R Abdallah

V Taly

S Zhao

D Pietrasz

J B Bachet

D Basile

L Mas

A Zaanan

P Laurent-Puig

Prof Julien Taieb
email: jtaieb75@gmail.com

Plasma circulating tumor DNA in pancreatic adenocarcinoma for screening, diagnosis, prognosis, treatment and follow-up: A Systematic Review

Keywords: circulating tumor DNA, screening, diagnosis, prognosis, treatment monitoring, pancreatic adenocarcinoma

Background:

While no biomarker is currently recommended for the management of pancreatic adnocarcinoma (PA) Circulating tumor DNA (ctDNA) seems promising but little is known on how it may help to manage our patients in the near future.

Materials and methods:

This systematic review of literature was designed to explore the current knowledge on ctDNA as a screening, diagnostic, prognostic, predictive and theranostic biomarker in the management of PA.

Results:

We retrieved 82 publications, including 62 full-text articles, 3 meta-analyses, 2 clinical trials and 1 abstract. The results were categorized into sections about screening, diagnosis; prognosis and follow-up of localized and advanced PA together with possible theranostics applications. Although its specificity is excellent, the current sensitivity of ctDNA remains a limitation especially in patients without metastatic disease. Therefore, this biomarker cannot be currently used as a screening or diagnostic tool. Increasing evidence suggests that ctDNA is a relevant candidate biomarker to assess minimal residual disease after radical surgery, but also a strong independent biomarker linked to a poor prognosis in advanced PA. Some recent data also indicates that ctDNA is an attractive biomarker for longitudinal follow-up and possibly early treatment adaptation. Its role in tumor profiling in advanced disease to decide targeted treatments remains to be explored.

Conclusion:

CtDNA appears to be a reliable prognostic tool. Though romising results have been reported, further studies are needed to define exactly how ctDNA can help physicians in the screening, diagnosis and treatment, as PA is expected to become a major cause of cancer-related deaths in the forthcoming decade.

HIGHLIGHTS:

• Detection of ctDNA is indicative of an unfavorable prognosis in patients with localized and advanced PA.

• The low sensitivity of ctDNA limits its interest for screening and diagnosis to date.

• Early ctDNA increase during the first month of chemotherapy could reflect treatment resistance.

• CtDNA for tumor molecular profiling in daily practice remains to be explored.

INTRODUCTION:

Pancreatic adenocarcinoma (PA) is the 7th leading cause of cancer-related death worldwide, with 458 918 deaths in 2018 (GLOBOCAN estimates). Its 5-year survival rate is one of the worst in the field of oncology and is <10% for all stages taken together [START_REF] Rawla | Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors[END_REF] . GLOBOCAN predicts a trend towards increased PA incidence (+77.7%, with 356 358 new cases) and mortality (+79.9%, with 345 181 deaths) from 2018 to 2040 (http://gco.iarc.fr/tomorrow/home). PA may even become the 3rd leading cause of death from cancer in the European Union for men and women after lung and colorectal cancers by 2025 [START_REF] Ferlay | More deaths from pancreatic cancer than breast cancer in the EU by 2017[END_REF] . Medical management of PA is challenging as around 80% to 90% of patients are diagnosed at an unresectable tumor stage. Moreover, PA diagnosis requires invasive procedures and may be difficult. Medical treatment consists of chemotherapy and radiotherapy depending on tumor stage. The treatment decision is not currently made according to tumor biology, unlike for many other cancers, with the exception of rare (1% to 5%) cases of PA with a micro-satellite instable (MSI) phenotype or a BRCA1/2 germline mutation. During the last decade, significant therapeutic advances have been made, with an increase in effective combination chemotherapeutic regimens, first in the metastatic setting and more recently in the adjuvant setting. However, patient survival remains disappointing, with a median overall survival (OS) less than one year in metastatic patients [START_REF] Conroy | FOLFIRINOX versus Gemcitabine for Metastatic Pancreatic Cancer[END_REF] and a 3-year rate of relapse-free survival below 40% in resected PA treated with adjuvant mFOLFIRINOX [START_REF] Conroy | FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer[END_REF] . We still lack reliable blood biomarkers for PA management. Carbohydrate antigen 19-9 (CA 19-9) is the most used biomarker for prognosis and monitoring, but has several limitations [START_REF] Ballehaninna | The clinical utility of serum CA 19-9 in the diagnosis, prognosis and management of pancreatic adenocarcinoma: An evidence based appraisal[END_REF] . With a consensual cutoff of 37 IU/mL, its performance does not allow its use for diagnostic purposes. The sensitivity (Se) and specificity (Sp) of CA 19-9 for diagnosis in symptomatic patients is 79-81% and 82-90% respectively, but its positive predictive value (PPV) for screening purposes in asymptomatic populations remains insufficient [START_REF] Chang | Low efficacy of serum levels of CA 19-9 in prediction of malignant diseases in asymptomatic population in Taiwan[END_REF][START_REF] Kim | Clinical usefulness of carbohydrate antigen 19-9 as a screening test for pancreatic cancer in an asymptomatic population[END_REF] . Moreover, CA 19-9 assay frequently yields falsepositive results in patients with cholestasis (whereas cholestasis has other frequent benign etiologies such as cholelithiasis), diabetes mellitus (which can also be induced by PA), cirrhosis, chronic pancreatitis (CP) and other gastrointestinal cancers. False negatives also represent a serious limitation since 5-10% of the Caucasian population of the Lewis-null phenotype are always negative for CA 19-9. Furthermore, there is controversial guidelines recommendation for the management of patients with advanced disease when invasive procedures fail to provide cytological confirmation of diagnosis. Some authors suggest to treat rapidly these patients with chemotherapy in case of poor general condition (ECOG-PS2) and CA 19-9 level higher than 10 times the upper limit of normal value (in patients without cholestasis), even without pathological proof [START_REF] Neuzillet | Pancreatic cancer: French clinical practice guidelines for diagnosis, treatment and follow-up (SNFGE, FFCD, GERCOR, UNICANCER, SFCD, SFED, SFRO, ACHBT, AFC)[END_REF] . For PA prognosis, CA 19-9 level at diagnosis seems to be associated with less R0 resection for localized PA if concentration is > 150 IU/mL, and with a metastatic disease if concentration is above 1000 IU/mL. In localized disease, normal preoperative CA 19-9 and clearance of CA 19-9 after resection reflect a good prognosis with prolonged survival [START_REF] Distler | Preoperative CEA and CA 19-9 are prognostic markers for survival after curative resection for ductal adenocarcinoma of the pancreasa retrospective tumor marker prognostic study[END_REF][START_REF] Kondo | Comparison of the prognostic impact of pre-and post-operative CA19-9, SPan-1, and DUPAN-II levels in patients with pancreatic carcinoma[END_REF][START_REF] Schlieman | Utility of tumor markers in determining resectability of pancreatic cancer[END_REF] . In metastatic disease, the decreasing of CA 19-9 level during chemotherapy is associated with better oncological outcomes [START_REF] Ishii | CA 19-9 in evaluating the response to chemotherapy in advanced pancreatic cancer[END_REF] . In the era of personalized medicine for patients with cancers, the identification of tissue biomarkers for diagnosis, prognosis and treatment is crucial but require invasive procedure at baseline but also during the follow-up. In this context, the translational research in PA is currently focused on the promising non-invasive option of plasma biomarkers, within the so-called "liquid biopsy" field, including cell-free circulating tumor DNA (ctDNA), circulating tumor cells (CTC) and circulating microRNAs. The objective of our review is to understand to what extent ctDNA can help clinicians in the screening, diagnosis, prognosis, follow-up and prediction of PA.

MATERIALS AND METHODS: Definition of the outcome

The aim is to evaluate the current clinical potential of ctDNA in PA management.

Data sources and search strategy

This systematic review was performed in January 2020 using several sources of human studies in the English language: PubMed database, unpublished data presented at international congresses as abstracts (American Society of Clinical Oncology ASCO and European Society of Medical Oncology ESMO) and ongoing clinical trials available on EudraCT (EU), clinicaltrial.gov (USA) and anzctr.org (Australia and New Zealand). Inclusion criteria included ctDNA measurement in plasma of patients at all PA stages and its correlation with screening, diagnosis, treatment response or oncological outcomes. Articles on patients with pancreatic cancers other than adenocarcinoma were excluded.

RESULTS

We screened 295 articles for inclusion in the review: 172 in PubMed using the following MeSH in January 2020: ("liquid biopsy" OR "cell free tumor dna" OR "cell free dna" OR "circulating tumor dna") AND ("pancreas/cancer" OR "adenocarcinoma/pdac" OR "pancreas/adenocarcinoma" OR "pancreatic/tumor" OR "pdac" OR "pancreatic cancer"); 29 in EudraCT, 41 in Clinicaltrial.gov, and 53 in anzctr.org. Finally, we excluded 24 records dealing exclusively with CTC, exosomes or miRNA. Eleven studies concerned biomarkers not assayed in peripheral blood (pancreatic juice, tissue, portal-vein blood samples). Thirty-five articles involved biomarkers other than ctDNA, and 17 studies focused on tumors other than PA (including upper gastrointestinal cancers and benign tumors such as intraductal papillary mucinous neoplasms (IPMN)). Four case reports, 2 editorials, 2 trials including < 15 patients and 8 reviews were not taken into account. Eighty-two records were finally eligible and included in the present review: 3 meta-analyses, 2 published trials, 62 full-text articles on translational studies, 1 ASCO abstract, 1 recommendation and 13 ongoing ctDNA clinical trials.

