Gianluca Cassese

Roberto Ivan Troisi

Salah Khayat

François Quenet

Federico Tomassini

Fabrizio Panaro
email: f-panaro@chu-montpellier.fr

Boris Guiu

Francois Quenet

Liver venous deprivation versus associating liver partition and portal vein ligation for staged hepatectomy for colo-rectal liver metastases: a comparison of early and late kinetic growth rates, and perioperative and oncological outcomes

Keywords:

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Liver resection is the cornerstone of treatment of colorectal liver metastases (CRLM) with low morbidity and mortality in highly experienced centers [START_REF] Choti | Trends in long-term survival following liver resection for hepatic colorectal metastases[END_REF][START_REF] Rees | Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients[END_REF]. Surgical therapy can achieve 5-year survival rates higher than 50%, compared to only about 5% for patients medically treated [START_REF] House | Survival after hepatic resection for metastatic colorectal cancer: trends in outcomes for 1,600 patients during two decades at a single institution[END_REF]. Currently, the standard of surgical care is the Parenchymal-Sparing Hepatectomy (PSH). This strategy is a balance between the potential to achieve a complete tumor resection with curative intent (R0 resection) and the preservation of as much liver parenchyma as possible [START_REF] Deng | Parenchymal-sparing versus extended hepatectomy for colorectal liver metastases: a systematic review and meta-analysis[END_REF]. Considering that up to 75% of patients develop a recurrence within 2 years [START_REF] Lintoiu-Ursut | Recurrence after hepatic resection in colorectal cancer liver metastasis -Review article[END_REF], PSH showed to improve 5-years Overall Survival (OS) in case of recurrence (salvageability) [START_REF] Mise | Parenchymal-sparing hepatectomy in colorectal liver metastasis improves salvageability and survival[END_REF]. The key point of such strategy is the possibility to repeatedly perform the hepatectomy, that can offer long-term disease control without increasing perioperative mortality and morbidity rates [START_REF] Oba | Survival benefit of repeat resection of successive recurrences after the initial hepatic resection for colorectal liver metastases[END_REF][START_REF] Alvarez | Parenchymal-sparing liver surgery in patients with colorectal carcinoma liver metastases[END_REF]. On the other hand, huge tumors or close vascular relationships could indicate major hepatectomies. The major limitation for performing extensive hepatectomies is the risk of post-hepatectomy liver failure (PHLF), that still remains the main cause of mortality after major liver resection [START_REF] Pulitano | Preoperative assessment of postoperative liver function: the importance of residual liver volume[END_REF]. In order to minimize the risk of PHLF, a correct preoperative work-up is mandatory by investigating both the quantity and the quality of the Future Liver Remnant (FLR). It is generally accepted that the volume of the FLR (FLR-V) must be higher than 25% of the volume of a normal liver, 30% of a liver with steatosis or after several chemotherapy cycles, and at least of 40% in case of cholestasis or cirrhosis [START_REF] Cassese | Future remnant Liver optimization: preoperative assessment, volume augmentation procedures and management of PVE failure[END_REF].

With the intention of optimizing the FLR, several techniques have been developed to induce hypertrophy. Since its introduction in 1984, portal vein embolization (PVE) is generally considered the standard technique for inducing increase in FLR [START_REF] Makuuchi | Preoperative portal embolization to increase safety of major hepatectomy for hilar bile duct carcinoma: a preliminary report[END_REF][START_REF] Van Lienden | Portal vein embolization before liver resection: a systematic review[END_REF][START_REF] Glantzounis | The role of portal vein embolization in the surgical management of primary hepatobiliary cancers. A systematic review[END_REF]. However, PVE is hampered by an substantial failure rate, with a 20% unsuitable rate for resection due to either insufficient FLR growth or tumor progression during the post-PVE period, that may be sometimes very long (6 weeks or more) to gain enough residual volume [START_REF] Alvarez | Natural history of portal vein embolization before liver resection: a 23-year analysis of intention-to-treat results[END_REF]. To overcome these limitations, associating liver partition and portal vein ligation (ALPPS), with also several modified versions, has been recently introduced. These techniques showed greater and faster hypertrophy than PVE, but at the cost of significantly higher morbidity and mortality [START_REF] Kang | Hypertrophy and liver function in ALPPS: correlation with morbidity and mortality[END_REF][START_REF] Hasselgren | Health economic evaluation of patients with colorectal liver metastases randomized to ALPPS or TSH -analysis from the LIGRO trial[END_REF].

In the past 5 years, a new interventional radiological technique has emerged: the liver venous deprivation (LVD). It has been proposed as an alternative radiological technique to induce hypertrophy of the FRL [START_REF] Guiu | Simultaneous trans-hepatic portal and hepatic vein embolization before major hepatectomy: the liver venous deprivation technique[END_REF]. It consists in the simultaneous embolization of the portal vein and one or two hepatic veins, to increase the damage to the liver leading to an increase in hypertrophy of the contralateral parenchyma, with a reported kinetic growth rate of 16 ± 7 cc/day according to the first reports [START_REF] Guiu | Simultaneous trans-hepatic portal and hepatic vein embolization before major hepatectomy: the liver venous deprivation technique[END_REF][START_REF] Guiu | Extended liver venous deprivation before major hepatectomy induces marked and very rapid increase in future liver remnant function[END_REF][START_REF] Guiu | Liver venous deprivation: a bright future for liver metastases-but what about hepatocellular carcinoma?[END_REF]. Recently, Guiu et al. reported the FLR volume increase rate after LVD is reduced from the 37,8% of the first 7 days to the 2% of the last 7 ones [START_REF] Guiu | Liver venous deprivation versus portal vein embolization before major hepatectomy: future liver remnant volumetric and functional changes[END_REF]. Thus, it seems unfair to compare KGR between LVD and ALPPS only after a long interval, as previously published in the only comparison article [START_REF] Chebaro | Liver venous deprivation or associating liver partition and portal vein ligation for staged hepatectomy?[END_REF]. . Yet, preliminary data reporting great early volume and functional FLR increase call for comparing LVD and ALPPS during this early period with the ultimate goal to operate the patient as early as possible to prevent further risk of tumor progression [START_REF] Zhang | New evidence for liver venous deprivation: safety and feasibility for extended liver resections[END_REF].

The aim of this retrospective study is therefore to compare, in patients with CRLM, the early and late FLR kinetic growth rate (KGR), perioperative and oncological outcomes following ALLPS vs. LVD before major hepatectomies in patients with CRLM.

Materials and methods

Study design

This is a tri-institutional retrospective observational study, conducted according to the STROBE guidelines (Strengthening and reporting observational studies in epidemiology) of the EQUATOR network [START_REF] Cuschieri | The STROBE guidelines[END_REF]. Informed consent was obtained prior to both radiological procedures and surgery.

Consecutive patients who underwent LVD and ALPPS prior to right hemi-hepatectomy or extended right hepatectomy (tri-sectionectomy) for colorectal liver metastases between December 2015 and December 2019 in Montpellier University Hospital (for LVD procedures), Montpellier Institute of Cancerology and at the Division of HPB Minimally Invasive and Robotic Surgery at the Federico II University Hospital of Naples (for ALPPS procedures), were retrospectively analyzed. All cirrhotic patients were excluded, to reduce heterogeneity, since the role of LVD in cirrhotic patients is still not clear [START_REF] Guiu | Liver venous deprivation: a bright future for liver metastases-but what about hepatocellular carcinoma?[END_REF] The presence of an underlying liver disease was based on the histo-pathologic results on the surgical specimen.

The therapeutic indications for all patients were discussed in a multidisciplinary oncology meeting. The decision to perform a liver augmentation procedure was based on FLR volume. The procedure was performed when the expected FLR was <25% in normal liver, <30% in liver after chemotherapy, <40% in case of underlying liver disease (cholestasis or fatty degeneration).

