]. However, so far, that has not been demonstrated

for CRCLM.

Trans Arterial Radio-Embolization (TARE) Y90 Radiation(59)

Trans Arterial Radio-embolization consists in intra-hepatic administration of yttrium 90 (Y90) labeled microspheres. When administered within the liver arterial network, the Y90 labeled microspheres lodge themsel es into tumor neo ascularization. The β-radiation emitted from the Y-90 microspheres is then delivered directly to the tumor core while the healthy li er tissue is preser ed [START_REF] Rees | Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients[END_REF]. β-radiation interacts with the DNA while cells are in the S or their G2 mitosis phase cycle, and creates DNA abnormalities responsible of cells death. In this approach, beads are the carrier/vector of the radiation, without theoretically playing any embolization role. Noteworthy, radiation needs to be done in normoxia condition, while hypoxemia or ischemia does not favor radiation therapy.

Hepatic intra-arterial chemotherapy (HIAC)

The concept of intra-arterial chemotherapy is to deliver into the liver tumor, a drug at higher dose that could not be obtainable systemically. It does not combine any embolization except the one performed during catheter insertion (flow redistribution, hepatic artery skeletonisation). The main hypothesis is that the sensitivity of metastases is directly related to the concentration of the drug inside the tumor. This technique is also free from the hepatic first-pass effect of the drug. A silicone catheter is implanted directly from the common femoral artery into the common hepatic artery. Its distal end is placed downstream of the gastro-duodenal artery to prevent chemotherapy reflux in the digestive tract. Its tunneled proximal end is connected to an implantable port placed on the iliac crest so as to be easily connected to an injection system with a Huber needle. Chemotherapy is delivered directly into the hepatic arterial system in several sessions eg. once every 2 weeks. In a number of cases, intra-hepatic hemodynamic circumstances do not permit good distribution of chemotherapy. For these reasons, the interventional radiologist who places the catheter proceeds with hepatic artery skeletization by multiple embolization (gastroduodenal, right gastric, sickle artery...) in order to produce a selective redistribution of blood flow to the liver targeted territories. An angiography is routinely performed prior to intervention to check the permeability of hepatic arterial network and drug distribution territories. The technical success of implantation reach's between 92 and 100% [START_REF] Deschamps | Percutaneous femoral implantation of an arterial port catheter for intraarterial chemotherapy: feasibility and predictive factors of long-term functionality[END_REF]. Patency rates at 6, 12

and 24 months are 91%, 81%, and 58%, respectively [START_REF] Tanaka | Radiologic placement of side-hole catheter with tip fixation for hepatic arterial infusion chemotherapy[END_REF]. The most frequent complication is peptic ulcer that may occur when reflux chemotherapy appears in the digestive tract. This side effect can be greatly reduced by the routine prophylactic embolization of the gastroduodenal and right gastric arteries. The chemical cholecystitis remains an exceptional event that does not require the cystic artery embolization [START_REF] Herrmann | Liver intraarterial chemotherapy: use of the femoral artery for percutaneous implantation of catheter-port systems[END_REF]. Thrombosis of the hepatic artery is a rare event and also is directly related to the size of the catheter placed in the hepatic artery. Port and catheter infection rate is about 2% (60).

ND CHAPTER: EVIDENCE AND PITFALLS: VALUE OF C-ARM CBCT IN DETECTING COLORECTAL LIVER METASTASES

Role of conventional angiography and potential role for C-arm CBCT

All Intra-arterial liver directed treatment need catheters insertion into the vascular network for navigation from the skin arterial access to the target lesion. C-arm X-ray generator/X-ray detector systems allow 2-Dimensional (2D) imaging such as fluoroscopy and digital subtraction angiography (DSA) images. Fluoroscopy gives a real-time image useful for navigation, as DSA frame provides static image suitable to tumor and vascular network detection. In addition, DSA is performed to assess procedure endpoint. Embolization endpoints are usually defined as tumor blush decrease and a contrast motion slow-down or total occlusion of the target arteries [START_REF] Orth | Technology Assessment Committee of the Society of Interventional R. C-arm cone-beam CT: general principles and technical considerations for use in interventional radiology[END_REF].

C-arm cone-beam computed tomography (C-arm CBCT)

Modern C-arm systems provide a 3-Dimentional (3D) CT X-ray imaging, in addition to [START_REF] Busse | Design and performance of a high-quality cardiac flat detector[END_REF]. Projection acquisition includes the use of onedimensional anti-scatter grids to suppress scattered photons.

The projections of the rotational scan are transferred directly to the reconstruction computer to produce volumetric data. The C-arm CBCT reconstruction uses a modified Feldkamp filtered back-projection. That is a straightforward generalization of the 2D filtered back-projection to cone-beam geometry [START_REF] Feldkamp | Practical cone-beam algorithm[END_REF]. Tomographic reconstruction on flat panel Carm systems strictly requires an accurate measurement of the true system position, which must be taken into account during back projection. This is determined from the geometric calibration. Parker weighting is applied to compensate line integrals measured more than once and a source attenuation correction is applied to correct for non-equiangular sampling during acceleration and deceleration [START_REF] Parker | Optimal short scan convolution reconstruction for fanbeam CT[END_REF].

Therefore, although the pixel size in flat-panel detectors theoretically allows for voxel sizes of 0.008 mm 3 , blur caused by the x-ray converter and reconstruction filter, protracted reconstruction times, patient dose considerations, and general lack of clinical necessity result in practical C-arm CBCT spatial resolutions similar to, if not slightly larger than, those of multidetector CT.

Data acquired from the rotational acquisition are automatically reconstructed and displayed on the workstation as a 3D rendering and/or in axial, coronal, sagittal, or oblique slice. C-arm CBCT images can be post-processed and manipulated on a separate 3D workstation. Post-processing software's are a ailable for different applications to enhance the spatial resolution, to correct for photon starvation or artifacts due to metal objects, or to quantify anatomic information such as vascular length and diameter or the size of a lesion [START_REF] Wallace | Threedimensional C-arm cone-beam CT: applications in the interventional suite[END_REF].

Value of C-arm CBCT

Since its introduction in 2005, the utility of C-arm CBCT has been demonstrated in multiple interventions and especially in interventional oncology [START_REF] Wallace | Threedimensional C-arm cone-beam CT: applications in the interventional suite[END_REF][START_REF] Wallace | Three-dimensional C-arm cone-beam CT: applications in the interventional suite[END_REF]. Because it allows to create 3D visualization of the vascular structures (high-contrast imaging) as well as softtissue tomography (low-contrast imaging), it has opened the door to several new applications. In particular, C-arm CBCT has improved the feasibility, effectiveness, and safety of many image-guided procedures, thereby leading Interventional Radiologists to perform procedures that were not possible by using traditional fluoroscopy or digital subtraction angiography (DSA) alone. In the setting of liver cancer, C-arm CBCT has been extensively studied for TACE especially for tumor detection, depiction of tumor arterial feeders, intraprocedural navigation, and assessment of treatment success [START_REF] Wallace | Threedimensional C-arm cone-beam CT: applications in the interventional suite[END_REF]. For example, C-arm CBCT has been shown to affect diagnosis and treatment in up to 81% of HCC hepatocellular carcinoma lesions during TACE, resulting in improvement of local progression free survival and overall survival [START_REF] Loffroy | Intraprocedural C-Arm Dual-Phase Cone-Beam CT: Can It Be Used to Predict Short-term Response to TACE with Drug-eluting Beads in Patients with Hepatocellular Carcinoma? Radiology[END_REF].

In practice, CBCT can be used to see the tumor, to reach the tumor and to assess treatment success during TACE.

C-arm CBCT to "See" the Tumor

Pre-procedural diagnostic imaging provides information to plan the intervention and confirms the indication, while intra-procedural imaging guides the intervention. Images obtained during the procedure are compared with those from pre-procedural diagnostic imaging, including contrast-enhanced MR imaging and/or multiphasic multidetector CT to confirm that the initial plan matches with the intra-procedural plan. In the setting of highly hyper-vascular tumor like HCC, it is recognized that high dose TACE is optimally delivered using hyper-selective catheterization. Accurate tumor localization increases the selectivity of drug delivery into the targeted tumors and limits non-target embolization, thereby preserving healthy liver tissue and optimizing tumor response [START_REF] Bouvier | Transarterial chemoembolisation: effect of selectivity on tolerance, tumour response and survival[END_REF][START_REF] Miyayama | Ultraselective transcatheter arterial chemoembolization with a 2-f tip microcatheter for small hepatocellular carcinomas: relationship between local tumor recurrence and visualization of the portal vein with iodized oil[END_REF]. In literature, the ability of C-arm CBCT to enable visualization of HCC lesions has been evaluated for its sensitivity and specificity in HCC detection, when compared with other diagnostic imaging modalities. C-arm CBCT is superior to DSA in the detection of HCC and can achieve a degree of intra-procedural HCC detection comparable to pre-procedural diagnostic imaging including contrast-enhanced MR imaging and multiphasic multidetector CT [START_REF] Meyer | Visualization of hypervascular liver lesions During TACE: comparison of angiographic C-arm CT and MDCT[END_REF][START_REF] Higashihara | Diagnostic accuracy of C-arm CT during selective transcatheter angiography for hepatocellular carcinoma: comparison with intravenous contrast-enhanced, biphasic, dynamic MDCT[END_REF][START_REF] Iwazawa | Detection of hepatocellular carcinoma: comparison of angiographic C-arm CT and MDCT[END_REF]. Meyer BC. et al. (72) [START_REF] Higashihara | Diagnostic accuracy of C-arm CT during selective transcatheter angiography for hepatocellular carcinoma: comparison with intravenous contrast-enhanced, biphasic, dynamic MDCT[END_REF][START_REF] Iwazawa | Detection of hepatocellular carcinoma: comparison of angiographic C-arm CT and MDCT[END_REF].

C-arm CBCT to "Reach" the Tumor

Determination of tumor arterial supply is critical to achieve a complete tumor treatment and to avoid non-target embolization [START_REF] Virmani | Effect of Carm angiographic CT on transcatheter arterial chemoembolization of liver tumors[END_REF][START_REF] Wang | Cystic artery localization with a three-dimensional angiography vessel tracking system compared with conventional two-dimensional angiography[END_REF]. The post processing of the C-arm CBCT image allows a comprehensive visualization of hepatic arterial anatomy and tumor arterial feeders. Thus it is useful to determine the degree of selectivity of the chemoembolization to the targeted tumors, to reduce the risk of non-target embolization. Several navigation softwares have been developed by the industry. These applications proceed with segmentation of the tumor and its feeders, while this arterial mesh is overlaid on the fluoroscopy screen [START_REF] Miyayama | Identification of small hepatocellular carcinoma and tumor-feeding branches with conebeam CT guidance technology during transcatheter arterial chemoembolization[END_REF]. This can result in better depiction of feeders and thanks to some software, guiding the IR in the super selective catheterism can be obtained. This type of application has been prospectively evaluated. Miyayama S. et al. (78) evaluated prospectively the performance of guidance software in identifying small HCC tumors and their feeders in 68 HCC <30mm during cTACE. Manual feeder vessel detection software was used. The authors compared the detectability of tumors and tumor-feeding sub-subsegmental arteries to nonselective digital subtraction angiography (DSA). Technical success of cTACE was divided in 3 grades according to 1-week CT findings: entire tumor embolized with at least a 5-mm-wide margin (i.e., complete), tumor embolized without a margin in parts (i.e., adequate), or entire tumor not embolized (I.e., incomplete). As a result, C-arm CBCT depicted all tumors. Non selective DSA (injection into the proper hepatic artery) depicted 49 tumors (72.1%). Among 100 tumor-feeding vessels, 81 were identified with guidance software and 38 with nonselective DSA.

Detectability of tumors with C-arm CBCT and tumor-feeding branches with guidance software was significantly better than that obtained with nonselective DSA (both p<.001).

Fifty-nine tumors (86.8%) were graded as completely embolized and nine (13.2%) were graded as adequately embolized.

Deschamps F. et al [START_REF] Deschamps | Computed Analysis of Three-Dimensional Cone-Beam Computed Tomography Angiography for Determination of Tumor-Feeding Vessels During Chemoembolization of Liver Tumor: A Pilot Study[END_REF] evaluated computed analysis of 3D C-arm CBCT of the liver for determination of sub-segmental tumor-feeding vessels. In this pilot study 18 consecutive patients (12 HCC and 6 NET) underwent cTACE. Feeding arteries were independently identified using automatic arterial segmentation software and compared to DSA (2D) and manual inspection on C-arm CBCT images (3D). Authors found a higher sensitivity of 3D (73%) than 2D (64%) images for identification of feeders (p = 0.036). The sensitivity of the automatic software (93%) was higher than 2D (P = 0.02) and 3D (P = 0.005) imaging. The duration for review of 3D imaging was longer than that for 2D imaging (187 vs. 94 s, p = 0.0001) or for automatic software (135 s, p = 0.0001). The degree of agreement between the 3 operators using 2D and 3D imaging were 54% and 62%, respectively, whereas it was 82% between the three operators using automatic segmentation software. All together, these data show that computed determination of arterial feeders is both accurate and sensitive.

Iwazawa J. et al. [START_REF] Iwazawa | Identifying feeding arteries during TACE of hepatic tumors: comparison of C-arm CT and digital subtraction angiography[END_REF] compared the diagnostic accuracy of C-arm CBCT to DSA in identifying tumor-feeding arteries during cTACE. For this purpose 33 consecutive patients with 58 HCC referred for cTACE were studied. The DSA and C-arm CT were sequentially performed for each studied artery. Four independent observers independently reviewed the DSA and C-arm CT images and used a 5-point grading scale to determine whether a studied artery supplied the target tumor. Diagnostic performance was compared using receiver operating characteristic (ROC) analysis. Sensitivity, specificity, and accuracy were calculated for arteries rated as definite or probable tumor feeders. Iodized oil accumulation on followup MDCT was the reference standard. Fifty-eight possible feeding arteries in 33 patients were examined. Among the studied arteries, follow-up CT confirmed that 33 were true tumor-feeding arteries, and the remaining 25 were not at all. C-arm CBCT resulted in a significantly larger area under the ROC curve (Az = 0.995) compared with DSA (Az = 0.841).

The sensitivity, specificity, and accuracy of C-arm CBCT (96.9%, 97.0%, and 96.9%, respectively) were significantly higher than those for DSA (77.2%, 73.0%, and 75.4%).

Wang X. et al. [START_REF] Wang | Cystic artery localization with a three-dimensional angiography vessel tracking system compared with conventional two-dimensional angiography[END_REF], compared the performance of vessel tracking software based on 3D angiography versus standard 2D (DSA) for identifying the cystic artery, in a prospective cohort of 25 patients referred to TACE. This team showed that cystic artery arterial take off was correctly located in tracking with the software in 96% of the cases. By contrast, DSA allowed the correct location of the cystic artery in 48% (p < .001).

In summary, regarding identification of arterial feeders of HCC lesions, the sensitivity, the specificity and the accuracy of C-arm CBCT in the detection of the tumor-feeding artery are all superior to DSA (97% vs. 77% and 97% vs. 73%, 97% vs. 75% respectively) [START_REF] Miyayama | Identification of small hepatocellular carcinoma and tumor-feeding branches with conebeam CT guidance technology during transcatheter arterial chemoembolization[END_REF][START_REF] Deschamps | Computed Analysis of Three-Dimensional Cone-Beam Computed Tomography Angiography for Determination of Tumor-Feeding Vessels During Chemoembolization of Liver Tumor: A Pilot Study[END_REF][START_REF] Iwazawa | Identifying feeding arteries during TACE of hepatic tumors: comparison of C-arm CT and digital subtraction angiography[END_REF].

So far, liver colorectal cancer metastases have not been studied with the same objective.

C-arm CBCT to Assess Treatment Success in an intraprocedural manner

Context: Evaluation of tumor response using cross sectional imaging

Assessing treatment success during TACE is critically important as it affects treatment endpoints and consequently tumor outcomes, local progression-free and, finally, overall survival [START_REF] Miyayama | Identification of small hepatocellular carcinoma and tumor-feeding branches with conebeam CT guidance technology during transcatheter arterial chemoembolization[END_REF][START_REF] Iwazawa | Survival after C-arm CTassisted chemoembolization of unresectable hepatocellular carcinoma[END_REF]. The usual timing for evaluation of local treatment success of HCC and NETs tumors is one month as it is two months for CRCLM with MRI or MDCT.

In contrast, immediate imaging during TACE can provide immediate assessment of tumor coverage and offer the possibility to change catheter positioning to ensure complete treatment of tumor burden and even to predict tumor response [START_REF] Loffroy | Intraprocedural C-arm dual-phase cone-beam CT: can it be used to predict short-term response to TACE with drug-eluting beads in patients with hepatocellular carcinoma?[END_REF][START_REF] Riaz | Imaging response in the primary index lesion and clinical outcomes following transarterial locoregional therapy for hepatocellular carcinoma[END_REF][START_REF] Monsky | Semiautomated segmentation for volumetric analysis of intratumoral ethiodol uptake and subsequent tumor necrosis after chemoembolization[END_REF]. Incomplete tumor treatment negatively impacts survival [START_REF] Jeon | Iodized oil uptake assessment with cone-beam CT in chemoembolization of small hepatocellular carcinomas[END_REF][START_REF] Iwazawa | C-arm CT for assessing initial failure of iodized oil accumulation in chemoembolization of hepatocellular carcinoma[END_REF]. The imaging characteristics of different chemo-embolic material differ substantially, requiring thus specific post processing as summarized here.

Imaging modalities include ultrasound, contrast-enhanced ultrasound, computed tomography (CT), and magnetic resonance imaging (MRI) and positron emission tomography (PET-CT). The best methods for staging are CT and MRI. A review of the literature indicates that MRI is more sensitive than CT for liver lesions detection [START_REF] Scharitzer | Preoperative evaluation of colorectal liver metastases: comparison between gadoxetic acidenhanced 3.0-T MRI and contrast-enhanced MDCT with histopathological correlation[END_REF][START_REF] Rojas Llimpe | Imaging in resectable colorectal liver metastasis patients with or without preoperative chemotherapy: results of the PROMETEO-01 study[END_REF][START_REF] Lee | Diagnostic performance of gadoxetic acid-enhanced liver MR imaging in the detection of HCCs and allocation of transplant recipients on the basis of the Milan criteria and UNOS guidelines: correlation with histopathologic findings[END_REF].

Multi-phasic contrast-enhanced MRI is accepted as a gold standard for diagnosing liver tumors and assessing treatment response after locoregional therapy, especially following TACE [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF][START_REF] Lee | Hepatic tumor response evaluation by MRI[END_REF]. Measuring changes in tumor size (Response Evaluation Criteria in Solid Tumors, RECIST), enhancement (European Association for the Study of the Liver, EASL), and tumor enhancement size (modified RECIST, mRECIST) on MRI are the three accepted methods to assess response to TACE (Figure 4) [START_REF] Kamel | Unresectable hepatocellular carcinoma: serial early vascular and cellular changes after transarterial chemoembolization as detected with MR imaging[END_REF].

RECIST evaluates response to treatment based tumor size changes and thus constitutes an indicator of treatment response. Targeted lesions are measured using a single linear summation, and specific attention is given to reproduce all measurements at the largest tumor diameter during follow-up. Tumor enhancement or necrosis is not used to assess treatment efficacy.

EASL evaluates response to treatment based on changes in tumor enhancement change since tumor enhancement or necrosis is reflecting treatment efficacy. The tumor enhancement is evaluated on a representative axial slice of the tumor. The enhancement is quantifying based on visual inspection of and grouped into quartile brackets [START_REF] Kamel | Unresectable hepatocellular carcinoma: serial early vascular and cellular changes after transarterial chemoembolization as detected with MR imaging[END_REF][START_REF] Liu | EASL-and mRECIST-evaluated responses to combination therapy of sorafenib with transarterial chemoembolization predict survival in patients with hepatocellular carcinoma[END_REF].

mRECIST evaluates response based on changes in size of tumor enhancement, exclusively in a single axial slice [START_REF] Lencioni | Modified RECIST (mRECIST) assessment for hepatocellular carcinoma[END_REF]. Although these three methods are widely used, they have some limitations when applied to TACE for primary and secondary liver tumor.

In the setting of HCC, TACE results in inhomogeneous tumor necrosis, and as a consequence, tumor enhancement becomes heterogeneous and changes in tumor size are inhomogeneous [START_REF] Lim | Hepatocellular carcinoma: evaluation of therapeutic response to interventional procedures[END_REF]. This makes current methods of tumor response assessment difficult after TACE. Furthermore, RECIST, mRECIST, and EASL are applied to one representative axial slice of the tumor [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF][START_REF] Lencioni | Modified RECIST (mRECIST) assessment for hepatocellular carcinoma[END_REF][START_REF] Lim | Hepatocellular carcinoma: evaluation of therapeutic response to interventional procedures[END_REF][START_REF] Bruix | Clinical management of hepatocellular carcinoma. Conclusions of the Barcelona-2000 EASL conference. European Association for the Study of the Liver[END_REF][START_REF] Miller | Reporting results of cancer treatment[END_REF][START_REF] Brown | Transcatheter therapy for hepatic malignancy: standardization of terminology and reporting criteria[END_REF]. Subsequently, different slice selection can lead to different response assessments. Additionally, in the case of RECIST (Response Evaluation Criteria in Solid Tumor (RECIST), the measurements comprise the longest diameter without accounting for the volume of the tumor [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF]. This assessment can be particularly inaccurate if the enhancement percentage attains the threshold between two quartiles. Recently, mRECIST, has been described as a more refined criterion because it takes into account the size of the enhancing tumor (somehow a mix between RECIST and EASL) [START_REF] Edeline | Comparison of tumor response by Response Evaluation Criteria in Solid Tumors (RECIST) and modified RECIST in patients treated with sorafenib for hepatocellular carcinoma[END_REF].

As endpoint for assessing cancer treatments is usually overall survival. Nonetheless, tumor responses as measured by RECIST (although one would not expect to see pure volume changes immediately after trans-catheter arterial chemo-embolization), EASL, and time to progression have been considered surrogate markers of treatment efficacy. RECIST [START_REF] Eisenhauer | New response evaluation criteria in solid tumours: revised RECIST guideline (version 1.1)[END_REF][START_REF] Miller | Reporting results of cancer treatment[END_REF], mRECIST [START_REF] Lencioni | Modified RECIST (mRECIST) assessment for hepatocellular carcinoma[END_REF], and EASL ( 96) have been especially designed for reporting study results in an effort to standardize the results of treatment and to allow comparisons of different types of treatment. These guidelines enable conversion of radiologic image observations into a quantitative and statistically trackable variable for measuring the response to therapy.

However, these methods in their current form analyze tumors in only the axial plane.

Despite strict standardized rules, all these methods present numerous drawbacks.

In Authors reported an excellent inter-reader agreement between EASL/mRECIST (κ = 0.905), but a weaker inter-reader agreement RECIST/mRECIST and EASL/RECIST (κ = 0.638 and 0.598, respecti ely). oreo er they demonstrated significant differences in Kaplan-Meir and Cox regression survivals between responders and non-responders with all the three methods (p < 0.0001). ulti ariate analysis identified REC ST response, tumor extent, performance status, concomitant chemotherapy, and prior surgery/ablation as independent prognostic factors. EASL response and mRECIST response were not found to be independent prognostic factors.

RECIST is superior to mRECIST/EASL given its ability to stratify survival benefit according to response category and to pinpoint independent prognostic significance.

C-arm CBCT to "Assess" Treatment Success with cTACE

Lipiodol® is a radiopaque contrast agent, and a biomarker of HCC response [START_REF] Miyayama | Ultraselective transcatheter arterial chemoembolization with a 2-f tip microcatheter for small hepatocellular carcinomas: relationship between local tumor recurrence and visualization of the portal vein with iodized oil[END_REF].

Lipiodol® deposition in the tumor is a prognostic factor affecting local recurrence of HCC and may be determined directly during the procedure using unenhanced CBCT, which offers equivalent Lipiodol® detection accuracy to unenhanced MDCT imaging [START_REF] Jeon | Iodized oil uptake assessment with cone-beam CT in chemoembolization of small hepatocellular carcinomas[END_REF][START_REF] Iwazawa | C-arm CT for assessing initial failure of iodized oil accumulation in chemoembolization of hepatocellular carcinoma[END_REF](101)(102)(103).

Iwazawa J. et al [START_REF] Iwazawa | C-arm CT for assessing initial failure of iodized oil accumulation in chemoembolization of hepatocellular carcinoma[END_REF] Because Lipiodol® tumor staining is a surrogate marker of tumor response, it is known recognized that C-arm CBCT should be used in order to depict residual or untreated lesions [START_REF] Monsky | Semiautomated segmentation for volumetric analysis of intratumoral ethiodol uptake and subsequent tumor necrosis after chemoembolization[END_REF]. Authors concluded C-arm CBCT as predictive of tumor response when compared to 1-month follow-up multiphasic MDCT or MR imaging.

C-arm CBCT to Assess Treatment Success with drug eluting beads

Radiolucent drug-eluting beads, loaded with doxorubicin, are mixed with contrast agent during delivery. These beads occlude tumor-feeding arteries from where the chemotherapy diffuses locally into the tumor (104). Assessment of DEB-TACE therefore requires the visualization of tumor-feeding vessel de-vascularization or tumor contrast agent saturation features on C-arm CBCT images [START_REF] Loffroy | Intraprocedural C-arm dual-phase cone-beam CT: can it be used to predict short-term response to TACE with drug-eluting beads in patients with hepatocellular carcinoma?[END_REF]105). The value of immediate postprocedural C-arm CBCT has been studied in several reports as follows.

Loffroy R. et al. [START_REF] Loffroy | Intraprocedural C-Arm Dual-Phase Cone-Beam CT: Can It Be Used to Predict Short-term Response to TACE with Drug-eluting Beads in Patients with Hepatocellular Carcinoma? Radiology[END_REF] 2 weeks before DEB-TACE. Two radiologists rated the likelihood of an image to be an HCC in using a 5-point confidence scale focused on the degree of arterial enhancement and the shape of the lesion on C-arm CBCT. They also graded the presence of HCC on MRI based on the signal intensities on T1-and T2-weighted images, arterial enhancement, and hypointensity on both the portal phase and the late phase. Diagnostic accuracy was evaluated using the alternative free-response receiver operating characteristic curve (AFROC) method.

The predictive value of C-arm CBCT was evaluated with a multivariate logistic regression analysis between the good-response and non-response of HCC lesions for TACE.

The authors demonstrated that the MRI accuracy was overall greater than that of Carm CBCT (0.890 vs. 0.681, respectively; p<0.001). Howe er, in small HCCs (≤1 cm), C-arm CBCT showed a higher sensitivity than MRI (90.9% vs. 70.5%, respectively; p=0.023) and a lower positive predictive value than MRI (40.8% vs. 57.4%, p=0.073).

Moreover these authors showed that a tumor with well-defined strong arterial enhancement on C-arm CBCT would have a better response to DEB-TACE (odds ratio=8.08, p=0.05).

C-arm CBCT to "Assess" Treatment Success as a function of perfusion parenchymal blood volume

Considering the limits of Dual-phase C-arm CBCT that we have previously presented, the potential a more sophisticated approach has been evaluated.

This approach so called Parenchymal Blood Volume (PBV) provides to the reader a surrogate of tumor perfusion. PBV calculation is based on the assumption that with an adequate contrast injection and timing, the contrast concentration in the liver tumor is constant (steady state) for the duration of a single C-arm CBCT acquisition. Dedicated software displays color maps that allow calculation of PBV. This approach is developed in section 4.2.

Syha R. et al (107) studied the clinical utility in HCC of PBV as derived from C-arm-CBCT, for immediate post treatment assessment of drug-eluting bead trans-arterial chemoembolization ((DEB-TACE). For that purpose 24 patients (52 HCC) were treated with DEB-TACE and PBV was evaluated before and after DEB-TACE. This was compared to RECIST criteria as measured on MDCT at 12 weeks. Authors reported that all treated lesions showed significant decrease of PBV after DEB-TACE (mean difference, -15.61 mL/100 mL, P < 0.0001).

However, 11 lesions that showed residual tumoral perfusion according to PBV had an Subgroup analysis showed these findings to be consistent except in patients with initial PBV values of 50-100 ml/1000 ml: HCC showed mean initial PBV values of 98.5 ml/1000 ml, CRCLM 64.0 ml/1000 ml and breast cancer liver metastases 108.5 ml/1000 ml. After DEB-TACE, PBV decreased by 11.7 % in HCC, by 36.4 % in CRCLM, and by 30.7% in breast cancer liver metastases; of note, the PBV changes between the different tumor groups were not statistically significant (p>0.05).

In summary the PBV measurement of malignant liver tumors using C-arm CT is feasible. Following TACE, PBV decreased significantly. Patients with low initial PBV elicited low local response rates and further increase in blood volume, whereas high initial tumor PBV showed better response to TACE.

As a conclusion from this literature review, C-arm CBCT appears to be a valuable adjunctive tool in the treatment of HCC lesions in using both cTACE and DEB-TACE.

However, up-to now literature does not report similar work focused on colorectal cancer liver metastases.

We will further explain the step-by-step approach that we have developed in order to evaluate the interest of C-arm CBCT in order to improve the intra procedural strategy as well as clinical results from liver intra-arterial treatment that is targeted to CRCLM.

We have previously stressed that TACE results in inhomogeneous tumor necrosis, and subsequently heterogeneous tumor enhancement and irregular changes in tumor size [START_REF] Lim | Hepatocellular carcinoma: evaluation of therapeutic response to interventional procedures[END_REF] that may reduce the performance of current methods of tumor response assessment. This limit of evaluation of tumor response is at least in part related to the single plane image analysis and would likely be overcome by volumetric assessment. However, due to the lack of automated 3D software, so far this approach is not clinically applicable.

Therefore were decided to evaluate the feasibility and reliability of semi-automatic tumor segmentation software. This was summarized in our manuscripts We found that tumor volume segmentation was feasible in less than one minute, and can assess the tumor change after DEB-TACE. Regarding evaluation of tumor enhancement we found that this semi-automated software was able for each lesion to quantify qEASL in less than 1 min.

The major finding of this study of feasibility is that it is possible to calculate tumor volume and enhancement pattern measurements in a time-efficient manner (< 4 minutes) on a voxel by voxel basis, thus providing a true 3D volumetric assessment.

Galizia et al. in a study using semi-automatic software to measure the volume of tumor necrosis after radio-embolization reported that 3 D tumor segmentation was more reproducible that 2 D measurements to assess tumor necrosis (110).

In practice, RECIST is subjected to considerable inter-observer and intra-observer variability of lesion measurement; Suzuki et al (111) reported up to 0.53 (95% confidence interval 0.33-0.72) inter-observer variability rate, and intra-observer variation ranged from 0.76-0.96 owing to some of the aforementioned reasons (112,113).

Volumetric measurement appears as a logical evolution because a change in tumor volume measurement is better assessed than only a change in axial measurement (114,115). Volumetric evaluation provides a more accurate depiction of tumor burden compared with one-dimensional and two-dimensional measurements (116). Volumetric measurement is also able to define tumor size changes earlier than two-dimensional measurements (117-119) with higher reproducibility (120,121). This is a crucial point in the follow-up of patients.

Histopathology, also found to be a predictor of survival, correlates better with tumor volume than with axial measurements (122, 123) and volumetric evaluation of treatment response (122,124,125).