We organized our results into categories according to the potential clinical role of ctDNA in PA: screening, diagnosis and differential diagnosis, prognosis and prediction of treatment response in localized and advanced disease, and finally the theranostic impact. These data are summarized in Tables 1-3 and Table 4 presents ongoing trials.

Technical considerations:

Circulating cell-free (cfDNA) is present in plasma, urine and other bodily fluids. cfDNA may be detected in patients with cancers but also increase in benign conditions such as infections or inflammatory process. For patients with cancer, a fraction of this cfDNA contains specific tumor alterations (mostly point mutations) and is called circulating tumor DNA (ctDNA) [START_REF] Haber | Blood-based analyses of cancer: circulating tumor cells and circulating tumor DNA[END_REF] . In PA, ctDNA is widely detected by codon 12 KRAS mutations (KRAS mut ) (G12V and G12D), which are found in more than 90% of PA tissues [START_REF] Almoguera | Most human carcinomas of the exocrine pancreas contain mutant c-K-ras genes[END_REF] . Less frequently, ctDNA is detected by means of other KRAS mutations (G12R, G12C, G12S, G12A, G13D, codons 59, 60 and 61) or mutations in other genes (mainly TP53, CDKN2A, SMAD4, NRAS, PIK3CA, STK11 and ERBB4). However, other methods have been developed for the ctDNA detection based on epigenetics modifications through the methylation profile of gene promoters. Hypermethylation of CpG islands in tumor suppressor gene promoters leads to gene silencing. As mentioned previously, the identification of ctDNA may be difficult because its potential dilution among total cfDNA. According to the tumor stage, ctDNA can represent less than 1% of the total cfDNA (especially in earlier tumor stage) and its detection therefore requires highly sensitive techniques [START_REF] Yong | Cancer biomarkers: Written in blood[END_REF] . The most common techniques are schematically divided into targeted, such as digital droplet-based PCR (ddPCR), or non-targeted, such as Next-Generation Sequencing (NGS), with a good correlation between these 2 techniques [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF][START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF] . NGS offers the possibility to amplify a large number of mutations through a gene panel, whereas ddPCR often limits the detection to a few mutations in parallel. The ddPCR is usually more sensitive than NGS (0.001% vs 2%), even if recent NGS-based techniques such as Base-Position Error Rate analysis have enhanced ctDNA detection [START_REF] Taly | Detecting biomarkers with microdroplet technology[END_REF][START_REF] Forshew | Noninvasive identification and monitoring of cancer mutations by targeted deep sequencing of plasma DNA[END_REF][START_REF] Pécuchet | Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA[END_REF] . These innovative detection techniques for the detection of ctDNA in a noninvasive way could provide rapidly informations regarding the genetic profil of cancer and also explore the torah molecular heterogeneity between different locations such as primary and metastatic site. These strategies based on ctDNA detection and analysis could have some potential clinical application, including screening, diagnosis and prediction of tumor response or prognosis.

ctDNA as a biomarker for screening and early detection (Table 1)

There is currently no consensual validated screening program in PA, even for high-risk patients (hereditary pancreatitis, family history of PA), highlighted the urgent need for a tool able to screen or detect early this tumor [START_REF] Us Preventive | Screening for Pancreatic Cancer: US Preventive Services Task Force Reaffirmation Recommendation Statement[END_REF] . Nowadays, the best preventive strategy relies on reduction in PA risk factors (tobacco, alcohol and unhealthy diet) [START_REF] Rawla | Epidemiology of Pancreatic Cancer: Global Trends, Etiology and Risk Factors[END_REF] . The carcinogenesis of PA has a long infra clinical period during which ctDNA detection could be used to screen for PA. It is estimated that the first mutational event initiating preneoplasic lesion (PanIN) occurs more than 10 years before the appearance of symptoms [START_REF] Yachida | Distant metastasis occurs late during the genetic evolution of pancreatic cancer[END_REF] . The diagnosis and treatment of PA at an early asymptomatic stage is associated with dramatically improved outcomes [START_REF] Conroy | FOLFIRINOX versus Gemcitabine for Metastatic Pancreatic Cancer[END_REF][START_REF] Conroy | FOLFIRINOX or Gemcitabine as Adjuvant Therapy for Pancreatic Cancer[END_REF] . In this context, ctDNA brings several advantages of PA screening, including high reproducibility, noninvasiveness and acceptability as a simple blood test. KRAS mut blood detection seemed to be the best candidate as KRAS gene alterations are early molecular events in PA carcinogenesis [START_REF] Algül | Mechanisms of Disease: chronic inflammation and cancer in the pancreas-a potential role for pancreatic stellate cells?[END_REF] . The high Sp (>96%) of KRAS mut ctDNA seems to be a promising marker for PA screening in asymptomatic patients [START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF][START_REF] Cohen | Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers[END_REF] . However, the low Se of ctDNA has limited its use in screening and early diagnosis among asymptomatic patients, as a negative result may not exclude early PA. KRAS mut ctDNA detection rates range between 10% and 45% for localized PA, with similar results reported with NGS and ddPCR [START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF][START_REF] Cohen | Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers[END_REF][START_REF] Takai | Clinical Utility of Circulating Tumor DNA for Molecular Assessment and Precision Medicine in Pancreatic Cancer[END_REF][START_REF] Brychta | Detection of KRAS Mutations in Circulating Tumor DNA by Digital PCR in Early Stages of Pancreatic Cancer[END_REF][START_REF] Allenson | High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients[END_REF][START_REF] Sausen | Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients[END_REF] . Higher detection rates (45%) were reported by Allenson et al. [START_REF] Allenson | High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients[END_REF] but were obtained at the price of lower Sp with up to 15% of false positives in healthy controls. However, the definition of ctDNA positivity according to a cutoff of the mutant allelic fraction (MAF) could allow optimal Se and Sp to be reached. Increasing plasma sample size could also improve KRAS mut detection Se (43% with 4 mL of plasma 31 vs 35% with 2 mL using the same technique [START_REF] Brychta | Detection of KRAS Mutations in Circulating Tumor DNA by Digital PCR in Early Stages of Pancreatic Cancer[END_REF] ). However, the best performances observed to date are 45% for Se and 99.5% for Sp, which are insufficient for screening. Besides, as KRAS mut occurs in the oncogenesis of other cancers (colorectal 32 , lung [START_REF] Freidin | Circulating Tumor DNA Outperforms Circulating Tumor Cells for KRAS Mutation Detection in Thoracic Malignancies[END_REF] ), its detection is not specific to PA. The combination of several biomarkers, detection of other mutations and different protocols have been studied in an attempt to improve the low Se of ctDNA. Combining KRAS mut ctDNA detection with 4 protein biomarkers (CA 19-9, carcinoembryonic antigen, hepatocyte growth factor and osteopontin) increased Se for early PA detection to 64% [START_REF] Cohen | Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers[END_REF] , but other target mutations (e.g.TP53) did not. Adding NGS-base gene panel for SMAD4, CDKN2A and TP53 mutations to KRAS mut led to a higher ctDNA detection in PA [START_REF] Adamo | Profiling tumour heterogeneity through circulating tumour DNA in patients with pancreatic cancer[END_REF] . Finally, methylation-based biomarkers such as promoter methylation of ADAMTS1 and BNC1 were respectively associated with an Se of 87% and 64%, and a comparable Sp of around 94% in detection of resectable PA [START_REF] Eissa | Promoter methylation of ADAMTS1 and BNC1 as potential biomarkers for early detection of pancreatic cancer in blood[END_REF] . While the combination with CA 19-9 did not improve Se, combining ADAMTS1 and BNC1 methylation status led to promising performances (Se 94.8%, Sp 91.6%). These promising strategies of combined biomarkers to improve ctDNA-based screening accuracy have now to be confirmed in larger series and compared to find the ideal strategy to move forward to screening programs.

ctDNA as a diagnostic tool (Table 1)

PA diagnosis remains invasive as histological samples are collected by surgical resection, endoscopic ultrasound-guided fine needle aspiration/biopsy (EUS-FNA/B) or biopsy of accessible metastases. These procedures are linked to the risks of tumor dissemination, acute pancreatitis and bleeding which may delay the treatment and therefore impact the clinical outcomes [START_REF] Fujii | Basic techniques in endoscopic ultrasound-guided fine needle aspiration for solid lesions: Adverse events and avoiding them[END_REF] . Moreover, EUS-FNA remains inconclusive in approximately 20% of cases [START_REF] Alizadeh | Diagnostic potency of EUS-guided FNA for the evaluation of pancreatic mass lesions[END_REF] . PA is responsible for desmoplastic reaction and most pancreatic material after EUS-FNA is composed of stromal cells that contribute to these false-negative results. In this context , liquid biopsy for diagnosis using ctDNA has been evaluated in a recent metaanalysis of 369 patients from 14 studies. ctDNA detection was correlated with molecular genetic alterations of PA tissue samples (from EUS-FNA or surgical resection). Patients without PA (healthy volunteers, chronic pancreatitis, IPMN) were excluded. Overall Se and Sp were 70% and 86%, respectively. Considering the 11 studies of this meta-analysis evaluating KRAS mut only, Se was lower (65%) but Sp was higher (91%) than with NGS gene panel methods. Here again, a better Se is needed to move to ctDNA as a routine practice tool for PA diagnosis.