An ALPPS procedure was indicated in case of right hemihepatectomy or tri-sectionectomy and a left lobe disease not susceptible of percutaneous therapy, and where LVD procedure was not available.

LVD procedure was performed at Montpellier University Hospital in case of right hemi-hepatectomy or tri-sectionectomy when the expected FLR volume and/or function were not sufficient, according to the center experience, within study protocols.

The follow-up policy after LVD and ALPPS was based on contrastenhanced computed tomography (CT) at one and three weeks following LVD, and at 8-12 days after ALPPS stage one.

The indication to completion surgery was based on volume data, according to underlying liver parenchyma and patient condition, according to the aforementioned criteria.

Radiological procedure

The LVD technique has been described in detail elsewhere [START_REF] Guiu | Simultaneous trans-hepatic portal and hepatic vein embolization before major hepatectomy: the liver venous deprivation technique[END_REF]. In summary, the right hepatic vein (and accessory when present) was cannulated under ultrasounds guidance. PVE was performed using the right transhepatic access. The right portal vessels were embolized using a compound of n-butyl cyanoacrylate and lipiodol (ratio 1: 6). The micro guidewire placed in the hepatic vein(s) was then used to deploy an oversized Amplatzer II vascular plug. The plug is placed at a distance of about 1.5 cm from the ostium of the inferior Vena Cava (IVC), in order to facilitate surgical ligation of the hepatic vein. Finally, all distal venous branches were embolized using a mixture of n-butyl cyanoacrylate and lipiodol (ratio 1: 6). Some patients also received an embolization of the middle hepatic vein, the so-called extended liver deprivation (eLVD) [START_REF] Guiu | Extended liver venous deprivation before major hepatectomy induces marked and very rapid increase in future liver remnant function[END_REF], depending on the baseline FLR and the type of surgery (resection of segment IV).

Surgical intervention

All patients included underwent a laparotomic right hemihepatectomy (segments 5-8), or extended right hepatectomy (S5-8, S4), with or without additional wedge resections in the left liver [START_REF]The brisbane 2000 terminology of liver anatomy and resections[END_REF]. An intraoperative ultrasound was performed to confirm the surgical feasibility of the procedure and to guide the resection. The right hepatic artery and portal vein were systematically ligated and dissected before the parenchymal section with an anterior approach. The hepatic veins were closed and divided with a vascular staple; the Amplatzer plug was not found to be an obstacle to the surgical ligation of the hepatic vein. If necessary, Pringle's maneuver with intermittent clamping and right hepatic vein control was performed.

For ALPPS procedure the first stage was carried out by treating eventual lesions of the lobe to preserve, by resection or thermal ablation. Then the transection line was performed by an ultrasound cavitation device and the right branch of the portal vein was ligated. The second stage consisted in the division of the ipsilateral artery and bile duct and hepatic vein in order to complete the hepatectomy [START_REF] Tomassini | Hemodynamic changes in ALPPS influence liver regeneration and function: results from a prospective study[END_REF].

CT-volumetry

Multiphase contrast-enhanced CT was carried out during preoperative assessment and after LVD or ALPPS stage 1. The portal-venous phase was used for volumetric assessment. The liver was outlined on an axial scan in a semi-automated fashion with manual adjustment to ensure that all extra-hepatic structures were excluded in order to calculate the total liver volume (TLV; cc). The tumor volume (TV; cc) and FLRV (cc) were calculated by manual delineation according to Couinaud's functional segmentation of the liver, as described by Heymsfield [START_REF] Lim | CT volumetry of the liver: where does it stand in clinical practice?[END_REF][START_REF] Heymsfield | Accurate measurement of liver, kidney, and spleen volume and mass by computerized axial tomography[END_REF]. For volumetric reconstructions in Montpellier University Hospital was used a PACS workstation (Carestream Vue PACS, Carestream Health), while in Naples the Synapse Vincent (Fuji film Medical, Tokyo, Japan), was currently used for complex PSH procedures and volume calculation.

The volume share of the FRL (FRL-V; %) was calculated using from the manual reconstruction, according to the formula described by Vauthey et al. [START_REF] Vauthey | Standardized measurement of the future liver remnant prior to extended liver resection: methodology and clinical associations[END_REF]: [START_REF] Choti | Trends in long-term survival following liver resection for hepatic colorectal metastases[END_REF] FLR-V share = FRL-V/(eTLV-TV) x 100, where eTLV: = -794.41 + 1,267.28 x body surface area.

Hypertrophy parameters

To compare the hypertrophy responses, the kinetic growth rates (KGR) for FRLV share were calculated by dividing the point differences of the FRLV share between preoperative and interstage assessments by the number of days after PVE or ALPPS stage 1. KGR was analyzed as both percentage growth per day as well as cc growth per day, as follows:

[3] KGR = (FLR interstage -FLR baseline )/days [START_REF] Rassam | Comparison of functional and volumetric increase of the future remnant liver and postoperative outcomes after portal vein embolization and complete or partial associating liver partition and portal vein ligation for staged hepatectomy (ALPPS)[END_REF]. The increase of the FRLV share was calculated using the following formula: [START_REF] Rees | Evaluation of long-term survival after hepatic resection for metastatic colorectal cancer: a multifactorial model of 929 patients[END_REF] FLR increase = FLR interstage -FLR baseline /FLR baseline x 100% [START_REF] Rassam | Comparison of Functional and Volumetric Increase of the Future Remnant Liver and Postoperative Outcomes after Portal Vein Embolization and Complete or Partial Associating Liver Partition and Portal Vein Ligation for Staged Hepatectomy (ALPPS)[END_REF].

Considering the long interval between radiological procedures and surgery, volumes were compared after the first post-procedural imaging and the last imaging made before surgery.

Post-operative follow-up

The Post-operative and follow-up data were analyzed. The Postoperative complications were classified according to the Clavien-Dindo classification [START_REF] Clavien | The Clavien-Dindo classification of surgical complications: five-year experience[END_REF]. PHLF, biliary leaks (BL) and post-hepatectomy hemorrhage (PHH) have been diagnosed and classified according to the International Study Group of Liver Surgery (ISGLS) [START_REF] Rahbari | Posthepatectomy liver failure: a definition and grading by the international study group of liver surgery (ISGLS)[END_REF][START_REF] Rahbari | Post-hepatectomy haemorrhage: a definition and grading by the international study group of liver surgery (ISGLS)[END_REF][START_REF] Koch | Bile leakage after hepatobiliary and pancreatic surgery: a definition and grading of severity by the International Study Group of Liver Surgery[END_REF]. Ascites was defined according to the International Ascites Club [START_REF] Moore | The management of ascites in cirrhosis: report on the consensus conference of the International Ascites Club[END_REF].

All patients were examined within one month after discharge from the surgery department and received clinical, biological and imaging evaluation every 3 months after discharge for the first two years according to oncological protocols. Then outpatients' controls were scheduled every 12 months if no relapse was found. In case of tumor recurrence, the case was re-examined by the Multi-Disciplinary Team (MDT) with the aim of carrying out curative treatment as much as possible.

Statistical analysis and endpoints

Continuous data are expressed as mean and standard deviation (SD) or median and interquartile range (IQR) depending on whether they have a normal distribution. Comparisons between groups were performed using student's T test or Wilcoxon's rank test, depending on the distribution of the variable.

Categorical data are expressed as associated frequencies and percentages. Comparisons between groups were performed using Pearson's Chi Square test.

The primary endpoint was KGR. Secondary outcomes were oncological outcomes and peri-operative results. All survival analyzes were performed using the Kaplan-Meier method to calculate the median and 95% CIs. Comparisons were made using the Log Rank test.

The Median follow-up was calculated using the inverse Kaplan-Meier methodology [START_REF] Betensky | Measures of follow-up in time-to-event studies: why provide them and what should they be?[END_REF]. Statistical analysis was conducted using SPSS software (version 26.0).