After this initial work and given the remaining questions, the merging key question ;2021The purpose of this study was to evaluate the accuracy of semiautomatic tumor segmentation software with C-arm CBCT and CT scanner and compare these measurements to pathologic volume-based measurements in a VX2 rabbit hepatic tumor model.

Manuscript #2: Comparison of semi-automatic volumetric VX2 hepatic tumor segmentation from cone beam CT and multi-detector CT with histology in rabbit models. Pellerin O, et al. AcadRadiol. 2013 Jan

In this work (Manuscript #2) we could performed the semiautomatic tumor segmentation in all tumors with an average time of 72 ± 28 seconds (range, 50-300 seconds)

for C-arm CBCT versus 88 ± 34 seconds (range, 68-250 seconds) for CT scanner of (p = 0.01).

We observed a strong correlation of the tumor size not only between C-arm CBCT and CT scan (rho = 0.993, P < .001) but also between CBCT and pathology (rho = 0.996, p< .001).

In addition, C-arm CBCT provided both a high precision and a strong agreement with a minimal bias when compared to CT scanner or pathologic samples. In general the C-arm CBCT tended to overestimate tumor volume, with a mean difference of 0.3 cm 3 (limits of agreement, 0.2 and 0.3 cm 3 ) from the true volume as sized by the pathologic specimens (Bland-Altman plots analysis). Finally, using the Sheiner and Beal test, high precision (0.674) and a small bias (0.368) were obtained with an excellent inter-observer reproducibility of the measurement (intraclass coefficients = 0.934).

These findings confirmed the precision of C-arm CBCT as a tool for intraprocedural tumor measurement. However some limits should be considered in the case of the VX2 tumor model. Indeed this tumor model generates a hypervascular tumor tissue closer to HCC or neuroendocrine liver metastases than that to CRCLM (127,128). Indeed, the VX2 tumor stroma is usually necrotic instead of sclerotic as it is the case for liver mCRC (129,130). The hypervascular corona surrounding the center of the tumor is a simple landmark to determine the limits of the tumors. The density ratio between the tumor stroma and its corona simplify the VX2 tumor identification. Therefore it might be that the quality of our findings could not been reproduced in human liver metastases.

In this manuscript the tumor enhancement was not quantified by the qEASL method since C-arm CBCT images were acquired with a non-standardized injection protocol that did not allow reproducible image.

Intra-procedural analysis of tumor enhancement change and prediction of tumor

response.

Dual-Phase C-arm CBCT

Once established the precision and the accuracy of the C-arm CBCT for primary liver tumor detection and measurement, we aimed at studying the value of observed tumor attenuation change in an intraprocedural manner in the case of secondary liver tumor. To get a better understanding of intraprocedural consequences of TACE or TARE at the tumor and tissue level, we decided to study the variation of tumor attenuation before and after TARE procedure to determine whether an elective micro-embolic effect could be visible. The level of embolization by the embolic material was studied in using small (30μm diameter) and radiolucent embolic beads injected into the hepatic arterial network. In The isible conse uences of 30μm beads injected in li er tumors resulted in a peri tumoral attenuation decrease visible at the late arterial phase. By contrast we observed in normal hepatic tissue an increase of the enhancement at the early arterial phase, which was magnified at the late arterial phase. Such an evidence should be explained by a subtotal occlusion of the tumor micro vascular environment, responsible of contrast media stagnation into the native arteries (observed at the early arterial phase) as well as retention into the normal hepatic tissue. This hypothesis has been already proposed, when tumor and tissue are exploring by intravital video-microscopy and intratumoral pressure measurement during TACE procedure [START_REF] Kan | Ethiodized oil emulsions in hepatic microcirculation: in vivo microscopy in animal models[END_REF]133,134). The authors suggest that the saturation of the microvascular tumoral network by Lipiodol® induces the opening of arterio-port shunts by a buffer effect that, in our study, was very likely responsible of the increase of the normal liver enhancement (135). This process is probably similar to the one observed with Lipiodol® when this product ends-up into the tumor peripheral portal branches [START_REF] Miyayama | Ultraselective transcatheter arterial chemoembolization with a 2-f tip microcatheter for small hepatocellular carcinomas: relationship between local tumor recurrence and visualization of the portal vein with iodized oil[END_REF].

Visualization

As we previously discussed, CRCLM evaluation of treatment success after intraarterial treatment is almost as important as the one of HCC, but differ in radiologic presentation and in intra-arterial treatment. CRCLM are lesser hyper-vascular than HCC and the Intra-arterial therapy are performed in a lobar manner rather that a super selective catheterization of tumor arterial feeder (130-132). . Both described specificities represent a challenge for the use of C-arm CBCT.

In practice, the micro-catheter must be adequately placed into the hepatic arterial network with the goal to reach all vascularized tumors. To permit the micro-catheter to accurately deliver therapeutic compound requires thus an optimal identification of tumor location, sizing and a clear-cut of tumor boundaries.

We have previously demonstrated that NETs tumor attenuation can be evaluated using both phases of the C-arm CBCT before and after TARE. The next question, pertaining to secondary liver lesions was to better understand if the same approach can be used for sizing, locating, and diagnosing CRCLM to finally assess chemo-embolization success.

Manuscript #4: Is dual-phase C-arm CBCT sufficiently accurate for the detection of colorectal cancer liver metastasis during liver intra-arterial treatment?

Next, we addressed the accuracy of Dual-Phase C-arm CBCT in a CRCLM patients prospective cohort referred to intra-arterial treatment (hepatic intra-arterial chemotherapy or irinotecan eluting beads).

≈-≈-≈-≈-≈--≈-≈-≈-≈-≈
This manuscript is currently in review before submission to publication.  The early arterial phase of CBCT yielded a poor sensitivity and specificity (Se = 58%, Sp = 51%, p < 0.0001), whereas the late arterial phase yielded a sensitivity of 84% and a specificity of 70% (p = 0.02).

≈-≈-≈-≈-≈--≈-≈-≈-≈-

Implication for Patient Care

It is not possible to rely on the early arterial phase during intra-arterial liver treatment to correctly diagnose a CRCLM. Only the late arterial phase can be proposed to operators for the correct placement of the microcatheter to allow correct intra-arterial hepatic treatment.

Summary statement

It is not possible to rely on the early arterial phase during intra-arterial liver treatment to correctly diagnose a lesion. Only the late arterial phase can be proposed to operators for the correct placement of the microcatheter to allow correct intra-arterial hepatic treatment.

ABSTRACT Purpose

This study aimed to compare the accuracy of dual-phase CBCT with that of multidetector computed tomography (MDCT) for the detection of colorectal cancer liver metastases (CRCLM).

Materials and methods

Between March 2012 and December 2015, 49 consecutive patients referred for intraarterial treatment for CRCLM were enrolled in a single center, prospective, observational study approved by our institutional review board. All patients were examined using the triphasic MDCT protocol and dual-phase CBCT before intra-arterial treatment. Two blinded observers independently reviewed all examinations. The sensitivity (Se) and specificity (Sp) of diagnosis using Dual-Phase CBCT were calculated using a 6-cell matrix method and a "worst-case scenario" defining a "real-life" situation. The Se and Sp of both phases were compared using cNemar's test.

Results

The early arterial phase (eAP) of Dual-Phase CBCT yielded a Se of 93% and a Sp of 85% and the late arterial phase (lAP) a Se of 99% and a Sp of 88%, using the 6-cell matrix method. The probability to correctly diagnose a CRCLM at the eAP and lAP was 60% and 90%, respectively. The worst-case scenario yielded a Se and a Sp of 58% and 51% at the eAP (p < 0.0001), and 84% and 70% at the lAP, respectively (p = 0.02).

Conclusion

The eAP had limited accuracy for the intra-procedural detection of CRCLM. Only the lAP appeared to have sufficient accuracy to be proposed to operators for the correct placement of the microcatheter to allow correct intra-arterial hepatic treatment.

MAIN BODY Introduction

C-arm cone beam computed tomography (CBCT) is a valuable adjunct to conventional angiography. It provides useful cross sectional imaging for patients undergoing intra-arterial liver treatment [START_REF] Meyer | Visualization of hypervascular liver lesions During TACE: comparison of angiographic C-arm CT and MDCT[END_REF]136). Dual-phase CBCT imaging uses two successive rotations of the Carm CBCT, allowing the study of contrast enhancement in the liver at two sequential time points, in the same region, during hepatic arterial injection. The hyper-acute nature of the arterial contrast medium injection provides accurate information on the number, distribution, and size of tumors [START_REF] Deschamps | Computed Analysis of Three-Dimensional Cone-Beam Computed Tomography Angiography for Determination of Tumor-Feeding Vessels During Chemoembolization of Liver Tumor: A Pilot Study[END_REF]132,137). However, most reports using dual-phase CBCT have focused on hepatocarcinoma (HCC) detection (136,138,139).

Colorectal cancer liver metastases (CRCLM) are increasingly treated by various intraarterial approaches such as irinotecan eluting bead trans-arterial chemoembolization, hepatic intra-arterial chemotherapy, and radioembolization [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF]. The therapeutic compound in CRCLM is delivered lobe by lobe and differs from HCC chemoembolization in which super selective chemoembolization is required (140-142). Positioning of the microcatheter in the hepatic arterial network needs to be adequately determined to reach all downstream vascularized tumors. Thus, these approaches require accurate identification of tumor location, size, and boundaries to ensure correct delivery of the therapeutic compound to the tumor.

The purpose of this work was compare the accuracy of dual-phase CBCT with multi detector computed tomography (MDCT) for the detection of CRCLM in patients undergoing intra-arterial treatment.

Material and Methods

Patient Study Selection

This was a single center, prospective, observational study running between March 2012 and December 2015. All consecutive patients with CRCLM referred for intra-arterial hepatic treatment (DEBIRI, TARE, Hepatic intra-arterial chemotherapy) were screened by the liver board conference for intra-arterial therapy according to the clinical indications and contraindications listed in Table 2. The local ethics committee approved the study, and all patients provided informed consent before inclusion.

Dual-Phase CBCT Technique

Images were acquired using a flat panel angiographic system (Artis Zee; Siemens Healthcare, Forchheim, Germany). Dual-phase CBCT allows sequential acquisition of two, back-to-back CBCT scans, providing early (eAP) and late (lAP) arterial phases using only one The two-dimensional projection images were reconstructed using Feldkamp back projection into 3D volumetric images with an isotropic voxel of 0.5 mm 3 for a 30 × 40 cm field of view (FOV) (matrix size 512×512) [START_REF] Feldkamp | Practical cone-beam algorithm[END_REF]. One millimeter-thick "CT-like" axial images were obtained and reconstructed to 3mm for observation of the CBCT images on a Syngo® (Siemens Health care, Forchheim, Germany) workstation.

Image Analysis

The three phases of the MDCT images and two phases of the CBCT images (including eAP and lAP) were retrieved from the PACS and anonymized. Two experienced readers independently interpreted the images on a Syngo workstation (Siemens Healthcare, Forchhiem Germany). The readers were blinded to the number and location of the liver lesions.

Each reader evaluated the MDCT images and recorded, the number, location, and maximum diameter (at the portal phase) of each hepatic lesion, segment by segment, on the MDCT portal phase (Couinaud segmentation) (143).

Then, they classified each lesion as: undoubtedly CRCLM, undoubtedly not a CRCLM (biliary cyst, benign li er tumor, other…), or uncertain. The following criteria were used to define a tumor as "undoubtedly CRCL ": spheroid lesion with the following characteristics: hypodensity relative to normal liver parenchyma on unenhanced CT; hypodense on hepatic arterial phase CT with a thin hyper-enhanced corona; a cockade appearance at the portal venous phase (central hypodensity surrounded by thick enhanced corona) (144).

One week later, each reader performed the Dual-Phase CBCT reading session in a random order different than that used for the reading of the MDCT images to avoid learning bias. CRCLM were defined as round and delineated hypodense areas surrounded by hyperdense capillaries at the eAP together with thin enhanced septa surrounded by a thick rim enhancement at the lAP (Figure 6).

After completing the reading of the two CBCT phases, each reader returned to the MDCT of the same patient and classified lesions that the CBCT missed as: missed lesion

Statistics

Statistical analysis was performed using SAS software, version 9.4 (SAS Institute, Cary, NC, US).

Analysis of tumor size measurement: The inter-observer agreement of tumor size was studied using intra-class correlation coefficients (ICC) as described by Shrout and Fleiss (145).

Intra class correlation values were classified as follows: ICC < 0. Analysis of tumor nature: The diagnostic accuracy of dual-phase CBCT was studied following two separate methods. First, the specificity (Sp), sensitivity (Se), predictive values, and likelihood ratios (LR) for tumor diagnosis were calculated using a 6-cell matrix as described by Simel et al. (147). The 95% confidence intervals (CI) of proportions were calculated using the Wilson method and the CIs of LRs were calculated, using the log method (148). Second: A "real-life" situation during the intra-arterial treatment, defined as the "worst-case scenario", was applied. n the worst-case scenario, all negative results of the readings, regardless of the reason (e.g. missed lesion because of streak artifacts, the absence of tumor enhancement, or liver truncation) were pooled with the uncertain results leading to the negative results in the worst-case scenario. The Se and Sp of both phases were compared using cNemar's test.

Results

Patient demographic data

Between March 2012 and December 2015, 103 consecutive patients were screened. Fifty patients were not included because the time between MDCT and the intra-arterial treatment was > 15 days for 36, and the MDCT technique was not appropriate for 18 (slice thickness > 3mm). Dual-phase CBCT failed for four patients and they were excluded from the study. In total, 49 patients were analyzed and 24 (49%) were assigned to DIBIRI and 25 (51%)

to hepatic intra-hepatic chemotherapy. All patients were free of biotherapy at the time of the procedure. The flow diagram for participation in the study and the patient baseline characteristics are shown in Figure 7 and Table 3, respectively. Albumin (g/dL) 34 ± 4.6 [25 -45] Total bilirubin (µmol/L) 12 ± 5 [4 -20] AST (UI/L) 130 ± 45 [28 -133] ALT (UI/L) 75 ± 44 [31 -120] Alkaline phosphatase (UI/L) 238 ± 58 [88 -449] GGT (UI/L) 49 ± 21 [1 -118] INR 1.2 ±21 [23 -118] Table 3: patient baseline characteristics.

Tumor diagnostic and measurement

All Dual-Phase CBCTs were successfully performed before intra-arterial therapy. Both readers identified a total of 307 hepatic lesions (8 ± 4 [3 -16) lesion/patient) on the MDCT portal phase with a mean maximum diameter of 32 ± 16 mm (Table 3). Reader 1 identified 240 lesion at the eAp (36 mm ± 16 [12 -98]) and 277 lesions at the lAP (33 mm ± 15 [12 -97]) on the CBCT images. Reader 2 identified 246 lesion at the eAp (37 mm ± 16 [11 -99])

and 281 lesions at the lAP (34 mm ± 16 [11 -98]) on the CBCT images.

Inter Reader Variability of tumor size

The tumor size evaluated by both readers using the two image modalities was intercorrelated (p < 0.001). The intra-class correlation coefficient was excellent (> 0.74) as the ICC for all measurements was between 0.949 and 0.992 (Table 5).

Inter modality Variability of tumor size

The Bland-Altman plots showed good agreement between dual-phase CBCT and MDCT for both readers, as well as both phases of dual-phase CBCT for each reader (Figure 8). The degree and the variance of measurement overestimation showed a relationship between the difference in the MDCT measurement and the eAP and lAP. Both readers significantly (p = 0.003) overestimated the mean diameter measurement by 1.9 mm and 2.02 mm at the eAP and the lAP, respectively, with a very small proportion of variance (0.21 and 0.19 for each phase, respectively). Diagnostic accuracy of tumor nature using Dual-Phase CBCT Among 307 lesions seen on MDCT, 264 (86%) were classified by both readers as undoubtedly CRCLM (Table 3). Forty-three (14%) were classified as undoubtedly not CRCLM (biliary cyst n = 39, adenoma n = 3, angioma n = 1). Reader 1 classified 157 (51%) and 226 (74%) lesions as undoubtedly CRCLM at the eAP and the lAP, respectively. Reader 2 classified 156 (51%) and 231 (75%) lesions as undoubtedly CRCLM at the eAP and the lAP, respectively (Table 6 Table 7).

We calculated the accuracy using only the results from reader 1, because the kappa test (κ = 0.84) showed good concordance between the two readers. The eAP yielded a Se of 93% and a Sp of 85% and the lAP yielded a Se of 99% and a Sp of 88% as assessed using the 6-cell matrix accuracy method (table 5 and6). The positive likelihoods (LR+) ratios, reflecting the odds of a positive test in favor of disease, were 6 and 9 for the eAP and lAP, respectively.

Thus, there is a 6 greater and 9 greater chance to correctly identify CRCLM with Dual-Phase CBCT when CRCLM is diagnosed by MDCT. In other words, the probability to correctly diagnose a CRCLM at the eAP and the lAP is 60% and 90%, respectively.

In the worst-case scenario we calculated a Se and a Sp of 58% and 51% at the eAP (p<0.0001), and 84% and 70% at the lAP, respectively (p = 0.02) (Table 7). Sensitivity (%) 93 [88 -96] 99 [97 -99] 58 [59 -64] 84 [79 -3] <0.0001 Specificity (%) 85 [67 -94] 88 [73 -95] 51 [37 -65] 70 [55 -81] 0.0215 Positive predictive value (%) 97 [94 -99] 98 [95 -99] 88 [82 -92] 94 [91 -97] Negative predictive value (%) 65 [48 -78] 94 [80 -98] 17 [11 -24] 42 [31 - 7: Accuracy of Dual-Phase C-arm CBCT. p Value for the early and the late arterial phase. LR+ is the conditional likelihood ratio of a positive test; the odds of a positive test in favor of disease. LR-is the conditional likelihood ratio of a negative test; the odds of a negative test in favor of disease. LR± is the conditional likelihood ratio of a non-positive, non-negative result; the odds of a non-positive, non-negative test in favor of disease. YD+ is the probability of a positive or a negative result when disease is present. YD-is the probability of a positive or a negative result when disease is absent.

Discussion

Here, we report a good inter-reader correlation coefficient for both phases of dual-phase CBCT regarding tumor size, with a small overestimation of 1.9 mm at the eAP and 2.02 mm at the lAP (p = 0.003) relative to MDCT. A preclinical study using the VX2 rabbit liver tumor has already shown that tumor sizing by C-arm CBCT results in a slight overestimation of lesion size (126). This finding may be related to the hyperacute nature of the arterial contrast injection medium, which magnifies the visibility of the transitional zone between healthy liver tissue and the tumor. According to previous work , this corresponds to the dense mesh of capillaries that surrounds the metastasis, resulting in a peri-tumoral corona that traps the contrast media [START_REF] Dezso | Development of arterial blood supply in experimental liver metastases[END_REF]. [START_REF] Higashihara | Diagnostic accuracy of C-arm CT during selective transcatheter angiography for hepatocellular carcinoma: comparison with intravenous contrast-enhanced, biphasic, dynamic MDCT[END_REF]. This approach is also an accurate method to predict tumor response after TACE or depict the micro-embolic effect after radioembolization of hypervascular tumors [START_REF] Loffroy | Intraprocedural C-arm dual-phase cone-beam CT: can it be used to predict short-term response to TACE with drug-eluting beads in patients with hepatocellular carcinoma?[END_REF]132).

In our study to assess the ability of dual-phase CBCT to diagnose the nature of a liver lesion, we found that the 6-cell matrix method yielded a Se/Sp of 93%/85% at the eAP and 99%/88% at the lAP. This demonstrates the lAP provides better accuracy than the eAP. This finding was more striking when considering the worst-case scenario in which we found a Se/Sp of 58%/51% at the eAP and 84%/70% at the LAP. This resulted in a low negative predictive value of 42%. This result suggests that, although dual-phase CBCT provides an accurate measurement of CRCLM, it is not possible to rely on the eAP during intra-arterial treatment to correctly diagnose lesions. There is 37% risk of being unable to conclude between a CRCLM and other types of lesions. There is also a risk of under-estimating the number of CRCLMs.

The poor Se and Sp at the eAP is mostly due to streak artifacts (which may have masked adjacent CRCLMs) and poor contrast enhancement. Indeed, there were more streak artifacts at the eAP than at the lAP (4% vs 0%) and the tumor-to-liver enhancement ratio was too poor to delineate the tumor more often in the eAP than in the lAP (12% vs 4%). In contrast, the eAP was almost as accurate as the lAP to identify HCC tumors (72% vs 87% Se) [START_REF] Higashihara | Diagnostic accuracy of C-arm CT during selective transcatheter angiography for hepatocellular carcinoma: comparison with intravenous contrast-enhanced, biphasic, dynamic MDCT[END_REF]149), and streak artifacts and insufficient tumor enhancement led to only 13% misdiagnosed tumors (149).

Here, we observed that all CRCLM do not appear to behave in the same manner: 12%

were not visible at the eAP and only 67% of these 12% were visible at the lAP. At least 4% were not visible in either phase. Shady et al. (150) reported similar findings in a study that evaluated enhancement patterns of CRCLM with hepatic MDCT arteriography. The CT acquisitions were triggered four seconds after intra-arterial injection of the contrast media.

They found that 60% of CRCLM were fully enhanced, 24% partially enhanced, and 16% nonenhanced. There are two possible explanations for these findings. First, CRCLMs have heterogeneous patterns of vascularization as they are variably supplied by both the hepatic artery and portal vein (151,152). Second, the phenomenon known as ''late enhancement'', which corresponds to progressive accumulation of contrast agent in the interstitial spaces contained within the fibrosis, could explain the 67% of CRCLMs that are visible at the lAP that are not at the eAP (153). This may account for differences in response to treatment. Our study was not designed to evaluate this specific point but further research may be informative concerning this question. CRCLM lesions are mostly fibrotic and display thin peripheral enhancement on arterial phase MDCT images with slow tumor enhancement on subsequent portal and delayed phases due to their portal vein input (144,155). The authors report better performance at the lAP than at the eAP, detecting 90% of the lesions seen by pretreatment MRI. Similar to our results, both the lack of adequate tumor enhancement and streak artifacts at the eAP were the two limiting factors for tumor detection.

The 30/40 cm flat panel detector also has significant intrinsic limitations as 18 (6%) of the lesions were missed in truncated liver segments. This limitation was even more apparent when applying the worst-case scenario, showing the impact of truncated segments on accuracy. This specific flaw of the CBCT has been report but has not been integrated into the accuracy calculations [START_REF] Higashihara | Diagnostic accuracy of C-arm CT during selective transcatheter angiography for hepatocellular carcinoma: comparison with intravenous contrast-enhanced, biphasic, dynamic MDCT[END_REF]149,154). The use of a larger flat panel detector could, at least partially, overcome this limitation.

Clinical Utility

The clinical relevance of using dual-phase CBCT to diagnose and size tumors should be weighed against the fact that intra-arterial treatments for CRCLM are overwhelmingly administered in a lobar approach and it is not recommended to perform selective catheterization of tumor feeders (140). Indeed, CRCLM arterial feeders are much smaller than those of HCC or NETs (156). CRCLM are also often more diffuse and numerous than can be detected by imaging (so called "missing metastasis"), a finding that explains the high recurrence rate after curative hepatic surgery (60% within two years) (157) and underlines the interest of intra-arterial therapy delivered to the lobe rather than only to metastatic liver segments. For example, a total cure of missing metastases has been histologically proven when hepatic surgery was performed after irinotecan drug eluting chemoembolization iterated in a lobar approach (158, 159).

C-arm CBCT at the lAP could become a valuable clinical tool for daily intra-arterial work when there will be further imaging refinements or progress in intra-arterial treatment and the interventional radiologist requires confirmation that a microcatheter irrigates a specific metastasis.

Conclusion

These findings suggest that, despite good inter-reader correlation and a good correlation in tumor sizing, it is not possible to rely on the eAP during intra-arterial liver treatment to correctly diagnose a lesion. There is 37% risk of being unable to conclude between a CRCLM and another type of lesion and a risk of underestimating their number. We can only propose the lAP to operators for the correct placement of the microcatheter to allow correct intra-

arterial hepatic treatment. ≈-≈-≈-≈-≈--≈-≈-≈-≈-≈ ≈-≈-≈-≈-≈--≈-≈-≈-≈-≈
Despite good inter readers' correlation and good correlation in tumor sizing, the early arterial phase of the Dual-Phase CBCT had limited accuracy in the intra procedural detection of CRCLM. Only the late arterial phase appears to be relevant and can be proposed to operators within the goal to correctly place the microcatheter allowing a correct intraarterial hepatic treatment. This study highlights the fact that all CRCLM do not behave enhancement in the same manner. In fact we found also that approximately 12 % of them are strictly not visible at the early arterial phase but became visible at the late arterial phase. This discrepancy is related to a different pattern of CRCLM vascularization as the CRCLM are supplied by both the hepatic artery and the portal vein, with variable ratio (151, 152); likely, it may explain difference in response to treatment.

In summary the poor diagnostic accuracy for of the early arterial phase and the average accuracy of the late arterial phase narrow the power of Dual-Phase C-arm CBCT to evaluate results after DEB-TACE in CRCLM patient. This limitation hampers a good evaluation of treatment response and, given late enhancement of CRCLM, point out the requirement of an optimal protocol of injection.

So we addressed the question whether the tumor enhancement decrease reflects modification of tumor perfusion parameters? Today, the objective analysis of organ perfusion is available on most of CT scanner or MRI units. This analysis is becoming a gold standard, and, more and more routinely used for patients follow-up under targeted therapies (160). Furthermore, it has recently been demonstrated that perfusion parameters can predict treatment response to antiangiogenic therapy [START_REF] Loffroy | Intraprocedural C-Arm Dual-Phase Cone-Beam CT: Can It Be Used to Predict Short-term Response to TACE with Drug-eluting Beads in Patients with Hepatocellular Carcinoma? Radiology[END_REF]. Because embolization as antiangiogenic drug is acting directly on tumor vessel, perfusion changes should be detected with CT scanner or MRI (161,162). Some preliminary studies have shown a good correlation between perfusion change and response to DEBTACE treatment at one month (163-167). However, it must be stressed that none of those preclinical and clinical studies exploring perfusion parameter with a meantime of seven days and one month, respectively, were done immediately after embolization procedure (one hour after at least).

In the following pilot study, our team explored the feasibility of immediate post-

Oral communication: Dynamic first-pass area-detector perfusion CT for the evaluation of perfusion changes following intra-arterial therapy in the liver VX2 model Mirpour S, Pellerin O, Geschwind JF, Liapi E. (168)

Our work aimed at prospectively evaluating whether dynamic first-pass area-detector CT imaging can depict perfusion changes following intra-arterial therapy in the liver VX2 model.

For that purpose, 17 VX2 bearing rabbits were scanned for their liver in using a 320detector row CT scanner (Acquilion One, Toshiba, Japan) before and one day after intraarterial therapy (DEBTACE or bland embolization). The volumetric scanning parameters were: FOV=22 cm, kV=120, mA=80, slice thickness = 0.5 mm, scan delay = 6 sec, intermittent scanning for = 67 sec (arterial phase q 2 sec, portal venous phase q 3 sec). Rabbits were injected with 1.5 ml/kg of iso-osmolar CM at 1 ml/sec, followed by a saline flush. CT data were analyzed using the Toshiba CT body perfusion software. Perfusion parameters were calculated within tumor, as well as within ipsi-and contra-lateral to tumor liver tissue.

Hepatic perfusion was established using the maximum slope model. Blood samples for liver function tests (LFTs) were obtained at baseline prior euthanasia. All rabbits were euthanized after the final CT scanner; then, tumor and liver samples were evaluated for percentage of tumor necrosis, presence of peri-tumoral fibrous capsule and microsphere count per tumor slide.

Following treatment, arterial flow (AF) of the tumor was significantly reduced (AF pre = 130.03, AF post = 78.15, p_0.0009), while tumor portal flow (PF) and perfusion index (PI) remained unchanged (p=0. 49 and 0.31, respectively). AF to the ipsi-lateral liver lobe was significantly reduced after treatment (AF pre = 132.412, AF post = 74.09, p = 0.04). PF of the ipsi-lateral liver lobe was also slightly reduced after treatment (p = 0.07). AF, PF and PI to the contra-lateral liver lobe remained unchanged after treatment (p = 0.87, 0.41 and 0.41, respectively) (Figure 9). No statistically significant difference was observed between baseline and final LFTs. At a histo-pahological level, all tumors were successfully embolized, with an average of 7 beads / tumor slide. Tumors had a fibrous capsule with a mean tumor necrosis of about 68%. No correlation was found between the percentage of tumor necrosis and changes in the tumor AF. the hepatic artery blood supply as a fraction of the total blood supply. They found that the arterial enhancement fraction was statistically significantly greater in tumors with a complete or partial metabolic response than in tumors with no metabolic response (i.g., those with stable disease or disease progression) (p = 0.038). As an Arterial enhancement fraction of less than 0.4 was associated with a 40% response rate, whereas an Arterial enhancement fraction greater than 0.75 was associated with a 78% response rate.

However, functional modifications induced by embolization or chemo-embolization stay unknown immediately after procedure. A functional analysis of perfusion change by embolic material is necessary.

Parenchymal blood volume C-arm CBCT

The new angiographic systems equipped with flat panel detector have been shown to produce parametric images that permit gaining functional information such as blood flow and mean time transit as well as blood volume (170)(171)(172)(173).

Through this approach, tumor perfusion appears feasible. Two main avenues of research are currently explored. The 1st is a "dynamic approach" that uses parametric imaging (generated by digital subtract angiography, e.g., iFlow® with time-attenuation curves (TAC's) and color-coded images. Parametric image allows to explore blood flow and mean time transit in a "2D" manner (174). The 2 nd one is a static approach with the use of the steady state parenchymal contrast media staining following injection. In this application two consecutive rotational acquisitions are performed and specific computation is demanded.

In 2009, Zellerhoff et al. described the use of C-arm CBCT for parenchymal blood volume evaluation (PBV) (175). PBV calculation is based on the assumption that with an adequate contrast injection, the contrast concentration in the imaged liver tumor is constant for the duration of a single C-arm CBCT acquisition (Figure 10). This leads to the calculation of the PBV from two measurements: a baseline (mask scan) acquired before contrast administration and the contrast distribution after contrast injection (fill scan). The C-arm CBCT PBV acquisition consists of two runs: an initial rotation (mask scan) followed by a second rotation after contrast medium injection (fill scan) (Figure 11). The following parameters are currently used: acquisition time approximately 5 s, 90 kV, 616×480 matrix, projection on 30×40 cm flat panel, 200° total angle, 0.8°/frame, 248 frames total, detector entrance dose 0.36 μGy/frame. Contrast injection starts immediately after the mask run and is maintained until the end of the fill run. The C-arm rotates back, which takes another 5-6 s, then the fill run starts. Thereby, the contrast has been distributed through the arteries, and the parenchyma has reached a homogeneous enhancement. A total volume of 36 mL of contrast medium (Iomeprol 350) diluted to 25% was injected into the hepatic artery at a rate of 3 mL/s using a power injector at 300 psi, so the total contrast administered is only 9 mL.

