Jean-Baptiste B Bachet

Hélène Blons

Pascal Hammel

Iman El Hariry

Fabienne Portales

Laurent Mineur

Jean-Philippe Metges

Claire Mulot

Camille Bourreau

Jason Cain

Jérôme Cros

Prof Pierre Laurent-Puig
email: pierre.laurent-puig@parisdescartes.fr

Circulating tumor DNA is prognostic and potentially predictive of eryaspase efficacy in second-line in patients with advanced pancreatic adenocarcinoma

Keywords: L-asparaginase, circulating tumor DNA, phase 2, prognostic, predictive

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Statement of translational relevance

Eryaspase is composed of L-asparaginase encapsulated in erythrocytes. It targets asparagine used by non-canonical pathways constitutively activated by KRAS signaling pathway in 80 to 90% of pancreatic cancers. In combination with chemotherapy, eryaspase was associated with a promising activity in a randomized phase 2 trial. Circulating tumor DNA (ctDNA) was assessed from plasma samples of patients included in this trial.

At baseline, ctDNA was detectable in two thirds of patients and was a strong prognostic factor. Early change of ctDNA level was significantly correlated with all oncologic outcomes. A significant interaction was observed between the presence of ctDNA and eryaspase efficacy.

We confirm in a phase 2 trial the interest of ctDNA, at baseline and in monitoring under treatment. Moreover, presence of ctDNA could be a predictive biomarker of eryaspase efficacy and had to be assessed in the ongoing phase 3 trial.

Introduction

Pancreatic adenocarcinoma (PAC) incidence increases in many countries and is expected to become the second leading cause of cancer death in 2030 [START_REF] Rahib | Projecting Cancer Incidence and Deaths to 2030: The Unexpected Burden of Thyroid, Liver, and Pancreas Cancers in the United States[END_REF]. Despite some progress, its prognosis remains poor with five-year survival rate of 9% all-stages combined [START_REF] Siegel | Cancer statistics, 2019[END_REF]. During the last 10 years, two chemotherapy regimens have demonstrated a clinical benefit in comparison to gemcitabine alone in first-line in metastatic patients: the FOLFIRINOX and the gemcitabine plus nab-paclitaxel regimen [START_REF] Thierry | FOLFIRINOX versus Gemcitabine for Metastatic Pancreatic Cancer[END_REF][START_REF] Hoff | Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine[END_REF]. The benefit of a second-line chemotherapy has been formally demonstrated in the CONKO-003 trial [START_REF] Pelzer | Best supportive care (BSC) versus oxaliplatin, folinic acid and 5-fluorouracil (OFF) plus BSC in patients for second-line advanced pancreatic cancer: a phase III-study from the German CONKO-study group[END_REF], and the combinations of 5-Fluorouracil (5-FU) plus nanoliposomal irinotecan and of 5-FU plus oxaliplatin have been validated as standard treatments after progression under a gemcitabine based first-line chemotherapy [START_REF] Wang-Gillam | NAPOLI-1 Study Group. Nanoliposomal irinotecan with fluorouracil and folinic acid in metastatic pancreatic cancer after previous gemcitabine-based therapy (NAPOLI-1): a global, randomised, openlabel, phase 3 trial[END_REF][START_REF] Ducreux | ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF].

New drugs developments are needed to improve our therapeutic possibilities and increase the overall survival of patients. One of the promising areas of research is to target some metabolites, such as glutamine or asparagine, used by non-canonical pathways constitutively activated by KRAS signaling pathway in 80 to 90% of pancreatic cancers [START_REF] Son | Glutamine supports pancreatic cancer growth through a KRAS-regulated metabolic pathway[END_REF][START_REF] Kawada | Targeting metabolic reprogramming in KRAS-driven cancers[END_REF].

Eryaspase is a novel approach to deliver asparaginase that is encapsulated within erythrocytes and administered as intravenous infusion. Eryaspase is well tolerated as monotherapy [START_REF] Bachet | Asparagine Synthetase Expression and Phase I Study With L-Asparaginase Encapsulated in Red Blood Cells in Patients With Pancreatic Adenocarcinoma[END_REF], and in combination with chemotherapy (gemcitabine or FOLFOX) in a randomized phase 2b trial [START_REF] Hammel | Erythrocyte-encapsulated asparaginase (eryaspase) combined with chemotherapy in secondline treatment of advanced pancreatic cancer: an open-label, randomized Phase IIb trial[END_REF]. Significant increases of progression free survival (PFS) (HR=0.56; 95% CI: 0.37-0.84; p= 0.005), and overall survival (OS) (HR= 0.60; 95% CI: 0.41-0.87; p= 0.008), and a good safety profile were observed. A randomized phase 3 trial is currently ongoing (EndraCT: 2018-000572-15).