Results

Patients and tumor characteristics

Overall, data regarding 32 patients from 3 centers were collected. The overall median follow-up was 36 months (CI 95% 8-52). Patients and tumors' characteristics are available in Table 1. Median diameter of the biggest lesion was slightly larger in ALPPS group, 50 mm vs 40 mm in LVD group, but without statistical significance (p 0.06), as well as for the median number of tumor nodules (6 vs 4, p = 0.24). The percentage of patients treated by chemotherapy was equal (100%). In ALPPS cohort, 11 patients presented synchronous CRLM, while 4 had a metachronous presentation; in the LVD group they were 14 and 3, respectively (p = 0.52). Among the patient with synchronous presentation, 8 patients (72%) had undergone a colon first resection strategy in ALPPS group, compared to 4 in LVD group (29%, p = 0.02).

Volume analysis

Median pre-procedural tumor volumes were comparable between the two groups (83 cc in ALPPS vs 51 cc in LVD, p = 0.24). The mean preprocedural FLRV-share was 29,7 in the ALPPS group vs 29,3 in the LVD one (p = 0.53). All LVD patients underwent volumetric evaluation after 7 days and after 21 days. ALPPS patients underwent first imaging followup after a median of 9 days (IQR 5). Early KGR did not significantly differ between both techniques (0.8% per day vs 0.3% per day, p = 0.70; 23 cc/day vs 26 cc/day, p = 0.31). On the contrary, late KGR was greater after ALLPS (20 cc/day vs 10 cc/day p = 0.02), with a lower difference in percentages (0.6% per day vs 0.2% per day, p = 0.27). The preoperative mean FLRV-share increase was comparable in the two groups

Peri-operative outcomes

The results were stratified according to liver volume augmentation procedure (LVD and ALPPS stage 1, Table 2) and surgical procedures (completion surgery and ALPPS stage I, Table 3) and surgical procedures (completion surgery and ALPPS stage I and II, Table 4). There was a significant difference in Length of Stay (LoS) in favor of LVD (3 vs 9 days, p 0.0001). After ALPPS stage 1, two patients were not discharged, being hospitalized until second stage. When comparing first procedures, intraprocedural blood loss and post-operative major complications were in favor of LVD procedure. The only shown difference in favor of ALPPS stage 1 was the number of left lobe treatment, due to an higher number of bilobar disease in ALPPS group. Surgical results couldn't be compared between first procedures, since LVD is a radiological intervention while ALPPS stage I is a complex surgery. Thus, surgical outcomes were compared also between LVD completion surgery and ALPPS stage I, showing a lower operation duration in the completion surgery (p 0.05), with similar blood losses and post-operative complications (Table 4a).

The successful resection rate was higher for LVD group (p = 0.27), where all patients underwent completion surgery, while one patient in ALPPS group failed because of complications after stage 1. Four patients received a right hepatectomy as ALPPS stage 2 surgery and 11 received a right tri-sectionectomy, whereas in LVD group 7 and 10 underwent these two procedures, respectively. The median delay before the hepatectomies was significantly inferior in ALPPS group 14 ± 13 vs 39 ± 31 (p 0.002). The median LoS after the completion surgery was similar in the compared groups, as well as the total incidence of postoperative complications (6 vs 9, p = 0.50). The median intraoperative estimated blood losses were lower in ALPPS groups (150 ± 600 vs 600 ± 1000, p = 0.004), as well as post operative hemorrhages (0 VS 7, P = 0.005), even if they were all grade A. Seven patients received intraoperative blood transfusions in LVD group, while only 2 during ALPPS stage 2 procedures (p = 0.05).

One patient died in the firsts 90 postoperative days in ALPPS group.

Survival analysis

In the ALPPS group 13 patients died during follow-up, with one death linked to stage 1 procedure. In the LVD group, 6 patients died during the follow-up, without deaths related to the procedure. The OS at 1 year was 72,7% in the ALPSS group (CI 95% 59-85) and 81,3% in LVD group (CI 95% 72-90); 3-ys OS was 27,4% (CI 95% 18-36) and 54,7% (CI 95% 46-63) respectively, without statistically significative difference (p 0.10) (Fig. 1). The Median DFS was comparable between both techniques: 6.1 months in the ALPPS cohort and 5.9 for LVD patients; p 0.66) (Fig. 1). During follow-up, in the ALPPS group, 15 patients developed liver recurrence (100%), while in LVD group 9 patients (52.9%) (p = 0.002). Among LVD patients 6 developed pulmonary metastasis and 4 patients a peritoneal carcinomatosis. In the second group, a pulmonary metastatic progression occurred in two patients, two patients developed a lombo-aortic lymph node metastasis, one patient a peritoneal carcinomatosis.

Discussion

To our knowledge this is the first study that compares early and late KGR after ALPPS and LVD for colorectal liver metastases, and that deals with the oncological outcomes of the two techniques, with a median follow-up of 36 months.

Previously, Chebaro et al. published a multicentric comparison between these two procedures, including different indications [START_REF] Chebaro | Liver venous deprivation or associating liver partition and portal vein ligation for staged hepatectomy?[END_REF]. This previous work was not focused on volumetric results, since it evaluated KGR at different and, thus, non-comparable time points, whereas previous literature showed that LVD presents very different kinetics during the post-procedural weeks [START_REF] Guiu | Liver venous deprivation versus portal vein embolization before major hepatectomy: future liver remnant volumetric and functional changes[END_REF]. Herein, early KGR did not significantly differed between both groups. Indeed, Guiu et al.previously showed both higher volumetric and functional gain in the first days after LVD procedure [START_REF] Guiu | Extended liver venous deprivation before major hepatectomy induces marked and very rapid increase in future liver remnant function[END_REF]. In the same study, the majority of FLR functional growth after PVE was also obtained as early as 7 days after preparation in line with previous literature that had shown that the volumetric gain underestimates the functional gain [START_REF] Sparrelid | Dynamic evaluation of liver volume and function in associating liver partition and portal vein ligation for staged hepatectomy[END_REF][START_REF] Olthof | Hepatobiliary scintigraphy to evaluate liver function in associating liver partition and portal vein ligation for staged hepatectomy: liver volume overestimates liver function[END_REF]. In a recent paper comparing LVD to PVE, it has been claimed that LVD could trigger fast FLR regeneration and be considered as a "radiological ALPPS", but unfair comparison of KGR have been reported so far since no early volumetric data were overlooked [START_REF] Laurent | Radiological simultaneous portohepatic vein embolization (RASPE) before major hepatectomy: a better way to optimize liver hypertrophy compared to portal vein embolization[END_REF].

Another substantial difference with the previously published multicentric comparative study, was the high heterogeneity in LVD technique, with many patients that received only proximal occlusion, while our work deals with a standardized procedure, providing a careful proximal and distal embolization in order to limit veno-venous collateral development in the resected liver in order to obtain a more effective growth. Such a difference could minimize heterogeneity in the results and explain another key difference in our outcomes: the 100% successful rate of LVD procedures, intended as the percentage of treated patient who were able to undergo liver surgery. In ALPPS group, one patient failed to undergo the hepatectomy because of a left hepatic vein thrombosis with hepatic decompensation and ascites, with postoperative complications that lead to death after 2 months. The result is coherent with previous experiences on ALPPS [START_REF] Lang | Associated liver partition and portal vein ligation for staged hepatectomy (ALPPS) registry: what have we learned?[END_REF], that could let imagine an higher part of ALPPS treated patients failing to undergo the planned intervention, even if very good results were specifically reported in CRLM patients [START_REF] Petrowsky | First long-term oncologic results of the ALPPS procedure in a large cohort of patients with colorectal liver metastases[END_REF]. Such a high successful rate for LVD must be confirmed in further and wider studies, but it could be ascribed also to the low complication rate of the radiological procedure, as already widely reported in the literature, which may significantly reduce the risk of drop-out [START_REF] Panaro | Perioperative impact of liver venous deprivation compared with portal venous embolization in patients undergoing right hepatectomy: preliminary results from the pioneer center[END_REF]. Indeed, the safety of the volume augmentation procedure appears to the authors to be a key issue in the comparison (the patients subjected to only one surgery), as well as the potential reduction of direct and indirect costs to be incurred for the health system. Thus, a further cost-effectiveness analysis will be also desirable in the future.