Figure 11:

Flow chart shows the acquisition protocol used to generate the parenchymal blood volume images of the liver using an Artis Zee Q System (Siemens). The patient is told to hold his breath shortly before the first rotation (mask run) starts [START_REF] Ferlay | Estimates of cancer incidence and mortality in Europe in 2008[END_REF]. Right after finishing the mask run, automatically triggered contrast injection is started [START_REF] Ferlay | Estimates of worldwide burden of cancer in 2008: GLOBOCAN 2008[END_REF] to ensure a constant contrast flow in the liver parenchyma. The C-arm rotates back and immediately starts the second rotation (fill run) [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF]. The overall scanning time is of about 19 s.

The processing steps during calculation of BV maps were shown in Figure 12. They were performed using a specific software installed in a Syngoworkstation (syngo XWP, Siemens Healthcare). Mask and fill run are reconstructed separately. A non-rigid registration algorithm is applied that accounts for organ motion and deformation to register mask and fill volume before the two volumes are subtracted. An algorithm is applied to segment out air and bone. The arterial input function value is calculated from an automated histogram analysis of the vessel tree. This arterial input function value is then applied as a scaling factor to obtain the quantitative BV map. The quantitative BV values are given in mL/1000 mL and stored as a DICOM (Digital Imaging and Communications in Medicine) gray value volume. In order to facilitate the windowing of the PBV maps, the units differ from MDCT perfusion, which are typically given in mL/100g. Assuming a liver tissue density of 1.06 g/mL, the values can be easily converted. In a final step, a smoothing filter is applied to reduce pixel noise.

The BV map is visualized as a color map (176-178).

Introduction

Rational and background

Liver metastases are the leading cause of death in patients with colorectal cancer.

They occur in 50 -75% of patients during the course of the disease but only 10-15% of them are suitable for surgical removal [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF]. Systemic chemotherapy is able to improve long term survival with a median overall survival time of 3.5 years, by increasing the resectabilty of liver metastases to 20% of patients (179). However, in many cases, systemic chemotherapy fails to downstage enough metastases to allow for surgical resection and relapses are frequent. In these cases, systemic treatment may have only a limited effect, and current strategies are often disappointing, particularly for KRAS-mutated patients who cannot receive anti-EGFR therapies. Irinotecan drug-eluting beads (DEBIRI) chemo-embolization have been developed as a local treatment for colorectal liver metastases (180, 181).

Localized and controlled drug release combined with embolization improves localized cytotoxicity with 93% of disease control rate and 8 and 13 months of liver progression free survival and overall survival, respectively (182).

There is no imaging endpoint to indicate TACE success in CRCLM, in contrast to HCC, in which the complete disappearance of tumor enhancement on final Dual-Phase C-arm CBCT as endpoint is a linked to success [START_REF] Loffroy | Intraprocedural C-arm dual-phase cone-beam CT: can it be used to predict short-term response to TACE with drug-eluting beads in patients with hepatocellular carcinoma?[END_REF]. Completion of TACE in CRCLM is based only on the injection of the total irinotecan eluting beads of each liver lobe and the treatment outcomes can only be assessed at the 2 months imaging follow-up (100). Therefore, there is The goal of our study was to test the hypotheses that the pre-post-chemoembolization PBV are correlated to the 2 months tumor response rate and can be used to predict tumor response and liver response at two months in CRCLM patients treated with DEBIRI chemo-embolization.

Material and Method

Study Cohort

This was a single-institution prospective observational study. The study was compliant with the Health Insurance Portability and Accountability Act and was approved by the institutional review board. All patients provided written informed consent before inclusion.

Patients enrolled in the study were referred to DEBIRI chemo-embolization for dominant CRCLM after failure to oxaliplatin and irinotecan based chemotherapy. Patients were considered eligible for DEBIRI according to the following clinical indications and contraindications (Table 8).

Clinical indication:

Colorectal cancer pathologically proven

Multiple Detector Computed Tomography (MDCT) acquisition

All patients underwent liver MDCT at baseline and at two months after the chemoembolization. The patients were imaged with a Siemens somatom 128 detectors CT unit (Siemens Health Care; Forchheim Germany). A bi-phasic imaging protocol was applied. This included non-enhanced and enhanced contrast at the portal phase. Images were acquired in 0.625 mm slice thickness reconstructed in 3mm.

DEBIRI chemo-embolization

All Chemo-embolizations were performed by an interventional radiologist with 10 years of expertise as described by Lencioni et al. (140). After selective catheterization of the right and left hepatic artery using a conventional microcatheter, each liver lobe was treated by 3 ml of DC-eads™ 1 70-150 μm ( iocompatibles, UK) loaded with 100 mg of irinotecan.

Both lobes were treated in the same session and no additional embolization was performed.

C-arm CBCT acquisition

Pre-procedural images were acquired using a flat panel angiographic system (Artis Q with the pure platform Siemens Healthcare, Forchheim, Germany). C-arm CBCT acquisition for blood volume evaluation were performed during the course of the chemo-embolization.

The acquisition for the PBV consisted of two consecutive spins. A first spin (mask run) without contrast enhancement was followed 7s later by a second spin after contrast medium injection (fill run). Both spins ran during a single breath hold (17s). The following parameters were applied to the system for data acquisition: acquisition time 5 s, 90 kV, 616 × 480 matrix, projection on 30 × 40 cm flat panel size, 200° total angle, 0.8°/frame, 248 frames total, detector entrance dose 0.36 μGy / frame.

Contrast media was injected into the proper hepatic artery through a 2.7Fr microcatheter (Progreat™ Terumo, Leu en, elgium) using a power injector ( ark 7 Arterion®, Medrad Inc®, Pittsburgh, Pennsylvania, USA). In order to obtain homogeneous enhancement of the whole liver we started the injection 7 s before acquisition of the second rotation. Twelve of Xenetix 350® (Guerbet, Roissy, France) mixed with 24 ml of saline serum were injected at 3ml/s resulting in an injection time of 12s for the whole 36 ml solution.

Parenchymal Blood Volume (PBV) calculation

The C-arm CBCT imaging batchs were transfered to a Syngo workstation (Siemens Healthcare, Forchheim, Germany) equipped with PBV application for data analysis. PBV is a post-processing algorithm that enable to determine at the contrast enhancement steady state the blood volume (175). The prolonged/slow injection allows to obtain a steady state of contrast in the liver; that allows to derive/measure enhancement values for the tissue, artery and vein independently of time (175). First the mask and fill volumes were registered with a non-rigid motion-correction algorithm then the 2 batches were subtracted. A segmentation algorithm was applied to select only the liver parenchyma and the arterial input function value was calculated from an automated histogram analysis of the vessel tree. This arterial input function value is then applied as a scaling factor to obtain the quantitative blood-volume map. As a final step, a smoothing filter is applied to reduce pixel noise. The blood-volume pixel value is presented to the reader into a parametric color-coding image that allows direct blood volume calculation.

Image analysis

The study coordinator retrieved from our PACS and anonymized all MDCT images (including non-contrast, and portal venous phase) and PBV image batch on the Syngo Work station. Five representative lesions per patient were labeled on both image modalities. Two readers interpreted independently the images.

First, each reader sized the five-labeled hepatic lesion on the base line and 2 months MDCT according to RECIST 1.0 (184). A week later the two readers performed the PBV reading session. The pre-post-chemo-embolization PBV batch were presented separately to both readers. For all the 5 labeled tumors per patient, they drew a region of interest (ROI) covering the tumor and reported the blood volume value in ml of blood/1000ml of tissue. The relationship between the ariable "Responder / non-Responder" and the blood volume was assessed using a logistic regression model. Patients were considered "Responder" if REC ST1.0 status was complete response (CR), partial response (PR) or stable disease (SD) and "Non Responder" if the REC ST1.0 status was progressi e disease (PD).

Statistics

Results

Patient demographics

Between March 2014 to December 2015, 45 CRCLM patients were screened at our multidisciplinary liver tumor board for intra-arterial therapy (Figure 13). Thirty-four patients (19 men) with a mean age of 63 years [43 ; 80] were referred to irinotecan eluting beads chemoembolization for CRCLM. All patients had multiple liver lesions in a progressive disease status (RECIST 1.0). The 9 screened patients were treated with radio-embolization (n=2) hepatic intra-arterial chemotherapy (n=7). Two patients were excluded because they did not complete the whole PBV protocol because of painful DEBIRI administration.

Stable lung metastases (<3 nodules, < 2cm) were observed at baseline in 4 (12%).

Patients had previously undergone a median of 3 [2 -6] chemotherapy lines and all were refractory to both irinotecan and oxaliplatin. No patients had previously undergone liver surgery and/or percutaneous ablation. The ECOG-PS was 0, and 1 in 28 (82%), and 6 (18%) patients respectively. The mean size of target tumor was at base line 35 ± 23 mm [11 ; 191], accounting for a mean base line RECIST 1.0 sum of 174 ± 66 mm [92; 380] per patient (Table 9). All 34 patients successfully completed both pre-and post-chemo-embolization PBV acquisition. Both readers correctly identified all the 170 target lesions. the mean RECIST 1.0 sum was 147 ± 82 mm [60 ; 392]. Thirty patients (88%) were in liver disease-controlled status (PR = 11 (32%), SD = 19 (56%)). Four (12%) patients were in liver progression status. The mean RECIST 1.0 tumor response rate was -13% ± 26 (-65 ; 49) (Table 9). We observed an excellent agreement between the two readers (ICC >0.74; p < 0.001).

Parenchyma Blood Volume and prediction of tumor response PBV measurement

The mean PBV value of all targets CRCLM was estimated at 62 ± 47 ml/1000ml [10 ; 195] and at 17 ± 8 ml/1000ml [1 ; 59] Classification of tumors according to their initial PBV resulted in 44 tumors in Group 1 (initial PBV < 20 ml/ 1000 ml), 41 tumors in Group 2 (initial PBV of 20 to 50 ml/1000 ml), 43

in Group 3 (initial PBV of 51 to 100 ml/1000 ml) and 42 on Group 4 (initial PBV >100 ml/ 1000 ml) (Table 10). In Group 4, DEBIRI resulted in tumor diameter reduction of 51 % and 84 % PBV decrease, with a moderate correlation between diameter reduction and PBV reduction (rho = -0.5469; p = 0.00018). In Group 3, DEBIRI resulted in diameter reduction of 24 % and 70 % PBV decrease, with a moderate correlation between diameter reduction and PBV reduction (rho = -0.4871; p = 0.00093). In Group 2 DEBIRI resulted in diameter increase in +1 % with 51 % PBV decrease, with a moderate correlation between diameter reduction and PBV reduction (rho = 0.0290; p = 0.85695). In Group 1, DEBIRI resulted in diameter increase in +23% with a 15% PBV decrease, with a moderate correlation between diameter reduction and PBV reduction (rho = -0.4046; p = 0.00645).

Prediction of tumor shrinkage

The relationship between the tumor size change and the PBV was assessed using a mixed model considering the individual deviations (several lesions per patient). This method shows that the increase of 1 ml/1000ml in the blood volume of the tumor before DEBIRI leads to a decrease of 0.54 mm in tumor diameter at 2 months follow-up (p<0.005). In addition, the decrease of 1 % in blood volume after DEBIRI leads to a decrease of 0.75 mm in the diameter change of the tumor, at the 2 months follow-up (p<0.005).

Through quartile analysis the mixed model shows that tumor with a PBV >100 ml/ 1000 ml before DEBIRI (Group 4), the decrease of 1 % in blood volume after DEBIRI leads to a decrease of 7 mm in the diameter change of the tumor at the 2 months follow-up (p<0.005).

Tumor from Group 3: the decrease of 1 % in blood volume after DEBIRI leads to a decrease of 2 mm in the diameter change of the tumor at the 2 months follow-up (p<0.005). Tumor from Group 2: the decrease of 1 % in blood volume after DEBIRI leads to a decrease of 1 mm in the diameter change of the tumor at the 2 months follow-up (p<0.005). And finally tumor from Group 1: the decrease of 1 % in blood volume after DEBIRI leads to a increase of 2 mm in the diameter change of the tumor at the 2 months follow-up (p<0.005).

Prediction of patient response according RECIST 1.0

Patients with a 1% increase in the mean PBV decrease after DEBIRI had a 10% lower likelihood of achieving disease control (OR=0.9, 95% confidence interval [CI]: 0.81-1; p=0.0493). Table 11

Discussion

Our results show that PBV allows quantification of changes in tumor blood volume during chemo-embolization and can be a predictor of tumor response to DEBIRI. The higher the blood volume before DEBIRI, the highest the tumor shrinks, if the % of PBV decrease reach more than 70 %. However this results moderately impacts the liver response rate along RECIST 1.0, because in the same patient all CRCLM do not behave in the same manner.

These results confirm that PBV is a valuable surrogate biomarker of tumor response/failure to DEBIRI.

PBV technique

In

Perfusion as a surrogate biomarker of tumor response

Dynamic contrast enhancement approach with CT or MRI; have shown to exhibit early change within the CRCLM after intra-arterial therapies such as trans-arterial radioembolization (TARE). Reiner CS. et al. (185) studied the value of the arterial perfusion of CRCLM before and after TARE and compared results to tumor response at 4 months. Arterial perfusion was defined as the arterial blood flow (in mL/min/100mL) in their study. They showed that responders had significant higher arterial perfusion before TARE and they exhibited a higher decrease in arterial perfusion after TARE. Conversely, non-responders had no arterial perfusion significant reduction. This was strong enough to influence the 1-year overall survival, which was significantly higher for patients who have significant decrease of arterial perfusion coefficient after TARE. Boas FE. Et al. (169) in lesser sophisticated perfusion approach showed that an arterial enhancement fraction (hepatic artery blood volume divided by total blood volume) of less than 0.4 was associated with a 40% response rate, whereas an arterial enhancement fraction greater than 0.75 was associated with a 78% response rate in CRCLM treated with TACE.

These results are in lines with ours.

Clinical utility of early recognition

Early recognition of therapeutic failure is crucial to guide patient treatment decision after chemo-embolization, especially when other therapeutic options such as percutaneous ablation (for <3 cm lesions) or hepatic intra-arterial chemotherapy can be applied. Ruers T. et al. (186) have demonstrated in a phase II trial that iterative percutaneous ablation of CRCLM, when technically feasible, combined to chemotherapy in non restectable patient was associated with improved long-term overall survival, compared to chemotherapy alone.

Thus our work opens the gate of a tumor-by-tumor approach, in which non-responders can be referred to ablative technique.

Limitation

Our study had several limitations. First, the patient sample was small, only including patients who were undergoing their first DEBIRI procedure. Findings in previously treated patients may have been different and may not correlate. Second, as the study coordinator choose only well delineated lesions as target lesion, lesion surrounding by streak artifacts from catheters or located in truncated liver segment were not considered for the study. This accounts in potential limited accurate estimation of tumor PBV, especially in small lesions.

Third limitation: this technology is owned by Siemens healthcare and is not available with others units. Forth limitation, the optimum timing for assessing CRCLM response after DEB-TACE remains unclear. We choose two months follow-up as an in-between early evaluation (e.g., 1 month) and as later evaluation (e.g., 4 months). The shortest indwelling follow-up tends to underestimate tumor response rate, as the later evaluation tend to miss the deepest response or early progression. Finally, we do not yet have long-term follow-up evaluation of patient outcomes and thus we cannot claim that patients with better tumor response have better clinical outcomes. The clinical significance for these findings must be validated in future with larger multicenter study. A longer follow-up is also required to determine whether tumor PBV can reliably help prediction of patient prognosis.

Conclusion

Our study highlights the utility of quantitative parameters of blood volume in an intra procedural manner. PBV reliability serves as a surrogate biomarker to predict early success/failure of CRCLM after DEBIRI. PBV should be used for further treatment optimization of DEBIRI, with regard to more individualized treatment schedules

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Our results show a strong correlation between PBV before the chemo-embolization and the tumors diameter reduction at 2 months (rho=-0.8587; p=0.00001) (Spearman correlation). More specifically it means that the increase of 1 ml/1000ml in the blood volume of the tumor before chemo-embolization leads to a decrease of 0.54 mm in the diameter change of the tumor. When studying at the % of PBV change after DEBIRI and tumor diameter reduction we also found a strong correlation (rho=0.8027; p=0.00001). So, the decrease of 1 % in the blood volume of the tumor after chemo-embolization leads to a decrease of 0.75mm in the diameter change of the tumor at the 2 months follow-up.

Then in analyzing the data, patients with a 1% decrease in the mean PBV after DEBIRI elicited a 10% lower likelihood of achieving disease control (OR=0.9, 95% confidence interval

[CI]: 0.81-1; p=0.0493).

To sum up, PBV in monitoring and quantifying changes in tumor blood volume during chemo-embolization provided accurate prediction of response to therapy.

Our study opens the gate of an early recognition of therapeutic success/failure with patient referred to DEBIRI. This approach is helpful to guide patient treatment decision after chemo-embolization especially when other therapeutic options such as per-cutaneous ablation for <3 cm lesions or hepatic intra-arterial chemotherapy can be applied. It is important to notice that liver directed treatments can play a significant role in patient outcomes. Ruers T. et al. (186) have demonstrated in a phase II trial that iterative percutaneous ablation of MCRCLM, when technically feasible, combined to chemotherapy in non restectable patient was associated with improved long-term overall survival compared to chemotherapy alone. In our future work we will aim at studying patient outcomes when tumor with low PBV have to be managed following per-cutaneous ablation, when feasible.

Conclusion

First the feasibility of tumor segmentation with semi-automatic software on MRI image was then applied to a VX2 liver tumor model. We found a strong correlation of tumor size between C-arm CBCT and CT scan (rho = 0.993, p < .001) but also between CBCT and pathology (rho = 0.996, p < .001); not to mention a minimal bias (0.3 cm3), and high precision (0.674) of when tumors sizing with C-arm CBCT.

The C-arm CBCT precision allowed us to quantify tumor enhancement before and after trans-arterial chemo-embolization (TACE), as a surrogate biomarker of tumor response with the goal to predict tumor response at an earlier stage. In performing Dual-phase C-arm CBCT we got for neuroendocrine tumor a significant enhancement decrease after embolic injection; this accounts for a noticeable micro-embolic effect at the tumor capillary bed level. In another study with CRCLM Dual-phase C-arm CBCT failed to demonstrate its utility in detection because of a lack of accuracy (Sp.58% Se. 51%). The discrepancy between NETS and CRCLM is likely due to the different vascular pattern of the both tumors; it is well known that NETS are hypervascular. Then in surveying intra-procedural evaluation of the parenchymal blood volume (PBV) in CRCLM, we showed that initial PBV was strongly correlated to tumor shrinkage after chemoembolization (rho=-0.8587; p=0.00001).

Moreover the increase of 1 ml/1000ml in the blood volume of the tumor before TACE lead to a decrease of 0.54mm in the diameter change of the tumor. Finally, from our study, CRCLM patients with a 1% decrease in PBV after DEBIRI had a 10% lower likelihood of achieving disease control (OR=0.9, 95% confidence interval CI: [0.81 -1]; p=0.0493).

Our Right now we anticipate the need for a dedicated clinical study that would evaluate prospectively the tumoral and patient outcome based on PBV as triage method. We plan to design a prospective clinical trial in which liver progression free survival and overall survival will be studied. In this study, after the DEBIRI, the tumors with poor outcomes as determined by PBV will be managed with percutaneous ablation (when feasible).

RD CHAPTER: DEVELOPPEMENT OF A NEW INTRA-ARTERIAL LIVER DIRECTED THERAPY

Given the above limitations of all intra-arterial therapies today, we decided to make a significant contribution to a new approach based on suicide gene therapy directed towards a hepatic cancer animal model.

Introduction: Gene strategy in oncology

Gene therapy, born after the Second World War, was really developed in the 1990s, while the human genome sequencing was initiated. Gene therapy concept is the use of nucleic acids (DNA or RNA) to prevent or treat diseases. The first clinical gene therapy protocol was proposed in 1989 by Rosenberg et al, and since, more than 400 clinical trials have been conducted worldwide, 50% of them dealing with oncology (188).

In cancer patients, the majority of treatments are based on cytotoxic molecules: the so-called "chemotherapy". They act more or less selectively on tumor cells by blocking for example, cell division cycle leading to cell apoptosis. The main drawback of systemic chemotherapy is their lack of selectivity. Indeed, the drugs are distributed both in the tumor and in the healthy tissue via the bloodstream, killing all dividing cells and being responsible for numerous side effects including cardiotoxicity, nephrotoxicity, neurotoxicity, myelosuppression... (189).

Since the last 10 years, the search of antitumor drugs that target a pathway specific to the tumor tissue has been the option adopted to enhance selectivity thanks to biologic compounds (monoclonal antibodies) or, more recently, those based on genetic tumor characteristics [START_REF] Markowitz | Molecular origins of cancer: Molecular basis of colorectal cancer[END_REF].

Gene therapy implies any procedure intended to treat or alleviate a disease by modifying genetically the tumor cell of a patient. The material to be transferred into patient's tumor cells may be genes, gene segments, or oligonucleotides. The targeted cells are approached directly, by means of intra-tumoral or intra-arterial injection in the tumor tissue. Initial efforts were concentrated on the oncogene deactivation and the replacement of non-operating tumor suppressor genes (190,191). These approaches were barely successful. Subsequently, the transfer of genetic materials (transgenes), directly into target cells, was carried out with the aim to change cells phenotype. Target cells may be normal cells, cancerous cells, immune mediated cells, or pluripotent stem cells. Once the transgene enters a cancer cell, it can either participate to the tumor cells death process or restore normal cellular functions. Regarding normal cells, the transgene can protect them from drug-induced toxicities, or activate immune cells to get rid specifically of the cancer cells.

Gene and vector-based molecular therapies in cancer include a wide range of treatment modalities to modify cancer cells, normal cells, and/or a tumor microenvironment (192).

Gene therapy proceeds in using four main types of genes, which can be introduced into tumor cells: 1. Gene, which suppresses the expression of an oncogene. 2. Gene, which restores a defective tumor suppressor gene (replacement gene therapy). 3. Gene, which enhances immune surveillance (immunotherapy). 4. Gene that activates a pro-drug into a cytotoxic agent (gene directed enzyme prodrug therapy) (GDEPT).

Transferring the genetic material directly into cells is referred to as "transfection", while moving them into cells carried by a vector ( irus, SCs…) is termed "transduction".

Non-viral approaches have the advantage of safety and easy modifiability, but have a lower transfection efficiency compared to viral vectors.

Several methods have been developed to facilitate the entry of genetic materials (transgenes) into target cells, using various vectors. They are grossly divided into four major categories: viral, non-viral, bacterial vectors and mesenchymal stem cells (MSCs).

Viruses are small particles that contain either ribonucleic acid (RNA) or deoxyribonucleic acid (DNA), and may be single-stranded or double-stranded. The viral structure consists of a genome surrounded by a protective protein coat (viral capsid), which helps the virus to attach to host cell receptors, and prevents from viral destruction by cell nuclease enzymes. A complete viral particle by itself is unable to replicate. To be propagated, the virus needs to introduce its genetic material into a host cell, in order to acquire metabolic and biosynthetic products for viral transcription and replication. Viruses can be modified genetically to be noninfectious. They usually bind to target cells and introduce their genetic materials into the host cell as part of their replication process. As they enter target cells, they can carry a load of other genetic material called "transgenes" (193). The Table 12 summarizes the various viral vectors used for gene therapies according to their advantages and drawbacks.

Non-viral vectors are synthetic compounds using physical, chemical specificities to transduce the gene into the cancer cells. The Table 13 summarizes the various non-viral vectors for gene therapy, their mechanism as well as advantages and drawbacks.

Gene Directed Enzyme Pro-drug Therapy (GDEPT) state of the art

Cancer accounts for more than 60% of gene therapy clinical trials; in about 10% of these trials, suicide gene holds as therapeutic gene (196,197). In this respect, two approaches are proposed: either oncolytic gene therapy, or Gene Directed Enzyme/Prodrug Therapy (GDEPT). The GDEPT consists in the delivery to tumor cells of an engineered suicide gene coding for a mutated enzyme responsible for the metabolism of a pro-drug into its cytotoxic agent. This novel therapeutic strategy follows two sequential steps (Figure 16).

Firstly, the therapeutic gene coding for the mutated enzyme is delivered to the tumor through a vector (lentivirus, plasmid or mesenchymal stem cells (MSCs)). Secondly a prodrug is given (intravenous or orally). The prodrug is converted by the mutated enzyme into the therapeutic agent which induces the tumor cells death (197). This therapeutic approach needs to master four parameters: i) the choice of the therapeutic gene, ii) the gene vectorization to the host cells, iii) the cell expression level of therapy, and finally iv) the evaluation of the therapeutic effectiveness of the method. Since expression of the suicide gene does not operate in all tumor cells, it is advantageous that cytotoxic metabolites diffuse through the cell membrane to elicit death of neighboring tumor cells leading to the so-called "by-stander" effect. Finally, the host immune system plays an important role through an immunogenic cell mechanism (198)(199)(200)(201).

Figure 16: Gene directed enzyme pro-drug therapy steps

Therapeutic gene choice

GDEPT implies a therapeutic tandem composed of a pro-drug and its related cytochrome. The cytochrome is the enzyme that metabolized a pro-dug into its active metabolites.

Therefore, the first step is to identify the therapeutic couple. The specific mechanism of a few therapeutic tandems such as ganciclovir/thymidine Kinase, irinotecan/carboxylesterase, or cyclophosphamide/cytochrome P450-2B6 is well documented (202). Our team focused on this later tandem that exhibits suitable properties for an efficient GDEPT. The CYT2B6 isoenzyme, present at a low level in normal tissue, electively activates CPA. This cytochrome causes CPA hydroxylation in the human liver into phosphoramid mustard and acroleine. CPA by itself does not induce itself cytotoxicity, while the phosphoramid mustard is a powerful anticancer drug, even at low concentrations (203). This molecule, which is freely distributed within the tumor, shows chemical stability and displays suitable pharmacological and pharmacokinetic properties.

Gene vectorization

The gene vectorization represents an important step in a gene therapy protocol; in that respect, efficient vectors have been surveyed to efficiently deliver the transgene to the target cell. The ideal vehicle is the one, when administered by a non-invasive way, able to electively penetrate into target cells for delivering the gene. It should also allow the transgene to be translated into the converting enzyme at a sufficient amount to finally produce significant therapeutic effects.

A wide range of vectors has been developed: from the simpler (eg.naked DNA) to the complex one (eg. genetically modified cells via viral vectors). Today, about 70% of worldwide clinical gene therapy trials use of viral vectors such as retroviruses, adenoviruses, adenoassociated viruses, lentiviruses (LV) as carriers of transgenes.

Noteworthy, retroviral vectors used to treat complex and severe immunodeficiencies had resulted in leukemia; indeed the insertion of the transgene in the proto-oncogene regions may cause severe mutagenesis (204), aside effect that has greatly hindered the development of gene therapy. However, there are now safer vectors such as lentivirus used for a successful gene therapy of adreno-leuko-distrophies and β-thalassemia (205,206).

Lentiviruses are retroviruses such as HIV-1, consisting of a lipid envelope and two molecules of identical single-stranded RNA-reverse transcriptase required for their reverse transcription to DNA. The HIV genome contains two regulatory genes, tat and rev, which are necessary for viral replication and 4 accessory genes, vif, vpr, vpu and nef, not necessary for replication or growth in-vitro but which are crucial for replication in-vivo (207). A transgene encoding a specific protein can be added via their co-transfection of plasmid by calcium phosphate precipitation. These viral vectors, increasingly proposed, may, in some cases, lack tissue specificity, thus limiting their use.

Mesenchymal stem cells (MSCs), as named by Arnold Caplan in 1991 (130), stand up as a new generation gene therapy vector. In adults, these undifferentiated cells originated from embryologic mesenchyme tissue are present in very small amounts in bone marrow and adipose tissue. They are able to renew and to differentiate towards many cell types.

Moreover, they possess the ability to migrate to tumors and establish themselves in the tumor microenvironment, the so-called "homing" (195). Such a biological process is initiated by the presence of inflammation mediators in the tumor microenvironment (208,209).

MSCs cells grow rapidly in culture and can be efficiently transduced with viral vectors (lentiviruses, or retroviruses) containing therapeutic genes. Once transfected, the suicide gene is expressed stably (?) in all MSCs. A minimal expression of the major histocompatibility complex, MHC-I and MHC-II, explains the low immunogenicity of MSCs (210,211). Albeit a low level of MHC class II was reported in MSCs, pro-inflammatory mediators, such as interferon (IFN)-gamma are able to activate the MSC antigen presenting cell properties and may induce a CD8+ T-cell response (210, 212, 213).

Cytotoxicity mechanism of CPA

Cyclophosphamide (CPA) is a bi-functional alkylating agent that requires its activation by cytochrome P450-2B6 (CYP2B6) to exert its cytotoxic effects. CYP2B6 leads to a4hydroxylation reaction that converts CPA into phosphoramid mustard and acrolein. The phosphoramid mustard interacts directly with DNA to form covalent bonds with the nucleophilic substrate via its alkyl radicals. The formation of these alkyl intra-strand or interstrand bonds in DNA, leads to the inhibition of DNA transcription and replication and, finally, to cell destruction. This process operates during all phases of the cells cycle but respect cells in G0 phase, thus avoiding resting cells (214, 215).

In patients with cancer, the CPA is activated primarily in the liver because CYP2B6 is exclusively present in this organ. Once produced, the active metabolite circulates in the whole body, and reaches both tumor and healthy tissues, resulting thus in numerous side effects (cardiotoxicity, nephrotoxicity, neurotoxicity, myelosuppression) (216). Further, the CPA displays immunosuppressive properties as it reduces the cell count of CD3 +, CD4 + and CD19 + lymphocytes, on the one hand, and it increases the rate of lymphocyte CD8 + T, on the other hand (217).

The "bystander" effect

As first described by [START_REF] Huyan | Immunosuppressive effect of cyclophosphamide on white blood cells and lymphocyte subpopulations from peripheral blood of Balb/c mice[END_REF], the "bystander" effect occurs when the cytoxic metabolite diffuses through the membrane of the transduced cells and reaches neighboring tumor cells that are deprived of transgene ( 218). As a result, most of the tumor cells die (205). In 1994, Huber et al. observed that after treatment with 5-fluoro-cytosine (5-FC), complete tumor regression was achieved while only 4% of tumor cell population were transfected with a therapeutic gene, hence pointing out the key role of the "bystander" effect in the success of GDEPT strategies (219).

Two major types of "bystander" effect are distinguished. First, the destruction of neighboring cells is due to the transfer of toxic metabolites through the "gap junctions" by means of apoptotic vesicles, or by diffusion of soluble toxic metabolites (220). Second, the tumor cells contact with dead or dying tumor cells or "therapeutic-SCs", the so-called "kiss of death" resulting from the transfer of apoptotic factors (221).

Immune response

The tumor microenvironment and the immune system likely play a key role in the modulation of tumor progression and response to treatment (222). Indeed, following GDEPT treatment, a significant immune response mediated by CD8 + T cells in immune-competent mice has been reported ( 223). This immune response is not related to an immunization against the fusion protein (CYT2B6-TM) expressed by the transduced MSCs, but rather to the dying tumor cells and "therapeutic" stem cells induce a host immune response mediated by natural killer cells and T-cells. This evidence gives thus a major advantage of the GDEPT strategy to prevent the metastases occurrence. This is magnified by the use of Treg lymphocyte inhibitor, such as the CPA (224,225). Thus Treg suppresses immune responses whatever systemic or micro tumor environment origin and may play a major role in vaccination and immunotherapy (226,227).