Factors influencing ctDNA diagnostic performances. ctDNA Se and Sp for PA diagnosis have been related to tumor stage. ctDNA detection has better Se in advanced cancers than in early-stage cancers, possibly due to higher tumor burden in the bloodstream. Among 410 malignancies including 155 PA, Bettegowda et al. [START_REF] Bettegowda | Detection of Circulating Tumor DNA in Earlyand Late-Stage Human Malignancies[END_REF] detected ctDNA in 48% and 75% of patients with localized and metastatic cancers, respectively. ctDNA detection rate and concentration similarly increased from stages I to IV. Studies focusing on PA confirmed this trend [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF][START_REF] Cohen | Combined circulating tumor DNA and protein biomarker-based liquid biopsy for the earlier detection of pancreatic cancers[END_REF][START_REF] Ako | Utility of serum DNA as a marker for KRAS mutations in pancreatic cancer tissue[END_REF] with a KRAS mut ctDNA detection rate of 10%, 17.5% and 33% for resectable, locally advanced and metastatic tumor stages, respectively [START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF] . The mean number of genetic alterations was higher in advanced than in resectable PA [START_REF] Patel | Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer[END_REF] . A high KRAS mut ctDNA detection rate was correlated with undifferentiated tumor [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF] . Beyond ctDNA detection, its quantification was also correlated with tumor stage [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF][START_REF] Chen | Ultrasensitive plasma ctDNA KRAS assay for detection, prognosis, and assessment of therapeutic response in patients with unresectable pancreatic ductal adenocarcinoma[END_REF][START_REF] Kim | Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma[END_REF] . Plasma KRAS mut concentrations and mutant allele fractions (MAF, proportion of mutated alleles among all alleles of a gene) were higher in metastatic than in locally advanced [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF] and resectable PA [START_REF] Patel | Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer[END_REF][START_REF] Kim | Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma[END_REF][START_REF] Wang | KRAS Mutant Allele Fraction in Circulating Cell-Free DNA Correlates With Clinical Stage in Pancreatic Cancer Patients[END_REF] . The KRAS mut MAF also increased with tumor size (sum of lesions' maximal diameters) [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] and 3Dmeasured tumor volume [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF] . Interestingly, ctDNA detection rates differed according to the location of metastases as reported in other cancers, such as colorectal cancer in the RASANC study [START_REF] Bachet | RAS mutation analysis in circulating tumor DNA from patients with metastatic colorectal cancer: the AGEO RASANC prospective multicenter study[END_REF] . KRAS mut ctDNA MAF were significantly higher in case of liver metastases compared to lung and peritoneal metastases [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF] , and for "liver or lung" compared to "peritoneal metastases" in another study [START_REF] Sugimori | Quantitative monitoring of circulating tumor DNA in patients with advanced pancreatic cancer undergoing chemotherapy[END_REF] . Furthermore, the capacity of ctDNA detection may be affected by previous anti-cancer treatments. In resectable PA, neoadjuvant chemotherapy was associated with a lower preoperative ctDNA detection rates than chemo-naïve resectable PA (21% vs 69%) [START_REF] Groot | Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer[END_REF] . Another study described similarly low ctDNA detection rate (29%) in patients with advanced PA previously treated by chemotherapy [START_REF] Pishvaian | A pilot study evaluating concordance between blood-based and patient-matched tumor molecular testing within pancreatic cancer patients participating in the Know Your Tumor (KYT) initiative[END_REF] . To our knowledge, there is no study evaluating the impact of radiotherapy on ctDNA detection rate.

How to improve ctDNA performance. ctDNA false-positive results for healthy patients would have disastrous psychological, medical and economic consequences, and conversely, ctDNA falsenegative results would mean that patients will not benefit from adequate treatment in due time. In this context, some strategies have been developed in order to increase the ctDNA performance for diagnosis by using (i) combination markers, (ii) methylated marker and/or (ii) new advanced techniques. Some studies have shown that adding a cancer-associated target genes (including notably TP53, CDKN2A, ROS1 or SMAD4) to KRAS mut detection was associated with a better Se for detection of ctDNA for the diagnosis of patients with PA [START_REF] Berger | Targeted deep sequencing of circulating tumor DNA in metastatic pancreatic cancer[END_REF][START_REF] Park | Utility of targeted deep sequencing for detecting circulating tumor DNA in pancreatic cancer patients[END_REF] . Other techniques such as ultrasensitive assays (able to detect degraded DNA with lower molecular weight assays and/or combining PCR and NGS) provided high detection rates (86-94%) [START_REF] Chen | Ultrasensitive plasma ctDNA KRAS assay for detection, prognosis, and assessment of therapeutic response in patients with unresectable pancreatic ductal adenocarcinoma[END_REF][START_REF] Liu | Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer[END_REF][START_REF] Pratt | Multiplex Enrichment and Detection of Rare KRAS Mutations in Liquid Biopsy Samples using Digital Droplet Pre-Amplification[END_REF] . The main limitation consisted of a 30% false-positive rate in healthy controls [START_REF] Liu | Enrichment of short mutant cell-free DNA fragments enhanced detection of pancreatic cancer[END_REF] .

ctDNA in differential diagnosis with benign pancreatic diseases

When KRAS mut ctDNA positivity provided high Sp to distinguish PA from healthy controls, it was not reliably accurate in differentiating benign pancreatic disease (pancreatitis, pancreatic cysts, benign tumors) from PA. Up to 20% of chronic pancreatitis (CP) [START_REF] Kinugasa | Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer[END_REF][START_REF] Rashid | Progression of Chronic Pancreatitis to Pancreatic Cancer: Is There a Role of Gene Mutations as a Screening Tool[END_REF] , 8% of benign pancreatic tumors [START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF] , 16% of pancreatic cysts and 17% of non-neoplastic pancreatic masses [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] had detectable KRAS mut ctDNA. Among undetermined solid pancreatic tumors referred for EUS-FNA, CA 19-9 had even better Se (79% vs 65%) and Sp (93% vs 75%) than KRAS mut ctDNA for PA diagnosis [START_REF] Sefrioui | Diagnostic value of CA19.9, circulating tumour DNA and circulating tumour cells in patients with solid pancreatic tumours[END_REF] . Quantitative ctDNA assessments combining biomarkers and methylation techniques enhanced the modest Sp of qualitative analysis of ctDNA and discriminated benign from malignant pancreatic diseases. ctDNA concentrations were lower in side-branch IPMN (without worrying features) and CP than in PA [START_REF] Berger | A Blood-Based Multi Marker Assay Supports the Differential Diagnosis of Early-Stage Pancreatic Cancer[END_REF] . KRAS mut MAF was also lower in benign pancreatic tumors (pseudocyst, IPMN, serous and mucinous cystadenoma) [START_REF] Wang | KRAS Mutant Allele Fraction in Circulating Cell-Free DNA Correlates With Clinical Stage in Pancreatic Cancer Patients[END_REF] and in CP [START_REF] Calvez-Kelm | KRAS mutations in blood circulating cellfree DNA: a pancreatic cancer case-control[END_REF] than in PA. Combining other biomarkers with ctDNA was more controversial. Cumulating high levels for at least 2 biomarkers (between CA 19-9, ctDNA and CTC) led to better Sp for patients with cholestasis (100% vs 86% for patients without cholestasis) [START_REF] Sefrioui | Diagnostic value of CA19.9, circulating tumour DNA and circulating tumour cells in patients with solid pancreatic tumours[END_REF] , while combining ctDNA (> 16 ng/mL) with CA 19-9 and thrombospondin-2 increased Se for early-stage PA detection from 70% to 87%, and Sp from xx to 92% in differentiating PA from CP and IPMN [START_REF] Berger | A Blood-Based Multi Marker Assay Supports the Differential Diagnosis of Early-Stage Pancreatic Cancer[END_REF] . Nevertheless, the combination of KRAS mut ctDNA and CA 19-9 did not perform better than CA 19-9 alone 26,45. Methylation biomarkers gave interesting results and were able to distinguish PA from benign pancreatic disorders, but not from healthy controls [START_REF] Melnikov | Methylation profile of circulating plasma DNA in patients with pancreatic cancer[END_REF] . The number (8 vs 5) of hypermethylated genes and their integration into a clinical and laboratory diagnostic model (AUC=0.86, Se 76%, Sp 83%) reliably differentiated PA from benign pancreatic diseases (chronic and acute pancreatitis, cystadenomas) independently of the stage of PA (unlike ctDNA alone) [START_REF] Henriksen | Cell-free DNA promoter hypermethylation in plasma as a diagnostic marker for pancreatic adenocarcinoma[END_REF] . Plasma methylation profile of 14 gene promoters significantly differed between CP and PA (Se 91.2%, Sp 90.8%, p<0.01) [START_REF] Liggett | Differential methylation of cell-free circulating DNA among patients with pancreatic cancer versus chronic pancreatitis[END_REF] .

ctDNA use in patients with resectable PA (Table 2)

Beyond ctDNA screening and diagnostic use, which is currently not recommended in clinical practice because of insufficient accuracy, the prognostic value of ctDNA before and after surgery, but also before and after (neo)adjuvant treatments, has been studied in resectable PA.

Preoperative ctDNA detection is a strong negative prognostic biomarker in resectable PA. Preoperative ctDNA positivity was associated with poorer OS (HR=2.3, 95% CI: 1.1-4.6), and with a non-significant trend to higher recurrence risk (HR=1.96, 95% CI: 0.65-5.9) in a meta-analysis including 375 patents with resectable PA from 5 retrospective studies [START_REF] Lee | Circulating tumor DNA as a prognostic indicator in resectable pancreatic ductal adenocarcinoma: A systematic review and meta-analysis[END_REF] , which is consistent with a prospective study involving 39 resected patients [START_REF] Watanabe | Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer[END_REF] .