The median delay between LVD and completion of surgery was 39 days. This delay can be regarded as particularly long for a technique able to provide fast FLR increase, but so far LVD has been regarded as a PVElike technique: the longer the waiting-time the greater the FLR augmentation. Thus, the introduction of this novel technique has been initially linked to a protocol aiming to 4-6 week interval before completion surgery, similar to PVE, the current standard of care. As suggested in a previous report, all the previously published results call for a strong reduction of this delay, and for applying an ALPPS-like strategy [START_REF] Guiu | Liver venous deprivation versus portal vein embolization before major hepatectomy: future liver remnant volumetric and functional changes[END_REF]. Further studies and efforts should focus on interval time reduction when applying LVD procedures, thanks to the excellent patients' tolerance and to the reported efficacy in terms of KGR [START_REF] Zhang | New evidence for liver venous deprivation: safety and feasibility for extended liver resections[END_REF][START_REF] Guiu | Liver venous deprivation: a bright future for liver metastases-but what about hepatocellular carcinoma?[END_REF].

Interestingly, intraoperative bleedings and transfusions, and posthepatectomy hemorrhage (even if all grade A) were higher in the LVD group. These results could be partially explained by the significative venous congestion developed after LVD, together with the presence of veno-venous shunts after the hepatic vein occlusion [START_REF] Panaro | Perioperative impact of liver venous deprivation compared with portal venous embolization in patients undergoing right hepatectomy: preliminary results from the pioneer center[END_REF], potentially leading to such an increase in the risk of intraoperative bleeding and post-operative hemorrhage. These hypotheses remain to be verified.

The fear of tumor progression while waiting after volume augmentation procedures is still a hot topic [START_REF] Hoekstra | Tumor progression after preoperative portal vein embolization[END_REF][START_REF] Pamecha | Effect of portal vein embolisation on the growth rate of colorectal liver metastases[END_REF][START_REF] Kokudo | Proliferative activity of intrahepatic colorectal metastases after preoperative hemihepatic portal vein embolization[END_REF]. One of the most accredited theories considers increased arterial blood flow as the main possible cause of tumor progression, and this is because colorectal metastases are mainly nourished by it [START_REF] Nagino | Immediate increase in arterial blood flow in embolized hepatic segments after portal vein embolization: CT demonstration[END_REF][START_REF] Archer | Vascularization of small liver metastases[END_REF]. However, the enrolled patients didn't experience a tumor progression after LVD before the completion surgery, similarly to the ALPPS cohort. The 1-yr and 3-ys survival and disease-free recurrence rates were not statistically different between the two groups, despite the limiting factors of the study design and the small sample size. As regard to the comparison between the oncological outcomes, we must also underline how ALPPS patients in our study had a slightly larger median tumor size. However the difference didn't reach the statistical significance, and could be ascribed to the tendency to carry out a more aggressive and more morbid surgical procedure in cases with more advanced disease. Since we are in an initial phase of diffusion of the LVD procedure, it is almost impossible to have two cohorts perfectly equal from this point of view, if not by setting up a prospective randomized study, that could be stimulated precisely by our results.

A higher proportion of patients underwent a clearance of the left lobe after ALPPS during the first stage procedure: 11 patients received an intra-procedural treatment of the left lobe (9 additional resections and 2 thermal ablations). It must be underlined that LVD also gives the opportunity to percutaneously treat the left lobe. Simply, ALPPS patients had higher percentage of bilobar disease, indicating once again a different strategy for a more advanced disease. These data, together with our results, confirm that the two techniques are basically not mutually exclusive but could be proposed by tailoring them specifically to case by case.

In our experience and according to the results of this study we believe that LVD could replace PVE in the future, both because it has shown an high safety and patients tolerance, and because it has excellent early KGR. On these assumptions is based the ongoing randomized trial of ultrafast FRL preparation comparing LVD and PVE, namely the HYPER-LIV 01. On the other hand, ALPPS can be a viable salvage option, as already proposed, after PVE failure [START_REF] Lopez-Lopez | ALPPS for hepatocarcinoma under cirrhosis: a feasible alternative to portal vein embolization[END_REF], or in the case of a patient requiring extensive left lobe tumor clearance not feasible by percutaneous technique. Once more, the correct patient and procedure selection is the way.

This study has several limitations. Firstly, its retrospective nature and the relatively small sample size, can't lead to definitive conclusions. Because of the new introduction of LVD technique, despite the multicenter nature of the study, the small sample size could also have decreased the statistical significance of our results. However, our aim was to compare for the first time the different KGR after the two procedure and stimulate new further studies that would take into account the different KGR growth trends. The difference in the disease course among the two cohorts according to the previously published literature, is another limitation [START_REF] Sasaki | The tumor burden score: a new "Metroticket" prognostic tool for colorectal liver metastases based on tumor size and number of tumors[END_REF]. It could explain the approximately 28% of lower overall survival and the higher percentage of liver-only recurrence in the ALPPS group. However, since LVD is still in the initial phase of diffusion and ALPPS is not a risk-free procedure, we are convinced that our paper should be read as a promising first comparison of the volumetric data of the new radiological procedure with respect to the "surgical competitor", also in order to understand the different growth kinetics and the perioperative and long term outcomes. At the same time, there are also some strengths in this study. Montpellier University Hospital is the pioneer of LVD procedure, with a consolidated technical experience possibly leading to better outcomes and a lower learning curve effect. Initial data on the differences between ALPPS and LVD were obtained from centers with a lot of experience in ALPPS and little in LVD, furthermore mixing for different indications. Herein we have considered only CRLM patients, therefore without taking into consideration patients with cholestatic or cirrhotic livers, as the place of LVD and ALPPS in the management of cirrhotic patients remains unclear [START_REF] Guiu | Liver venous deprivation: a bright future for liver metastases-but what about hepatocellular carcinoma?[END_REF][START_REF] Enne | ALPPS as a salvage procedure after insufficient future liver remnant hypertrophy following portal vein occlusion[END_REF]. Similarly, the good results of ALPPS in terms of safety and survival outcomes are in line with reports from experienced centers, that propose CRLM as the best indication [START_REF] Petrowsky | First long-term oncologic results of the ALPPS procedure in a large cohort of patients with colorectal liver metastases[END_REF].

In conclusion, despite its retrospective design and small sample sizes, this study shows very interesting results that may guide additional prospective studies, as well as help clinical practice for CRLM patients. LVD and ALPPS showed comparable daily KGR in the early period when performed in centers with high expertise and a perfected procedure. LVD allows a slightly higher successful resection rate than ALPPS, with a significantly lower LoS and severe postoperative complications. Additional randomized prospective studies are thus needed to adequately confirm such results.

Research or publication funding

None.

Declaration of competing interest

Nothing to declare.

Fig. 1 .

 1 Fig. 1. a) Overall Survival and b) Disease Free Survival for ALPPS and LVD group.

Table 1

 1 Patients and tumor characteristics. SD: standard deviations; BMI: body mass index; IQR: inter-quartile range; RECIST: response evaluation criteria in solid tumors; CI: confidence intervals. Data about volume analysis are shown in Table2.