The presence of an intense inflammatory infiltrate was described in tumors developed in immune-competent animals treated by GDEPT approach (228). In addition, a significant reduction of "bystander" effect in immunodeficient mice likely demonstrates how important is the host immunity on "bystander" effect for a successful GDEPT (229). The immune stimulation enhances tumor destruction not only locally, but also at distance, an evidence which foresees a better prognosis of patients with microscopic metastases (230).

The GDEPT experience of the unit INSERM 1147

Our team, the INSERM UMR-S1147 Unit, has gained for several years a strong experience on GDEPT preclinical application using the triple mutant gene CYP2B6TM-RED / CPA. In this therapeutic model, CPA was used as a pro-drug and a mutated CYP2B6 gene fused with reductase gene as a suicide gene, which converts CPA in cytotoxic metabolites. To reach therapeutic success, our team has delineated four essential requisites:

1-A highly efficient suicide gene coding for a protein able to bioactivate CPA into cytotoxic metabolites. This optimized suicide gene was built by fusing a CYP2B6 triple mutant with NADPH cytochrome P450 reductase (RED) to supply electrons required for CYP activity. The resulting fusion gene (CYP2B6TM-RED) was shown to be 13 times more active than CYP2B6 wild type and sufficient to achieve full CPA activation without involvement of endogenous reductase, which is not expressed in most of tumors (patent PCT/EP2012/058219, US-2014-0127180A1). given intra peritonealy 3 days after the MSCs injection. This vectorization system induced a 33% total healing of mice after completion of the GDEPT. Moreover the complete therapeutic cascade of the GDEPT, including bystander effect and immune response after re-challenge, was demonstrated (232).

The main reason of the partial failure of gene targeting was probably the inefficient transfer of the suicide gene to the tumor. Indeed, the 3 first critical requisites of our technical approach were clearly optimized in the mouse models. Meanwhile, one preliminary clinical trials has been published by others authors using the GDEPT and showed a lack of toxicity, with a limited efficacy (201). Thus our fourth requisite, the delivery/targeting of gene therapy, has emerged as mandatory to gain a full therapeutic efficacy.

Towards a new therapeutic option for P450 GDEPT: transduced MSCs, a way to improve hepatic metastasis targeting

Therefore, to foresee human applications, the above-described limitations in gene delivery need to be overcome. The intra-arterial (IA) approach, offers some advantages that could overcome some rule out our limitations. The IA approach takes advantage of the tumor physio-pathological condition. As liver tumors are exclusively vascularized by the hepatic artery, a selective catheterization of tumor arterial feeders is feasible for the deposit of a therapeutic compound; indeed, this approach is daily proposed for treatment of patients with liver tumor by injection of lipiodol®, drug eluting beads or direct intra-arterial chemotherapy (234-236). Moreover, the IA delivery favors pharmacokinetic properties leading to a higher therapeutic compound deposit and long lasting exposition to tumor [START_REF] Collins | Pharmacologic rationale for regional drug delivery[END_REF]237). This method has been proven to be very efficient in humans and animals. Indeed, the IA injection allows the delivery of a high level of the therapeutic agent very close to the tumor by selective catheterization (238,239). More specifically in the field of cancer gene therapy, the IA route has been evaluated; mainly with adenoviral agents as cell repair or cell destruction mechanism (240).

Because rats and mice are especially difficult to selectively catheterize (inappropriate devices), rabbit is the favorite animal model used for this purpose. Since the 1930' a cancer model, the VX2 tumor, has been developed in the rabbit by heterotopic implantation of anaplastic squamous cell carcinoma (papilloma virus-induced) (127). This rabbit tumor model is characterized by hyper-vascularity, rapid growth, and easy propagation in implanted tissues as well as rapid metastatic behavior (lung and peritoneal carcinomatosis) all features that well mimicked human hepatocarcinoma. Since its introduction, this tumor has been extensively used as a liver cancer model. The high tumor growth rate and the relatively large size of rabbit vasculature make the model particularly well suited for IR approach to deliver efficiently our MSCs-CYP2B6TM-RED. Indeed, the VX2 model is extensively used in numerous studies for loco-regional treatment of hepatic tumors even in cancer gene therapy (241,242).

Five in-vitro and one in-vivo steps to fulfill were required before envisioning the proof of concept of the IA delivery of transduced MSCs in the VX2 rabbit tumor model.

MSCs homing properties with VX2 cells

To assess homing properties of MSCs with VX2 tumor cells, the transwell technique was applied to MSCs and VX2 cells. This migration assay is commonly used to test to the migratory ability of MSCs through a membrane mimicking the endothelial barrier to tumor (Figure 17).

CPA response of the VX2 cells

An in-vitro screening study was performed in using three different tumor cell lines (VX2, TC1, and TC1-TM5) that were incubated with CPA up to 3 mM. Results indicated that CPA had no direct cytolytic activity on VX2 tumor cells as compared to TC1 and TC1 -TM5.

Thus, the chemoresistance of VX2 tumor cells to CPA was confirmed and allowed its use as a valuable tumor model (Figure 20).

In-vitro validation of murine MSCs as vectors for suicide gene and evaluation of a by-stander effect

As In-vitro VX2 tumor cells were insensitive to CPA up to 3 mM, we treated cocultures of murine MSCs-CYP2B6TM-RED clone and VX2 cells at different cell ratios by increasing doses of CPA (from 0 to 3mM) to (Figure 21). Cytotoxic metabolites produced by transduced MSCs diffused out of these cells and were able to kill VX2 tumor cells, thus demonstrating in-vitro an efficient by-stander effect. Cytochrome P450 hepatocytes of rabbit has no specific activity CYP2B6, but is expressed in the form of two isoenzymes, CYP2B4 and CYP2B5 showing iso CPA hydroxylase activity from 10 to 35 times lower than the CYP 2B1 of the rat, and 5 to 18 times lower than that of human. Km and Vmax of rabbit CYP2B4 and CYP2B5 of human CYP2B6, and rat CYP are reported in Table 14 (214,243). These data provide us with a marginal CPA hydroxylase activity of rabbit hepatocytes and therefore a specificity of treatment, upon systemic administration of CPA. So we can expect that following systemic administration of the CPA, MSCsCYP2B6TM-RED cells primarily metabolize CPA to its active metabolite. [START_REF] Tol | Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer[END_REF][START_REF] Kennedy | Recommendations for radioembolization of hepatic malignancies using yttrium-90 microsphere brachytherapy: a consensus panel report from the radioembolization brachytherapy oncology consortium[END_REF].

Km (mM) Vmax (min

Assessment of MSC distribution after their intra-arterial (IA) delivery

To assess the outcome of MSCs after IA injection into the hepatic artery, we studied 2

New Zealand White rabbits as follows: 1 rabbit bearing VX2 liver tumor and 1 rabbit without

Figure 24:

Photoluminescence assay performed 72 hours after rabbit sacrifice on frozen sample of different organs and tumor. This graph demonstrates a significant higher luminescence in the VX2 tumor and the surrounding tissues wherein the rabbit MSCs-Luc were intra-arterially injected.

Proof of concept of the intra-arterial delivery of transduced MSCs for GDEPT

We performed the proof of concept of a GDEPT with IA injection of transduced-MSCs in the VX2 rabbit liver tumor. For this purpose, 6 rabbits with VX2 liver tumor received once (3 rabbits) or twice (3 rabbits), intra-arterial injection of transduced-MSCs expressing the fusion gene. These injections were followed 48 hours later by intra venous injection of 80mg/Kg CPA. Twenty-five days later, animals were sacrificed. This study demonstrated a significant difference in tumor size in treated animals strongly associated to about 78% of tumor necrosis as compared to animal control (p<0.05). Indirect evidence of fusion gene efficacy was observed in the treated animal group by a significant decrease of the neutrophil count

Advances in knowledge

 It is feasible to proceed to intra-arterial delivery of transduced mesenchymal stem cells to elicit gene directed enzyme prodrug therapy.

 Our pilot study demonstrates sustainable tumor control rate in VX2 rabbit tumor model upon GDEPT.

Implication for patient care

This pilot study firstly demonstrated the feasibility and the efficacy of an intra-arterial injection of transduced MSCs for playing gene directed enzyme pro-drug therapy. Before envisioning human application further experiments are needed.

Summary statement

Hepatic intra-arterial injection of transduced MSCs followed by intravenous injection of CPA is feasible, allows significant tumor volume control, as assessed by tumor necrosis observed by histopathology examination and diminished the observed metastasis.

Introduction

The treatment of primary and secondary liver tumor remains challenging (187).

Currently, trans-arterial chemoembolization is mostly proposed as salvage treatment. The trans-catheter approach for the treatment of liver tumor is based on the fact that tumor blood supply arises from the hepatic artery, while normal liver parenchyma is vascularized by the portal vein system (234). These conditions favor local delivery of therapeutic agent (239). The selective intra-arterial injection of therapeutic agent allows a very limited systemic passage, leading thus to a decrease of systemic toxicity, with an enhanced local effect [START_REF] Collins | Pharmacologic rationale for regional drug delivery[END_REF]. Nonetheless, the overall clinical outcomes remain limited due to various reasons non optimal local drug release, absence of specific appropriate chemotherapy or hypovascular nature of certain type of li er tumors… Since two decades, several intra-arterial gene delivery approaches have been evaluated (240). Among the different therapeutic strategies, the gene directed enzyme / pro-drug therapy (GDEPT) consists in the delivery to tumor cells of a suicide gene coding for an enzyme responsible for the in-situ conversion of a pro-drug into cytotoxic metabolites.

This therapeutic strategy follows two sequential steps (Figure 25). Firstly the therapeutic gene is delivered to the tumor through a vector (i.e. lentivirus, plasmid or mesenchymal stem cells (MSCs) (244). Secondly a prodrug is administered using the IV route and is locally converted into the therapeutic agent by the suicide enzyme, which induces the death of tumor cells trough the by stander effect (197). Cyclophosphamide (CPA) is a widely used chemotherapeutic agent that requires activation by the cytochrome P450 2B6 (CYP2B6), to exert its cytotoxic effects. However, CYP2B6 has a low affinity for CPA and CPA elicits a very modest effect in liver tumors and its use is precluded by serious side effects (245). To overcome these limitations, a triple mutant gene has been engineered; this gene encodes the cytochrome CYP2B6TM that is fused to NADPH-cytochrome P450 reductase. This optimized suicide gene (CYP2B6TM-RED) is able to metabolize CPA 13-fold more efficiently than CYP2B6 wild type (223,246,247).

In order to deliver the engineered gene to the tumor, MSCs has been used as the vector because MSCs can be easily isolated, efficiently transduced to express the therapeutic gene and possess a high ability to migrate toward tumors and home into them (233). The proof of concept of this therapeutic strategy was previously obtained in a murine model with TC1 tumor cells. Transduced MCSs were firstly injected into the tumor, and then CPA was intraperitoneally given 72 hours later. This therapeutic sequence was repeated 4 times once a week. A complete eradication of the tumor in 33% of the mice without any recurrence 24 months later was achieved. However, one serious limitation of this approach was the very limited reliability of percutaneous intra-tumoral injection of MSCs, thus probably explaining the failure in the 66% uncured mice (248).

On these grounds, it appeared logical as a next step to develop an intra-arterial (IA) delivery approach. Beside the advantages described above, such an IA injection would enable an accurate delivery close to the tumor. To evaluate this approach we used the heterotopic VX2 tumor in rabbits [START_REF] Conrad | In patients with colorectal liver metastases, can we still rely on number to define treatment and outcome?[END_REF].

The goal of our pilot study was to assess the feasibility and efficacy of GDEPT using murine transduced MSCs to be intra-arterially delivered into a VX2 liver rabbit tumor model.

Materials and Methods

This animal study was fully compliant with the European Community guidelines for the care and use of laboratory animals and approved by our ethical committee (# 07430).

Experimental procedures were conducted at the Center for Research in Interventional Imaging (CR2i, APHP-INRA, France). Preliminary experimental works to assess the bystander effect and the IA distribution of MSCs have been performed and report in appendix.

Transduced mesenchymal stem cell preparations.

Murine MSCs-CYP2B6TM-RED were obtained as previously described by Amara I. et al. ( 248). Briefly, murine MSCs from bone-marrow of C57BL/6 mice (Life technologies, GIBCO, France) were transduced using lentiviral particles expressing the optimized suicide gene CYP2B6TM-RED (223).

Animal model

Vx2 cell liver implantation

Tumor carriers were induced by intramuscular injection of 10 7 VX2 cells in each hind leg of adult New Zealand White male rabbits. Tumor was allowed to grow for 2 weeks. Then the carrier tumors were excised and minced. Aliquots of fresh tumor chunks (~1mm 3 ) were then implanted in the left hepatic lobe of recipient animals as previously described by Lee K. et al. (249). Two weeks following implantation, a 15 cm 3 single VX2 liver tumor was identified in the left liver lobe by US scans examination, and treatment was initiated.

Effects of intra-arterial delivery of murine MSCs expressing the suicide gene (CYP2B6TM-RED) on tumor growth

Eleven New Zealand White rabbits weighing 3.8-4.3 kg bearing VX2 tumors were randomly assigned into 3 groups. Control group A (1 rabbit) free of any treatment; Control group B, 2 rabbits receiving only intravenous injection of 80mg/Kg CPA at day 2 (D2) and D9;

and Group C was composed of 8 rabbits receiving the GDEPT twice. The GDEPT treatment combined two steps. At D0, transduced-MSCs were injected intra-arterially into the hepatic left artery as close as possible to the tumor vessels. Secondly, at D3, CPA was intravenously injected (80mg/Kg in 30 min) through the marginal ear vein. The same protocol was repeated 7 days later. In case of severe toxicity transduced MSCs intra-arterial injection and CPA was postponed two days later, and a CPA dose reduction (40mg/Kg) was applied in all animals.

Hepatic angiography and MSCs-CYP2B6TM-RED injection.

Trans-catheter hepatic artery catheterization and MSCs-CYP2B6TM-RED injection were performed using fluoroscopy guidance. The animals were anesthetized (buprenorphine 30µg/kg, and Isoflurane 3% + oxygen 97%) and ventilated through endotracheal tube.

Surgical cut down was performed to gain access into the common femoral artery, through a 4-French sheath. The celiac axis and the left hepatic artery were selectively catheterized with a 2-French Progreat™ α microcatheter (Terumo Europe Leu en elgium). Following hepatic artery injection of contrast media (Xenetix 350 Guerbet, France), the tumor was visualized as a region of hyper-vascular blush located on the left side of the liver and 4.10 6 MSCs-CYP2B6TM-RED/rabbit mixed in 2 ml solution (PBS) were slowly infused into the left hepatic artery over a 1 min period. The technical procedure end point was the full injection of the MSCs-CYP2B6TM-RED. No embolic material was used.

Animal housing, well-being, follow-up and sacrifice

During the study, animals were housed under the supervision of a trained veterinarian team. The overall health and wellbeing of each animal was monitored daily until animal sacrifice at D25.

Blood examination (blood cells count, liver function (ALT, ALP, bilirubin, albumin), and renal function were determined at D0, D7, D19, and D25

The tumor size was evaluated by ultrasound (Voluson E8 Expert; GE Healthcare, France) D0, D7, D11, D19, and D25. Three orthogonal diameters were registered for volumetric tumor evaluation.

All animals were sacrificed at D25 using general anesthesia (Isoflurane 5% + oxygen 95%) and an over dosage of sodium pentobarbital (182mg/Kg).

Macroscopic observation and histology

For each animal a summary of the key observations was recorded at necropsy. Rabbit liver (right, left and caudate lobe), lungs, kidneys, stomach, and bowel were removed and fixed in 20/1 acetic formalin. Then a cut was done into the liver tumor at the maximum diameter. The longest tumoral diameter was recorded and 5 slices of 5 mm thickness were then prepared for pathologic examination: two into the tumor, one into the normal liver tissue adjacent to tumor (≈1 cm); one in the left lobe (>1cm away from the tumor), and one in the right lobe. All samples were embedded in paraffin and stained with hematoxylin eosin saffron. Tumor viability was estimated by visual inspection and expressed as a percentage of viable tumor area for each slice.

Statistics.

Statistical analysis was performed using Xlstat version 12.0 (Addinsoft, France).

Results were expressed as mean/median, standard deviation (SD) values. Kruskal-Wallis non-parametric tests were used for comparisons of continuous variables between the groups. Results were considered as statistically significant if p < 0.05. At D11, 3 out of the 6 surviving animals were in a general status suitable for a second GDEPT session (group C2). These 3 animals received intra-arterial injection of 4.10 6

Results

Feasibility of Intra-arterial delivery of

transduced MSCs at D11, followed at D14 by half a dose of CPA (40 mg/Kg) to overcome toxicities. In addition, N-acetyl cysteine (225 mg/Kg IV) was used as liver and renal protector against CPA metabolites toxicities. No further toxicity was observed after the second GDEPT session in all these 3 animals. The 3 other animals (group C1) were kept alive in the animal facility and underwent complete follow-up.

At Day 25, all surviving rabbits were sacrificed. In total, 3 animals completed two full GDEPT sessions and 3 received only one GDEPT due to severe toxicity.

Follow-up and toxicities

In all treated rabbits from group B and C, we observed food and water intakes reduction together with a mild polyuria and hematuria. An increase of alanine aminotransferase and alkaline phosphatase levels were observed in groups B and C as compared to the non-treated animal at D7 and at a lesser extent at D19. All values returned to normal values at D25 (Table 15). No other biological abnormality was observed (bilirubin, albumin and serum creatinine).

Neutropenia was also observed in groups B and C, but was significantly more severe in the C2 group receiving twice the GDEPT (p<0.05). At D25 this neutropenia disappeared in all treated groups (Table 15). 10.7 ± 1.7 [9.6-11.9] 10.2 ± 1 [9.5 -10.9] 4.3 ± 0.8 [3.7 -4.8] 14 ± 7.8 [8.5 -19.5] Group C group C1 6.1 ± 0.5 [5.8 -6.6] 3 ± 0.3 [2.8 -3.4] 5.8 ± 0.2 [5.7 -6] 12.9 ± 2.9 [9.6 -14.9 38 ± 9 [28 -46] 263 ± 234 [20 -487] 43 ± 20 [26 -65] 35 ± 11 [27 -48] group C2 40 ± 8 [35 -49] 53 ± 24 [33 -80] 35 ± 6 [29 -40] 42 ± 14 [33 -58] Table 15: Blood examination results performed at D0, 7, 19, and 25.

Tumor volume follow-up

A significant antitumoral effect of the GDEPT was observed. A significant tumor volume difference between group B and group C at D7 (p=0.024), D11 (p=0.024), D19 (p=0.048) and D25 (p=0.048) was observed.

Macroscopic examination and histopathology

At sacrifice, in the control groups (A & B), gross examination yielded a median of 4 [3 -5] peritoneal carcinomatosis nodules with a median of lung metastases of 12 [12 -15] per animal. In group C peritoneal carcinomatosis and lung metastases were observed in 4

(median 3 nodules) and 3 (median 3 nodules) animals, respectively. Strikingly, 2 rabbits out of 3 that received twice the GDEPT protocol had neither peritoneal carcinomatosis nodules nor lung metastases.

Liver macroscopic examination showed in control animals (group A and B) the largest tumors, with major core liquefaction. The normal liver from group A, B and C did not display any necrotic areas or other abnormalities. In Group C, tumors appeared necrotic with a fibrotic core in some instance.

Histopathological examination showed 78 ± 9 % tumor necrosis, 12 ± 7 % fibrosis and 10 ±5 % residual tumors group C, with no intravascular tumoral embolus. This was associated to a moderate inflammatory reaction in the portal space. In the control groups a mean of 28% ± 22 of tumor necrosis was observed, associated with 22% ± 12 of fibrosis and 50% ± 10 of viable tumors (Figure 28). Moreover all animals from the control groups exhibited venous tumoral embolus either in the peri-tumoral area both in the right/medial liver lobe, reflecting the aggressive nature of the VX2 tumor model. Significantly less residual tumor was observed in the the GEDPT treated animals compare to the control groups (p<0.05). Multiple VX2 nodules can be seen with large viable (vT) and limited necrotic (nT) tumor tissue surrounded by fibrotic (F) linear area. The Figure 28b shows Representative histology section stained with haematein-eosin-saffron of a tumor from group C2 (original magnification × 2.5). VX2 tumor is massively necrotic (nT) with only few cluster of viable tumor (vT) limited by fibrotic (F) linear area.

Discussion

In this study, we demonstrated the feasibility and the sustainable efficacy of intraarterial delivery of a suicide gene carried by transduced MSCs. We obtained significant volume reduction of VX2 liver tumors thanks to the by stander effect compared to control groups.

This experiment follows preliminary works in which was developed a successful active gene, a gene vector, a transgene expression in the tumor microenvironment, a by-stander effect and an immune response (223,248).

We also demonstrated the feasibility and the higher benefit of repeating intraarterial delivery of transduced-MSCs (D0 and D9) as vector for GDEPT to a liver tumor model.

Indeed the VX2 model tumor treated by GDEPT as compared to the control group showed a marked reduction of growth along the treatment at Days 7,[START_REF] Takayama | Colorectal cancer: genetics of development and metastasis[END_REF]19 and 25 Histopathology examination confirmed the results by demonstrating a larger significant tumor necrosis in the treated group while lung metastases were not found in two animals over three that received the GDEPT twice.

In this work we also demonstrated that intra-arterial delivery of MSC is a valuable approach, being able to overcome limitations of alternative administration route.

Despite apparently easier to use, IV administration or direct injection into the tumor failed to allow proper antitumoral effect because MSC are exposed to undesirable interactions between blood components, non-target cells, such as those in the reticuloendothelial system (240, 242). We observed previously that percutaneous injection result in highly variable and overall insufficient gene delivery in the tumors. In fact, we observed in this previous work that only 33 % of animals were cured and we suggested that this could be related to insufficient MSC accessing into the tumor (248). In fact maximal transgene expression occurs along the needle tract, and in addition, the expression of the therapeutic gene decreases with the increase in distance from the injection site to the tumor (250).

Among gene delivery vectors, mesenchymal stem cells (MSCs) are particularly suitable for suicide gene therapy due to their ability, to migrate towards tumors and to not express major histo-compatibility complex (251). This property was well demonstrated in our preliminary luciferase assay where we were able to demonstrate the specific MSCs homing within hepatic tumor without any "leakage" in others organs (see appendix).

Therefore, it seems very likely that an efficient GDEPT could result from hepatic intra-arterial injection of transduced-MSCs while no embolic material is needed to maintain the gene vector in the tumor micro-environment.

Transduced-SCs will thus allow a "Trojan-horse" suicide gene strategy. Noteworthy using transfected-MSCs, no tumor cells gene transfection is necessary as they are in-situ the "first actor" of the GDEPT. Thereafter tumor cells death is promoted thanks to the diffusion to the cytotoxic metabolites so called by-stander effect.

Literature indicate MSCs are weakly immunogenic as they elicit a minimal expression of the major histo-compatibility complex. Pro-inflammatory mediators (such as interferongamma) have been proposed to activate the MSCs antigen-presenting cell and could induce a valuable CD8+ T cell response (T lymphocyte killer) (248,252). Interestingly these features may explain the low occurrence of lung metastases and peritoneal carcinomatosis in the animal group C2 which received the 2 scheduled GDEPT.

Cyclophosphamide is a pro-drug that is catabolized by cytochrome P450-2B6 (CYP2B6) in two compounds i.e., acroleine and phosphoramide mustard which is an alkylating agent that causes intra-and inter-DNA cross-links blocking cells mitosis and promotes apoptosis (253). The fusion gene (so-called CYP2B6TM-RED) codes a mutated cytochrome that is 13 times more active than CYP2B6 wild type and is sufficient to achieve full CPA activation even in absence of endogenous reductase that is not expressed in most tumors cells (215,246,254). An inside-out passive diffusion mechanism of phosphoramide mustard from transduced MSCs to tumor cells though GAP junction allows cell death through the by-stander effect [START_REF] Manfredi | Epidemiology and management of liver metastases from colorectal cancer[END_REF].

Our in vitro tests showed that VX2 cells were insensitive to CPA (see appendix). When MSCs expressing CYP2B6TM-RED were treated with CPA, the produced supernatants containing cytotoxic metabolites can kill VX2 tumor cells. These data demonstrate, thus, an in vitro efficient by-stander effect.

As, so far, literature has reported few CPA metabolism in rabbit, one could expect a lack of CPA metabolism and subsequence toxicities in this animal species (243). The neutropenia and liver toxicity in the group control clearly point out an endogenous CPA metabolism. However it must be noted that the neutropenia was deeper in the test group than in the control group, thus indicating an indirect proof of CPA metabolism by transduced-MSCs.

The above-described proof of concept study demonstrates the feasibility and efficacy of intra-arterial GDEPT in rabbit; however, toxicity was significant and should be improved.

Regarding toxicity, based on literature data, in rabbit, CPA at 80 mg/kg of led to unwanted liver and renal toxicities leading to determine the CPA optimal efficacy/toxicity ratio; in addition the protecting agent MESNA could be used to limit this toxicity as shown in humans. Concerning efficacy, the increase of anti-tumoral response could be obtained by optimizing the amounts of injected transduced-MSCs, the number of injections the doses of CPA and the delay between transduced-MSCs injection and CPA treatment.

Altogether our study demonstrates the feasibility and the efficacy of a GDEPT using mesenchymal stem cells transfected with mutated gene coding for a hyper-active cytochrome P450 (modified CYP2B6). Hepatic intra-arterial injection of transduced MSCs followed by intravenous injection of CPA is feasible, allows significant tumor volume control, as assessed by tumor necrosis observed by histopathology examination and diminished the observed metastasis.

Appendix

Before envision the proof of concept of a GDEPT with transduced MSCs intra arterially delivered in a VX2 rabbit liver tumor, two experiments were needed. The first experiment was performed to assess in-vitro the by-stander effect, as the second one was to study MSCs homing into the tumor microenvironment when MSCs are intra arterially injected.

Evaluation of in-vitro by-stander effect

To determine if the by-stander effect operate with VX2 carcinoma cells, murine MSCs-CYP2B6TM-RED clone and VX2 carcinoma cells were seeded in triplicate into 96-well plates at different ratios, in MSCs culture medium. Twenty-four hours later, cells were exposed for 3 days to CPA (0 to 1 mM) and cell viability was measured by MTS assay.

In-vitro, VX2 tumor cells were insensitive to CPA up to 1 mM. We added increasing

Assessment of MSCs distribution after intra-arterial (IA) delivery

To assess the outcome of MSCs after IA injection into the hepatic artery, we evaluated 2 New Zealand White rabbits as follows: 1 rabbit bearing VX2 liver tumor and 1 rabbit without any tumor underwent single IA delivery in the left hepatic artery of rabbit MSCs expressing luciferase. Rabbit MSCs-luciferase were obtained as previously described by Amara et al. (submitted manuscript). Briefly, rabbit MSCs from bone-marrow of New-Zealand white rabbits (Clinisciences, Nanterre, France) were transduced by lentiviral particles expressing either luciferase or the optimized suicide gene CYP2B6TM-RED, respectively [START_REF] Takayama | Colorectal cancer: genetics of development and metastasis[END_REF]. We checked that the pool of rabbit MSCs expressed luciferase at high level by the luminescence assay system (Promega, Madison, Wisconsin); this pool was further used to study MSCs distribution after intra-arterial injection.

Seventy-two hours later, both animals were sacrificed. Tumor and various organs (liver, kidneys, spleen, stomach, duodenum, lung, heart) were removed, homogenized in PBS and centrifuged at 10 000g for 3 min. The luminescence of 20 µl of the supernatant was measured using the luciferase assay system. The results are expressed as arbitrary units of luminescence per g of protein.

The photo-luminescence assay performed 72 hours later clearly showed in the rabbit bearing a VX2 tumor a very high expression of luciferase essentially near and into the tumor with almost no expression in other hepatic lobes and organs. In the control rabbit without tumor, luciferase was weakly detectable near the injection site without diffusion in other organs (Figure 30 appendix). Somme small and limited ischemic areas likely related to an embolic effect of the rabbit SCs, gi en their relati ely large size (200 × 13 μm) were observed. To overcome this drawback during GDEPT and to avoid this confounding factor, we went on with smaller MSC. Therefore and given the absence of major histocompatibility complex in MSCs, we chose mice MSCs for the next steps of our experiment. ≈-≈-≈-≈-≈--≈-≈-≈-≈-≈

Discussion

The efficacy of novel strategies of gene therapy in cancer depends on i) their ability to target selectively the tumor and, ii) to deliver the genetic material into adequate quantities to tumor cells (255)(256)(257)(258)(259)(260). In this respect, the large part of success greatly depends on achieving high tumor transfection, -in using the best vector-, in addition to an effective anticancer strategy (259,260). Noteworthy, it was shown that direct intra-tumoral delivery leads to a maximal transgene expression along the needle tract, that gradually decreases as a function of the distance between the injection site and the tumor (250).

Intra-arterial delivery of gene vector represents a very valuable route to consider as shown by experimental and clinical studies (261-263). Indeed, two evidences have to be kept in mind. First, tumors derive their blood supply from the arterial circulation, indicating arterial delivery may target the cancer relative to the detriment of normal tissues (234).

Second, when therapeutics are delivered through an artery, the first pass clearance of gene therapy vectors by the liver stays minimal and hence reduces systemic side effects [START_REF] Collins | Pharmacologic rationale for regional drug delivery[END_REF]264).

In previous studies, when gene therapy vectors when administered via the hepatic artery, about 98% of the injected vector were identified in the liver tumor (242,264,265). In other words, when IA pathway is chosen, the majority of gene therapy vector is cleared by the organ, and gives a strong pharmacologic rational to our approach.

However, the lack of success of some intra-arterial gene therapy trials may be related to delivery issues rather than a payload bioactivity. A lot of variables that affect tumor transfection via the intra-arterial route in a cancer gene therapy context had been previously surveyed in order to optimize bioavailability (255,266). As delivered into the arterial blood stream, the gene vector has to cross the vessel wall, before moving across the extracellular matrix and to, finally, reach the tumor cell (255)(256)(257)(258)(266)(267)(268). In addition to these anatomic impediments, 3 physiological barriers may explain the possibly poor bioavailability of treatment in tumors: 1) a heterogeneous tumor vascular supply limits the delivery of therapeutics to well vascularized or well perfused regions of the tumor; 2) elevated tumor interstitial pressure reduces the penetration of the therapeutic macromolecules whereby a high pressure outward radial gradient leaves tumor regions without drug; and 3) large transport distances within the tumor interstitium slow down the transit of the treatment and thus make distal regions of a tumor hard to reach (266). Within the goal to increase the gene to tumor cells transfection, embolic material such as Lipiodol® has been proposed and showed a significant increase of gene transduction to tumor when comparedto a free delivery of gene vector into the arterial tumor network (242). This is an approach that will be thoroughly evaluated in our lab in the following months.

MSCs are particularly suitable for GDEPT since, thanks to their remarkable properties, they can overcome the above-delineated limitations. Indeed, MSCs spontaneously migrate towards tumors and do not express histo-compatibility complex (251). When they are intraarterially delivered, MSCs express their specific homing properties within hepatic tumor.

MSCs nest themselves into the tumor microenvironment and remain in large amount 72 hours after their deposit, while no embolic material is needed to maintain the gene vector in the tumor microenvironment. Moreover in case of unwanted deposition of MSCs in others organs (eg. lung, heart, brain, kidney…), mesenchymal cells did not sur i e o er 72 hours.