Postoperative ctDNA was clinically relevant as a surrogate for "minimal residual disease" (MRD) in PA, as described in hematologic malignancies. If ctDNA levels fall after surgery [START_REF] Groot | Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer[END_REF] , immediate postoperative ctDNA KRAS mut positivity has been associated to poorer recurrence-free survival (RFS) and OS [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Groot | Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer[END_REF][START_REF] Lee | Circulating tumor DNA as a potential marker of adjuvant chemotherapy benefit following surgery for localised pancreatic cancer[END_REF] , which is consistent with a recent meta-analysis (OS: HR=3.7, 95% CI: 1.45-9.3, and RFS: HR=2.2, 95% CI: 0.99-4.9) [START_REF] Lee | Circulating tumor DNA as a prognostic indicator in resectable pancreatic ductal adenocarcinoma: A systematic review and meta-analysis[END_REF] . The shift from preoperative KRAS mut negativity to postoperative KRAS mut positivity assessed by ctDNA was also an important independent poor prognostic factor for OS (HR=9.4; 95% CI: 2-44; p=0.004) in another study with 45 patients [START_REF] Nakano | KRAS mutations in cell-free DNA from preoperative and postoperative sera as a pancreatic cancer marker: a retrospective study[END_REF] .

Postoperative ctDNA monitoring could also help clinicians to detect and treat early PA relapse. Prospective postoperative emergence of KRAS mut ctDNA during monitoring (including at least 3 serial liquid biopsies) significantly predicted worse OS (HR=54, 95% CI: 6.6-447, p<0.001) in 39 cases of PA [START_REF] Watanabe | Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer[END_REF] . Moreover, ctDNA follow-up predicted relapse 84 days (Se 90%, Sp 88%) 48 to 6.5 months [START_REF] Sausen | Clinical implications of genomic alterations in the tumour and circulation of pancreatic cancer patients[END_REF] earlier than CT imaging. However, it is at present unknown if this early detection could increase OS through early treatment approaches. The clinical utility of ctDNA to guide our treatment strategies needs to be clarified as data are currently scarce. In one study, KRAS MAF variation after neoadjuvant therapy did not help clinicians to indicate surgery [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] . Adjuvant treatment with doublet chemotherapy seemed better than gemcitabine alone in a postoperative ctDNApositive cohort of 13 patients [START_REF] Lee | Circulating tumor DNA as a potential marker of adjuvant chemotherapy benefit following surgery for localised pancreatic cancer[END_REF] . Another study (DYNAMIC-Pancreas) is evaluating adjuvant strategies according to ctDNA testing. Resected patients are randomized after surgery to a biomarker-driven arm (de-escalation treatment if "ctDNA-negative" and escalation chemotherapy with FOLFIRINOX or gemcitabine Nab-paclitaxel if "ctDNA-positive"), or to a standard-of-care adjuvant chemotherapy. However, the impressive results of adjuvant FOLFIRINOX published last year seriously compromised the DYNAMIC-Pancreas relevance.

ctDNA use in advanced PA

Baseline ctDNA as a prognostic biomarker in advanced PA (Table 3)

The prognostic role of ctDNA in locally advanced and metastatic PA has also been largely studied. Pre-chemotherapy KRAS mut detection clearly predicted worse progression-free survival (PFS) and 2to 4-fold worse OS compared to ctDNA-negative patients in all studies focusing on advanced PA [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF][START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Chen | K-ras mutational status predicts poor prognosis in unresectable pancreatic cancer[END_REF][START_REF] Tjensvoll | Clinical relevance of circulating KRAS mutated DNA in plasma from patients with advanced pancreatic cancer[END_REF] , which is consistent with prospective phase I/II studies results in advanced PA [START_REF] Van Laethem | Phase I/II Study of Refametinib (BAY 86-9766) in Combination with Gemcitabine in Advanced Pancreatic cancer[END_REF][START_REF] Semrad | Pharmacodynamic separation of gemcitabine and erlotinib in locally advanced or metastatic pancreatic cancer: therapeutic and biomarker results[END_REF] and with a meta-analysis of 18 articles (PFS: HR=2.31 [1.47, 3.64], OS: HR=2.57 [1.95, 3.38]) with 1243 patients (also including non-metastatic patients) [START_REF] Chen | Prognostic value of circulating cell-free DNA in patients with pancreatic cancer: A systemic review and meta-analysis[END_REF] . Among KRAS mutations, G12V KRAS mut seemed particularly associated with poorer OS compared to the other KRAS mut (HR=2.62 [1.32, 5.20]) [START_REF] Ako | Utility of serum DNA as a marker for KRAS mutations in pancreatic cancer tissue[END_REF][START_REF] Kinugasa | Detection of K-ras gene mutation by liquid biopsy in patients with pancreatic cancer[END_REF][START_REF] Chen | Prognostic value of circulating cell-free DNA in patients with pancreatic cancer: A systemic review and meta-analysis[END_REF][START_REF] Cheng | Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients[END_REF] . Cumulating plasma KRAS copy number gain and KRAS mut predicted worse outcomes compared to KRAS mut only [START_REF] Mohan | Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer[END_REF] . Studies testing ctDNA detection as a prognostic biomarker in PA are summarized in Table 3.

KRAS mut ctDNA quantitative detection approaches are also related to prognosis in advanced PA [START_REF] Lapin | Fragment size and level of cell-free DNA provide prognostic information in patients with advanced pancreatic cancer[END_REF] , but are still subject to controversy, unlike KRAS mut ctDNA qualitative detection. OS significantly decreased in patients with the highest KRAS mut MAF [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Mohan | Analysis of circulating cell-free DNA identifies KRAS copy number gain and mutation as a novel prognostic marker in Pancreatic cancer[END_REF] , KRAS mut concentrations [START_REF] Kim | Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma[END_REF] , and ctDNA levels (including other mutations such as TP53) [START_REF] Patel | Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer[END_REF] . Combined with CT scan tumor volume, ctDNA MAF has also been reported to be an independant predictor for OS [START_REF] Strijker | Circulating tumor DNA quantity is related to tumor volume and both predict survival in metastatic pancreatic ductal adenocarcinoma[END_REF] . In contrast, other studies didn't find any correlation between KRAS mut median MAF [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] , and KRAS mut fraction abundance [START_REF] Kim | Prognostic Implications of Multiplex Detection of KRAS Mutations in Cell-Free DNA from Patients with Pancreatic Ductal Adenocarcinoma[END_REF] with PFS and/or OS in metastatic patients.

Biomarkers other than KRAS mut have also been described as unfavorable prognostic biomarkers in advanced PA, such as total cell-free plasma DNA levels [START_REF] Singh | High levels of cell-free circulating nucleic acids in pancreatic cancer are associated with vascular encasement, metastasis and poor survival[END_REF] , ERBB2 exon 17 mutation [START_REF] Cheng | Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients[END_REF] , and methylated ctDNA (less expensive and providing more rapid results than NGS) [START_REF] Pietrasz | Methylated circulating tumor DNA (Met-DNA) as an independent prognostic factor in metastatic pancreatic adenocarcinoma (mPAC) patients[END_REF][START_REF] Henriksen | Promoter hypermethylation in plasma-derived cell-free DNA as a prognostic marker for pancreatic adenocarcinoma staging[END_REF][START_REF] Henriksen | Cell-free DNA promoter hypermethylation in plasma as a predictive marker for survival of patients with pancreatic adenocarcinoma[END_REF] .

ctDNA as an early predictive marker for treatment response in advanced PA ctDNA is a consensual prognostic biomarker, but is also a promising treatment monitoring tool, even if the literature provides less evidence for PA than for other tumors. On the top of baseline ctDNA prognostic value, the issue for unresectable patients consists of early adaptation of toxic and futile systemic treatment without waiting 1 or 2 months for confirmation of radiologic progression. Moreover, interpretation of imaging can be difficult after administration of chemotherapy, as CT scans cannot easily differentiate viable carcinoma and fibrosis. In patients with a discordant response (mixed response and progression in different tumor lesions, or progression on CT contrasting with a clinical and biological response or conversely), ctDNA could help to better identify a real efficacy (or not) to treatment. In order to overcome CT-scan follow-up limits, concomitant KRAS mut ctDNA was evaluated every 8 weeks. ctDNA detection rate reliably coincided with radiological treatment response for 10 of 13 metastatic patients in a first study [START_REF] Cheng | Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients[END_REF] . The emergence of ctDNA positivity predicted earlier progression under treatment [START_REF] Cheng | Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients[END_REF] . Similar results for PFS and OS were found in a prospective study of 39 unresectable patients [START_REF] Watanabe | Longitudinal monitoring of KRAS-mutated circulating tumor DNA enables the prediction of prognosis and therapeutic responses in patients with pancreatic cancer[END_REF] . Conversely, KRAS mut ctDNA clearance during chemotherapy provided better PFS than remaining positive KRAS mut ctDNA [START_REF] Sugimori | Quantitative monitoring of circulating tumor DNA in patients with advanced pancreatic cancer undergoing chemotherapy[END_REF] . Nevertheless, ctDNA monitoring failed to predict disease progression in another study [START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] .