		ALPPS	LVD	p-
				value
	Age, mean (SD)	59.2	58.7	0.91
	Sex (male, %)	7 (35)	13 (65)	0.13
	BMI, mean (SD)	26.4 (3.9)	25.9 (3.4)	0.91
	Tumor size, median in mm	50 (40-101.5)	40 (21-52.5)	0.06
	(IQR 25-75 )
	Tumor nodules, median	6 (5-10)	4 (1-4,5)	0.24
	(IQR 25-75 )
	Mutated K-Ras, n (%)	6 (40)	6 (35)	0.54
	Liver metastasis	11 (73)	14 (83)	0.53
	presentation,n (%)	-3 (27)	-10 (71)	0.02
	Synchronous	-8 (72)	-4 (29)
	-Liver first approach	4 (27)	3 (17)
	-Colon first approach
	Metachronous
	Neoadjuvant	15 (100)	17 (100)	ns
	Chemotherapy, n (%)
	Chemotherapy cycles	12 (8-15)	6 (4-14)	0.13
	before liver surgery,
	median (IQRs 25-75 )
	Preoperative	FOLFOX 1 (6)	FOLFOX 1 (6)	0.99
	chemotherapy schemes,	FOLFIRI 1 (6)	FOLFIRI 3 (17.5)	0.60
	n (%)	FOLFIRI +	FOLFIRI +	0.60
		CETUXIMAB 1 (6)	CETUXIMAB 3	0.46
		FOLFOX +	(17.5)	0.46
		CETUXIMAB 1 (6)	FOLFOX +	0.33
		XELOX +	CETUXIMAB 0 (0)	0.22
		BEVACIZUMAB 1	XELOX +	0.38
		(6)	BEVACIZUMAB	0.32
		FOLFIRINOX 1 (6)	0 (0)
		FOLFOXIRI +	FOLFIRINOX 4 (24)
		BEVACIZUMAB	FOLFOXIRI +
		0 (0)	BEVACIZUMAB 3
		FOLFOX +	(17.5)
		BEVACIZUMAB 4	FOLFOX +
		(24)	BEVACIZUMAB 2
		FOLFIRI +	(12)
		BEVACIZUMAB 4	FOLFIRI +
		(24)	BEVACIZUMAB 1
			(6)
	Response to preoperative	4	5	0.86
	chemotherapy according	11	12
	to RECIST criteria, n (%)
	Partial Response
	Stable Disease
	Follow-up, median (CI	38 (31-43)	32 (16-46)	0.07
	95%)

Table 2

 2 Volumetric analysis. SD: standard deviations; IQR: inter-quartile range; TLV: Total liver volume; FLRV: Future liver remnant volume; KGR: kinetic growth rate.

		ALPPS	LVD	p-
				value
	Pre-procedural Tumor Volume, Median	83	51	0.24
	(IQR 25-75 )	(44-186)	(35-121.5)
	Pre-procedural FLRV-share %, mean (SD)	29.7 (16)	29.3 (6.8)	0.53
	Early Evaluation
	Delay procedure-first imaging in days,	9 (5)	7 (0)
	median (IQR)
	TLV gain in cc, mean (SD)	308 (298)	290 (261)	0.83
	FLRV share %, mean (SD)	40 (13)	34 (8)	0.11
	FLRV increase %, mean (SD)	62 (36)	40 (38)	0.09
	KGR %, mean (SD)	0.8 (0,9)	0.3 (1.7)	0.31
	KGR in cc, mean (SD)	23 (15)	26 (25)	0.70
	Late Evaluation
	Delay procedure-last imaging in days,	9 (9)	21 (0)
	median (IQR)
	TLV gain in cc, mean (SD)	306 (298)	183 (271)	0.24
	FLRV share %, mean (SD)	41 (13)	39 (9)	0.69
	FLRV increase %, mean (SD)	60 (34)	49 (29)	0.32
	KGR %, mean (SD)	0.6 (0.9)	0.2 (0.2)	0.21
	KGR in cc, mean (SD)	20 (15)	10 (8)	0.02

Table 3

 3 First liver volume augmentation procedures. Comparison of the outcomes between ALPPS stage 1 vs LVD. IQR: Inter-quartile range.

	ALPPS (n =	LVD (n =	p-value
	17)	15)


Stephen Ferrière

Peter J Lenting
email: peter.lenting@inserm.fr

Antibodies in the treatment of Haemophilia A, a biochemical perspective

Keywords: Hemophilia, Factor VIII, antibody

Replacement therapy has been proven effective in the management of bleedings in hemophilia A. Nevertheless, this approach comes with a number of shortcomings, like the need for frequent intravenous infusions and the development of neutralizing antibodies in 20-30% of the patients with severe hemophilia A replacement. This has led to the development of novel strategies to expand the spectrum of treatment options, some of which are based on antibody-technology. These include a bispecific antibody that bridges enzyme Factor IXa and substrate Factor X, monoclonal antibodies that block the function of tissue factor pathway inhibitor, and a factor VIII-nanobody fusion protein with strongly enhanced von Willebrand factor binding. In this review, functional and mechanistical considerations on the use of these antibody variants will be discussed.

Introduction

The activated derivative of factor VIII (FVIIIa) participates in the coagulation cascade as a non-enzymatic cofactor for activated factor IX (FIXa) in the generation of activated factor X (FXa). [START_REF] Lenting | The life cycle of coagulation factor VIII in view of its structure and function[END_REF] Its physiological relevance in this enzymatic reaction is illustrated by the notion that the functional absence of FVIII predisposes to a severe bleeding diathesis, known as hemophilia A.

For decades, the treatment of hemophilia A has mainly consisted of replacement therapy using FVIII concentrates of varying purity. As of today highly purified plasma-derived or recombinant FVIII concentrates are broadly used for the prophylactic treatment of hemophilia A, thereby not only markedly reducing spontaneous bleeding events but also enabling home therapy. [START_REF] Nilsson | Twenty-five years' experience of prophylactic treatment in severe haemophilia A and B[END_REF][START_REF] Manco-Johnson | Prophylaxis versus episodic treatment to prevent joint disease in boys with severe hemophilia[END_REF] Although effective, replacement therapy comes with a number of disadvantages, like the need for frequent intravenous applications and the development of anti-FVIII antibodies in 20-30% of the patients with severe haemophilia A. [START_REF] Gouw | Factor VIII products and inhibitor development in severe hemophilia A[END_REF][START_REF] Calvez | Recombinant factor VIII products and inhibitor development in previously untreated boys with severe hemophilia A[END_REF][START_REF] Peyvandi | A Randomized Trial of Factor VIII and Neutralizing Antibodies in Hemophilia A[END_REF] It is not surprising therefore that a search for alternative strategies to improve hemophilia A treatment has been ongoing. In this review, we will focus on antibody-based treatment options, and three examples will be discussed. First, a bispecific antibody (Emicizumab) that combines FIXa and FX in order to enhance FXa generation; second, antibodies that block the function of tissue factor pathway inhibitor (TFPI), thereby promoting activity of the tissue factor/factor VIIa (TF/FVIIa) and prothrombinase complex; and third, a FVIII-nanobody fusion protein that displays prolonged survival and reduced immunogenicity. [START_REF] Peterson | Targeting TFPI for hemophilia treatment[END_REF][START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF][START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation[END_REF] Importantly, this review will limit itself to the functional and mechanistic aspects of the antibodies, and will not discuss clinical efficacy.