Consequently, this evidence points out no target damage when GDEPT will be set at the CPA injection.

Transduced-SCs will thus allow a "Trojan-horse" suicide gene strategy. To play GDEPT with lentivirus or plasmids gene vector, the suicide gene transfection of tumors cells requires a high level of gene expression to be expressed. By contrast and noteworthy using transfected-MSCs, no tumor cells gene transfection is necessary as they are in-situ the "first actor" of the GDEPT. Thereafter tumor cells death is promoted due to the diffusion to the cytotoxic metabolites, according to the so-called by-stander effect. In this line, our preclinical study demonstrates the feasibility and the efficacy of intra-arterial delivery of transduced-MSCs as vector for GDEPT to a liver tumor model leading to tumor control rate.

The amount of administered therapeutic cells necessary to home to the tumor was estimated to less than 10% of the tumor mass (269,270). Therefore, it is reasonable to use The pilot rabbit models gave us many information on the feasibility of the approach, on the initial doses to use and was partially successful in terms of tumor reduction and impairment of metastasis; however four main issues need to be addressed.

-Toxicity: 80 mg/kg of CPA (based on literature data) led to liver and kidney toxicities (preliminary experiments). It will be thus essential to determine the CPA ideal dose in terms of efficacy/toxicity ratio.

-Efficacy: to reinforce the anti-tumoral effect we will increase.

- -Immune response: to describe the interaction of our therapy and the immune system.

-The immune response will be tested by re-challenge experiments in previously treated rabbits.

-Evaluation: to evaluate tumor response rate and animal survival -To compare tumor response rate of our strategy to conventional TACE in VX2 liver tumor model.

-To measure animal survival when GDEPT strategy is applied in canine showing hemangiopericyotma (spontaneous tumor) hepatic tumor. This will be performed in a veterinary facility.

Annex I

Colorectal cancer genetic and biomarkers:

Colorectal cancer carcinogenesis mechanisms

Colon carcinogenesis can result from three types of mechanisms: chromosomal instability (CIN), genetic instability (microsatellite instability MSI), and CIMP phenotype. 16: pathways of carcinogenesis CIN + and MSI +: main features and differences.

Chromosomal instability (CIN phenotype)

This phenotype is present in 80% of sporadic colorectal cancers. It is defined by the occurrence of recurrent losses on certain chromosome arms, leading to loss of heterozygosis some of which are associated with mutations of tumor suppressor genes (APC and TP53) (274).

Genetic instability (MSI phenotype)

The instability of microsatellite loci is due to a failure of repair, i.g, the DNA mismatch repair (DNA MMR) mechanism. This secondary mechanism corresponds to hypermethylation of the promoter region of the hMLH1 gene in sporadic cancers. In the Lynch syndrome a constitutional mutations of hMSH2, hMLH1, hMSH6, and PMS2 gene inactivate the MMR. The tumors in this group have a phenotype called MSI + and represent 15% of colorectal cancers (275).

CIMP phenotype

The CIMP phenotype corresponds to the cytosine methylation of CpG islands of the promoter regions genes. This results in an inactivation of the transcription, such as the tumor suppressor gene CDKN2A. Part of CIMP + tumors are MSI tumors, with hypermethylation of the hMLH1 gene promoter (276). Figure 31

Main altered signaling pathways in colorectal cancer

Four mains signaling pathway could be altered in colorectal cancer.

-The APC pathway. The constitutional alteration of the APC gene is responsible of the familial adenomatous polyposis. APC is a tumor suppressor gene, which is mutated in 60-80% of colonic cancers with CIN phenotype. This phenomenon is associated to the loss of the other allele leading to a complete inactivation of the APC gene. APC protein normally provides a negative control on cell cycle through interaction with betacatenin ( 14).

- -The PI3K signaling pathway. This pathway that is important for cell cycle control, is frequently altered in colorectal cancers at various levels. PI3K protein is composed of two subunits. The PIK3CA gene encodes the p110 catalytic subunit whose mutation concerns32% of colorectal cancers. P110 subunit allows the generation of phosphatidylinositol [START_REF] De Baere | Arterial therapies of colorectal cancer metastases to the liver[END_REF][START_REF] Jemal | Global cancer statistics. CA: a cancer journal for clinicians[END_REF]5triphosphate (PIP3). A cascade of phosphorylation of different proteins: PDK-1, AKT (or PKB), mTOR (mammalian target of rapamycin), activates the p70S6 kinase (p70S6K) and the EIF4E (eukaryotic translation initation factor-4). Phosphatase PTEN (phosphatase with tensin homology) represents a negative regulator of the PI3K pathway, by dephosphorylating PIP3.

PTEN gene is a tumor suppressor gene, having inactivating mutations in 15 to 30% of case.

Another actor of this pathway is GSK3 beta (glycogen synthase kinase-3) which is a kinase regulated by both the Wnt / betacatenin and PI3K (278).

-The transforming growth factor TGF-ß and the "genome guardian" p53, two suppressor genes, are the key partners of two others signaling pathway that negatively control the cell cycle. These two genes are frequently inactivated in colorectal cancer. TGF-ß pathway is deregulated in 20 to 30% of colorectal cancers CIN+ phenotype by inactivating mutations of the gene as well as SMAD4 SMAD2 which encode proteins that transduce the signal induced by the interaction between the ligands and the receptor type I or II TGF ß (TGF-TGFßRI or ßRII). TGFb-SSII gene is inactivated in 60 -80% of the phenotype MSI [START_REF] Takayama | Colorectal cancer: genetics of development and metastasis[END_REF]. The protein p53 allows either the repair of DNA damage by blocking the cell cycle so that they are not repaired or induction of apoptosis of cells for which the DNA repair could not take place. [START_REF] Rosen | Colorectal cancer screening disparities related to obesity and gender[END_REF]:1043-52 [START_REF] Perkins | Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer[END_REF].

Annex II Treatments of metastatic colorectal cancer (mCRC): state of the art

Although the prognosis for patients with mCRC without specific treatment remains limited, the deciphering of signaling pathways described in Annex I has led to multiple new treatment options, over the last two decades. As a result, median survival has increased from approximately 6 months in the 1990s to nearly 30 months in the latest large randomized study [START_REF] Venook | CALGB/SWOG 80405: Phase III trial of irinotecan/5-FU/leucovorin (FOLFIRI) or oxaliplatin/5-FU/leucovorin (mFOLFOX6) with bevacizumab (BV) or cetuximab (CET) for patients (pts) with KRAS wildtype (wt) untreated metastatic adenocarcinoma of the colon or rectum (MCRC)[END_REF][START_REF] Scheithauer | Randomised comparison of combination chemotherapy plus supportive care with supportive care alone in patients with metastatic colorectal cancer[END_REF]. Such an improvement in survival has been driven by the sequential deployment of a variety of seminal chemotherapy combined with the so-called targeted therapeutic agents. This latter class includes monoclonal antibodies directed to various growth factors and their enzymatic machineries: vascular endothelial growth factor (VEGF) (bevacizumab) and epidermal growth factor receptor (EGFR) (cetuximab and panitumumab), aflibercept, a recombinant fusion protein also directed against VEGF, and regorafenib, an active inhibitor of multiple tyrosine kinases. Finally, a small but substantial minority of patients with isolated sites of metastases may potentially be curable with surgery and liver-directed therapies. The availability of multiple therapeutic agents for the treatment of mCRC therefore requires a strategic approach to maximize patient benefit, in terms of both life expectancy and quality of life. When determining initial treatment, the first step is to evaluate whether the patient is potentially curable by a surgical resection of metastases, either at the time of diagnosis or after chemotherapy. This approach will guide the choice and timing of chemotherapy. If the patient does not seem curable, treatment regimens that offer the longest progression-free survival (PFS) and overall survival (OS) and that maintain quality of life as long as possible are to be preferred.

Treatment algorithm

The Figure 1 summarizes the patient algorithm that combines available clinical and molecular data, for selection of systemic treatment and integration with surgical resection in patients with mCRC.

Liver surgery for mCRC

Out of the patients diagnosed with colon and rectal cancer, 50% will develop metastases and a large proportion of these will be liver metastases (CRLM) [START_REF] Tzeng | Colorectal liver metastases[END_REF].

Unresectable disease is the norm in these cases, but among the estimated 20% who are able to achieve complete resection, there is a related overall 5-year survival of 50% to 60% and associated with a hazard ratio of 1.3 for disease recurrence [START_REF] Tzeng | Colorectal liver metastases[END_REF]. In fact, liver resection is the sole treatment associated with long-term survival in patients with liver mCRC [START_REF] Fong | Clinical score for predicting recurrence after hepatic resection for metastatic colorectal cancer: analysis of 1001 consecutive cases[END_REF][START_REF] Stangl | Factors influencing the natural history of colorectal liver metastases[END_REF]. The surgical management of CRLM is complex. The rationale for hepatic resection is based on the fact that patients are cured by surgery at a rate similar to that for some primary nonmetastatic malignancies. The goal of surgery is to obtain a complete tumor resection with safe margin (R0) and, although non-anatomic wedge resections are sometimes sufficient, wider segmental or lobar resections may provide a better chance of surgical success. Overall, the rate of operative resectability is high and a small minority of patients undergoes nontherapeutic laparotomy. Predictors of survival include size and number of tumors, short disease-free interval, node-positive primary tumor, positive margins, and the presence of extra hepatic disease. These factors do not always exclude patients from resection given the possibility of long-term survival, even in the presence of poor prognostic factors. Overall mortality after hepatic resection for liver mCRC is less than 1%, likely resulting from improved operative technique and patient selection [START_REF] Tzeng | Colorectal liver metastases[END_REF]. Systemic chemotherapy has improved for patients with unresectable liver mCRC, but randomized trials to date have not shown a significant improvement in outcomes with modern regimens given in the perioperative setting [START_REF] Nordlinger | Perioperative chemotherapy with FOLFOX4 and surgery versus surgery alone for resectable liver metastases from colorectal cancer (EORTC Intergroup trial 40983): a randomised controlled trial[END_REF]. Response to neoadjuvant therapy is not considered as prognostic factor as even patients with progression who remain resectable can exhibit long-term survival after resection. Conversion to complete resection can be obtained in patients with unresectable CRLM using a combination of systemic and Hepatic Arterial Injection (HAI) therapeutic approach [START_REF] Goere | Prolonged survival of initially unresectable hepatic colorectal cancer patients treated with hepatic arterial infusion of oxaliplatin followed by radical surgery of metastases[END_REF].

Systemic regimens for metastatic colorectal cancer

In patients with unresectable mCRC representing most cases, systemic treatment focuses on tumor control. Occasionally the treatment may be symptoms directed (palliative)

and not with a curative aim. The treatment goals are to increase life expectancy while maintaining quality of life for as long as possible. In this context, the model of predefined successive lines of chemotherapy is being abandoned in favor of a continuum-of-care approach, case by case, similar to that taken in other chronic illnesses [START_REF] Goldberg | The continuum of care: a paradigm for the management of metastatic colorectal cancer[END_REF]. The 3 active conventional chemotherapeutic agents for mCRC are fluoropyrimidines (including intravenous 5-fluorouracil or its oral prodrug equivalent, capecitabine), irinotecan, and oxaliplatin (Table 1). Patients clearly benefit from access to all active agents [START_REF] Goldberg | The continuum of care: a paradigm for the management of metastatic colorectal cancer[END_REF][START_REF] Grothey | Overall survival of patients with advanced colorectal cancer correlates with availability of fluorouracil, irinotecan, and oxaliplatin regardless of whether doublet or single-agent therapy is used first line[END_REF]. A variety of targeted therapeutic agents are also available (see Table 1), which are incorporated into conventional chemotherapy regimens at various time points across the continuum of care.

Initial Treatment: the Chemotherapy Backbone

Data from head-to-head comparisons suggest that outcomes with first-line oxaliplatin, 5-fluorouracil, and leucovorin (FOLFOX) and irinotecan, 5-fluorouracil, and leucovorin (FOLFIRI) are similar [START_REF] Colucci | Phase III randomized trial of FOLFIRI versus FOLFOX4 in the treatment of advanced colorectal cancer: a multicenter study of the Gruppo Oncologico Dell'Italia Meridionale[END_REF]. The choice between initial FOLFIRI and FOLFOX should be based on expected treatment-related toxicity in the context of coexisting co-morbidities for any given patients. For instance, a patient with long-standing diabetes mellitus or preexisting neuropathy may be recommended FOLFIRI rather than the neuropathy inducing FOLFOX regimen. For patients who are not candidates for an intensive oxaliplatin-based or irinotecan-based regimen because of comorbidities, performance status, or personal preference, fluoropyrimidine therapy alone (with or without a targeted therapy agent) can be considered. In early studies, high rates of successful resection and favorable long-term survival rates for patients with initially unresectable liver metastases have been reported for a triplet regimen that combines all 3 classes of conventional chemotherapy (FOLFOXIRI), and this regimen may be considered in highly select patients until additional randomized data are available [START_REF] Masi | Long-term outcome of initially unresectable metastatic colorectal cancer patients treated with 5fluorouracil/leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) followed by radical surgery of metastases[END_REF]281).

For patients receiving a FOLFIRI-like regimen after progression on FOLFOX, expected response rates are between 4% and 20%, and PFS of 2.5 to 7.

Incorporating Anti-Vascular Endothelial Growth Factor Agents

Bevacizumab is an anti-VEGF monoclonal antibody. It does not have significant single agent activity in metastatic colorectal cancer (285). However, multiple clinical trials have shown that bevacizumab adds benefits to first-line fluoropyrimidine-, oxaliplatin-and irinotecan based regimens given across the continuum of care in patients with metastatic disease [START_REF] Hurwitz | Bevacizumab in combination with fluorouracil and leucovorin: an active regimen for first-line metastatic colorectal cancer[END_REF][START_REF] Kabbinavar | Combined analysis of efficacy: the addition of bevacizumab to fluorouracil/leucovorin improves survival for patients with metastatic colorectal cancer[END_REF][START_REF] Vincenzi | Bevacizumab in association with de Gramont 5-fluorouracil/folinic acid in patients with oxaliplatin-, irinotecan-, and cetuximab-refractory colorectal cancer: a single-center phase 2 trial[END_REF]. In the randomized TREE-2 trial, for instance, adding bevacizumab to oxaliplatin and 5-fluorouracil-containing regimens in previously untreated patients resulted in a median OS of 23.7 months versus 18.2 months for the combined non-bevacizumabtreated groups (286). The addition of bevacizumab to oxaliplatin-based regimen in previously treated patients with metastatic colorectal cancer (with 5-fluorouracil or irinotecan) also led to improved PFS (7.3 vs 4.7 months) and median OS (12.9 vs 10.8 months) compared with FOLFOX alone in the ECOG 3200 trial conducted in the second-line setting (285). Thus, regarding an individual patient for initial or subsequent treatments, the accepted consensus in the management of metastatic colorectal cancer is to add bevacizumab to the chosen chemotherapy backbone. Bevacizumab is associated with increased rates of grade 3 or 4 hypertension, bowel perforation, impaired wound healing, arterial thromboembolism, and bleeding events (287). So careful patient selection and monitoring for toxicity is mandatory, when surgical intervention is to be considered. Given that the half-life of bevacizumab is approximately 3 weeks, the general recommendation of authors is to hold bevacizumab for a period of 4 to 6 weeks before and after surgery to avoid postsurgical complications such as wound dehiscence. Traditional teaching with conventional treatments has been to discontinue all classes of drugs when patients progress.

It is unclear, however, whether a similar strategy should be adopted with biological targeted agents, particularly those with antiangiogenic activity. The efficacy of continuing bevacizumab beyond progression was tested in the ML18147 phase III trial, which demonstrated a significant improvement in PFS (5.7 vs 4.1 months) and OS (11.2 vs 9.8 months). Bevacizumab-related adverse events were not increased in comparison with historical data of first-line bevacizumab treatment (288).

Aflibercept is a recombinant fusion protein that binds with higher affinity to VEGF-A than does bevacizumab in a cell-free system (289). In the VELOUR trial, the median OS was significantly longer in patients treated with aflibercept (13.5 vs. 12.1 months) and the median PFS was 6.9 vs. 4.7 months. Treatment benefit was similar regardless of prior bevacizumab exposure (about 30% of patients in the trial) (290). However, there are no head-to-head trials comparing continuation of bevacizumab beyond progression with switching to aflibercept in this setting; hence, both antiVEGF agents are considered as appropriate.

Incorporating Anti-Epidermal Growth Factor Receptor Agents: Role of Precision Therapy

Anti-EGFR agents can improve outcomes in metastatic disease, both as single agents and in combination regimens, pending biomarker analysis (research of tumor mutations in tumor biopsies) to select appropriate patient. With regards to colon cancers and so far, this so-called precision or personalized medicine leans on the EGFR pathway functionality, which strickly depends on RAS genes status (K RAS & NRAS). Activating mutations in KRAS, which induce constitutive activation of the RAS-RAFERK pathway, result in resistance to anti-EGFR therapy (291). In 2009, the American Society of Clinical Oncology recommended that all patients being considered for anti-EGFR therapy must undergo KRAS mutation testing of their tumors, and that treatment with these agents be restricted to those with wild-type (WT) KRAS, defined as an absence of mutations in exon 2 of KRAS gene by qualitative realtime polymerase chain reaction (292). Emerging data suggest that resistance to anti-EGFR therapies can also be mediated by lower-frequency mutations in KRAS outside of exon 2 and in NRAS [START_REF] Vincenzi | Bevacizumab in association with de Gramont 5-fluorouracil/folinic acid in patients with oxaliplatin-, irinotecan-, and cetuximab-refractory colorectal cancer: a single-center phase 2 trial[END_REF][START_REF] Tol | Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer[END_REF][START_REF] Hecht | A randomized phase IIIB trial of chemotherapy, bevacizumab, and panitumumab compared with chemotherapy and bevacizumab alone for metastatic colorectal cancer[END_REF][START_REF] Grothey | Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial[END_REF] In 2013, the PRIME study demonstrated that within the so-called classic WT KRAS population (without mutations in exon 2), patients with mutations in other KRAS exons (exons 3 and 4) or in NRAS (exons 2 and 3) did not benefit from the addition of panitumumab to FOLFOX. Of concern, such patients had a non-significantly worse PFS (7.3 vs 8.0 months, p=5 .33) and OS (hazard ratio 1.39, p=5 .12) with the addition of anti-EGFR therapy (293). Furthermore, the addition of panitumumab to chemotherapy increased OS significantly in patients with so-called extended WT RAS (no mutations in exons 2, 3, and 4 of KRAS and NRAS) (277). Other analyses have confirmed these findings. The emerging consensus is that all patients with metastatic colorectal cancer should be tested for extended RAS mutations and that those with such mutations should not be recommended anti-EGFR therapy. First-line cetuximab was explored in the CRYSTAL trial in previously untreated metastatic colorectal cancer; patients were randomly assigned to FOLFIRI with or without cetuximab. Among patients with WT KRAS, response rates were significantly higher in those who received cetuximab (57% vs 40%), as was median PFS and OS (23.5 vs 20 months) (294). The EPIC trial randomly assigned 1298 oxaliplatin-refractory patients to irinotecan with or without cetuximab. PFS was significantly higher with combined therapy (4 vs 2.6 months), as were rates of objective response (16% vs 4%) and overall disease control (61% vs 46%) (295). Increasing data also support the efficacy of first-, second-, and third-line panitumumab in combination with oxaliplatin-based or irinotecan-based regimens in patients with WT RAS tumors (296). Cetuximab and panitumumab appear to have comparable efficacy when used as single agents for salvage therapy in patients with chemotherapy-refractory metastatic colorectal cancer, and when used for initial or subsequent therapy for metastatic colorectal cancer in conjunction with an irinotecan-based chemotherapy regimen (293). The choice of the anti-EGFR agent largely depends on provider comfort with specific agents, concerns regarding infusion reactions, and logistics (cetuximab is a weekly regimen whereas panitumumab is every other week). Although response rates in individual studies were higher with the addition of cetuximab to chemotherapy than by adding bevacizumab in patients with WT KRAS status, the median survival benefit is similar (293). When there is likelihood of converting patients to resectable metastatic disease, the improved response rates supports using anti-EGFR therapy in the first-line setting. Dual antibody therapy targeting both VEGF and EGFR has been tested and found to lead to worsened outcomes, and is therefore not recommended 19114685 [START_REF] Tol | Chemotherapy, bevacizumab, and cetuximab in metastatic colorectal cancer[END_REF]. Among patients with WT RAS, an important issue is whether to add bevacizumab or anti-EGFR therapy to the chemotherapy backbone as initial therapy. Two initial trials with small sample sizes suggested benefit for an "anti-EGFR first" approach. Howe er, definiti e results from the largest such study, C80405, a US Intergroup study, were presented in 2014 and showed no survival difference for either cetuximab or bevacizumab when combined with a chemotherapy backbone in the initial treatment setting (29.9 vs 29 months, P 5 .34).

Therefore, either antibody can be used in the initial therapy setting, with choice driven again by toxicity, patient preference, and logistics. It should be noted that C80405 did include patients who may have had extended RAS mutations (only classic KRAS mutants were excluded); results of this subgroup analysis may alter the final conclusions in the future.

The Regorafenib targets a variety of kinases implicated in angiogenic and tumor growth promoting pathways. Its activity in refractory metastatic colorectal cancer demonstrated in the CORRECT trial [START_REF] Grothey | Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial[END_REF]. Patients who had progressed after multiple standard therapies assigned to regorafenib showed a modest, although statistically significant, improvement in median OS (6.4 vs 5 months), and PFS (1.9 months vs 1.7 months). At present, regorafenib is reserved for patients whose cancers have progressed on the other standard chemotherapeutic and targeted therapy agents [START_REF] Grothey | Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial[END_REF].

Maintenance regimens

Approximately 75% of patients discontinue first-line chemotherapy in trials for reasons other than progressive disease, and face the question of whether to maintain chemotherapy or to take a break. The OPTIMOX trials showed that oxaliplatin can be safely stopped after 6 cycles in a FOLFOX regimen, and that complete discontinuation of chemotherapy had a negative impact on PFS, compared with maintenance therapy with 5fluorouracil (297). Results from CAIRO-3 showed that maintenance therapy with bevacizumab plus capecitabine after 6 cycles of CAPOX plus bevacizumab was associated with a significant longer PFS (8.5 vs 4.1 months) [START_REF] Simkens | Maintenance treatment with capecitabine and bevacizumab in metastatic colorectal cancer (CAIRO3): a phase 3 randomised controlled trial of the Dutch Colorectal Cancer Group[END_REF]. Decisions regarding maintenance therapy versus treatment breaks must also take into account patient preferences and cost.

Potentially curable advanced colorectal cancer

The liver is the first site of hematogenous dissemination in most patients with colorectal cancer. Approximately 10% of patients can live past 5 years even with metastatic disease. The definition of resectable metastatic disease is evolving, and there is not an accepted standard: even bilobar metastases and extrahepatic disease are no longer considered contra-indications. Decision-making in this setting requires multidisciplinary collaboration: a practical approach requires that patients should be medically fit for surgery, existing liver disease should be resectable with adequate remnant liver, and extra-hepatic disease should be controlled. Surgical resection is the preferred treatment in patients with oligometastatic disease primarily in the liver, with 5-year survival rates of approximately 50% to 60% in patients with favorable prognostic features [START_REF] Rees | Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients[END_REF]. Approximately one-fifth of such patients survive beyond 10 years in some series, and this applies also to non-hepatic disease in certain series [START_REF] Tomlinson | Actual 10-year survival after resection of colorectal liver metastases defines cure[END_REF][START_REF] Chau | A VELOUR post hoc subset analysis: prognostic groups and treatment outcomes in patients with metastatic colorectal cancer treated with aflibercept and FOLFIRI[END_REF]. When the primary tumor site is controlled and the metastatic disease is limited in lungs without extra-pulmonary location (except for resectable or resected hepatic lesion), resection of isolated pulmonary metastases can increase survival rates up to 40% at 5 years [START_REF] Chau | A VELOUR post hoc subset analysis: prognostic groups and treatment outcomes in patients with metastatic colorectal cancer treated with aflibercept and FOLFIRI[END_REF]. Select patients with initially unresectable liver metastases may become eligible for resection if the response to chemotherapy is sufficient. This approach has been termed "con ersion" therapy to distinguish it from neoadjuvant therapy.

Conversion therapy allows 12% to 33% of initially unresectable or borderline resectable metastases to become eligible for metastasectomy [START_REF] Masi | Long-term outcome of initially unresectable metastatic colorectal cancer patients treated with 5fluorouracil/leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) followed by radical surgery of metastases[END_REF]. Five-year survival rates average 30% to 35%, which is substantially better than expected with chemotherapy alone.

However, the choice of regimen is not well established. The triplet chemotherapy regimen FOLFOXIRI combined with bevacizumab was associated with significantly higher response rates (65% vs 53%) and PFS (median 12.2 vs 9 months) in comparison with the combination of FOLFIRI plus bevacizumab in the phase III TRIBE trial [START_REF] Masi | Long-term outcome of initially unresectable metastatic colorectal cancer patients treated with 5fluorouracil/leucovorin, oxaliplatin, and irinotecan (FOLFOXIRI) followed by radical surgery of metastases[END_REF]. However, FOLFOXIRI did not result in a significantly higher secondary complete (R0) liver resection rate (15% vs 12%), and was associated with greater adverse effects (298). Combination of anti-EGFR inhibitor with either irinotecan-based or oxaliplatin-based regimens has shown modestly improved resection rates in patients with WT KRAS status (37).

Liver-Directed Treatments

Several other regional therapies, including regional hepatic intra-arterial chemotherapy or chemo-embolization, and radio-embolization, had been proposed as options for liver-isolated mCRC patients who are not candidate for surgery [START_REF] Goere | Prolonged survival of initially unresectable hepatic colorectal cancer patients treated with hepatic arterial infusion of oxaliplatin followed by radical surgery of metastases[END_REF][START_REF] Fiorentini | Intra-arterial infusion of irinotecan-loaded drug-eluting beads (DEBIRI) versus intravenous therapy (FOLFIRI) for hepatic metastases from colorectal cancer: final results of a phase III study[END_REF]. These therapies are often incorporated with initial hepatic resection or used as alternatives in patients who present not medically fit enough to undergo surgical resection. Although these methods can provide excellent local control, long-term survival outcomes have not been studied in depth, so far [START_REF] Pellerin | Intra-arterial treatment of liver metastases from colorectal carcinoma[END_REF]. Intra-arterial liver directed treatment clinical outcomes are presented in annex III.

Drug Eluting Beads Trans Arterial Chimio-Embolization (DEBTACE)

The recent development of drug eluting beads loaded with irinotecan has changed the place of chemoembolization in CRCLM application. Three different applications of Irinotecan Drug eluting beads (DEBIRI) in CRCLM have been currently studied: -salvage situation, -chemo naive patients, -neo adjuvant to hepatic surgery. [START_REF] Fiorentini | Intra-arterial infusion of irinotecan-loaded drug-eluting beads (DEBIRI) versus intravenous therapy (FOLFIRI) for hepatic metastases from colorectal cancer: final results of a phase III study[END_REF].

To overcome the possible progression in body while the liver was controlled, DEBIRI efficacy was studied in association with systemic chemotherapy (capcetamine, be acizumab…). This approach allows 80% of disease control rate at three months with 8.1 to 10 months PFS and 21 months OS (306-308).

First line treatment

As a first line treatment in association with FOLFOX + bevacizumab, the DEBIRI had also been used in a phase III randomized study. The FOLFOX-DEBIRI arm versus the FOLFOX + bevacizumab arm show at 2 months (78% vs 54%, p=.02), 4 (95% vs 70%, p=.03), and 6 months (76% vs 60%, p=.05), respectively, shows a significant tumor control (309, 310).

There was significantly more down-sizing to resection in the FOLFOX-DEBIRI arm versus the FOLFOX/bevacizumab arm (35% vs 16%, p=.05), and there was improved median progression-free survival (15.3 vs 7.6 months) (158).

Neo adjuvant prior to surgery

Neo adjuvant DEBIRI prior to hepatic surgery was similarly reported in the PARAGON study. This study aimed at evaluating the tumor resectability at surgery as a primary goal and the report of safety outcomes. Today, early results show a high rate of tumor destruction, with complete tumor response, thus allowing surgery (159).

Hepatic Intra-Arterial Chemotherapy (HIAC)

Historically HIAC was mostly proposed to patient who failed to Irinotecan and oxaliplatin based chemotherapy. Numerous phase III studies and meta-analysis showed a response rate of 42.9% vs. 18.4% after HIAC versus intravenous 5-FU respectively (OR: 2.26; p <0.0001). However, no significant difference on overall survival has been observed as the HIAC is used without systemic chemotherapy (319).

More recently HIAC was combined to intravenous chemotherapy. The OPTILIV07

Phase II trial studies the efficacy of a triple therapy CIAH (5-FU, irinotecan, oxaliplatin) with cetuximab IV in unresectable WT-KRAS pretreated patient. The authors reported a high response rate and 46% of secondary hepatic resections. In other words, the OPTILIV07 protocol multiplies by six the probability of observing a complete pathological response, which is recognized to be linked to prolonged survival (320).

In summary the HIAC is proposed to patients with extensive hepatic involvement.

HIAC is potentially indicated in the 3 following types of therapeutic strategy: i) an attack treatment to ensure a better chance of control and secondary resection, ii) a "closure" treatment after an excellent response in patients remaining inaccessible to surgical resection of their hepatic metastases to achieve maximum control tumor, or iii) a salvage therapy in selected patients with advanced intra-venous chemotherapy.

Trans-Arterial Radio-Embolization (TARE or Y90)

The majority of Y90 trials enrolled patients failing to oxaliplatin and irinotecan based chemotherapy. In this population a disease control rate of 24% to 33%, associated to median PFS of 8.3 to 12.6 months was mainly reported (p<0.005).

For patient with liver dominant CRCLM and naïve of any chemotherapy, Y90 had been evaluated in association to Folfox 6 in a randomized phase III trial (Sirflox trial), compared to Folfox 6 alone (321). 530 patients were randomly assigned to treatment (control,263;SIRT,267). Despite negative results of the study (primary objective not reached), (Median PFS at any site was 10.2 v 10.7 months in control versus SIRT (hazard ratio, 0.93; 95% CI, 0.77 to 1.12; P = .43)) a significant different of liver PFS was reported in the patient group receiving Y90 + Folfox compared to the Folfox alone group. The median liver PFS was 20.5 months and 12.5 months for Y90+folfox and for Folfox respectively (HR: 0.69; 95% CI 0.55-0.90; p = 0.002). The overall response rate (PR + CR) was 76.4% and 68.0%

for Y90+folfox arm and for Folfox respectively (p = 0.113)(322). implantation of Vx2 fibroblastic tumors and has become the reference model for pre-clinical evaluation of intra-arterial chemo embolization.

So we decided to evaluate the feasibility, efficacy and safety of intra-arterial GDEPT delivery in a rabbit model of cancer. As a first step, our need was to understand the ability of MSCs to cross the vasculature barrier, and thereafter to nest and to properly grow in the tumor environment. In addition, we planned to evaluate the putative adverse ability of MSCs to move to other vital organs where they could eventually induce local toxicity than the tumor itself.

Study goal.