Early KRAS mut ctDNA level variations were also associated with tumor response to chemotherapy in patients with advanced disease. An increase of KRAS mut ctDNA between baseline and day 14 was significantly associated with worse PFS and OS [START_REF] Del Re | Early changes in plasma DNA levels of mutant KRAS as a sensitive marker of response to chemotherapy in pancreatic cancer[END_REF] and was able to detect progressive disease with an Se of 83% and an Sp of 100% [START_REF] Kruger | Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer[END_REF] . Another study [START_REF] Perets | Mutant KRAS Circulating Tumor DNA Is an Accurate Tool for Pancreatic Cancer Monitoring[END_REF] found significant association between KRAS mut ctDNA slope (ΔctDNA/Δtime) and OS with a sharp and deep decline correlated with longer OS. In contrast, the kinetics of other protein-based tumor markers (CA 19-9, CEA and CYFRA 21-1) had no predictive value for treatment response [START_REF] Kruger | Repeated mutKRAS ctDNA measurements represent a novel and promising tool for early response prediction and therapy monitoring in advanced pancreatic cancer[END_REF] . Mutations other than KRAS have finally been tested in 2 studies and seem to reliably reflect chemotherapeutic efficacy. In a study of 15 patients 52 , KRAS mut allele frequency was significantly higher in progressive patients but also for other target genes. Combined KRAS and TP53 MAF levels also significantly decreased during treatment and increased at progression, contrary to CA 19-9 analyses [START_REF] Berger | Targeted deep sequencing of circulating tumor DNA in metastatic pancreatic cancer[END_REF] .

These results could in the future open the way to the early adaptation of chemotherapy in patients with early ctDNA increase. However, if the interest of ctDNA for treatment monitoring seems thus promising, it needs further validation in large prospective cohorts to be used in clinical practice.

ctDNA as a promising tool for personalized treatment

Beyond prognostic and treatment monitoring concerns, ctDNA may also be interesting for molecular screening of specific subtypes of PA in the future. ctDNA can indeed be complementary to tissue biomarkers in exploring PA genetic profiling in order to screen actionable molecular alterations for targeted treatments. Luchini et al. [START_REF] Luchini | Liquid Biopsy as Surrogate for Tissue for Molecular Profiling in Pancreatic Cancer: A Meta-Analysis Towards Precision Medicine[END_REF] in their meta-analysis reported 32% concordance between all the mutations detected with NGS multi-gene panels in tissue specimens vs blood samples. In this work, 38% of mutations were detected in tissue specimens only, while 30% were detected in liquid biopsy only. These results can probably be explained by intratumor heterogeneity and by the emergence of different clones in the primary tumor and the metastases. Another study even concluded that ctDNA was more representative of tumor molecular heterogeneity than tissue specimens in colon and pancreatic cancers [START_REF] Vietsch | Circulating cell-free DNA mutation patterns in early and late stage colon and pancreatic cancer[END_REF] . Indeed, 78% of mutations detected in ctDNA were not detected in primary tumor samples, and new mutations were detected in blood compared to previous ctDNA when the tumor progressed.

Actionable molecular alterations have already been described in PA tissue with current clinical therapeutic applications. For example, approximately 1% of PA have mismatch repair deficiency (dMMR, MSI) and could be eligible for checkpoint inhibitor trials [START_REF] Hu | Evaluating Mismatch Repair Deficiency in Pancreatic Adenocarcinoma: Challenges and Recommendations[END_REF][START_REF] Marabelle | Efficacy of Pembrolizumab in Patients With Noncolorectal High Microsatellite Instability/Mismatch Repair-Deficient Cancer: Results From the Phase II KEYNOTE-158 Study[END_REF] . Four to 7% of PA harbor tissue germline BRCA1/2 mutation and may benefit from PARP inhibitor treatment [START_REF] Golan | Maintenance Olaparib for Germline BRCA-Mutated Metastatic Pancreatic Cancer[END_REF] . More recently, tissue NRG1 gene fusions were described in up to 6% of PA, which are targetable by afatinib with promising clinical outcomes [START_REF] Jones | NRG1 Gene Fusions Are Recurrent, Clinically Actionable Gene Rearrangements in KRAS Wild-Type Pancreatic Ductal Adenocarcinoma[END_REF] . Plasma MSI and BRCA2 mutation detection are feasible and were respectively reported to be 50% in resected [START_REF] Moriyama | Allelic imbalance and microsatellite instability in plasma DNA released from polyclonal pancreatic adenocarcinoma[END_REF] and 12% in metastatic PA [START_REF] Cheng | Analysis of ctDNA to predict prognosis and monitor treatment responses in metastatic pancreatic cancer patients[END_REF] , when no NRG1 ctDNA assessment seems available to date. Other tumor tissue targetable biomarkers have also been identified in plasma in up to 73% of PA such as ATM, PALB2 (indicating sensitivity to PARP inhibitors or platinum agents), CCND2, CDK4 and CDK6 (CDK inhibitors), AKT1, AKT2, ARID1A, PIK3CA PIK3CG (PI3K/mTOR inhibitors) and tyrosine kinase receptors AXL, EGFR, FGFR1, FLT3 and PDGFRA (tyrosine kinase inhibitors) [START_REF] Patel | Clinical correlates of blood-derived circulating tumor DNA in pancreatic cancer[END_REF][START_REF] Pishvaian | A pilot study evaluating concordance between blood-based and patient-matched tumor molecular testing within pancreatic cancer patients participating in the Know Your Tumor (KYT) initiative[END_REF] . Hence, ctDNA therapeutic screening seems feasible in clinical practice and could in the future represent a promising way to screen for targeted therapies, as obtaining sufficient tissue samples remains challenging in many patients. Nevertheless, ctDNA has currently no therapeutic application in PA because of the lack of consensual optimal technique, high costs and lack of prospective clinical trials evaluating the allocation of targeted therapies according to plasma mutation detection.

Conclusion:

ctDNA detection appears already to be a strong unfavorable prognostic biomarker of RFS/PFS and OS in resected and advanced PA. The high specificity of ctDNA could provide significant help in PA diagnosis if ctDNA is detected in advanced disease patients, to avoid repeated invasive procedures and treatment delay, but its low sensitivity prevents for now its screening and diagnostic use in clinical practice. Developing ultrasensitive techniques, detecting other mutations, methylation-specific profiles, or combination with other plasma biomarkers could enhance the diagnostic and screening performances of ctDNA in the future. ctDNA could also be combined with other liquid biopsy techniques such as exoDNA (additional source of plasma DNA in the form of microvesicles) to improve their complementary diagnostic and monitoring performances [START_REF] Allenson | High prevalence of mutant KRAS in circulating exosome-derived DNA from early-stage pancreatic cancer patients[END_REF][START_REF] Bernard | Circulating Nucleic Acids Are Associated With Outcomes of Patients With Pancreatic Cancer[END_REF] . Early ctDNA increase during chemotherapy seems to be relevant to assess early treatment efficacy, but needs further validation as studies were conducted in a very limited number of patients. Finally, less invasive procedures could even be developed in the future, as Terasawa et al. [START_REF] Terasawa | Utility of liquid biopsy using urine in patients with pancreatic ductal adenocarcinoma[END_REF] recently found similar KRAS mut detection rates in plasma and urine. To conclude, ctDNA is a promising biomarker for PA management. In the forthcoming years we will have to agree on consensual standardized protocols for the techniques used, the biomarker or combination chosen, and to validate them prospectively on large patients series. This, together with dedicated prospective trials accurately designed for each clinical situation, is the only way to potentially allow the use of ctDNA for daily practice PA patients' management in the future.

ACKNOWLEDGMENTS:

We thank AGEO for providing support for English language editing.

FUNDING: None

DISCLOSURE: JT has received honoraria as a speaker and/or in an advisory role from Merck KGaA, Sanofi, Roche Genentech, MSD, Lilly, Celgene, Servier, Pierre Fabre and Amgen. JBB has received personal fees from Amgen, AstraZeneca, Bayer, Merck Serono, Pierre Fabre, Roche, Sanofi, Servier, Shire, and non-financial support from Amgen, Merck Serono, and Roche. AZ has received honoraria from Baxter, Roche, Merck Serono, MSD, Amgen, Servier, Sanofi, Lilly.


MD Gonçalo Boleto

PhD David Saadoun

MD Patrice Cacoub
email: patrice.cacoub@aphp.fr

Prof Ap-Hp Patrice Cacoub

Smith Kline

Janssen

Glaxo Smith

Strategies to prevent persistent or relapsed mixed cryoglobulinemia

Keywords: cryoglobulinemia, vasculitis, therapy, chronic hepatitis C, rituximab

Highlights box

-Treatment of cryoglobulinemic vasculitis (CryoVas) may target either the viral trigger hepatitis C virus (HCV) or the downstream B-cell lymphoproliferation.

-Aggressive therapy with direct-acting antivirals should be systematically considered for HCV-CryoVas patients.

-Rituximab (RTX) is an effective and safe option for severe CryoVas patients.

-Management of rituximab-refractory CryoVas remains a major challenge.

-B-cell-targeted therapies other than RTX or low-dose interleukin-2 are the most promising strategies for the management of refractory CryoVas cases.

Abstract

Introduction

Mixed cryoglobulinemia (MC) are immune complexes that can deposit in small and medium size arteries and cause systemic vasculitis called cryoglobulinemic vasculitis (CryoVas). CryoVas shows different clinical manifestations i.e. purpura, arthralgia and/or arthritis, skin ulcers, peripheral neuropathy, nephritis, and may progress to more life threatening illness. Hepatitis C virus (HCV) infection is the more frequent condition to be assessed in patients with MC, followed by connective tissue diseases and B-cell non-Hodgkin's lymphoma. In HCV-related cases, the mainstay of CryoVas treatment is interferon free antiviral therapy. However, a significant proportion of patients who show HCV eradication will develop persistent CryoVas manifestations needing treatment intensification.