Bispecific antibody Emicizumab: comparison with FVIII

Emicizumab (also known as ACE910 or Hemlibra) is a bispecific antibody that interacts with FIX/FIXa and FX/FXa. [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF] The rationale behind this antibody is that it brings the enzyme FIXa and its substrate FX in close proximity, favoring conversion of FX into FXa. To some extent, this antibody resembles FVIIIa, the cofactor within the tenase complex that is also capable of simultaneously interacting with FIXa and FX. Nevertheless, Emicizumab and FVIII are very different proteins in terms of structure and regulation, and consequently they will act differently during coagulation. In order to understand these differences, it is perhaps convenient to first provide a reminder of how FVIII actually functions. We have recently described the mode of action of FVIII in great detail 10 , and will therefore only provide a summary of the key points in this regard:

1: On/off switch: FVIII circulates as a pre-cofactor that requires activation by thrombin and/or FXa before it becomes an active cofactor. However, because FVIIIa is a very labile protein, it will lose its function by just falling apart within minutes following activation. 11 Also, FVIIIa is an efficient substrate for activated Protein C, which is able to inactivate FVIIIa via site-specific proteolysis. [START_REF] Eaton | Proteolytic processing of human factor VIII. Correlation of specific cleavages by thrombin, factor Xa, and activated protein C with activation and inactivation of factor VIII coagulant activity[END_REF] FVIIIa activity is thus regulated by an on-and off switch.

2: Specific cofactor functions: FVIIIa's cofactor function consists of different explicit tasks.

FVIIIa initiates formation of the tenase complex by recruiting FIXa to the phospholipid surface, limiting the movement of the proteins to a two-dimensional space. [START_REF] Duffy | Binding of factor VIIIa and factor VIII to factor IXa on phospholipid vesicles[END_REF] While interacting with FIXa, FVIIIa stabilizes the protease domain of FIXa. [START_REF] Fay | The A2 subunit of factor VIIIa modulates the active site of factor IXa[END_REF] This interaction has two main functions: to reduce the flexible movements of the surface loops that surround the active center and to position the active center at the appropriate height from the phospholipid surface. Both functions are crucial to optimize the catalytic capacity of FIXa. Once in place, the complex is ready to receive FX. By interacting with FVIIIa, the activation peptide of FX is optimally aligned to the active site of FIXa, which further promotes FXa generation. [START_REF] Lapan | Localization of a factor X interactive site in the A1 subunit of factor VIIIa[END_REF] Thus, multiple aspects of FVIIIa cofactor function define its enhancing potential within the tenase complex.

3: Specificity: Although FIXa binds equally well to FVIII or FVIIIa (with this interaction only being limited by the presence of von WiIlebrand factor (VWF)), the opposite is untrue. FVIIIa (or FVIII) is unable to interact with the FIX zymogen. [START_REF] Lenting | Identification of a binding site for blood coagulation factor IXa on the light chain of human factor VIII[END_REF] This restricts the formation of the complex to one between the enzyme and cofactor, rather than between the zymogen and cofactor. Furthermore, FVIIIa is able to interact with FX, while less so with FXa. [START_REF] Lapan | Localization of a factor X interactive site in the A1 subunit of factor VIIIa[END_REF] Indeed, the low affinity for FXa compared to FX allows the release of FXa from the FIXa/FVIIIa complex, making the tenase complex available for a next substrate molecule. Hence, FVIIIa displays specificity towards both enzyme and substrate.

4: Stimulating activity: The magnitude by which FVIIIa enhances the catalytic activity of FIXa is often determined in assays using purified components. Accordingly, variables like phospholipid concentration and composition, as well as concentrations of the proteins are determinant for the catalytic efficiency that is measured. In general, FVIIIa enhances FIXa activity by 1,000-to 1 million-fold.

5: Limiting factor within the tenase complex:

To establish what is the limiting factor within the tenase complex, it is of relevance to consider the respective protein concentrations. It is obvious that FVIII concentrations (0.3-1 nM) are considerably lower than those of FIX (90 nM) or FX (135 nM). The actual concentrations of FVIIIa and FIXa during in vivo coagulation are difficult to establish, but even if we consider that 50% of FVIII is activated versus 5% of FIX, this would still mean 9-30-fold less FVIIIa than FIXa (0.15-0.5 nM versus 4.5 nM). It is thus fair to assume that FVIIIa is the limiting factor within the tenase complex.

In view of this complex mechanism of action and regulation of FVIIIa, it is obvious that the characteristics of the antibody-based protein Emicizumab will be rather different. A first important difference is that Emicizumab has similar affinity for FIX versus FIXa and for FX versus FXa. [START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF] This indicates that the specificity of FVIIIa towards FIXa and FX is no longer present in the bispecific antibody. It is therefore possible that the following ternary complexes will be formed in the circulation: Emicizumab/FIX/FX, Emicizumab/FIXa/FX (which is the one that is relevant for FXa generation), Emicizumab/FIX/FXa and Emicizumab/FIXa/FXa. How much of each complex is being formed is very much dependent on the respective concentrations of the individual constituents. Based on concentrations and respective affinities, it is possible to calculate how much of each complex is formed. In the normal circulation and at therapeutic Emicizumab concentrations of 55 µg/ml (370 nM), the Emicizumab/FIX/FX complex will be the most abundant, with about 0.8 nM ternary complex. [START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF] Interestingly, in normal individuals (and there is no reason to assume that this will be different in haemophilia A patients), there is about 120 pM of FIXa that is constitutively present in the circulation. [START_REF] Bauer | Factor IX is activated in vivo by the tissue factor mechanism[END_REF] Since Emicizumab does not make any distinction between FIX and FIXa, only a small portion of these 120 pM of FIXa will bind to Emicizumab. Indeed, the ternary complex relevant for FXa generation (Emicizumab/FIXa/FX) present under basal conditions in these treated patients can be calculated to be about 2 pM. Of note, since Emicizumab is not regulated by an on-or off-switch, this may indicate that there is a constitutive background activity of this Emicizumab/FIXa/FX complex. It is not to be excluded that such low background activity may act already during small subclinical bleeds that do not provoke a full trigger of the coagulation cascade.

An important question is of course: how efficiently does Emicizumab enhance FIXa activity in comparison to FVIIIa? The antibody configuration of Emicizumab will prevent it from capturing FIXa and FX to the phospholipid surface (something that does not exclude the activation reaction to be still phospholipid-dependent). Furthermore, with the Emicizumab binding site being located in the EGF1-like domain of FIXa, the bispecific antibody is also unlikely to modify the flexibility and position of the FIXa protease domain to a significant extent. It is therefore difficult to expect that Emicizumab to be as efficient as FVIIIa in stimulating FX activation. Experiments using purified components showed that Emicizumab was about 10-fold less efficient in enhancing the catalytic efficiency by which FIXa activates FX. [START_REF] Kitazawa | A bispecific antibody to factors IXa and X restores factor VIII hemostatic activity in a hemophilia A model[END_REF][START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF]

Emicizumab: monitoring using activity assays

To monitor Emicizumab-based therapy in the patients, two issues are of relevance. First, one might want to verify levels of Emicizumab during therapy. In current clinical trials, a specific ELISA-system using anti-idiotype antibodies has been applied. [START_REF] Uchida | A first-in-human phase 1 study of ACE910, a novel factor VIII-mimetic bispecific antibody, in healthy subjects[END_REF] Alternatively, one could also employ a chromogenic FVIII activity assay, with the necessity of such an assay to contain human FIXa and FX (for example Biophen FVIII:C by Hyphen Biomed) and uses Emicizumab as standard. The main difficulty is to find a so-called FVIII-equivalence using activity assays. Several assays are available to measure FVIII activity, like the aPTT, thrombin generation assays (initiated by TF or factor XIa) or clot waveform analysis. Each of these assays are based on FVIII being the limiting factor, while the amount of FIXa that is generated in these tests is quite variable from one test to the other, and even depends on the activating reagent that used. Knowing that Emicizumab is present in large excess over all other relevant components (FIXa and FX) and is always in its "on"-mode, it will thus be the amount of FIXa present that will determine the activity of Emicizumab. With the FIXa concentrations being different from one assay to the other, it will be complicated to assign a FVIII-like value to a certain Emicizumab concentration. Care should therefore be taken to extrapolate findings concerning Emicizumab in laboratory assays to the in vivo hemostatic potential of this molecule. We would like to emphasize that this does not mean that Emicizumab activity in vivo is per definition lower than what will be measured in laboratory assays. It cannot be excluded that Emicizumab has the intrinsic capacity to modulate the extent of FIXa formation in a way that goes unnoticed in the regular activity assays. For example, it is known that FXa is able to generate the FIX activation intermediate FIXa, which is a much better substrate than FIX for the TF/FVIIa complex. Akin to Emicizumab promoting formation of FXa by FIX, it is theoretically possible that it also promotes FXa-mediated FIXa formation, which in the presence of TF/FVIIa is then readily converted into FIXa. This pathway could eventually explain why Emicizumab seems to have higher activity in TF-based thrombin generation assays compared to FXIa-based thrombin generation assays.