This preliminary study aimed at evaluating the level of MSCs cells trapped into the tumor and its microenvironment as well as their potential dissemination in non-target organs upon IA administration into the liver in the VX2 rabbit model.

Material & method.

To correctly assess the distribution of MSCs cells while they were administered to the tumor through the tumor arterial network we chose the Vx2 rabbit tumor model. This model allowed a reliable catheterization of the main tumor arterial feeder, and a reliable MSCs delivery by the tumor. So, orthotropic Vx2 tumors were surgically implanted into the liver of rabbits and grew to a visible tumor within 2 weeks.

To evaluate the level of MSCs delivery to target and non-target organs, we have used rabbit MSCs cells expressing a high level of luciferase

In non-target organs (brain, lung heart, muscle, kidney, stomach and bowel), the level of luciferase was evaluated using a chemoluminescent assay. In the tumoral liver, MSCsluciferase cells were searched on tissue slices in order to evaluate their occurrence and delivery within the tumor tissue / microenvironment.

Experiment synopsis.

MSCs-Luciferase cells were injected intra-arterially to rabbit Vx2 tumor models once the liver tumor had reached a 2 / 2.5 cm diameter, as assessed by ultrasound. The animals were sacrificed48 hours after MSC injection. This timing was chosen because we anticipated to administer the pro-drug at D2 after MSCs injection.

In addition, we assessed the possible spontaneous migration of MSCs in the absence of liver tumor in normal rabbits and looked for potential spontaneous signal of luciferase + in normal rabbits

Experimental groups.

For this purpose, four New-Zealand White rabbits were used and divided in four groups:

 Vx2 rabbit + MSCs-luciferase (n=2),

 Normal rabbit + MSCs-luciferase (n=1),  Normal rabbit (n=1).

Cell preparation.

The rabbit-MSCs-Luc cells were prepared as required for an adequate luciferase expression suitable for the experiment. These cells were in vitro cultured and maintained in the INSERM 1147 laboratory (Paris).On the day of IA injection in rabbits, a suspension of 10 7 cells in 200µl PBS was prepared.

Animal Housing.

During the study, animals were housed at the CR2i research center (Jouy-en-Josas).

The overall health and well-being of each animal had been monitored daily; supply of food and water intake, posture observation, were in a total accordance with the ethical rules.

Vx2 culture and Implant.

The Vx2 tumor cells were prepared as described by Pascale F. et al., and maintained in the laboratory at Cr2i (Jouy-en-Josas), and finally injected in the left lobe of liver. For this purpose, all rabbits were anesthetized with a mixture of acepromazine (2.5 mg/kg) and ketamine hydrochloride (44 mg/kg) that was administered intra-muscularly. An intravenous access was gained via a marginal ear vein and sodium pentobarbital was given intravenously to maintain anesthesia. After shaving and povidone iodine brushing of the abdomen of each rabbit was, the liver was exposed with a midline subxiphoid incision, and the slurry (mixture of matrigel and Vx2 tumor cell 0.1-0.2mL) was implanted directly into the left lobe of the exposed liver of recipient rabbits using a 21-gauge angio-catheter. This procedure was done under ultrasound guidance without abdominal cut down. This approach allowed a better animal recovery after Vx2 implantation, and furthermore the growth of a single, solitary, well-demarcated tumor in the liver of each rabbit. The abdomen was closed in two layers, and a proper aseptic technique was rigorously observed during each implantation. After surgery, the animals were placed in cages and monitored in the animal laboratory under direct supervision of a physician or a technician until they recovered from anesthesia.

Analgesic buprenorphine (0.02-0.05 mg/kg) was administered intramuscularly for pain or distress. The tumors was allowed to grow for another 10 to 14 days, at which time they were expected to reach an ellipsoidal shape with dimensions ranging between 2.5 and 3.5cm diameter.

Intra-arterial injection of MSC in the rabbits.

First, rabbits were sedated with a mixture of acepromazine ( 2 After catheter removal, the common femoral artery was ligatured using resorbable suture material to obtain hemostasis. Animals were placed into cages and monitored in the animal laboratory under the direct supervision of a physician or a technician until they fully recovered from anesthesia. Analgesic buprenorphine (0.02-0.05 mg/kg) was administered intra-muscularly for pain or distress. The animals returned to animal facility and followed-up daily until the time of sacrifice.

Animal Sacrifice.

Animals were sacrificed at 48 hours after the beginning of the experiment. Animal sacrifice was done under general anesthesia (acepromazine 2.5 mg/kg and ketamine hydrochloride 44 mg/kg) by an over dosage of Pentobarbital Sodium.

Autopsy/necropsy.

Necropsy was performed by a qualified scientist on all died animals following entry into the study, regardless of whether the animals completed the allotted time interval or not. For each animal a summary of the key observations was registered at autopsy/necropsy and all results were recorded. Rabbit liver (right, left and caudate lobe), brain, heart, lungs, kidneys, muscle, stomach and bowel were removed. The following whole organs (Brain, heart, lungs, kidneys, muscle, stomach and bowel) were immediately frozen in nitrogen for further examination. A cut was done into the liver tumor at the maximum diameter. Then, 6 samples of 5 mm 3 were prepared and fixed in 20/1 acetic formalin: 2 into the tumor, 1 into the normal li er tissue adjacent to tumor (≈1 cm), 1in the left lobe (>1cm away from the tumor), 1 in the right lobe and 1 in the caudate lobe will be done.

Histology.

Two specific analyses had been performed.

1 st, each frozen orange was prepared for luciferase activity search in non-target organs.

2 nd , the 6 samples were paraffin embedded, hematoxylin and eosin stained, and incubated with specific luc-antibody to identify the location of MCSc-Luc cells. Moreover, JC/70 monoclonal antibody (Dako, Glostrup, Denmark) against CD31 was used for micro vessel staining using the LSAB method (DakoLSAB kit; Glostrup, Denmark). Quantification of MSCs was presented as the total of number of cells recovered from the tissue samples, and, to a possible extent, as the number of cells per volume or area of individual studied tissue samples. The locations where cells were discovered were considered. Results were presented in Table format showing all the tissues where MSCs-Luc cells were detected or not.

approach (248). To overcome the technical difficulties of intra tumoral injection, the intraarterial approach (IA) provides many advantages. As tumors are massively vascularized through an arterial network, IA injection allows a high quantity and concentration level of the therapeutic agent; furthermore the deposit could be produced as close as the tumor, by selective catheterization. Because rats and mice are especially difficult to selectively catheterized, the most frequently used animal model for intra-arterial treatment evaluation is the rabbit with heterotopic Vx2 tumor implantation.

We previously demonstrate, in DISTRILAP study, that following injection through the artery of the tumor arterial network, Rabbits-MSCs-Luc stay and home themselves in the tumor tissue. Seventy-two hours later, a large quantity of MSCs-Luc cells were found in the tumor environment and can be detected by luminescence assay, as well. No MSCs-luc cells in nontarget organ could be observed.

Based on DISTRILAP result study, we strongly believe that IA injection of rabbit MSCs-CYP2B6TM-RED will produce a full delivery of the suicide gene into the tumor leading thus to an efficient GEDEP strategy.

Study goal

The goal of our study is evaluate the GEDEP strategy in liver Vx2 hepatic rabbit tumor by the comparison to a group control.

Study endpoint

The primary endpoint of our study will be the tumor response after proceeding to the therapeutic sequence.

The secondary endpoint will be the report of observed toxicity.

Material & method

Tumor response rate was assessed by the percentage of tumor shrinkage from baseline to animal scarify/death. At base line, tumor diameter was determined by ultrasound scan and with contrast enhance CBCT. At sacrifice tumor diameter was evaluated by ultrasound scan (immediately before sacrifice) and on pathologic examination. Animal was considered in a complete response status if the tumor completely disappeared, i) in a partial response status if the tumor shrunk more than 20%, ii) in a stable disease if the tumor shrunk less than 20% or increase less than 30%, iii) in a progressive status if the tumor increased more than 30%. The appearance of any new liver lesion or extra hepatic lesion was associated to a progressive status.

Toxicities were reported for each animal, based on clinical observation, and blood examination (liver, hematologic, and renal analysis).

Experiment synopsis.

For this purpose, the Vx2 rabbit tumor model was used. Approximately 10 New-Zealand White rabbits were implanted with Vx2 tumor as described below. When tumor had a 2 / 2.5 cm diameter, rabbits were eligible for the study. The rabbit cohort was split in a control group (3 animals) and in the experimental group (7 animals).

In the control group, one rabbit rabbits (control B) did not receive any treatment, while two rabbits (control A) received CPA (80mg/m²) injection at day 2 and day 9.

In the experiment group, all the 7 animals received successively twice time the same therapeutic sequence. The 1 st IA injection of rabbit MSCs-CYP2B6TM-RED cells (10 6 cells in 200µl), at day 0, then the 2 nd intravenous injection of CPA (80 mg/m²) were performed at day 2. The same procedure sequence was repeated at day 7 and day 9. Then all the animals were followed until sacrifice at day 31. Figure 34 summarizes the experimental plan. The rabbit-MSCs-CYP2B6TM-RED cells were prepared as required for an adequate CYP2B gene expression suitable for the experiment and culture.

Vx2 Culture and Implant.

The Vx2 tumor cells were prepared as described by Pascale F. and injected in the liver left lobe. For this purpose all rabbits, were anesthetized with a mixture of acepromazine (2.5 mg/kg) and ketamine hydrochloride (44 mg/kg) administered intra muscularly. An intravenous access was gained via a marginal ear vein and sodium pentobarbital will be given intravenously to maintain anesthesia. The abdomen of each rabbit was shaved and brushed with povidone iodine. The liver was exposed with a midline subxiphoid incision, and the slurry (mixture of matrigel and Vx2 tumor cell 0.1-0.2mL) was implanted directly into the left lobe of the exposed liver of recipient rabbits using a 21-gauge angiocatheter. This procedure was done under ultrasound guidance without abdominal cut down. This approach allowed a better animal recovery after Vx2 implantation, and furthermore the growth of a single, solitary, well-demarcated tumor in the liver of each rabbit. The abdomen was closed in two layers, and proper aseptic technique was rigorously observed during each implantation. After surgery, the animals were placed in cages and monitored in the animal laboratory under direct supervision of a physician or a technician until they recover from anesthesia. Analgesic buprenorphine (0.02-0.05 mg/kg) was administered intramuscularly for pain or distress. The tumors were allowed to grow for another 10 to 14 days, at which time they were expected to reach an ellipsoidal shape with dimensions ranging between 2.5 and 3.5cm diameter.

Animal preparation for the study.

Before procedure, animals were anesthetized. Rabbits were sedated with a mixture of acepromazine (2.5 mg/kg) and ketamine hydrochloride (44 mg/kg) administrated intramuscularly. Then, rabbits were anesthetized via an intravenous access (gained via a marginal ear vein) by sodium pentobarbital injection. Anesthesia was maintained by injection of propofol (3 to 5 mg/kg).

Rabbits were placed in a supine position, on a specific Plexiglas rack. The whole legs were exposed and maintained to the rack with ropes. A surgical cut-down was performed at the common femoral artery to ensure arterial access. A 2Fr introduce sheath was inserted in it.

This arterial access was maintained until the last image acquisition required by the protocol was done. After completion of all the image modality acquisitions required by the protocol, the catheter was removed. Then the common femoral artery was ligatured using resorbable suture material to obtain hemostasis. Animals were placed into cages and monitored in the animal laboratory under the direct supervision of a physician or a technician until they fully recover from anesthesia. Analgesic buprenorphine (0.02-0.05 mg/kg) was administered intra-muscularly for pain or distress. The animals returned to animal facility and followed-up daily until the time of sacrifice.

Angiography and MSCs-CYP2B6TM-RED injection.

Trans-catheter hepatic artery angiography and embolization was done under fluoroscopy guidance. The animals were brought to the angiography suite and intubated ). The procedure technical end point was to fully inject the prepared dose of MSCs-CYP2B6TM-RED. This procedure will be repeated at day 7.

CPA administration.

The CPA injection was performed for the 7 animal involved in the test group and the 2 animals in the control group (sub group a) at day 2 and 9. Under sedation, (mixture of acepromazine (2.5 mg/kg) and ketamine hydrochloride (44 mg/kg) administrated intramuscularly). Through the marginal ear vein 80 mg/m² of CPA was injected, in 15 min.

Animal Housing.

During the study, animals were housed at the CR2i research center by Archimmed team. The overall health and well-being of each animal wase monitored daily: food and water intake, posture.

Animal follow-up.

For all animals a follow-up was undertaken until animal death or sacrificed at day 31.

Animal being was evaluated and reported on the preclinical research form.

Blood sampling: were performed at base line and every 7 days after the first procedure. In the collected blood samples liver function (AST, ALT, GGT, ALP, LDH, Bilirubin), red blood cell, white blood cells, platelets, PT, albumin were explored.

The tumor size was followed by ultrasound. Two orthogonal diameters were registered and report on preclinical research form. Ultrasound evaluation was performed at base line, and every 7 days.

Animal Sacrifice.

Animals were sacrificed at day 31. Animal sacrifice was done under general anesthesia (acepromazine 2.5 mg/kg and ketamine hydrochloride 44 mg/kg) by an over dosage of Pentobarbital Sodium.

Autopsy/necropsy.

Necropsy was performed by a qualified individual on all animals dying following entry into the study regardless of whether the animals completed the allotted time interval or not.

For each animal a summary of the key observations was made at autopsy/necropsy and all results were recorded. Rabbit liver (right, left and caudate lobe), brain, heart, lungs, kidneys, muscle, stomach and bowel were removed. A cut was done into the liver tumor at the maximum diameter. Tumor size (including longest diameter) was performed. Then 6 slices of 5 mm thickness were done. Two into the tumor, one into the normal liver tissue adjacent to tumor (≈1 cm); one in the left lobe (>1cm away from the tumor), one in the right lobe and one in the caudate lobe were done. All sample were fixed in 20/1 acetic formalin.

Two into the tumor, one into the normal li er tissue adjacent to tumor (≈1 cm); one in the left lobe, one in the right lobe and one in the caudate lobe. In a same manner, samples were taken from the lung, the heart, the stomach, the bowel, and the kidney. All samples were sending to the pathology for paraffin embedding and hematoxylin and eosin saffron staining, and immunohistochemistry.

Histology.

Hematoxylin and eosin saffron staining was performed first.

Mouse antibodies against rabbit CD44 were used for searching remaining Rabbit-MSCs-CYP2B6TM-RED. Quantification of MSCs was presented as the total number of cells recovered from the tissue samples, and, to the extent possible, as the number of cells per volume or area of individual tissue samples studied. The locations where cells have been discovered are of a particular interest for considerations. These results were presented in table format showing all the tissues where Rabbit-MSCs-CYP2B6TM-RED has been found and all negative.

The JC/70 monoclonal antibody against rabbit CD31 will be used for micro vessel staining using the LSAB method (DakoLSAB kit; Glostrup, Denmark). The histopathologic evaluation included assessment of tumor necrosis, location of luminar vessels in the tumor and border, changes of tumor borders including peritumoral desmoplastic reaction, lymphocytic infiltration, vascular proliferation, stasis of border vessels and adjacent liver sinuses, compression of hepatic parenchyma, and atrophy of the hepatic cords. Tumor boundaries were defined as the desmoplastic reaction zone. In each tumor sample, the histologic images of 5-10 regions of tumor border were taken at a magnification of ×25, and ×100, respectively. The thickness of tumor border wa measured on the images at a magnification of ×25. The mean and median of these 5-10regions will be then recorded. The extent of the blood stasis of the vessels in the tumor border vessels and adjacent liver sinuses will be evaluated in a semi quantitative fashion by using the following scales: +++ will indicate prominent; ++, moderate;+, mild; and -, none.

219

  es remerciements s'adressent tout d'abord mon Directeur de th se, le Professeur arc Sapo al. Tout au long de ce tra ail, il a su m'aider à définir et orienter ce travail par des conseils précieux et avis s la hauteur de ses comp tences. ls s'adressent aussi adame sabelle de aziers et au Professeur Philippe eaune, de l'unit NSER UR S 1147. Je vous remercie pour votre confiance en m'associant à vos travaux sur la thérapie par gène suicide. A os cotes j'ai progress dans un domaine d'a enir ui m' tait inconnu. ls s'adressent galement au Professeur Oli ier Cl ment directeur de l' uipe 2 NSER 970 magerie de l'angiogen se ui m'a accueilli dans le laboratoire. e tiens galement témoigner toute ma reconnaissance aux Professeurs Boris Guiu et Jean-Pierre Tasu, professeurs l'Uni ersit de ontpelliers et l'Uni ersit de Poitier respecti ement, pour l'honneur u'ils me font en prenant la charge de rapporteur et en si geant ce jury. es remerciements s'adressent aussi aux Professeurs al rie ilegrain, ulien Taieb, Thierry de aere, et au Docteur ulien Namur, pour l'honneur u'ils me font en si geant ce jury. 'exprime ma toute gratitude à mon oncle le Docteur Jean Bénard, sans ton oeil critique et avisé ce manuscrit n'aurait pas la même valeur. 'exprime galement adame Carole Déan ma gratitude, ton écoute attentive et tes conseils judicieux m'ont été utiles et ton bureau est un ha re de paix… 'adresse galement tous mes remerciements pour leur soutien uotidien mes coll gues du ser ice de Radiologie inter entionnelle de l'Hôpital Europ en Georges Pompidou, les Docteurs Amouyal, del Guidice, Beyssen, Pagny et Louail. A titre plus personnel, je remercie affectueusement mon épouse Sophie, pour sa grande patience, les encouragements et la confiance u'elle m'a témoignés pendant la rédaction du manuscrit. Enfin à mes parents Marie-Thérèse et Jean-François, mon frère Pierre et son épouse Agnès et ma soeur Anne Pellerin, je tiens vous exprimer ma profonde reconnaissance. Soyez remerciés pour votre affection et votre soutien ininterrompu. Et selon la devise familiale: «Ceux qui sèment dans le peine, Moissonneront avec chants d'allégresse». Psaume 126 :5.

Figure 5 :

 5 Figure 5: Scatter plots show correlation of tumor enhancement changes in (a) early or arterial phase and (b) delayed or venous phase after TACE with doxorubicin-eluting beads, as measured with cone-beam CT (CBCT) and MR imaging. A decrease in tumor enhancement at cone-beam CT showed a linear correlation with findings at MR imaging. Estimated correlation coefficients were excellent for both arterial (R = 0.89, p<0.0001) and venous (R = 0.82, p <0.0001) phases.....................................................................................................................

  Figure6a, b, c: MDCT at the portal phase (Figure6a), dualphase C-arm CBCT: eAP (Figure6b), and dualphase Carm CBCT: IAP (Figure6c) in a53 years old woman with widespread liver invasion by CRCLM. Figure6ashow 6 tumor involving segment II, III, IV, V and VI appeared as hypodens lesion surrounded by liver parenchyma enhanced. The eAP (Figure6b) show three tumors with moderate peripheral enhancement associated to minimal streak artifact from catheter or contrast media (white arrow head). All the three lesions were classify as uncertain by readers. Figure6cthe late arterial phase show 5 lesions with peripheral corona surrounding the tumor classified as undoubtedly CRCLM. Readers classified the segment II lesion as uncertain by (white arrow). At last lesion in segment 4 was missed at the both phases(Figure 6a white arrow). ..........................................

Figure 7 :

 7 Figure 7: flow diagram of the study participants..................................................................................................

Figure 9 :

 9 Figure 9: MDCT perfusion scan performed before (a and b) and one day after the chemo-embolization. These figures demonstrate the reduction of tumor arterial flow (AF) after the chemo-embolization compare to base line, while tumor portal flow and perfusion index remained unchanged. ..........................................................

Figure 10 :

 10 Figure 10: Injection and acquisition protocol for C-arm CBCT parenchymal blood volume measurements. ......

Figure 12 :

 12 Figure 12: Processing steps for reconstruction of the parenchymal blood volume (PBV) maps. ........................

Figure 13 :

 13 Figure13: patient flow chart ..............................................................................................................................

Figure 14 :

 14 Figure 14:Representative case of a 68 years old man with CRCLM referred to DEBIRI. Four index lesions are shown. Lesion 2 had a 181 ml/1000ml PBV before and doped down to 32 ml/100ml after the DEBIRI. This lesion totally shrink at the 2 monthsMDCT follow-up ..................................................................................................

Figure 15 :

 15 Figure 15: Non-linear regression of PBV before (a) after (b) % of PBV change (c) and % of tumor change at 2 months. ...............................................................................................................................................................

Figure 19 a

 19 Figure 19 a & b: MSCs survival in iodinate contrast media Iomeporl (Figure 19 a) or Lipiodol® (Figure 19 b) ...

Figure 20 :

 20 Figure 20: VX2, TC1, TC1 -TM5 cells survival test in CPA ...................................................................................

Figure 21 :

 21 Figure 21: VX2 cells survival in various concentration ofCPA .............................................................................

Figure 22 a

 22 Figure 22 a, b & c:Ultrasound Doppler scan before catheterization, the tumor is located in the left liver lobe(30 × 17 mm) well limited and hypervascular. Figure22cleft hepatic artery angiography showing the hypervascular tumor located in the left liver lobe. The catheter was advanced closer as possible to the tumor to inject the rabbitMSCs-Luc. .................................................................................................................................................

Figure 23 a

 23 Figure 23 a & b: Whole animal/organ photoluminescence assay performed 2 hours (Figure 23 a) and 72 hours (Figure 23 b) later after intra-arterial injection of rabbit MSCs-LUC. Rabbit received intra venous injection of luciferine and were examined.Figure 23 a shows a high and intense luminescence in the left abdominal wall corresponding to the VX2 liver tumor. Figure 23 b shows the whole liver examined after rabbit sacrifice, showing clear luminescence into the tumor. .....................................................................................................................

Figure 24 :

 24 Figure 24: Photoluminescence assay performed 72 hours after rabbit sacrifice on frozen sample of different organs and tumor. This graph demonstrates a significant higher luminescence in the VX2 tumor and the surrounding tissues wherein the rabbit MSCs-Luc were intra-arterially injected. ..............................................

Figure 25 :

 25 Figure 25: GDEPT concept of intra-arterial injections of transduced MSCs. The GDEPT follow several steps. Firstly the therapeutic gene is transduced to MSCs as a carrier, and then the transduced MSCs are delivered to the tumor. Secondly a prodrug is administered and is locally converted into the therapeutic agent by the suicide enzyme, which induces the death of tumor cells. The cell death stimulates an immune response mediated though CD8 lymphocyte accomplishing tumor cells death and "vaccination" against tumor recurrence. .........

Figure 26 :

 26 Figure 26:Experimental plan applied to animals. In group C, CPA major toxicities were responsible of two rabbit early sacrifice between D3 and D4 and 3 animals (group C1) did not underwent to the second in injection of GDEPT for mild CPA toxicities. For the 3 remaining rabbit (group C2) suitable for a second GDEPT, the injection of transduced MSC was post ponded atD11 and CPA at D14. ...........................................................................

Figure 27 :

 27 Figure 27: VX2 tumor volume as a function of time by group. A significant tumor volume difference between group B and group C at D7 (p=0.024), D11 (p=0.024), D19 (p=0.048) and D25 (p=0.048) was observed after completion of theGDEPT protocol. .....................................................................................................................

Figure 1 :

 1 Figure 1: A proposed algorithm for selection of systemic treatment and integration with surgical resection in patients with metastatic colorectal carcinoma, based on available clinical and molecular data. 5FU, 5-fluorouracil; CAPOX, capecitabine/oxaliplatin; FOLFIRI, irinotecan/5-fluorouracil/leucovorin; FOLFOX, oxaliplatin /5-fluorouracil /leucovorin; WT, wild-type.

Figure 2 :

 2 Figure 2: Liver and tumor vascular in-put 4.1.3. Pharmacokinetic advantage of arterial delivery[START_REF] Dedrick | Arterial drug infusion: pharmacokinetic problems and pitfalls[END_REF][START_REF] Eckman | A critical evaluation of the principles governing the advantages of intra-arterial infusions[END_REF][START_REF] Collins | Pharmacologic rationale for regional drug delivery[END_REF][START_REF] Blesing | Intra-hepatic arterial drug delivery[END_REF]

Figure 3 :

 3 Figure 3: Flow diagram illustrating the effect of intraregional differences in blood flow describe byDedrick RL. et. al.. (42).C is the drug concentration in the blood (μg/ml); V is the volume of distribution (ml); Q is the arterial blood flow rate (ml/min); I is the infusion rate of the drug μg/min); and the subscripts b, t and h refers, respectively, to the infused remainder of the body, the tumor and the healthy tissue.

  fluoroscopy and DSA. By use of a rotational acquisition around the patient, C-arm CBCT is able to reconstruct tomographic projection in CT-like image. C-arm CBCT imaging is based on a projection acquisition, whereby the X-ray source and detector are mounted on a C-shaped gantry capable to perform a motorized movement around the patient. During the movement of the system, X-ray projection images of the object are acquired along multiple angular directions, following a circular path covering an angular range of at least 210°. This is the minimum angular range to fulfill the 180° plus fan-angle criterion for data acquisition. The target area is positioned at the isocenter of rotation and C-arm CBCT volumes are then obtained by integrating information from the 2D two-dimensional high-resolution projection images. In contrast to classic multidetector CT acquisitions, the motion path includes acceleration and deceleration phases encompassing a phase of constant speed of 30°-60° per second and projection acquisition are performed in pulsed mode. Tube and detector settings depend on the clinical application. For abdominal imaging, typical tube parameters are 5-10 msec pulses per projection at 120 kVp tube voltage including copper filtration with frame rates of 30-60 frames per second. The use of flat detectors has been a corner stone in the development of C-arm CBCT imaging due to high resolution, high detector quantum efficiency, high frame rate, high dynamic range, small image lag, and excellent linearity. Flat detectors panel are based on a thin-film cesium iodide scintillator coupled to a chargecoupled device fabricated on a large-area panel. Recent detectors cover a planar region of 30 × 40 cm for a reconstructed volume of 30 × 30 × 40 cm at a high spatial resolution on the order of 150 × 150 µm 2 pixel size

Figure 4 :

 4 Figure 4: Illustration of the measurement methods of the three response criteria. The gray area within the nodules represents the enhanced viable lesions, and the white area represents non enhanced or iodized oil-retaining lesions. A, the maximum diameter of the entire tumor before treatment (pretreatment); A′, the maximum diameter of the entire tumor after treatment (post treatment); A″, the maximum diameter of the enhanced area of the tumor after treatment; B, the diameter perpendicular to A; and B″, the diameter perpendicular to A″

  the setting of CRCLM, Akinwande O. et al. (100) study the performance of the mRECIST, and the European Association for the Study of Liver (EASL) guidelines and correlated them with survival in patients with metastatic colorectal cancer (mCRC) treated with DEB-TACE or TARE in a historic cohort of 228 patient.

  studied the feasibility of C-arm CBCT detection of incomplete accumulation of iodized oil in hepatocellular carcinoma immediately after cTACE. In a retrospective manner this team reviewed 80 HCC in 55 patients who underwent cTACE with an unenhanced C-arm CBCT at the end of the procedure and they compared the results to unenhanced MDCT images at one month. Images were scored using a predefined detection scale for incomplete iodized oil accumulation. The accuracy for predicting residual lesions was compared using the area under the ROC. Contrast-enhanced CT findings obtained 1 month after TACE served as reference standards. Readers identify 18 viable lesions over the 80 studied lesions by contrast-enhanced MDCT. The accuracy of the C-arm CBCT (Az = 0.816) was not significantly different (p = 0.449) from that of the MDCT (Az = 0.841). In addition, sensitivity, specificity, and positive and negative predictive values for C-arm CBCT (80.5%, 74.2%, 47.5%, and 92.9%, respectively) and MDCT (86.1%, 75.0%, 50.0%, and 94.9%, respectively) did not differ significantly. Results indicated C-arm CBCT feasibility, nearly equivalent to MDCT, for detecting incomplete iodized oil accumulation after TACE; they also pointed out the immediate assessment of iodized oil accumulation without the need to perform follow-up unenhanced MDCT. Moreover, C-arm CBCT could help to target potential missed arterial feeders in the same session and increase the overall tumor response per patient. In a similar manner, Chen R. et al.(102) evaluated retrospectively, in a 15 patient cohort, the ability of unenhanced C-arm CBCT, performed immediately after cTACE to assess Lipiodol® deposition in HCC and compared it with unenhanced MDCT 24 h later. In this paper the author refined the detection of Lipodol® staining by the use of semi-automatic 3D volume segmentation software on both image modalities. The authors demonstrated that unenhanced C-arm CBCT was similar to MDCT in assessing Lipiodol® staining in HCC after cTACE (p = .214). In addition, a high correlation in the tumor volume and Lipiodol® between CBCT and MDCT (r = 0.919 and 0.903) was observed. The same author group published (103) that C-arm CBCT when compared to MDCT has a similar capability, to assess Lipiodol® deposition in 3D for HCC patients treated with cTACE. Always in comparing C-arm CBCT to MDCT, the Bland-Altman plots showed only minimal difference and high agreement.

  assessed the relation between arterial enhancement changes after DEB-TACE for HCC lesions and 1 month tumoral response (EASL criteria) with MR imaging. These authors defined the percentage of enhancement as the percentage of the total area of enhancement seen on the axial view of the largest tumor after DEB-TACE. Then they classified these findings in 5% increments, ranging from no (0%) enhancement to 100% enhancement. Data pointed out a significant relationship between residual tumor enhancement at C-arm CBCT after TACE and complete and/or partial tumor response (EASL) at MR imaging (odds ratio: 0.95 CI 0910, 0990 p=0.027). In differentiating the arterial and venous phase of MR images, results indicated the arterial (odds ratio, 0.95; 95% confidence interval [CI]: 0.91, 0.99; P = .023) and venous (odds ratio, 0.96; 95% CI: 0.93, 0.99; P = .035) phases were comparable using the multivariate logistic regression model. Caution should be used when talking about arterial (early arterial) and venous (late arterial) phase of the Dualphase C-arm CBCT in this work. In this respect, the reader may view in Figure5CBCT late arterial phase instead of CBCT venous phase. In fact the second acquisition time was at 18 seconds after the beginning of injection, which is at the best a late arterial phase. The following figure (Figure5) is an example of their findings, demonstrating good correlation between early or late C-arm CBCT phase with arterial or venous MRI phase.