Areas covered

This review highlights key advances, recent clinical trial updates and ongoing studies on the management of persistent and refractory CryoVas. Therapeutic strategies and treatment agents to manage the disease are described. A literature review was performed by searching for available research studies published before January 2020 on the Medline (PubMed) database.

Expert opinion

Antiviral therapy with direct antiviral agents is the mainstay of treatment for patients with HCV-associated CryoVas. B-cell depleting strategies, mainly with rituximab, is the main therapeutic option in severe and refractory cases of infectious and non-infectious CryoVas. Ongoing trials are currently exploring other targeted biological treatments in this setting.

Introduction

Cryoglobulinemia is defined as the persistent presence in the serum of circulating cryoglobulins which are immunoglobulin complexes that precipitate at cold temperature and redissolve at 37ºC. According to Brouet's classification [START_REF] Brouet | Biologic and clinical significance of cryoglobulins: A report of 86 cases[END_REF] three subtypes of cryoglobulins can be defined: type I (isolated monoclonal component), and type II and III also called mixed cryoglobulins (MC) which are immune complexes composed by a polyclonal immunoglobulin acting as antigen and an immunoglobulin either monoclonal (type II) or polyclonal (type III). Small-to-medium size vessel vasculitis also called cryoglobulinemic vasculitis (CryoVas) is related to MC and can cause an array of clinical manifestations (purpura, arthralgia and/or arthritis, skin ulcers, peripheral neuropathy, nephritis) and can be a life-threatening condition. The fundamental mechanism contributing to MC is aberrant autoantibody production by B cells and low grade B cell proliferation. Whereas type I cryoglobulins are almost always linked to B cell lymphoproliferative disorders, type II and III mixed cryoglobulins are related to chronic hepatitis C virus (HCV) infection in the majority of cases. Of note, cryoglobulinemia is found in 40% to 60% of HCV infected patients, and 10-35% of them will develop CryoVas manifestations [2]. The production of cryoglobulinemia is correlated with a long lasting HCV infection, older age and the degree of liver fibrosis [START_REF] Zignego | Expert opinion on managing chronic HCV in patients with mixed cryoglobulinaemia vasculitis[END_REF]. Other chronic infectious disorders caused by human immunodeficiency viruses (HIV) or hepatitis B virus (HBV) or systemic autoimmune disorders [in particular Sjögren's syndrome (SS) and systemic lupus erythematosus (SLE)] can also be associated with MC and CryoVas.

Five to 20% of patients with primary SS may present MC and CryoVas; such patients are at higher risk of B-cell lymphoma and have a relatively poor survival [START_REF] Zignego | Expert opinion on managing chronic HCV in patients with mixed cryoglobulinaemia vasculitis[END_REF][START_REF] Tzioufas | Mixed monoclonal cryoglobulinemia and monoclonal rheumatoid factor cross-reactive idiotypes as predictive factors for the development of lymphoma in primary Sjögren's syndrome[END_REF][START_REF] Quartuccio | Biomarkers of lymphoma in Sjögren's syndrome and evaluation of the lymphoma risk in prelymphomatous conditions: Results of a multicenter study[END_REF].

Treating patients suffering from CryoVas should be a priority. Even if symptoms are often initially mild, the disease may progress to life-threatening widespread vasculitis with glomerulonephritis, heart, gut or central nervous system involvement [START_REF] Terrier | Cryoglobulinemia vasculitis: an update[END_REF][START_REF] Cacoub | Cryoglobulinemia Vasculitis[END_REF]. HCVassociated CryoVas is linked to significant mortality with a 5-year survival rate of 75% [START_REF] Terrier | Prognostic factors in patients with hepatitis C virus infection and systemic vasculitis[END_REF]. This was confirmed in a study showing a significantly lower survival rate in HCV-CryoVas patients compared with the expected survival of age and sex-matched subjects in the general population [START_REF] Ferri | Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients[END_REF]. Aside from its specific symptoms, CryoVas patients also have an increased risk of developing B-cell lymphomas with an incidence rate 35 times higher than in the general population. Despite the success with interferon-free directacting antivirals (DAAs) the management of refractory HCV-CryoVas remains a challenge. In patients presenting with severe or relapsing disease an induction phase of immunosuppression if often necessary. Targeting both the B-cell arm of autoimmunity and the viral trigger with B-cell-depleting induction therapy with rituximab and optimal anti-HCV treatment appears logical in this setting. In non-HCV CryoVas the treatment depends of the severity of the clinical picture and of the associated aetiology. However, in a small proportion of CryoVas patients, no underlying cause can be identified and therefore therapy directed at the underlying aetiology cannot be performed [START_REF] Saadoun | Current and future therapeutic strategies for treating mixed cryoglobulinemia[END_REF].

Here, we review recent studies investigating therapeutic strategies to manage persistent or relapsed MC and CryoVas.

Methods

A literature review was performed by searching for available research studies published before January 2020 on the Medline (PubMed) database. The following English keywords were used: mixed cryoglobulinemia, mixed cryoglobulins, cryoglobulinemic vasculitis, and also abatacept, antiviral, azathioprine, belimumab, biological, biological diseasemodifying antirheumatic drugs, corticoid, corticosteroid, cyclophosphamide, directacting antiviral, glucocorticoid, immunomodulatory, immunosuppressive, immunosuppressant, interleukine-2, methotrexate, methylprednisolone, mycophenolate, mofetil, ofatumumab, plasma exchange, prednisone, rituximab, tocilizumab, tofacitinib, tumor necrosis factor-alpha. These words were combined using the Boolean operators 'AND' or 'OR'.

No limits were applied. Controlled clinical trials, original articles, and case series were analysed . When there were no controlled clinical trials, open studies, case reports or low quality studies were analyzed. We screened reference lists of relevant articles to ensure that no relevant publications were overlooked.

Treatment strategy

3.1.HCV-related cryoglobulinemic vasculitis

In HCV infected patients, CryoVas treatment should be initiated without delay and always include antiviral treatment. Management of these patients must take into consideration a case-by-case stratification according to the severity of vasculitis [START_REF] Retamozo | Introducing treat-to-target strategies of autoimmune extrahepatic manifestations of chronic hepatitis C virus infection[END_REF][START_REF] Ramos-Casals | Evidence-based recommendations on the management of extrahepatic manifestations of chronic hepatitis C virus infection[END_REF][START_REF] Liver | Electronic address: easloffice@easloffice.eu. EASL Recommendations on Treatment of Hepatitis C[END_REF].

Patients with mild-to-moderate vasculitis and non-life-threatening symptoms (i.e.

arthralgia, asthenia, purpura, sensory neuropathy) may be treated with antiviral therapy alone. Sustained suppression of HCV replication is associated with a significantly higher rate of complete clinical remission in patients with HCV-CryoVas [START_REF] Cacoub | Impact of sustained virological response on the extrahepatic manifestations of chronic hepatitis C: a meta-analysis[END_REF]. The recent advent of interferon-free direct-acting antivirals (DAAs), which have now the potential to induce sustained virological response rates greater than 95%, has dramatically changed the management of chronic HCV infection and HCV-CryoVas patients [START_REF] Cacoub | Long-term Efficacy of Interferon-Free Antiviral Treatment Regimens in Patients With Hepatitis C Virus-Associated Cryoglobulinemia Vasculitis[END_REF][START_REF] Landau | Causes and predictive factors of mortality in a cohort of patients with hepatitis C virus-related cryoglobulinemic vasculitis treated with antiviral therapy[END_REF]. An initial open-label study evaluating the effectiveness and safety of an oral interferon-free and ribavirin-free regimen with sofosbuvir plus daclatasvir in patients with HCV-CryoVas showed a complete clinical remission in 90.2% of patients at week 24. Complete remission rate at 12 months was 90%, with an event free survival rate at 2 years of 100%

(although the disappearance of cryoglobulin was noted in only 50% of cases) [START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF].

Gragnani et al have also showed very high rates of clinical and virological responses (93% and 100%, respectively) in patients treated by sofosbuvir based interferon-free regimens [START_REF] Gragnani | Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia[END_REF]. Bonacci et al reported 71% of complete clinical response, 94% of sustained virological response and 48% of cryoglobulin clearance in a series of 35 HCV-CryoVas patients treated by different DAAs [START_REF] Bonacci | Virologic, Clinical, and Immune Response Outcomes of Patients With Hepatitis C Virus-Associated Cryoglobulinemia Treated With Direct-Acting Antivirals[END_REF]. The most recently approved DAAs show improved pan-genomic and higher antiviral activities requiring no particular therapeutic guidance (i.e. duration of antiviral therapy, presentation and severity of CryoVas, HCV genotype, viral load and severity of fibrosis stage/hepatic function) [START_REF] Desbois | Cryoglobulinemia: An update in 2019[END_REF].