Emicizumab in combination with other procoagulant agents

Under certain conditions, Emicizumab has been or will be used in combination with other procoagulant proteins, such as FVIII concentrates or FVIII-bypassing agents like FEIBA or FVIIa. It is of interest to consider how the co-use of such products impacts coagulation. In case of FEIBA use, several issues need to be considered. First, FEIBA consists of many different proteins, including high concentrations of prothrombin, FX and FIX. [START_REF] Varadi | Pro-and anticoagulant factors facilitate thrombin generation and balance the haemostatic response to FEIBA((R)) in prophylactic therapy[END_REF] By adding FIX and FX to the circulation, one automatically increases levels of the ternary complexes. A dose of 100 U/kg of FEIBA will more than double the concentration of the ternary Emicizumab/FIX/FX complex in the circulation, assuming a therapeutic Emicizumab level of 370 nM. [START_REF] Kitazawa | Factor VIIIa-mimetic cofactor activity of a bispecific antibody to factors IX/IXa and X/Xa, emicizumab, depends on its ability to bridge the antigens[END_REF] Furthermore, FEIBA contains considerable amounts of FIXa and a dose of 100 U/kg may ultimately result in circulating FIXa levels that may reach 1 nM. [START_REF] Elodi | Activation of clotting factors in prothrombin complex concentrates as demonstrated by clotting assays for factors IXa and Xa[END_REF][START_REF] Hultin | Activated clotting factors in factor IX concentrates[END_REF] Since Emicizumab makes no distinction between FIX and FIXa, and doesn't require an on-switch to become active, the presence of FIXa may directly be used to trigger FXa generation: the ternary, FXa-generating Emicizumab/FIXa/FX complex will rise to a concentration of … pM.

With regard to the use of FVIIa, this will be rather different. The use of FVIIa will not change the basal FIX or FX levels, suggesting ternary complex formation remains unaltered.

However, FVIIa will cause the generation of both FIXa and FXa, thereby increasing the participation of these proteins in the complexes with Emicizumab. Primate studies using a dose of 50 µg/kg FVIIa revealed that such a dose doubles the FIXa concentration in the circulation from about 120 to 240 pM. [START_REF] Ten Cate | The activation of factor X and prothrombin by recombinant factor VIIa in vivo is mediated by tissue factor[END_REF] This suggests that that FVIIa may contribute to an increase in active Emicizumab/FIXa/FX complexes, albeit probably to a lesser extent compared to Feiba.

As for the combination with FVIII, two distinct situations should be considered. First, in patients having no anti-FVIII antibodies receiving regular doses of FVIII concentrates (25-50 U/kg), FVIII will be present together with Emicizumab and compete for the interaction with FIXa. Since FVIIIa displays much higher affinity for FIXa than Emicizumab (2-15 nM versus 1.5 µM), and will not be diluted to non-activated FIX zymogen, it seems likely that any FIXa present will first chose to partner with FVIIIa. However, also in these situations, FIXa will be present in excess over FVIIIa. The remaining FIXa thus remains available to interact with Emicizumab. Dependent on the excess of FIXa, there might therefore be the formation of ternary complexes of Emicizumab/FIXa/FX in addition to the tenase complex. However, these ternary complexes are 11-fold less efficient than the tenase complex in generating FXa. Will this be different under conditions where patients receive immune-tolerance therapy? Such patients have inhibitory antibodies against FVIII, and receive high doses of FVIII on a daily basis. Dependent on the nature of the inhibitory antibodies and their concentrations, they may prevent FVIII from interacting with FIXa. As such, FIXa remains available for binding to Emicizumab. Given the polyclonal character of the antibodies in most patients, it is probable that FVIII will not be interacting with FIXa in these inhibitor patients.

Emicizumab will therefore be able to assist FIXa during immune tolerance therapy.

Monoclonal antibodies targeting TFPI

TFPI is a Kunitz-type inhibitor, known to interfere with the initial phase of coagulation. [START_REF] Mast | Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein[END_REF][START_REF] Maroney | New insights into the biology of tissue factor pathway inhibitor[END_REF] Two isoforms are present in humans: TFPIa, which contains three Kunitz domains and a basic C-terminal region, and TFPIb in which the third Kunitz domain and the basic region are replaced by a glycosyl phosphatidyl-inositol-binding sequence. The mode of action of TFPI is perhaps less straightforward than many seem to appreciate. [START_REF] Mast | Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein[END_REF][START_REF] Baugh | Regulation of extrinsic pathway factor Xa formation by tissue factor pathway inhibitor[END_REF] At low concentrations, its Kunitz-2 domain interacts loosely with FXa, before isomerizing into a tight complex. This complex is then able to rapidly inhibit the activity of the TF/FVIIa complex, via interactions between the Kunitz-1 domain of TFPI and the FVIIa active site. TFPI alone is a less effective inhibitor of TF/FVIIa compared to FXa/TFPI. The fact that TFPI plays an important role in the regulation of the initial phase of coagulation has sparked interest related to its use as a target in the treatment of haemophilia. [START_REF] Peterson | Targeting TFPI for hemophilia treatment[END_REF] The rationale would be that by blocking TFPI, more FXa and FIXa will be generated by the FVIIa/TF complex, which in turn will lead to increased thrombin generation and subsequent fibrin deposition and platelet activation. Preliminary studies indeed revealed that anti-TFPI antibodies shorten the coagulation time of hemophilic plasma and reduce the bleeding tendency in a rabbit model of acquired hemophilia A. [START_REF] Nordfang | Inhibition of extrinsic pathway inhibitor shortens the coagulation time of normal plasma and of hemophilia plasma[END_REF][START_REF] Erhardtsen | Blocking of tissue factor pathway inhibitor (TFPI) shortens the bleeding time in rabbits with antibody induced haemophilia A[END_REF] As to our knowledge, three monoclonal antibodies targeting TFPI are currently under clinical development: PF-06741086 by Pfizer, BAY1093884 by Bayer Healthcare and Concizumab by Novo Nordisk. Results from efficacy studies using these antibodies are awaited with great anticipation. Meanwhile, it is of interest to reflect on the various aspects of how such monoclonal antibodies affect TFPI biology. Several issues seem to be of relevance in this regard. First, TFPI is not only an inhibitor of the FXa/FVIIa/TF complex, but is also recognized as a direct inhibitor of the prothrombinase complex. [START_REF] Wood | Tissue factor pathway inhibitor-alpha inhibits prothrombinase during the initiation of blood coagulation[END_REF] At present, it is unclear which of the three clinical candidates is able to interfere with TFPI-mediated inhibition of the prothrombinase complex, and how this affects the pro-hemostatic potential of such antibody.

A second issue relates to the biodistribution of TFPI. The majority of the TFPI molecules are located at the surface and within endothelial cells, while only a small percentage of TFPI is present in plasma, mostly associated to lipoproteins. [START_REF] Maroney | Expression of tissue factor pathway inhibitor by endothelial cells and platelets[END_REF] There is also a portion that is stored in platelets. [START_REF] Novotny | Platelets secrete a coagulation inhibitor functionally and antigenically similar to the lipoprotein associated coagulation inhibitor[END_REF] It will thus be difficult to monitor how much of the total TFPI population will be inhibited by the therapeutic antibodies, while having only access to the patients' plasma.