Figure 5 :

 5 Figure 5: Scatter plots show correlation of tumor enhancement changes in (a) early or arterial phase and (b) delayed or venous phase after TACE with doxorubicin-eluting beads, as measured with cone-beam CT (CBCT) and MR imaging. A decrease in tumor enhancement at cone-beam CT showed a linear correlation with findings at MR imaging. Estimated correlation coefficients were excellent for both arterial (R = 0.89, p<0.0001) and venous (R = 0.82, p <0.0001) phases. As a summary, a limited tumor enhancement changes on Dual-Phase C-arm CBCT images after DEB-TACE may suggest to the operator either the need for retreatment, or to search for additional feeding arteries. Commonly, the post-DEB-TACE Dual-Phase C-arm CBCT technique displays an arterial tumor enhancement and tumor-feeding arteries on the first scan (arterial phase, 3-15-seconds acquisition delay), and then parenchymal tumor enhancement on the second scan (parenchymal phase, 28-second acquisition delay).

  unfavorable outcome, with less tumor shrinkage over time(-0.02 ± 0.49 vs. -0.76 ± 0.38; P < 0.0001), compared with completely treated lesions. The authors concluded that PBV volume values derived from C-arm CT acquisitions are useful in post treatment assessment of DEB-TACE in HCC. Residual tumor perfusion in PBV maps has predictive potential for mid-term tumor response in HCC and could allow a more individualized treatment schedule for DEB-TACE in HCC patients. Vogl TJ. et al. (108) also studied PBV in a larger cohort of 111 patients, referred for DEB-TACE. They studied the correlation between the intra-procedural PBV and the tumorchange on MRI at 6weeks. The cohort was composed of a mix of primary and secondary liver tumors (colorectal, breast, pancreas…). When patients were classified by initial blood volume into 3 groups: group A with an initial blood volume >100 ml/ 1000 ml (n=37), group B with an initial blood volume 50-100 ml/1000 ml (n=37) and group C with an initial blood volume of <50 ml/1000 ml (n=37). Group A dropped 7.1 % in size and 47.2 % in blood volume; group B dropped 4.6 % in size and 25.7 % in blood volume; and group C decreased 2.8 % in size and increased 82.2 % in blood volume. The correlation analysis (Spearman and Kendall) showed a significant correlation between initial blood volume and the change in blood volume between the initial and the last PBV measurement (rho=0.61; p<0.0001).

First, Manuscript # 1

 1 deals with evaluation on MRI of the feasibility of semi-automatic tumor segmentation and its enhancement quantification thanks to dedicated software in a population of HCC patient treated with TACE. Secondly Manuscript #2 studies the precision and the bias of the semiautomatic tumor segmentation software applied to C-arm CBCT image from VX2 liver tumor model. Our purpose was to show that hepatic tumor volume and enhancement pattern measurements are possible in a time efficient and reproducible manner on a voxel-by-voxel basis, giving a true 3D volumetric assessment. For this purpose we used a semi-automatic tumor segmentation tool software to measure i) tumor volumes, ii) enhancement for the entire tumor volume, and iii) the percentage of enhancing tumor volume. We propose that these measures be called quantitative EASL (qEASL) and volumetric RECIST (vRECIST). The pre-and post-DEB-TACE MR images were analyzed with this prototype segmentation software in 17 consecutive patients with unresectable HCC. The vRECIST value was calculated before and after, a positive change value indicating a decrease in tumor volume-TACE. In addition tumoral enhancement on the MR image at the pre-and post-TACE (qEASL) to determine the viable tumor defined as a percentage of the total tumor volume and expressed as percent of qEASL. We found a positive change value in qEASL reflecting a decrease in viable tumor volume or tumor enhancement percentage after DEB-TACE. A percentage change was also calculated as before minus after qEASL values divided by before qEASL values.

  regarded the true volume of tumor since we could only obtain a relative change in dimensions. So we chosed VX2 liver tumor rabbit model to address this issue and to confirm the accuracy of the measured volumes. Indeed the VX2 rabbit model would permit to standardize better the methods. In the following paper, we have studied the tumor sizing precision and bias of the C-arm CBCT with semiautomatic tumor segmentation software.To assess the precision of the C-arm CBCT to detect and size tumors, we study a unique liver tumor in the rabbit VX2 hepatic tumor model with semiautomatic tumor segmentation software. Tumor size was compared to CT scanner at the arterial phase in keeping in mind that the pathologic examination was the gold standard. The VX2 hepatic tumor appeared on C-arm CBCT or CT scanner as a spontaneous hypodensity and, after contrast injection, as a hypodensity surrounded by a hyperdensity corona. This hyperdense corona delimits the tumor boundaries. The tumor was measured using automatic tumor segmentation and dedicated software that a previous pilot study from us had evaluated (109).

  Thus this limitation in the performance of C-arm CBCT peculiar to the rabbit model required specific and thorough study regarding measurement and classification of colorectal cancer liver metastases. For that purpose, we studied a cohort of consecutive patient with CRCLM treated by Irinotecan drug eluting beads (DEBIRI) chemo-embolization. Instead of a solitary, uniform, homogenous tumor into the liver such as VX2 tumor, multiple lesions with various diameters, shape, and random location were targeted. This study was designed to determine the accuracy of Dual-Phase C-arm CBCT in comparison with CT scanner at the portal phase as a gold standard. Herein, the predictive positive value, predictive negative value, sensitivity, specificity and inter-observer variability were calculated.In summary semi-automatic tumor segmentation software enabled tumor volumes sizing and quantitative of enhancement for with HCC as well as tumor model mimicking HHC (VX2).

  of tumor-feeding blood vessels and parenchymal space are important to determine catheter position and injection parameters for TACE, since they can ultimately impact on treatment outcomes. Image acquisition during two separate phases during a TACE procedure provides early and late arterial phases of the same structure. The completion of these two phases has been shown to provide superior conspicuous liver tumor than imaging a single phase alone[START_REF] Meyer | Visualization of hypervascular liver lesions During TACE: comparison of angiographic C-arm CT and MDCT[END_REF]. The novelty of this technology is the possibility for tumor imaging at both early and delayed arterial phases in using only one intra-arterial injection of contrast medium. This improvement allows visualization of Lipiodol® targeted regions and visualization of changes in temporal enhancement before and after drug eluting beads, as we reported in section 3. The simultaneous display of both phases side-by-side enables direct visual comparison between the two phases or between pre-and post-treatment images.The specificity of Dual-Phase C-arm CBCT is to capture two different enhancement phases of the same hepatic-tumor structure. The early arterial phase shows the native arterial network and the tumor feeding arteries, while the delayed phase shows tumor enhancement at the capillary level (131). In other words, the former visual comparison (early vs. late) could provide an evidence (or indication) of the vascular density of the tumor in relation to the feeding vessel while the second comparison (pre-vs. post-) could provide an evidence (or indication) of the enhancement change due to embolization and its clinical relevance.



  ABBREVIATED TITLE PAGE

  contrast injection as described byLin M. et al (131). For each C-arm spin, the liver was positioned in the system isocenter and during 8 s and 240 projection images (30 frames/s) were acquired with the motorized C-arm covering a 210° clockwise arc.The two scans were triggered at 3 and 18 s after a selective single injection of undiluted contrast medium through a 2.7Fr coaxial microcatheter (Progreat®; Terumo Leuven, Belgium) placed into the proper hepatic artery. We used a power injector (Medrad® Mark 7 Arterion® Bayer HealthCare Whippany NJ) with the following parameters to perform the injection: injection rate of 4 m L/s; injection volume of 15 mL contrast media (iobitridol 350 mg I/mL; Guerbet Roissy, France) flushed with 25 mL saline solution (total injected volume of 40 mL), at a PSI of 300. A 21-s apnea (single breath hold) was required during the two consecutive spins to avoid motion artifacts.

  because of truncated liver, missed lesion because of streak artifacts, or missed lesion because of the absence of tumor enhancement (Figures6).

Figure

  Figure 6a, b, c : MDCT at the portal phase (Figure 6a), dualphase C-arm CBCT: eAP (Figure 6b), and dualphase C-arm CBCT: IAP (Figure 6c) in a53 years old woman with widespread liver invasion by CRCLM. Figure 6a show 6 tumor involving segment II, III, IV, V and VI appeared as hypodens lesion surrounded by liver parenchyma enhanced. The eAP (Figure 6b) show three tumors with moderate peripheral enhancement associated to minimal streak artifact from catheter or contrast media (white arrow head). All the three lesions were classify as uncertain by readers. Figure 6c the late arterial phase show 5 lesions with peripheral corona surrounding the tumor classified as undoubtedly CRCLM. Readers classified

Figure 7 :

 7 Figure 7: flow diagram of the study participants.

Figure 8 a

 8 Figure 8 a, b, c, d : Bland and Altman plot for reader 1 at the early arterial phase (8a) and the late arterial phase (8b). Bland and Altman plot for reader 2 at the early arterial phase (8c) and the late arterial phase (8d).

  Dual-phase C-arm CBT was developed to increase HCC detection during TACE procedures (131). Higashihara et al. have demonstrated that dual-phase CBCT performs similarly to MDCT in terms of accuracy (MDCT, mean Az value 0.83; C-arm CBCT, 0.85; p = 0.032), sensitivity (MDCT, mean Az value 0.65; C-arm CBCT, 0.60; p = 0.040), and positive predictive value (MDCT, mean 0.98; C-arm CBCT, 0.97; p = 0.068)

  Schernthaner E.R. et al. reported the performance of Dual-Phase CBCT for the detection of cholangicarcinoma (154). Cholangiocarcinomas give a similar pattern to those of CRCLMs.

  DEBTACE perfusion analysis with CT scanner on the rabbit VX2 liver tumor model. (Oral communication at the Society of Interventional Radiology (SIR) Meeting, Chicago March 2013.

Figure 9 :

 9 Figure 9: MDCT perfusion scan performed before (a and b) and one day after the chemoembolization. These figures demonstrate the reduction of tumor arterial flow (AF) after the chemo-embolization compare to base line, while tumor portal flow and perfusion index remained unchanged.Altogether, our data demonstrated that dynamic first-pass area-detector CT imaging can successfully depict changes in liver and tumor perfusion, especially in the reduction of the arterial flow occurring one day after intra-arterial therapy in the liver VX2 model.

Figure 10 :

 10 Figure 10: Injection and acquisition protocol for C-arm CBCT parenchymal blood volume measurements.

Figure 12 :

 12 Figure 12: Processing steps for reconstruction of the parenchymal blood volume (PBV) maps.

a

  clearly need for a preoperative surrogate biomarker able to indicate treatment success/failure. Parenchymal blood volume (PBV) assessed by C-arm CBCT, is a quantitative parameter allowing to assess tumor perfusion. PBV has been shown to yield a strong correlation to CT perfusion in HCC tumor (rho = 0.0903 p <0.001) (183).

8 :

 8 Evidence of bilobar CRCLM involvement with CT scanner Progressive disease (PD) in the liver at the time of inclusion Oxaliplatin and Irinotecan based chemotherapy failure Non-resectable liver metastases < 4 stable extra hepatic lesions ECOG-PS < 2 Ade uate hematologic function (neutrophil ≥ 1.0 × G/L, platelets ≥ 75 × G/L) Adequate liver function (Child-Pugh classification A or B) Total bilirubin ≤ 20 µmol/L Albumin ≥ 25 g/L Adequate renal function (creatinine < 200 µmol/L) Life expectancy of more than 12 weeks Clinical contra-indication: Portal vein thrombosis/invasion or portal vein hypertension Any contraindication for hepatic embolization portosystemic shunt; biliary duct dilatation) Transaminase le els ≥5 times the upper limit of normal or >250 U /l Severe allergy to iodinate contrast mediaTable Clinical indication and contradiction for patient selection to DEBIRI

  Statistical analysis was performed with SAS software version 9.4 (SAS Institute, Cary, NC, US). Results are presented as mean or (median) ± standard deviation (SD). A p-value of <0.05 indicated a statistically significant difference for the used tests. As a first step, the interobserver agreement of tumor size at MDCT and PBV was studied with Intra-class Correlation Coefficients (ICC) as described by Shrout and Fleiss (145). Intra Class correlation values were classified as follows: ICC<0.40, poor agreement; ICC 0.40-0.60, moderate agreement; ICC 0.60-0.80, substantial agreement; ICC>0.80, good agreement (145). Baseline of tumor PBV values before, after and PBV rate of change after chemoembolization was correlated to the tumor response at 2 months using the Spearman rank correlation coefficient. In order to stratify the behavior of the CRCLM we classified the tumors by quartile according to their initial blood volume value. The relationship between the tumor size change, the pre-DEBIRI blood volume, and the % of PBV change after DEBIRI was also assessed using a mixed model considering the individual deviations (several lesions per patient).

Figure 13 :

 13 Figure 13: patient flow chart Tumor and Patients response rate with RECIST 1.0 criteria

  (p<0.0001) before and after chemo-embolization, respectively. These accounts for a PBV average reduction of -55 ± 29 % [-99 ; 0]. The figures 14a, b, c, d show a representative case.

Figure 14 :

 14 Figure 14: Representative case of a 68 years old man with CRCLM referred to DEBIRI. Four index lesions are shown. Lesion 2 had a 181 ml/1000ml PBV before and doped down to 32 ml/100ml after the DEBIRI. This lesion totally shrink at the 2 months MDCT follow-up Correlation between PBV and tumor size

Figure 15 :

 15 Figure 15: Non-linear regression of PBV before (a) after (b) % of PBV change (c) and % of tumor change at 2 months.

2 - 4 - 2 -

 242 A major by-stander effect (passive diffusion of CPA cytotoxic metabolites), which was clearly demonstrated both in-vitro and in-vivo. Indeed, in a mouse model, a complete eradication of tumors after CPA administration was observed even if only a quarter of tumor cells expressed the suicide gene (223). Another team observed this bystander effect in a similar model (231).3-A strong immune response against the tumor cells which was clearly demonstrated in immune-competent mouse models. In fact, in mice previously cured by GDEPT, the re-injection of tumor cells led to an initial tumor growth, which regressed spontaneously, completely, and quickly between day 7 and 10 without any CPA administration. No recurrence for more than 10 months was observed. We demonstrated that this effect was mediated by CD8+ T cells (232). An efficient targeting/delivery of suicide gene to tumors. Two gene vectors have been tested by four intra-tumoral injections as a delivery pathway, followed two days later by intra-peritoneal CPA injection.1-Recombinant lentivirus expressing our suicide gene (LV-CYP2B6TM-RED) was first tried. The transduced lentiviral method led to moderate effects mainly due to the low rate of lentivirus gene transfer to tumor cells. Moreover, producing the lentivirus that express at a high level of suicide gene has remained difficult. The limited tumor response induced by lentiviral approach led our team to turn to another vector of suicide gene likely mediated by cells to tumor tropism such asMSCs (223, 233). Mouse mesenchymal stem cells (MSCs) expressing our suicide gene after infection by the recombinant lentivirus (MSCs-CYP2B6TM-RED) were tried. Transduced MSCs were injected percutanously in the tumor and its boundaries 4 times while CPA was

  For that purpose MSCs are placed in an upper compartment separated to the lower compartment (containing the VX2 cells) by a microporous membrane. The MSCs used in that experiment were tagged with luciferase (MSCs-Luc). One hour later, MCSs-Luc migrates through the membrane and homes within the VX2 cells. The Figure 18 shows the luminescence assay (Promega, Madisson, US) performed one hour latter. Results indicate a high luciferase activity at the lower compartment, significantly different to control. This finding demonstrates that MSCs are able to migrate toward VX2 cells, thus highlighting the homing effect.

Figure 17 :Figure 18 :

 1718 Figure 17: Transwell migration assay

Figure 19 a

 19 Figure 19 a & b: MSCs survival in iodinate contrast media Iomeporl (Figure 19 a) or Lipiodol® (Figure 19 b)

Figure 20 :

 20 Figure 20: VX2, TC1, TC1 -TM5 cells survival test in CPA

Figure 21 :

 21 Figure 21: VX2 cells survival in various concentration of CPA

  any tumor underwent single IA delivery in the left hepatic artery of rabbit MSCs expressing luciferase (MSCs-Luc) (Figure 22 a, b & c). Two hours later, luciferine was given and animals were imaged in-vivo by photoluminescence assay. This test clearly shows a high expression of luciferase within the tumor surface (Figure 23 a). Seventy-two hours after the IA MSCs-Luc delivery rabbits received luciferine once again and were scarified. Tumor and various organs (liver, kidneys, spleen, stomach, duodenum, lung, heart) were removed, homogenized in PBS and centrifuged at 10 000g for 3 min. The luminescence of 20 µl of the supernatant was measured using the luciferase assay system. The results are expressed as arbitrary units of luminescence per g of protein. A very high expression of luciferase essentially near and inside the tumor with almost no expression in other hepatic lobes and organs was found. These results were confirmed by immune-histochemical staining (Figure 23 b & 24). The experimental protocol is developed in the Annex IV.

Figure 22 a

 22 Figure 22 a, b & c: Ultrasound Doppler scan before catheterization, the tumor is located in the left liver lobe (30 × 17 mm) well limited and hypervascular. Figure 22c left hepatic artery angiography showing the hypervascular tumor located in the left liver lobe. The catheter was advanced closer as possible to the tumor to inject the rabbit MSCs-Luc.

Figure 23 a

 23 Figure 23 a & b: Whole animal/organ photoluminescence assay performed 2 hours (Figure 23 a) and 72 hours (Figure 23 b) later after intra-arterial injection of rabbit MSCs-LUC. Rabbit received intra venous injection of luciferine and were examined.Figure 23 a shows a high and intense luminescence in the left abdominal wall corresponding to the VX2 liver tumor. Figure 23 b shows the whole liver examined after rabbit sacrifice, showing clear luminescence into the tumor.

  of the transduced MSCs and the CPA (Pellerin et al., submitted for publication.. ) The experimental protocol is developed in the Annex V. of a gene directed enzyme prodrug therapy with intra-arterial delivery of mesenchymal stem cells in a rabbit VX2 tumor hepatic model.

Figure 25 :

 25 Figure 25: GDEPT concept of intra-arterial injections of transduced MSCs. The GDEPT follow several steps. Firstly the therapeutic gene is transduced to MSCs as a carrier, and then the

  MSCs-CYP2B6TM-RED followed by CPA administration All 8 rabbits of group C underwent the first hepatic artery injection of transduced MSCs. At D3, the animals received intravenous 80mg/Kg of CPA. Between D3 and D4, all animals receiving CPA developed toxicities, leading to two early sacrificed in group C. The 6 remaining animals were successfully managed by rehydration and antibiotic therapy but the second session of GDEPT was postponed 4 days later. The Figure 26 summarizes the overall experimental plan.

Figure 26 :

 26 Figure 26: Experimental plan applied to animals. In group C, CPA major toxicities were responsible of two rabbit early sacrifice between D3 and D4 and 3 animals (group C1) did not underwent to the second in injection of GDEPT for mild CPA toxicities. For the 3 remaining rabbit (group C2) suitable for a second GDEPT, the injection of transduced MSC was post ponded at D11 and CPA at D14.

  Figure 27 shows the tumor volume of each group at baseline, D7, D11, D19 and D25. A tumor volume difference was showed between the group C1 receiving a single treatment of GDEPT and C2 receiving a double treatment at D19 and 25 (p>0.05).

Figure 27 :

 27 Figure 27: VX2 tumor volume as a function of time by group. A significant tumor volume difference between group B and group C at D7 (p=0.024), D11 (p=0.024), D19 (p=0.048) and D25 (p=0.048) was observed after completion of the GDEPT protocol.

Figure 28 :

 28 Figure 28: the Figure 28a shows representative tumor histology section stained with hematoxilin-eosin-saffron of the control rabbit (group A) (original magnification ×2.5).

  doses of CPA to co-cultures of murine MSCs-CYP2B6TM-RED clone and VX2 cells at different MSCs to VX2 cell ratios (Figure 29 appendix). Cytotoxic metabolites produced by transduced MSCs diffuse out of these cells and were able to kill VX2 tumor cells, thus demonstrating invitro an efficient by-stander effect.

Figure 29 :

 29 Figure 29: appendix-in-vitro by-stander evaluation.

Figure 30 :

 30 Figure 30: appendix -Photoluminescence assay performed 72 hours on frozen sample of different organs and tumor. This graph demonstrates a significant higher luminescence in the tumor and the surrounding tissues wherein the rabbit MSCs-Luc were intra-arterially injected.

  repeated injections of the therapeutic cells to achieve tumor regression. Multiple injections of transduced MSCs, exhibited stronger anti-tumoral and anti-metastatic effects as compared to a single injection (271) and Pellerin O et al, manuscript submitted to Radiology).The mesenchymal stroma is also a determining factor to consider in the control of tumor growth. As a common feature of tumor development, tumor cells dysregulate the expression of chemokines, growth factors and extracellular matrix proteins to mimic the environment of injured tissues and, in using many inflammatory mediators, they attract the MSCs. Not to afford that the interactions of the cytokines or chemokines with their corresponding receptors induce the migration of MSCs to the tumor microenvironment (272).Therefore, the effective treatment of tumors by transduced MSCs could include the killing not only of tumor cells present in the tumor mass but also of initiating cancer stem cells. In addition, dying cells of the treated tumor, in vivo, induce an immune reaction, which consists of tumor infiltration with T-cells, and macrophages, which is associated with high levels of various cytokines(223, 273). The combination of stem cell directed pro-drug therapy with immuno-stimulation via inoculation of genetically modified MSCs with potent cytokines or pro-apototic gene products could increase the therapeutic effects of MSCs.Literature indicates MSCs are weakly immunogenic as they elicit a minimal expression of the major histo-compatibility complex. Pro-inflammatory mediators (such as interferongamma), shown to activate the MSCs antigen-presenting cells, could induce a valuable CD8+ T cell response (T lymphocyte killer)(232, 252). Interestingly, in our study, these features may explain the low occurrence of lung metastases and peritoneal carcinomatosis in the animal subgroup C that received the 2 scheduled GDEPT.As a conclusion, MSCs is a promising vehicle given their high targeting efficiency and their long retention in tumor sites. Our in vitro and in vivo studies have clearly evidenced an efficient bystander effect between engineered stem cells, such as MSCs-CYP2B6TM-RED, and a large number of tumor cell types. It is known that the presence of cancer stem cells (CSCs) in the main tumor mass may contribute to classical therapeutic failures.

-

  The amount of transduced MSCs by IA injection, -The time of multiplication of transduced MSCs in the tumoral environment, by extending the delay between IA MSCs and IV CPA injections, The number of MSCs/CPA injections by repeating them 4 times instead of 2.

Figure 31 :

 31 Distribution of the three pathway of carcinogenesis. CIPM: CpG Island methylation phenotype; CIN chromosomal instability; MSI: microsatellite instability; MSS: microsatellite stability.

  The EGFR Signaling pathway. The epidermal growth factor receptor (EGFR) or the human epidermal receptor (HER-1) is a trans-membrane receptor that belongs to the family of tyrosine kinases receptors. The two main pathways of intracellular signaling resulting from EGFR activation are RAS / MAPK (mitogen-activated protein kinase), and PI3K (phosphatidylinositol inositol 3 kinase) / AKT. The activation of EGFR induces the recruitment of the RAS protein and the activation cascade of different protein kinase RAF, MEK and ERK.ERK proteins are then translocated into the nucleus, inducing transcription of genes involved in proliferation, resistance to apoptosis, angiogenesis, invasion and metastatic spread. The RAS family includes the protein KRAS, NRAS and HRAS, KRAS which the corresponding genes, HRAS and NRAS can be mutated. Historically, it was shown that the presence of an activating mutation of codon 12 and 13, located in exon 2 of the KRAS gene was predictive of resistance to treatment with anti-EGFR antibody. Recently, the addition of other mutations located in exons 3 and 4 and KRAS in exon 2, 3 and in exon 4 of NRAS gene improves the prediction of resistance to anti-EGFR antibody. The prevalence of mutations in the KRAS and NRAS genes in colorectal cancer is around 50%. The BRAF gene encodes RAF1 protein, and it is mutated in 9 to 11% of colorectal cancers in a mutually exclusive manner with SAR family gene mutations. It is frequently mutated in sporadic MSI+ tumors. The presence of a mutation of the BRAF gene is a bad prognostic factor (277).

  TP53 mutations occur in 50 to 75% of the CRC (279).A more integrative vision of CRC has recently been published by several research teams worldwide (280). This vision is based on the expression of several thousand genes. At least 4 groups of colon tumors were discriminated particular phenotypic characteristics. The first group corresponds MSI cancers, associated with frequent mutations of BRAF. The second group is characterized by chromosomal instability and a collapsed immune infiltrate. The third group is characterized by an over-representation of cancer with KRAS mutation and chromosomal instability phenotype. A signature suggesting a phenotype of stem cells and epithelial-mesenchymal transition characterizes the fourth group. The latter group is, apparently, of poor prognosis. This integrative vision allows an easier understanding of the genetic factors or CRC and pragmatic application patients.The figure32summarizes the different regulation pathway involved in CRC, and potential target for biotherapies.

Figure 32 :

 32 Figure 32: Biomarkers for prediction of response to anti-EGFR therapies. Presented are biomarkers that can influence response to anti-EGFR therapies. Pink are negative biomarkers and green are positive biomarkers. Numbers in percentage are mutation rates in metastatic colorectal cancer. ADCC acts only with cetuximab and not panitumumab. The four receptors (EGFR, HER2, HER3 and MET) can activate both pathways (RAS-MAPK and PI3K-AKT pathways). ADCC: Antibody-dependent cellular cytotoxicity; NK: Natural killer. Courtesy to Perkins G et al Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer. Pharmacogenomics. 2014 May;15[START_REF] Rosen | Colorectal cancer screening disparities related to obesity and gender[END_REF]:1043-52[START_REF] Perkins | Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer[END_REF].

Figure 33 :

 33 Figure 33: Conventional chemotherapy and Targeted therapeutic agents. Abbreviations: EGFR, epidermal growth factor receptor; IgG1, immunoglobulin G1; VEGF, vascular endothelial growth factor.

  .5 mg/kg) and ketamine hydrochloride (44 mg/kg) administrated intramuscularly. This anesthesia, via an intravenous access in a marginal ear vein, proceeded by sodium pentobarbital injection and was maintained by injection of propofol (3 to 5 mg/kg).Trans-catheter hepatic artery angiography and MSCs-Luc injection were done under fluoroscopy guidance. The animals were brought to the angiography suite and intubated using a size 3.0-mm endotracheal tube (Mallinckrodt Medical, St. Louis, MO) but not ventilated. Surgical cut down was done to gain access into the common femoral artery using a 4-French sheath (Cook, Inc., Bloomington, IN). A specially manufactured 2-French catheter with a tip in the shape of a hockey stick (JB1 catheter; Cook) was manipulated into the celiac axis, after which a celiac arteriogram was done to delineate the blood supply to the liver and confirmed the location of the tumor. The tumor was visualized as a region of hyper-vascular blush located on the left side of the liver near the gastric fundus. The left hepatic artery, which nearly exclusively provided flow to the tumor, was then be selectively catheterized off the common hepatic artery with the aid of a steerable guide wire (0.010-0.014 inch Transcend wire; Boston Scientific Oncology, Natick, MA). After having ensured an adequate positioning of the catheter, (the closer as possible to the tumor), the MSCs was carefully infused directly into the artery. The 3 animals received an injection of 10 M of MSCs and no complementary embolization was performed. Eventually, after MSCs injection a mild embolization was performed with resorbable embolic material such as gelfoam, starch microspheres). The procedure technical end point was to fully inject the prepared dose of MSCs.

Figure 34 :

 34 Figure 34: Experimental plan

using a size 3

 3 .0-mm endotracheal tube (Mallinkrodt Medical, St. Louis, MO) but not ventilated. Surgical cut down was done to gain access into the common femoral artery, after which a 4-French sheath (Cook, Inc., Bloomington, IN) was placed. A specially manufactured 2-French catheter with a tip in the shape of a hockey stick (JB1 catheter; Cook) was manipulated into the celiac axis, after which a celiac arteriogram was done to delineate the blood supply to the liver and confirm the location of the tumor. The tumor was normally visualized as a region of hyper-vascular blush located on the left side of the liver near the gastric fundus. The left hepatic artery, which nearly exclusively provided flow to the tumor, was then be selectively catheterized off the common hepatic artery with the aid of a steerable guide wire (0.010-0.014 inch Transsend wire; Boston Scientific Oncology, Natick, MA). After having ensured an adequate positioning of the catheter, (the closer as possible to the tumor), the Rabbit-MSCs-CYP2B6TM-RED were carefully infused directly into the artery. All the 7 animals received an injection of Rabbit-MSCs-CYP2B6TM-RED (10 -13 M of cells in 1000µl

Figure 35 :

 35 Figure 35: Animal daily chart.

  Photoluminescence assay performed 72 hours on frozen sample of different organs and tumor. This graph demonstrates a significant higher luminescence in the tumor and the surrounding tissues wherein the rabbitMSCs-Luc were intra-arterially injected. ...............................................................................

. VX2 tumor is massively necrotic (nT) with only few cluster of viable tumor (vT) limited by fibrotic

. Figure 30: appendix -Figure 31: Distribution of the three pathway of carcinogenesis. CIPM: CpG Island methylation phenotype; CIN chromosomal instability; MSI: microsatellite instability; MSS: microsatellite stability. ......................................

Figure 32: Biomarkers for prediction of response to anti-EGFR therapies. Presented are biomarkers that can influence response to anti-EGFR therapies. Pink are negative biomarkers and green are positive biomarkers. Numbers in percentage are mutation rates in metastatic colorectal cancer. ADCC acts only with cetuximab and not panitumumab. The four receptors (EGFR, HER2, HER3 and MET) can activate both pathways (RAS-MAPK and PI3K-AKT pathways). ADCC: Antibody-dependent cellular cytotoxicity; NK: Natural killer. Courtesy to Perkins

G et al Beyond KRAS status and response to anti-EGFR therapy in metastatic colorectal cancer. Pharmacogenomics. 2014 May;15

Table 1 :

 1 Hepatic arterial drug advantage (Rd) forIA injection of cytotoxic agents ..............................................

Table 2 :

 2 Indication and contradiction Criteria for intra-arterial treatment .........................................................

Table 3 :

 3 patient baseline characteristics.

Table 4 :

 4 image modalities lesions measurement and diagnostic. * biliary cyst, adenoma or focal nodular hyperplasia .

Table 5 :

 5 Intraclass correlation coefficients between readers versus MDCT at the portal phase. * p < 0.001 for the correlation coefficients between measurements of different observers. # Calculated for comparison of correlation coefficients betweenmethods. .........................................................................................................

Table 6 :

 6 appendix -6-cell matrix table of diagnostic result per image modality ................................................

Table 7 :

 7 Accuracy of Dual-Phase C-arm CBCT. p Value for the early and the late arterial phase. LR+ is the conditional likelihood ratio of a positive test; the odds of a positive test in favor of disease. LR-is the conditional likelihood ratio of a negative test; the odds of a negative test in favor of disease. LR± is the conditional likelihood ratio of a non-positive, non-negative result; the odds of a non-positive, non-negative test in favor of

disease. YD+ is the probability of a positive or a negative result when disease is present. YD-is the probability of a positive or a negative result when disease is

absent. ......................................................................................

Table 8 :

 8 Clinical

indication and contradiction for patient selection to

DEBIRI ....................................................

Table 9 :

 9 Change

in tumor size and PBV value before and after chemoembolization. Values are presented as mean ± standard deviation

[min ; max]

.* two months after

DEBIRI. ..................................................................

Table 10 :

 10 Values

are presented as mean ± standard deviation

[min ; max]. ......................................................

Table 11 :

 11 Results

of Logistic regression model used to predict the probability of disease control at 2 months as function of covariates..................................

........................................................................................................

Table 12 :

 12 Viral

vectors for gene

therapy. ............................................................................................................

Table 13 :

 13 Non

-viral and bacterial vectors for gene therapy. ..............................................................................

Table 14 :

 14 CPA

hydroxylase activity by type of species according to Chen C. et al

(29, 59). .................................

Table 15 :

 15 Blood

examination results performed at D0,

7, 19, and 25. ..............................

................................. Table 16: pathways of carcinogenesis CIN + and MSI +: main features and differences. ...................................