Immunosuppressant therapy remains useful in patients with severe HCV-CryoVas in order to rapidly control vasculitis [START_REF] Retamozo | Introducing treat-to-target strategies of autoimmune extrahepatic manifestations of chronic hepatitis C virus infection[END_REF][START_REF] Ramos-Casals | Evidence-based recommendations on the management of extrahepatic manifestations of chronic hepatitis C virus infection[END_REF][START_REF] Ramos-Casals | The cryoglobulinaemias[END_REF]. Of note, even in such severe CryoVas cases, DAAs should be always used to clear HCV infection. Intravenous methylprednisolone (0.5-1.0g/day for 3-5 days) in combination with plasma exchange (PLEX) should be used in life-threatening presentations such as severe kidney dysfunction, skin necrosis, involvement of the gastrointestinal tract or central nervous system [2]. The use of B-cell depleting agents such as rituximab has demonstrated greater efficacy than conventional immunosuppressants in HCV-CryoVas, even in cirrhotic patients [START_REF] Quartuccio | Retreatment regimen of rituximab monotherapy given at the relapse of severe HCV-related cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study[END_REF]. The best rituximab dosing regimen is 375mg/m 2 given weekly for 4 consecutive weeks [START_REF] Sneller | A randomized controlled trial of rituximab following failure of antiviral therapy for hepatitis C virus-associated cryoglobulinemic vasculitis[END_REF]. In a randomized controlled trial the authors compared rituximab versus conventional treatment (glucocorticoids; azathioprine or cyclophosphamide; or PLEX) in patients with severe HCV-CryoVas. Relapse-free survival at 3, 6 and 24 months was statistically higher in the rituximab group. Vasculitis activity as measured by the Birmingham Activity Score (BVAS) decreased only after treatment with rituximab. Interestingly, the median duration of response to rituximab was 18 months [START_REF] Quartuccio | Retreatment regimen of rituximab monotherapy given at the relapse of severe HCV-related cryoglobulinemic vasculitis: Long-term follow up data of a randomized controlled multicentre study[END_REF]. Rituximab provides long-term remission and besides its immunomodulatory effect, it also plays an important role by depleting CD19 positive-B cells, known to be HCV reservoirs [START_REF] Morsica | Replication of Hepatitis C Virus in B Lymphocytes (CD19+)[END_REF].

Some HCV-CryoVas patients successfully treated with anti-viral therapy and undetectable viral load may present with persistent CryoVas manifestations. This effect is likely due to the emergence of clonal B-cells that developed a neoplastic behaviour after prolonged HCV stimulation, acquiring an independent proliferative potential. These patients should be treated with rituximab alone [2]. Caution is nonetheless needed regarding the possibility of rituximab associated vasculitis flare due to the formation of immune complexes [START_REF] Ramos-Casals | The cryoglobulinaemias[END_REF]. A recent study reporting rituximab-associated autoimmune disease flares identified 3.4% vasculitis flares after a median of 8 days following rituximab infusion. All cases were observed in patients with type II MC after a median of 8 days following rituximab infusion. Vasculitis flares included acute kidney injury, purpura, gastrointestinal involvement and myocarditis. Among HCV-CryoVas patients, those who develop rituximab-associated CryoVas had more frequently renal involvement, B-cell lymphoproliferation, higher levels of cryoglobulin and lower C4 serum levels [START_REF] Desbois | Retrospective study reporting the incidence, predictors, and outcome of rituximab-associated CryoVas flares[END_REF].

A possible way to overcome the risk of CryoVas flare is to use rituximab at lower dosage, as it seems to be as effective as higher dosages [START_REF] Visentini | Efficacy of low-dose rituximab for mixed cryoglobulinemia[END_REF][START_REF] Visentini | Efficacy of low-dose rituximab for the treatment of mixed cryoglobulinemia vasculitis: Phase II clinical trial and systematic review[END_REF]. Interestingly, Saadoun et al showed that treatment with low-dose interleukin-2 lead to clinical improvement of HCV-CryoVas through regulatory T cells recovery, which might also represent a future approach to manage refractory cases [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF].

Cryoglobulinemic vasculitis unrelated to HCV infection

Other infectious disorders

Non-HCV related CryoVas secondary to other infectious disorders are rare. They are mainly represented by HIV and chronic HBV infection. MC can be detected in 17% and 9% of patients with HIV and HBV respectively [START_REF] Ferri | Mixed cryoglobulinemia: demographic, clinical, and serologic features and survival in 231 patients[END_REF][START_REF] Bonnet | Prevalence of cryoglobulinemia and serological markers of autoimmunity in human immunodeficiency virus infected individuals: a cross-sectional study of 97 patients[END_REF]. Only anecdotal cases of other infectious causes such cytomegalovirus, Epstein Barr virus, parvovirus B19, pyogenic bacterial and parasitic infections, have been reported [START_REF] Terrier | Non HCV-related infectious cryoglobulinemia vasculitis: Results from the French nationwide CryoVas survey and systematic review of the literature[END_REF][START_REF] Galli | HCV-unrelated cryoglobulinaemic vasculitis: the results of a prospective observational study by the Italian Group for the Study of Cryoglobulinaemias (GISC)[END_REF][START_REF] Mazzaro | Hepatitis B virus related cryoglobulinemic vasculitis: A multicentre open label study from the Gruppo Italiano di Studio delle Crioglobulinemie -GISC[END_REF]. In patients with CryoVas due to HBV or HIV, antiviral therapy should always be initiated before or at the same time as immunosuppressive therapy. Such patients are at higher risk for enhanced viral replication resulting in accelerated liver injury and worsening of extra-hepatic manifestations when treated with cyclophosphamide or rituximab [START_REF] Viganò | HBV-Associated Cryoglobulinemic Vasculitis: Remission after Antiviral Therapy with Entecavir[END_REF]. Rituximab should not be used in patients with active hepatitis flares, as fatal hepatitis cases have been reported [START_REF] Li | Prognostic analysis of acute exacerbations of hepatitis-B after chemotherapy in combination with rituximab in 19 patients with lymphoma[END_REF]. In patients with mild to moderate HBV-related CryoVas, antiviral agents (entecavir, tenofovir, alpha-interferon, lamivudine) should be used as first-line therapy.

The therapeutic goal in these patients is to obtain a negativation of serum HBV-DNA [START_REF] Mazzaro | Hepatitis B virus related cryoglobulinemic vasculitis: A multicentre open label study from the Gruppo Italiano di Studio delle Crioglobulinemie -GISC[END_REF]. In most severe vasculitic presentations, concomitant therapy with specific antivirals and rituximab and/or PLEX and/or conventional immunosuppressive agents (i.e. cyclophosphamide), in association with short-term glucocorticoids is recommended [START_REF] Evens | Rituximab-associated hepatitis B virus (HBV) reactivation in lymphoproliferative diseases: meta-analysis and examination of FDA safety reports[END_REF].

Autoimmune disorders

The widespread use of DAAs to treat HCV infection and HCV-CryoVas has deeply modified the spectrum of aetiologies causing CryoVas. From a large database of 15 970 patients screened for the presence of cryoglobulinemia between 2011 and 2018, it has been showed that the incidence of MC is decreasing steadily. HCV infection represents in recent years only 56% of cases, and as a consequence, connective tissue diseases in particular SS and SLE represent the main aetiologies of MC [START_REF] Boleto | Mixed Cryoglobulinaemia Since the Advent of New Direct-acting Antivirals for Hepatitis C Infection: Clinical Characteristics, Etiologies and Biological Features in 679 Patients[END_REF]. This is in line with another recent study of 242 patients with non-infectious CryoVas, showing underlying connective tissue disorders (SS, SLE and systemic sclerosis) in 30% of cases [START_REF] Terrier | Management of noninfectious mixed[END_REF]. Two previous studies [START_REF] Ramos-Casals | Cryoglobulinemia in primary Sjögren's syndrome: prevalence and clinical characteristics in a series of 115 patients[END_REF][START_REF] García-Carrasco | Cryoglobulinemia in systemic lupus erythematosus: prevalence and clinical characteristics in a series of 122 patients[END_REF] showed that MC were detected in the sera of 16% and 25% of patients with SS and SLE respectively. Moreover, patients with circulating cryoglobulins had more frequently CryoVas in particular cutaneous vasculitis. The presence of systemic vasculitis with cutaneous and peripheral nervous system involvement in patients with SS should prompt an evaluation for the presence of lymphoma. In the French database of 15 970 patients, SLE represented the main cause of non-related MC followed by SS; however, SS appeared as the first cause of CryoVas. In 2018, autoimmune disorders represented the main cause of MC (36.5% of all cases) as compared to HCV infection (32.6%) [START_REF] Boleto | Mixed Cryoglobulinaemia Since the Advent of New Direct-acting Antivirals for Hepatitis C Infection: Clinical Characteristics, Etiologies and Biological Features in 679 Patients[END_REF]. The approach for inducing clinical remission in these patients are derived from therapeutic strategies used in other systemic vasculitides. The therapeutic approach is mostly based on the use of high-dose glucocorticoids, and conventional immunosuppressive treatment such as cyclophosphamide and/or rituximab. Other agents such as azathioprine and mycophenolate mofetil are mainly used as remission maintenance agents. According to recent data, the use of glucocorticoids and rituximab may now be considered standard of care in patients with severe CryoVas secondary to autoimmune disorders, in particular connective tissue diseases [START_REF] Terrier | Safety and efficacy of rituximab in nonviral cryoglobulinemia vasculitis: data from the French Autoimmunity and Rituximab registry[END_REF][START_REF] Terrier | Predictors of early relapse in patients with noninfectious mixed cryoglobulinemia vasculitis: results from the French nationwide CryoVas survey[END_REF].