Moreover, in vivo studies using mice with a combined tissue-specific deficiency of TFPI and full deficiency of FVIII revealed that particularly platelet-stored TFPIa is of particular relevance to compensate for the absence of FVIII. [START_REF] Maroney | Absence of hematopoietic tissue factor pathway inhibitor mitigates bleeding in mice with hemophilia[END_REF] At this point, it is uncertain which of the three monoclonal antibodies in clinical development is able to specifically target plateletstored TFPI. A final Indeed, we know that the FVIIa/TF complex may exert functions beyond hemostasis, in part via PAR-2 mediated signaling processes. [START_REF] Mackman | The many faces of tissue factor[END_REF] For example, animal studies have revealed that cell-bound TFPI contributes to reduce tumor metastasis and atherosclerosis. [START_REF] Xiao | Conditional knockout of TFPI-1 in VSMCs of mice accelerates atherosclerosis by enhancing AMOT/YAP pathway[END_REF][START_REF] Wang | Endothelial cell-anchored tissue factor pathway inhibitor regulates tumor metastasis to the lung in mice[END_REF] Furthermore, TFPI may also inhibit other proteases that are not involved in haemostasis, and the FVII-Activating proteases has been reported to be among them. [START_REF] Stephan | Tissue factor pathway inhibitor is an inhibitor of factor VII-activating protease[END_REF] It would be important therefore to investigate how the prophylactic use of anti-TFPI antibodies modulates the extra-hemostatic activities of the TF/FVIIa complex and other proteases.

A Factor VIII-nanobody fusion protein

For our third example of how antibodies could be used in the development of novel strategies in the treatment of hemophilia, we would like to focus on a specific kind of antibodies, namely heavy chain-only antibodies. [START_REF] Hamers-Casterman | Naturally occurring antibodies devoid of light chains[END_REF] Such antibodies are found in members of the Camelid family, which include camels and llamas. These heavy chain-only antibodies lack the light chain that is present in classic IgG molecules, and consequently all the antigen-binding information is constrained within the variable heavy chain region of the heavy chain-only antibodies. This variable region (also known as VHH or nanobody) can thus be used similar to the single chain Fv fragments of classic IgGs. In isolated form, nanobodies have a molecular weight of about 15 kDa. Despite their small size, they can still display high affinity for their target antigen. In the present example of this review, nanobodies have been used as fusion partner to FVIII. [START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation[END_REF] Of course, many other possibilities to use these specific antibody fragments do exist (see for instance reviews: [START_REF] Beghein | Nanobody Technology: A Versatile Toolkit for Microscopic Imaging, Protein-Protein Interaction Analysis, and Protein Function Exploration[END_REF][START_REF] Manglik | Nanobodies to Study G Protein-Coupled Receptor Structure and Function[END_REF][START_REF] Vincke | Introduction to heavy chain antibodies and derived Nanobodies[END_REF] ).

The rationale behind the use of nanobodies as fusion partner for FVIII lies in the observation that although FVIII binds with high affinity to VWF, there is still 2-5% of FVIII that circulates as free protein. [START_REF] Noe | A mathematical model of coagulation factor VIII kinetics[END_REF][START_REF] Schambeck | High factor VIII (FVIII) levels in venous thromboembolism: role of unbound FVIII[END_REF] Free FVIII differs from VWF-bound FVIII in that it is cleared 6-10 fold more rapidly. Moreover, VWF modulates the anti-FVIII immune response by affecting the pathway by which FVIII is endocytosed by antigen presenting cells. [START_REF] Hartholt | To serve and protect: The modulatory role of von Willebrand factor on factor VIII immunogenicity[END_REF][START_REF] Oldenburg | Alloantibodies to therapeutic factor VIII in hemophilia A: the role of von Willebrand factor in regulating factor VIII immunogenicity[END_REF] The underlying hypothesis in the design of the FVIII-nanobody fusion protein was therefore to create a FVIII molecule that forms a tighter complex with VWF, thereby reducing the amount of free FVIII.

In order to do so, nanobodies were generated against the VWF D'-D3 domain, the region that contains the FVIII binding site. These nanobodies indeed display an approximately 50to 100-fold slower dissociation rate constant from VWF compared to FVIII. [START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation[END_REF] Two copies of one of these nanobodies, ie. KB-VWF-013, were cloned into the FVIII cDNA, replacing the non-functional FVIII B-domain. [START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation[END_REF] The final protein, designated FVIII-KB013bv, was produced in mammalian cell lines as a single chain protein, and analyzed for function in in vitro assays. In terms of activity, FVIII-KB013bv was having full cofactor activity in one stage-and chromogenic assays. FVIII-KB013bv further had a 25-fold higher apparent affinity for VWF compared to wild-type FVIII (13 pM versus 330 pM), confirming that the presence of the nanobodies considerably improves binding to VWF. This increased affinity means that at a concentration of 1 U/ml, more than 99.8% of FVIII-KB013 is bound to VWF, whereas this is about 96.7% for wild-type FVIII. [START_REF] Muczynski | A factor VIII-nanobody fusion protein forming an ultra-stable complex with VWF: effect on clearance & antibody formation[END_REF] The effect of improved VWF binding was evaluated in FVIII-deficient mice. When determining circulatory survival, it was observed that FVIII-KB013bv was cleared two-fold slower than wilt-type FVIII. The increased survival was further established by its capacity to correct bleeding in FVIII-deficient mice 24 hours after a single intravenous injection, whereas by using the same dose wild-type FVIII was no longer able to do so. 9 Thus, the FVIII-nanobody fusion protein is fully functional in vivo and has a prolonged half-life. With regard to the development of anti-FVIII antibodies, we observed that FVIII-KB013bv was less immunogenic than wild-type FVIII (15% of mice developed antibodies against FVIII-KB013bv versus 85% developed antibodies against FVIII). 9 The reason for this difference will require further studies, but may be related to the fact that upon binding of the classic FVIII/VWF complex to antigen presenting-cells, FVIII enters into these cells, whereas VWF remains mainly at the cell surface. [START_REF] Sorvillo | von Willebrand factor binds to the surface of dendritic cells and modulates peptide presentation of factor VIII[END_REF] By having a FVIII molecule that sticks to VWF much more tightly, it is possible that less FVIII is able to enter the antigen presenting-cells, resulting in the formation of fewer anti-FVIII antibodies. In comparison to Emicizumab and the anti-TFPI antibodies, FVIII-KB013bv is not yet ready to enter clinical evaluation. Nevertheless, it represents an interesting example of how antibodies can be used to optimize treatment in general and that of hemophilia A in particular.

Conclusion

This review has presented three different ways of how antibodies signify novel strategies to innovate the management of bleeding episodes in hemophilia. Importantly, the use of antibodies is not limited to a simple inhibition of the function of its target. Two of the three examples presented here, such as Emicizumab that has procoagulant cofactor activity, and the FVIII-nanobody fusion protein that ameliorates the molecular properties of the complex FVIII molecule represent these options. The future will teach us if other types of application will become available to further improve the clinical management of haemophilia.

Figure 1 :

 1 Figure 1: Antibodies in hemophilia treatment. The mechanism of action of three different types of antibodies that are or may be useful in the management of hemophilia have schematically been depicted.

Disclosures

SF has no relevant conflict of interest. PJL has received honorarium/speakers fee from Bayer Healthcare, CSL Behring, Grifols, Kedrion, LFB, Novo Nordisk, Octapharma, Roche, Spark Therapeutics, Shire and Sobi. He is also co-inventor on patent applications related to treatment options for bleeding disorders, including a patent application describing the FVIIInanobody fusion protein. PJL is co-founder/co-owner of Laelaps Therapeutics.