Table 17 :

 17 Major publications of

1 ST CHAPTER: LIVER COLORECTAL METASTASES, STATE OF THE ART AND ROLE OF INTRA-ARTERIAL THERAPY IN 2016 1. Incidence, mortality rate

	Colorectal cancer (CRC) is one of the most commonly diagnosed cancers in Europe
	and worldwide (1, 2). In 2008, CRC ranked the fourth most common cancer worldwide (1.23
	million new cases) and the second commonest cause of cancer-related mortality (more than
	600,000 CRC related deaths in the world (4). The life-time incidence of CRC in an average risk
	population is estimated at 5% and progressively increases after age 50 years old. Both
	genetic and environmental factors play a role in colon cancer formation (5). Despite better

understanding of the molecular mechanisms involved in colorectal carcinogenesis, little progress has been made in the successful management of advanced CRC. Data from the Surveillance, Epidemiology and End Result Program suggests that the 5 years survival rates have improved from 56.5% to 64.9% for patients diagnosed in the early 1980s for patients diagnosed in 2005. This trend is mostly due to early diagnosis and aggressive treatment.

Table 1 :

 1 Hepatic

arterial drug advantage (Rd) for IA injection of cytotoxic agents 4.1.4. Blood flow (45

arm cone-beam CT detect a micro-embolic effect after TheraSphere radioembolization of neuroendocrine and carcinoid liver metastasis? Pellerin O, et al. Cancer Biother. Radiopharm. 2013 Jul-Aug;28(6):459-65.) (132)

  addition we wanted to assess if imaging could allow seeing where the Y 90 beads ended into the tumor by measuring the contrast enhancement decrease after treatment.

	Manuscript #3:
	Can C-

Table 2 :

 2 Indication and contradiction Criteria for intra-arterial treatment 350 Guerbet, Roissy, France) at the arterial phase (20 s) and at the portal phase (70 s). Images were acquired in 0.6 mm thick slices and reconstructed in 3mm using the

	Indication for intra-arterial treatment:
	Pathologically proven mCRC
	Evidence of bilobar CRCLM involvement by CT scan
	Progressive disease (PD) in the liver at the time of screening
	Non-resectable liver metastases according to the multidisciplinary board
	< 4 stable extra hepatic lesions
	ECOG-PS < 2
	Ade uate hematological function (neutrophils ≥ 1.0 × 10 9 /L, platelets ≥ 75 ×
	Adequate liver function (Child-Pugh classification A or B)
	G/L) Total bilirubin ≤ 20 µmol/L
	Albumin ≥ 25 g/L
	Adequate renal function (creatinine < 200 µmol/L)
	Life expectancy > 12 weeks
	Contraindication for intra-arterial treatment:
	Liver invasion by tumor > 70%
	Portal vein thrombosis/invasion or portal vein hypertension
	Any contraindication for hepatic embolization
	Transaminase le els ≥ 5 times the upper limit of normal or > 250 U /l
	portosystemic shunt; biliary duct dilatation) Allergy to iodinated contrast media
	MDCT Technique
	Baseline MDCT, 15 days before intra-arterial therapy, was required for inclusion in
	the study. Patients were imaged using a Siemens Somatom definition AS+ 128 detectors CT

unit (Siemens Health Care; Forchheim Germany). A triphasic imaging protocol was applied and included the following: unenhanced acquisition, post contrast media injection (iobitridol,

Table 4 :

 4 image modalities lesions measurement and diagnostic. * biliary cyst, adenoma or focal nodular hyperplasia

Table 5 :

 5 Intraclass correlation coefficients between readers versus MDCT at the portal phase. * p < 0.001 for the correlation coefficients between measurements of different observers. # Calculated for comparison of correlation coefficients between methods.

Table 6 :

 6 appendix -6-cell matrix table of diagnostic result per image modality

	MDCT
	Undoubtedly	Undoubtedly	Total
	CRCLM	no CRCLM

Table 9 :

 9 Change

			Before	After	% of change after
	Feature					p value
			chemoembolization	chemoembolization	chemoembolization
	Tumor n = 170
	Tumor size (mm)	35 ± 23 [11 ; 191]	29 ± 23 [0 ; 191]	-13% ± 34 [-100 ; +85]	<0.0001
	PBV (ml/1000ml)	62 ± 47 [10 ; 195]	17 ± 8 [1 ; 59]	-55% ± 29 [-99 ; 0].	<0.0001
	Patient n = 34
	RECIST	1.0
			174 ± 66 [92 ; 380]	147 ± 82 [60 -392]	-13% ± 26 (-66 ; 49)	<0.0001
	sum*(mm)
	PBV (ml/1000ml)	62 ± 38 [13 ; 141]	17 ± 5 [10 -31]	-55% ± 22 [-90 ; 0]	<0.0001

in tumor size and PBV value before and after chemoembolization. Values are presented as mean ± standard deviation [min ; max].* two months after DEBIRI.

Table 10 :

 10 Values

are presented as mean ± standard deviation

[min ; max]

.

Table 11 :

 11 Results of Logistic regression model used to predict the probability of disease control at 2 months as function of covariates.

			Logistic regression
	Variable Odds ratio 95% wald CI p value
	Pre-DEBIRI PBV	1.121	0.978; 1.284 0.1003
	Post-DEBIRI PBV	5.765	0.619; 53.726 0.124
	% of PBV change after DEBIRI	0.9	0.811; 1	0.0493

presents the results of logistic regression model to estimate the odds and likelihood of disease control rate as a function of predictors: pre-DEBIRI PBV, post-DEBIRI PBV and PBV rate of change after chemo-embolization.

  Syha R. et al (107) demonstrated the clinical utility of a PBV, for immediate post treatment assessment of DEB-TACE in HCC patient. They showed that all treated lesions had significant decrease of PBV after DEB-TACE (mean difference, -15.61 mL/100 mL, P < 0.0001).

	When lesions exhibiting residual PBV, less tumor shrinkage over time was observed (-0.02 ±
	0.49 vs. -0.76 ± 0.38; p < 0.0001). Vogl TJ. et al. (108) studied PBV in a larger cohort of 111
	patients, of a mix of primary and secondary liver tumors referred for DEB-TACE. They
	reported that the mean PBV pre DEB-TACE for HCC, CRCLM, and breast cancer liver
	metastases were 98 ml/1000ml, 64ml/1000ml, 108ml/1000ml, respectively. They showed a
	correlation between initial PBV and the % of PBV change of after DEB-TACE (rho=0.61;
	p<0.0001), but without significance when considered a tumor-by-tumor analysis. They also
	reported that patients with initial PBV >100 ml/1000 ml had 7.1% tumor shrinkage; patient
	with 50-100 ml/1000 ml had 4.6% tumor shrinkage; and patient with PBV <50 ml/1000 ml
	had 2.8% tumor shrinkage. But authors failed to demonstrate significant correlation
	enhancement reaches a steady state. A non-rigid registration algorithm is applied to register
	mask and fill volume and then the two volumes are subtracted and bone and air are
	segmented out. The arterial input function value is calculated from an automated histogram
	analysis of the vessel tree. This arterial input function value is then applied as a scaling factor
	to obtain the quantitative PBV map. The quantitative PBV values are given in mL/1000 mL.

2009, Zellerhoff et al. described the use of C-arm CBCT for parenchymal blood volume evaluation (175). PBV calculation is based on the assumption that, with an adequate contrast injection, the contrast concentration in the imaged liver tumor is constant for the duration of a single C-arm CBCT acquisition. This leads to the calculation of the PBV when tumor (176-178). PBV have been mostly evaluated with HCC. between pre-DEB-TACE PBV or change of PBV and tumor size change.

  last work shows that local tumor response can be predicted in an intra-resulted in a noticeable increase in patient overall survival (187). But it is important to notice that the tumor perfusion status is not yet incorporated in patient algorithm selection to better address patient to the best therapeutic option. In the same lines, to date and unfortunately no meta-analysis has been performed including CRCLM patients. Such tremendous work is required to document enough patient selection criteria.

	Roughly and according to current reported data, DEBIRI in colorectal cancer liver metastases
	do not dramatically change patient outcomes. An additional weak effect in life expectancy
	(from 2 to 4 months) could be accounted for DEBIRI as compared to chemotherapies such as
	Regorafenib in patients at end stage (160, 161). Our works mainly delt to refine the patient
	selection and, thus, to improve patient outcomes. Thus we are not in a position to claim any
	impact on patient survival. Huge efforts are mandatory; to reach this goal, a multicenter
	study, involving large cohort of patients is needed.
	procedural manner. It merges up that through evaluation of the parenchymal blood volume
	within tumor, one can expect a better selection of tumor/patients who are the best
	responder to DEBIRI. Further developments calling larger patient cohorts are needed to
	firmly state our conclusion
	Since the 80', major improvement of HCC patient outcomes have been performed

thanks to an accurate patient selection and at earlier diagnosis. In this regard, the broad meta-analysis reported by

Llovet JM. et al. (158)

in 2003, that applied the well-known BCLC HCC patient selection algorithm, enabled to correctly allocate patient to TACE or other treatment and

  Some bacteria such as Escherichia coli, Salmonella typhimurium, Clostridium, and Listeria have the capability of specifically targeting tumor cells, leading to RNA interference and gene silencing with blockage of RNA functions(194). Bacterial vectors can deliver prodrug converting enzymes and cytotoxic agents into tumor cells, and can mediate the host immune response (Table13).

	Vectors	Specificity	Advantages	Drawbacks	Clinical trial
	Adenoviruses	Double-stranded DNA viruses. Cause mild respiratory, digestive and ocular infection in humans. Target both dividing and non-dividing tumor cells. Selectively target certain cellular receptors or molecular defects.	High transduction efficiency. (≈100%). Long-term sustained gene expression	Tendencies for viral shedding and latent infection. Genetic instability of carried genes. Subsequent chromosomal aberration may lead to the development of lymphoproliferative disorders. As nearly half of all humans have been exposed to these viruses during their life time,with the generation of neutralizing antibodies, they may lead to high immunogenicity, with shorter durationof adenoviral-mediated transgene expression	Astrocytoma combination with radiation and/or temozolomide. ONYX-015(Onyx Pharmaceuticals) management of refractory head and neck cancer in combination with cisplatinum. Ad5-D24, recombinantH103, Ad5-CD/TKrep, CG7870, KH901, and OBP-301 (Telomelysin). Oncolytic adenoviruses alone rarely produce dramatic tumor response; need combination to other therapy.
		Small Single-stranded DNA viruses.
		Do not cause infection without co-infection of a
	Adeno-associated	helper virus in human. Target both dividing and non-dividing tumor cells.	Long-term gene expression. Lacks immunogenicity Lack		NA
	virus	Stably integrated into the host DNA at a	of pathogenicity in humans
		predictable location within a chromosome-19 in
		cell culture,
	Herpes simplex virus	Large, enveloped double-stranded DNA virus. Naturally neurotrophic that infects humans particularly at the oral and genital mucosa, and ultimately spreads to sensory nerves to replicate or become dormant at the sensory ganglions. Carry an oncolytic gene it is non-integrating in targeting the cell genome.	Broad cell tropism administered (suicide gene) Capability to carry a large load of transgenes such as thymidine kinase enzyme gene enhancing tumor lysis when ganciclovir medication is subsequently	Viral reactivation may lead to oral or genital ulcerations, skin rashes, or even encephalitis. Can evade the host immune system in case of toxicities, effective antiviral therapies are available to control viral replication.	Talimogene laherparepvec (TVEC) either as a monotherapy, or in association with surgery, radiation therapy or chemotherapy, particularly on patients with high-grade glioma. Currently, some success has been reported.
		Single-stranded RNA retroviruses, caring a reverse	Efficient transduction of	Inadequate immune responses as well as antitumor
		transcriptase enzyme.	both dividing and non-	response.
	Lentivirus	The most known lentivirus is human	dividing cells. Modified	Risk of viral transformation into pathogenic HIV	NA
		immunodeficiency virus (HIV).	viruses have low antivirus	infection, especially in immunized individuals.
		The infectious parts of the virus have been	immunity, low potential for	Insertional mutagenesis of new cancer genes into the
		removed and replaced by the therapeutic gene.	genotoxicity.	host genome, with the risk of second malignancy.
		Non-enveloped double-stranded RNA oncolytic	The virus is considered
		virus.	relatively benign, with good		Because of its relative safety, the virus is presently
		Infects animals, rarely cause human illness.	safety records, does not		being used in several clinical trials, as oncolytic
	Reovirus	Oncolytic gene mechanism through immune system stimulation. Cause a bystander immune	require genetic alterations to become an oncolytic	NA	reovirus monotherapy, administered intratumoral, intravenously, or intra-peritoneally; or as
		activation, by the release of tumor-associated	virus, and is less expensive		polytherapy, in combination with radiation therapy
		antigens following cellular lysis further stimulates	to be produced		or chemotherapy.
		innate immunity against tumor cells.	commercially.
					139

Mesenchymal stem cells (MSCs) are undifferentiated cells originating from embryologic mesenchyme tissue. They are able to renew and to differentiate towards many cell types, to migrate to tumors and establish themselves in the tumor microenvironment

(195)

. MSCs properties will be developed later in the article.

Table 12 :

 12 Viral vectors for gene therapy.

		Vectors	Transfection process	Transduction process	Advantages	Drawbacks	Clinical trial
			Compressed air
			or fluid (gene
			gun),		They are best
		Gene DNA coated on gold nanoparticules	ultrasound, to into the target force the genetic material	NA	suited for gene of gene delivery into tissues or in case	Difficult process not reliable	NA
			cell to release		vaccination
	Physical		its DNA into
	mediated		nucleus.
	gene
	transfer				In combination
	system		The gene is		with	Lack of genome
			delivered to the		chemotherapeutic	integration with	Melanoma,
		Naked DNA gene transfer or plasmid DNA gene transfer	nucleus through nano-pores that have been created by	NA	agents. Effectiveness in a vast array of	potential oncogenic effect	Prostate cancer, Colorectal cancer, and
			reversible		tumor cell types,	Unwanted	leukemia
			electroporation.		ease of its	immunogenicity
					administration.
					Endothelial cells
	Chemical mediated gene transfer	Cationic liposomes are microscopic vesicles of synthetic phospholipids and cholesterol	The complex enters into cells by endocytosis nucleus gene to the and deliver the	NA	selectivity. High rate of gene transfer. the lack of severe many genes, and as carriers for Broad application	Lake of efficacy NA
					side effects.
			Bacterial	Tumor cells
			vectors can	targeting,
			deliver a pro-	leading to
	Bacterial mediated gene transfer	E.Coli, Salmonella typhimurium, Clostridium, and Listeria	drug converting enzymes and cytotoxic agents into tumor cells, and can mediate the host immune response. They selectively	RNA interference and gene silencing with blockage of RNA functions, including cellular	Effective, practical and cheaper to manufacture compared to viral vectors. disorders of gastrointestinal the management orally, that fit to Administered	The outcome has been far less pronounced techniques. silencing interference other RNA compared to	NA
			colonize and	metabolism
			grow within the	and protein
			tumor.	synthesis

Table 13 :

 13 Non-viral and bacterial vectors for gene therapy.

Table 14 :

 14 CPA hydroxylase activity by type of species according toChen C. et al

	-1 )	Vmax/Km

Table 1

 1 summarizes the characteristics of these two main mechanisms. A third mechanism involves epigenetic modifications: the phenotype CIMP (CpG Island methylation phenotype).

	CNi+

  1 months, respectively (282, 283). On the other hand, studies of oxaliplatin-based therapy in patients failing an initial irinotecan-based regimen describe response rates around 10%, and median time to progression (TTP) of 4 to 5 months (283, 284). The current standard approach to metastatic colorectal cancer includes either oxaliplatin-based therapy after progression on a FOLFIRIlike regimen, or irinotecan-based therapy after progression on a FOLFOX-like regimen.

	Category	Mechanism	Major Adverse Effects
			Thymidylate synthase
	chemotherapy	Fluoropyrimidines (5-fluorouracil/capecitabine)	inhibitors; the accumulated deoxyuridine monophosphate misincorporates into DNA, resulting in inhibition of DNA synthesis and function Converted by carboxylesterase to SN38, which prevents the	Myelosuppression, Mucositis and/or Diarrhea, Hand-foot syndrome. Diarrhea,
	Conventional	Irinotecan	relegation of DNA and further results in double-strand DNA breaks and cellular death A third-generation platinum	Myelosuppression, Alopecia. Neurotoxicity,
			compound, covalently	Acute cold-triggered,
		Oxaliplatin	binding to DNA, which	Sensory neuropathy,
			results in inhibition of DNA	Chronic cumulative,
			synthesis and	sensory neuropathy.
			Bevacizumab: a recombinant
			humanized monoclonal	Hypertension,
			antibody to VEGF-A	Bleeding,
			receptors.	Gastrointestinal
		VEGF inhibitors	Aflibercept: a VEGF receptor	perforation,
		(bevacizumab, aflibercept)	decoy fusion protein	Arterial
	agents therapy		consisting of extracellular domain components of VEGF-R1 and VEGF-R2 fused with the Fc region of IgG1	thromboembolism, (including stroke and myocardial,infarction), Infusion réactions,
	Targeted	Anti-EGFR antibodies (cetuximab, panitumumab)	Monoclonal antibodies against EGFR	Hypomagnesemia, Pruritus/dry skin, Pulmonary toxicity, Diarrhea
		Kinase inhibitor (regorafenib)	A small-molecule inhibitor of multiple cell-signaling kinases	Hand-foot syndrome, Fatigue, Diarrhea, Hypertension

Table 17 :

 17 Major publications of CRCLM cTACE. nr: non reported, * in months.

3.1. Salvage situation

	DEBIRI was first evaluated in salvage situation for patient showing mCRC limited to
	liver following failure of Oxaliplatin and Irinotecan based chemotherapy. In literature, a 69%
	of liver disease control, with a median hepatic PFS and OS of 8 and 25 months respectively,
	were reported (305). In addition, a Phase III randomized trial comparing DEBIRI versus
	FOLFIRI in patients refractory to oxaliplatin demonstrated a significant advantage of DEBIRI

over FOLFIRI in terms of liver damage control hepatic progression free survival (PFS) and overall survival (OS) (8 vs 5 months, and 25 vs 15 months respectively). Because of extra hepatic progression, the global PFS was poor (6 months) for the DEBIRI group

Table 4 summarizes

 4 DEBIRI publications.

		Authors	Study design	Primary objective	Number of patient	Clinical situation	Drugs	Embolic material	Concomitant systemic y chemotherap	Disease control weeks# 12 rate at	Median free survival* progression	Median survival* over all	Down staging rate
		Aliberti et al. 2006 (311)	Phase I	Toxicity feasibility	10	PD status after Failure to Oxaliplatin and Irinotecan based chemotherapy	Irinotecan 100mg per lobe	Dc beads 100 -300	No	RR = n.r. SD = n.r. PD = n.r.	n.r.	n.r.	n.r.
		Fiorentini et al. 2008 (312)	Observational Phase IIa	Toxicity	20	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	No	RR = 80 SD = n.r. PD = n.r.	n.r.	n.r.	n.r.
	Salvage situation DEBIRI alone	Martin et al. 2009 (313) Martin et al. 2010 (314)	Observational phase IIa Observational multicenter phase I	Toxicity Toxicity	30 84	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe Irinotecan 100mg per lobe	Dc beads 100 -300 Dc beads 100 -300	n.r. n.r.	#RR= 75 #SD= n.r. #PD= n.r. CR = n.r. PR = n.r. SD = n.r. PD = n.r.	n.r n.r.	n.r. n.r.	n.r. n.r.
		Martin et al. 2011 (315)	Observational multicenter phase IIa	Toxicity And efficacy	55	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	Yes	CR = 12 PR = 53 SD = 30. PD = 5	11	19	n.r.
		Authors	Study design	Primary objective	Number of patient	Clinical situation	Drugs	Embolic material	Concomitant systemic y chemotherap	Disease control weeks# 12 rate at	Median free survival* progression	Median survival* over all	Down staging rate
		Aliberti et al. 2011 (305)	Observational multicenter phase IIa	Response rate Toxicity QOL	82	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	No	RR = 78 SD = n.r. PD = n.r.	8	29	n.r.
										OR = 69
	Salvage	Fiorentini et al. 2012 (38)	Multicenter phase III DEBIRI vs Folfiri	Efficacy OS PFS	36 38	PD status after Failure to Oxaliplatin based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	No FOLFIRI for the control arm	SD = 11 PD = 20 OR = 20 SD = 34	8 5	25 15	n.r.
	situation									PD = 46
		Eichler et al. 2012 (316)	Phase I and PK study	PK and toxicity	11	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	Yes various chemotherapi es	CR = 0 PR =9 SD=64 PD=27	n.r.	n.r.	n.r.
		Narayanan et al. 2013. (317)	Observational Phase IIa	Toxicity	28	PD status after Failure to Oxaliplatin and Irinotecan based chemoterapy	Irinotecan 100mg per lobe	Dc beads 100 -300	n.r.	#CR = 15 #PR = 30 #SD= 20 #PD=35	4	13.3	n.r.

Table 18 :

 18 DEBIRI relevant publications

Table 5

 5

	summarized

Table 19 :

 19 Y90 studies results

Pellerin Olivier -Thèse de doctorat -2016

Manuscript #5:

Can Cone-Beam CT tumor blood volume predict the response to chemoembolization of colorectal liver metastases? O. Pellerin et al.

Abstract Purpose:

Gene directed enzyme prodrug therapy (GDEPT) is a cancer gene therapy using a gene (optimized by mutations) encoding an enzyme delivered to tumor cells, and followed by administration of a pro-drug, which is converted locally into a cytotoxic by the mutated enzyme. The goal of our study is to assess feasibility and efficacy of a suicide gene therapy using transduced mesenchymal stem cells intra-arterially delivered into a VX2 liver rabbit tumor model.

Materials and methods

11 rabbits with a VX2 liver tumor were randomly assigned into 3 groups. Control group A (1 rabbit) free of any treatment; Control group B (2 rabbits) received intravenous injection of 80mg/Kg cyclophosphamide at day 3 and 9; and Group C (8 rabbits) received the GDEPT treatment consisting in intra-arterial injection of transduced MSCs successively at day 0 and 11 followed by CPA injection at day 3 and 14. Animals were followed until sacrifice (day 25) by ultrasound scan and blood examination. All organ pathologic examination was performed after sacrifice.

Results

A significant tumor volume difference between control groups and group C was observed at D7 (p=0.024); D11 (p=0.024), and D25 (p=0.048). Tumor necrosis was significantly greater for rabbits who received the GDEPT (78% of total tumor surface) compared with control animals (22% of total tumor surface (p=0.006).

Conclusion

Intra-arterial delivery of transduced MSCs with a suicide gene is feasible and caused significant tumor necrosis in a model of VX2 liver tumor.

Annex III

Intra-arterial treatment of colorectal cancer liver metastases: Clinical outcomes, literature review (39)

HIA treatment for CRCLM: technical clues

The administration of HIA treatment in the setting of CRCLM is different from that of Trans Arterial Chimio-Embolization, the so called TACE applied for hepato-carcinoma. In order to perform a super selective catheterization of each tumor arterial feeders, a lobar/regional approach is proposed. Firstly because CRCLM arterial feeders are especially tiny as compared to those of HCC or NETs, thus they do not allow super-selective catheterization (156). Secondly, CRCLM are associated to a high recurrence rate after curative hepatic surgery (60% within 2 years), due to the presence of undetected micro metastases, the so-called "missing" metastases (157). The regional approach tends to overcome this pathological impediment. Indeed data clearly showed regional chemoembolization or intra-arterial chemotherapy for CRC metastases could unmask missing metastases. Moreover, some authors described totally cured missing metastases (historically proven) when hepatic surgery was performed (158, 159).

Conventional Trans Arterial Chimio-embolization (cTACE)

Historically, cTACE was tested before the wide availability of drug eluting beads. All the reported studies represent patients cohort study and focus on the use of cTACE within patient after failure of many chemotherapies. Generally, Lipiodol® is administered in water/oil emulsion with chemotherapy and followed by resorbable (gelfoam, starch beads) of definitive bland embolization (Poly vinyl alcohol, trisacyle microparticuls). Doxorubicin, cis-platin, mitomycin-C and gemcitabine are the most frequent drug used alone or in association. A median survival of about 25 months, with a probability of survival at 2 years of 66% was reported. At 3-months follow-up, cTACE yielded a complete response rate of 3%, a partial response rate of 24%, and 42% stable disease rate, while progression was observed in 12% (no time to progression was reported). Table 3 presents the most relevant published studies in this respect.

Annex IV

DISTRILAP:

Estimation of MSCs-Luc distribution after liver intra-arterial injection.

Study rational

Gene directed enzyme prodrug therapy (GDEPT); a novel anticancer therapeutic strategy consists in the delivery of an engineered suicide gene to tumor cells. The suicide gene is responsible for an in situ conversion of a prodrug into cytotoxic metabolites. To carry the gene to the tumor microenvironment a specific vector is needed. The mesenchymal stem cells (MSCs) have been chosen since they particularly suit for gene transduction, they exhibit remarkable migratory property towards tumors / metastases and they are weakly immunogenic.

Some preliminary works of INSERM UMR-S1147 team made the proof of concept of this therapeutic strategy. The suicide gene chosen was CYP450 which converts CPA in mustard phosphoramid and acroleine. The phosphoramid mustard promotes the inhibition of the DNA transcription and replication resulting in cellular apoptosis.

Two routes of administration of the transfected MSCs were tested in mice. First when IV injected the MSCs did not reach the tumor and were massively stuck into the pulmonary circulation (248). Secondly the transfected MSCs were directly injected into the tumor and its boundaries, which led to encouraging results, but the technical difficulty and the reliability of percutaneous tumor injection appeared as a major limit of this administration mode (248). On these grounds we intended testing the intra-arterial (IA) delivery of therapeutic agent.

Based onto the exclusive arterial vascularization of primary tumors or metastases IA has been used for years in the cancer treatment. Indeed the intra-arterial injection allows the delivery of a high level of the therapeutic agent (both in concentration and quantity) very close to the tumor thanks to selective catheterization. As rats and mice are especially difficult to selectively catheterized mainly because of too small vessels and lack of appropriate material, the most frequently used animal model to evaluate an IA treatment evaluation is the rabbit. A cancer model has been developed in the rabbit by heterotopic

Annex V EFFILAP:

Proof of concept of the GDEPT with intra-arterial delivery of MSCs-CYP2B6TM-RED in arabbitVx2 tumor hepatic model.

Study rational

Among the different therapeutic strategies in the genic therapy area, the gene directed enzyme / prodrug therapy (GDEPT) consists in the delivery to tumor cells of an engineered suicide gene responsible of a prodrogue in situ metabolism into a cytotoxic agent. This novel therapeutic strategy could be summarized in two steps. First the therapeutic gene is delivered to the tumor through a vector (lentivirus or mesenchymal stem cells MSCs), and then as a second step a prodrug is systemically administered. The drug is locally converted into the therapeutic agent carried by MSCs, which promote the death of both the tumor cells and the MSCs cells and by the liberation of the toxic metabolite in the tumor microenvironment. This mechanism is called bystander effect. This therapeutic approach induces an immune response by the activation lymphocyte T CD8+ that improves the tumor response.

The INSERM U1147 team engineered a triple mutant gene coding for the CYT2B6 (223,247). This cytochrome protein is responsible of the metabolism of cyclophosphamide (CPA) into its active metabolite. Through different methods, the engineered gene has been transfected into MSCs. The MSCs constitute a reliable vector for suicide gene strategies.

Because, they are particularly suited for gene transduction, they exhibit remarkable migratory property towards tumors / metastases and they are weakly immunogenic. Some preliminary works have made the proof of concept of this therapeutic strategy. The administration of the transfected MSCs were tested on mice in 2 different ways. Frist the MSCs were intravenously injected. Unfortunately the MSCs did not reach the tumor and were massively stuck into the pulmonary circulation, which is inappropriate for treatment (248). Secondly the transfected MSCs were directly injected into the tumor and its boundaries, then cyclophosphamide was intravenously given 24 hours after each MSCS injection. This experiment has shown encouraging results. However, the difficulty and the reliability of percutaneous tumor injection of MSCs is the major limit of this administration

Title :

Study of antitumor effects of hepatic intra-arterial therapy (chemoembolization and suicide gene therapy). Role of the Intraprocedural imaging: preclinical and clinical evaluation.

Abstract :

Colorectal cancer liver metastases (CRCLM) are a leading cause of death. They occur in 50 -75% of patients during the course of the disease. Intra-arterial treatments for CRCLM have led to continuous progress; but these treatments still face significant limitations regarding tolerance and results. We explored two different avenues in trying to improve the results of intra-arterial treatment. The first one consists in optimizing the intraprocedural imaging technique aiming at anticipating the tumoral response.

The second one consists in de eloping a completely new approach based on so called "gene directed enzyme prodrug therapy" (GEPD).

As a 1st part, we explored the intraprocedural response to chemo-embolization based on various approach using C-arm CBCT. We first demonstrated the feasibility of tumor segmentation with semi-automatic software on MRI image and then apply the same method a VX2 liver tumor model. We found a strong correlation of tumor size between Carm CBCT and CT scan (rho = 0.993, p < .001) but also between CBCT and pathology (rho = 0.996, p < .001). In addition, we found a minimal bias (0.3 cm3), and high precision (0.674) when measuring tumors with C-arm CBCT. Since the C-arm CBCT precision was demonstrated, the next work was focused on the quantification of tumor enhancement before and after trans-arterial chemo-embolization (TACE); as a surrogate biomarker of tumor response with the goal to predict tumor response at an earlier stage. Dual-phase C-arm CBCT was first used, showing significant neuroendocrine tumor enhancement decrease after embolic injection accounting for a noticeable micro-embolic effect at the tumor capillary bed level. In another study, Dual-phase C-arm CBCT failed to demonstrate its utility in CRCLM detection because of a lack of accuracy (Sp.58% Se. 51%). We then orientated our search on intraprocedural evaluation of the parenchymal blood volume (PBV). In this work we showed that initial PBV was strongly correlated to tumor shrinkage after chemoembolization (rho=-0.8587; p=0.00001). Moreover the increase of 1 ml/1000ml in the blood volume of the tumor before TACE leads to a decrease of 0.54mm in the diameter change of the tumor. Finally, patients with a 1% decrease in PBV after DEBIRI had a 10% lower likelihood of achieving disease control (OR=0.9, 95% confidence interval CI: [0.81 -1]; p=0.0493). In conclusion of this 1st part, we anticipate the need for a dedicated clinical study that would evaluate prospectively the tumoral outcome based on PBV, with possible triage of responding/non responding tumors PBV toward alternative liver directed technique.

As the 2nd part, thanks to a strong collaboration with INSERM U 1147 team we developed a new intra-arterial treatment based on a suicide gene therapy consisting of an engineered gene coding for a mutated cytochrome responsible of the metabolism of a prodrug. This new approach uses transduced mesenchymal stem cells (MSCs) as carrier of the suicide gene. Our work consisted in in-vivo experiments in rabbit VX2 liver tumor and showed significant tumor volume difference between control and test groups associated to tumor necrosis significantly greater for rabbits receiving the GDEPT (78% of total tumor surface) compared with control animals (22% of total tumor surface (p=0.006). These findings deserve further confirmations which are under way (refine the strategy, document toxicities…).

In summary, in this thesis we show the value of C-arm CBCT in assessing CRCLM response to chemoembolization and enlarge the scope of intra-arterial therapy to suicide gene therapies which appear promising.