Haematological disorders

In patients with non-infectious CryoVas, B-cell malignancy accounts for ~20% of cases [START_REF] Terrier | Management of noninfectious mixed[END_REF]. The most common haematological conditions associated with MC are B-cell non-Hodgkin lymphomas, mainly marginal zone and lymphoplasmacytic lymphomas. The overall risk of non-Hodgkin lymphoma in patients with HCV-CryoVas is 35 times higher than in the general population [START_REF] Monti | Incidence and characteristics of non-Hodgkin lymphomas in a multicenter case file of patients with hepatitis C virusrelated symptomatic mixed cryoglobulinemias[END_REF]. HCV-positive non-Hodgkin's lymphoma usually occurs following a long period of infection (>15 years) especially in the presence of cryoglobulin levels above 600mg/L, the presence of CryoVas manifestations and hypogammaglobulinemia [START_REF] Saadoun | Hepatitis C-associated mixed cryoglobulinaemia: a crossroad between autoimmunity and lymphoproliferation[END_REF]. Treating the underlying lymphoproliferative disorder should be a priority in the management of symptomatic CryoVas. The treatment usually consists of anthracycline-based chemotherapy coupled with rituximab (375 mg/m 2 given weekly for 4 consecutive weeks) [START_REF] Solimando | Targeting B-cell non Hodgkin lymphoma: New and old tricks[END_REF]. Plasma exchange therapy may deserve consideration in patients with severe vasculitic symptoms [START_REF] Vallisa | Role of Anti-Hepatitis C Virus (HCV) Treatment in HCV-Related, Low-Grade, B-Cell, Non-Hodgkin's Lymphoma: A Multicenter Italian Experience[END_REF].

Essential

In a small percentage of non-HCV-related MC, no underlying cause can be identified and patients are classified as having essential cryoglobulinemia [START_REF] Terrier | Management of noninfectious mixed[END_REF]. Since therapy directed at the underlying aetiology cannot be performed, these patients should be treated with immunosuppressants which usually includes a short course of glucocorticoids associated with rituximab (less frequently cyclophosphamide or mycophenolate mofetil) [START_REF] Roccatello | Long-term effects of anti-CD20 monoclonal antibody treatment of cryoglobulinaemic glomerulonephritis[END_REF][START_REF] Roccatello | Improved (4 Plus 2) Rituximab Protocol for Severe Cases of Mixed Cryoglobulinemia: A 6-Year Observational Study[END_REF].

PLEX can be considered in the most severe or refractory cases. Although rituximab has proven to be safer than conventional immunosuppressants and highly effective in this setting, some patients show no or only partial response to this treatment. Rituximabrefractory CryoVas can be defined as an insufficient response or no response within 4-6 weeks after the initial infusion. Before treatment is intensified, the clinician should always rule out CryoVas mimics (underlying infection or cancer-related comorbidity) and evaluate patient's adherence to medical prescriptions [2]. Some alternative approaches have been proposed in refractory cases. Ofatumumab (a potent anti-CD20 monoclonal antibody) or belimumab (an anti-B-cell activator antibody), are potential second-line therapies for patients who continue to show symptoms of CryoVas [START_REF] Ostojic | Managing refractory cryoglobulinemic vasculitis: challenges and solutions[END_REF]. While tumor necrosis factor inhibitors are not effective, other biologic disease-modifying antirheumatic drugs such as abatacept or tocilizumab seem to be promising [START_REF] Joseph | Treatment of rheumatoid arthritis in patients with concomitant chronic hepatitis C infection[END_REF][START_REF] Cohen | Efficacy of tocilizumab in rituximab-refractory cryoglobulinemia vasculitis[END_REF].

Expert opinion

The introduction of safe and highly effective HCV therapies has resulted in a paradigm shift in how we manage refractory and relapsed HCV-CryoVas. Despite the excellent efficacy of DAAs, 3% to 10% of patients who achieved viral eradication can still experience CryoVas relapses [START_REF] Saadoun | Efficacy and Safety of Sofosbuvir Plus Daclatasvir for Treatment of HCV-Associated Cryoglobulinemia Vasculitis[END_REF][START_REF] Gragnani | Prospective study of guideline-tailored therapy with direct-acting antivirals for hepatitis C virus-associated mixed cryoglobulinemia[END_REF][START_REF] Gragnani | Long-term effect of HCV eradication in patients with mixed cryoglobulinemia: a prospective, controlled, open-label, cohort study[END_REF]. Available data suggest that when the immune disorder is established, antivirals may not be able to interfere with the immune-mediated injury and the pathogenesis of CryoVas. The degree of reversibility of CryoVas is inversely correlated with the duration of the immune stimulation by HCV [START_REF] Zignego | [END_REF]. Lowgrade B-cell lymphoproliferation (i.e. persistence of B-cell clones) after HCV eradication probably explains persistent immunological abnormalities and vasculitic symptoms in some individuals with long lasting infection. A cohort study found that the effect of sustained virological responses against extrahepatic manifestations was lessened as the time to initiation of HCV therapy increased [START_REF] Zignego | Expert opinion on managing chronic HCV in patients with mixed cryoglobulinaemia vasculitis[END_REF]. In some patients, B-cell stimulation and proliferation might have passed a "no return" point. This emphasizes the importance of the early treatment of HCV infection and CryoVas. Clinicians should be aware of the possibility of the presence of underlying malignant lymphoma when patients develop a relapse of CryoVas despite sustained virological responses [START_REF] Landau | Relapse of hepatitis C virus-associated mixed cryoglobulinemia vasculitis in patients with sustained viral response[END_REF]. An early use of DAAs is therefore recommended in all HCV-CryoVas patients. DAAs should be used alone to treat non-severe cases of CryoVas to eradicate the immune-mediated injury and prevent B-cell lymphoproliferation. In more severe cases, especially in the presence of kidney involvement, DAAs should be associated to immunosuppressants with rituximab being the most widely used drug. Rituximab also plays an important role by depleting CD19 positive-B-cells which are known to be HCV reservoirs [START_REF] Dammacco | The expanding spectrum of HCV-related cryoglobulinemic vasculitis: a narrative review[END_REF]. According to several studies, rituximab is a safe medication in patients with CryoVas, including those with chronic HCV infection [START_REF] Ostojic | Managing refractory cryoglobulinemic vasculitis: challenges and solutions[END_REF]. However, clinicians should be aware that some patients might present CryoVas flares after rituximab therapy especially in the presence of renal involvement, high levels of circulating cryoglobulin and low C4 serum levels. Indeed, rituximab may form complexes with IgM and IgG1 leading to vasculitis flare [START_REF] Desbois | Retrospective study reporting the incidence, predictors, and outcome of rituximab-associated CryoVas flares[END_REF]. In patients with risk factors of rituximab-induced vasculitis flare, lower doses of rituximab should be used (250-500mg per infusion). We also recommend that all patients with CryoVas who are receiving immunosuppressive therapy should receive age-appropriate vaccination.

The management of rituximab-refractory CryoVas remains a major challenge in HCV and non-HCV patients. Refractory disease can be defined as the absence of CryoVas clinical response within 4-6 weeks after induction therapy, or less than 50% improvement after 12 weeks [2]. In the most severe and refractory cases, we recommend the use of plasma exchange daily for 5 to 14 sessions, followed by 3 PLEX per week for two to three weeks. Of note, replacement fluid should be warmed to prevent precipitation of circulating cryoglobulin.

The management of severe CryoVas can be especially challenging in immunocompromised patients. As stated before, a fair proportion of patients with HIV or B-cell lymphoproliferative disorders present MC. A reduction in the serum levels of polyclonal immunoglobulins (Ig) is frequent in the latter increasing the risk of infectious complications [START_REF] Vacca | Subcutaneous immunoglobulins in patients with multiple myeloma and secondary hypogammaglobulinemia: a randomized trial[END_REF]. Furthermore, secondary hypogammaglobulinemia is also a wellknown late complication after rituximab treatment [START_REF] Boleto | Predictors of hypogammaglobulinemia during rituximab maintenance therapy in rheumatoid arthritis: A 12-year longitudinal multi-center study[END_REF]. Interestingly, a previous study showed that a specific clonal population of B cells resembling the exhausted CD21(low) B cells seen in HIV or in primary immunodeficiency conditions can accumulate in HCVassociated CryoVas [START_REF] Visentini | Clonal B cells of HCV-associated mixed cryoglobulinemia patients contain exhausted marginal zone-like and CD21 low cells overexpressing Stra13[END_REF]. In the context of immunodeficiency, induction of regulatory T cells with low-dose IL-2 for the treatment of refractory CryoVas represents a promising strategy [START_REF] Saadoun | Regulatory T-cell responses to low-dose interleukin-2 in HCV-induced vasculitis[END_REF].

Due to the rarity of this disorder, it is worthwhile mentioning that the majority of the studies for the management of relapsing CryoVas discussed in this review come from low level evidence uncontrolled observational or case series studies.

LegendsFigure 1 .

 1 Figure1. Proposed algorithm for pharmacological treatment of cryoglobulinemia vasculitis (CryoVas). IV, intravenous; PLEX, plasma exchange; GC, glucocorticoids; IL-2, interleukin-2.1 Severe kidney dysfunction, skin necrosis, involvement of the gastrointestinal tract or central nervous system;2 In cases of hepatitis B or HIV infection antiviral therapy should always be initiated before or at the same time as immunosuppressive therapy;3 In the presence of risk factors for rituximab-induced vasculitis flare (i.e. renal involvement, high levels of circulating cryoglobulin and low C4 levels) lower doses of rituximab should be used (250-500mg per infusion).

Figure 2 .

 2 Figure 2. The main pathophysiology and potential therapy targets of cryoglobulinemic vasculitis. HCV, hepatitis C virus; Th cell, T-helper cell; BlyS/BAFF, B Lymphocyte Stimulator/ B-cell activating factor; IL-6, interleukin-6; IL-2, interleukin-2; Treg, regulatory T cells.

Based on these data we propose an algorithm that can aid clinical decision making and pharmacological management of CryoVas (Figure 1). Figure 2 illustrates the pathophysiology and potential therapeutic targets of refractory/relapsing CryoVas.