Circulating tumor DNA (ctDNA) can be applied to monitor response and resistance to systemic therapy and to capture tumor heterogeneity better than tissue biopsies [START_REF] Siravegna | How liquid biopsies can change clinical practice in oncology[END_REF]. Its testing is highly specific but its sensitivity is influenced by technical issues and by tumor characteristics [START_REF] Siravegna | How liquid biopsies can change clinical practice in oncology[END_REF][START_REF] Bettegowda | Detection of Circulating Tumor DNA in Early-and Late-Stage Human Malignancies[END_REF][START_REF] Wan | Liquid biopsies come of age: towards implementation of circulating tumour DNA[END_REF][START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF]. In patients with PAC at diagnosis, the detection of ctDNA and the level of ctDNA if detectable are highly prognostic all stages combined [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Lee | Circulating tumor DNA as a prognostic indicator in resectable pancreatic ductal adenocarcinoma: A systematic review and meta-analysis[END_REF]. Moreover, preliminary data suggests that the evolution of ctDNA under chemotherapy may be an early predictive biomarker of treatment efficacy [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][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].

In this study, we investigated the prognostic and predictive value of ctDNA in the randomized phase 2b trial that compared chemotherapy with or without eryaspase in second-line treatment of patients with advanced PAC.

Patients and Methods

Study design, patients and sample collection

This multicenter, open-label, randomized phase 2b trial was conducted by the GERCOR group ("Groupe coopérateur multidisciplinaire en Oncologie") and was sponsored by Erytech Pharma (NCT02502656). All the patients gave their written informed consent. The trial was conducted in accordance with the protocol and principles of International Conference of Harmonization Good Clinical Practices and Declaration of Helsinki and was approved by an independent ethics committee. Details of this study have been previously published [START_REF] Hammel | Erythrocyte-encapsulated asparaginase (eryaspase) combined with chemotherapy in secondline treatment of advanced pancreatic cancer: an open-label, randomized Phase IIb trial[END_REF].

Briefly, eligible patients were randomized in a 2:1 ratio to receive eryaspase plus chemotherapy (gemcitabine or mFOLFOX6 according to previously administered first-line) or chemotherapy alone. Tumor assessments were done every 8 weeks using RECIST 1.1 criteria.

As part of the study, plasma samples of included patients were collected at day 1 of first cycle (baseline), second cycle and third cycle.

Circulating tumor DNA assessment

ctDNA detection was performed by NGS as previously described [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF]. Briefly, Circulating cell-free DNA was extracted from plasma by using the Maxwell® RSC ccfDNA Plasma Kit (Promega, France) and sequenced using the AmpliSeq Colon and Lung Cancer Panel V2 (22 genes). Samples were analyzed after BAM recalibration using a specific algorithm developed to detect allelic ratios <2%, the BPER method.

Statistical analyses

The analysis plan included the evaluation of objective response rate (ORR), PFS and OS in the intent to treat (ITT), and as a function of the treatment arm (eryaspase vs control).

The median (range) or mean (standard deviation, SD) and frequency (percentage) were used to describe continuous and categorical variables, respectively. Medians and proportions were compared using the Wilcoxon-Mann-Whitney and chi-square test, respectively, or with Fisher's exact test if appropriate. PFS and OS were estimated using the Kaplan-Meier method.

As exploratory purpose univariate and multivariate Cox analyses were done to estimate hazard ratio with 95% confidence interval (CI).

For quantitative analysis of ctDNA we used variant allelic fraction (VAF). In case of several mutations identified in the same patient, the highest value was retained. The prognostic value of ctDNA was assessed by a Cox model. To draw survival curves, ctDNA value was divided in VAF tertiles.

To assess the early variation of ctDNA, we calculated a ratio between the VAF measured at day 1 of first cycle (day 1) and at day 1 of the second cycle (day 28). We used the maximal VAF (maxVAF) at day 1 of first cycle (P1); the corresponding mutation was selected and if the mutation was present in the plasma sampling at day 1 of the second cycle (P2) the ratio was calculated as follow: (maxVAF_P1mutA-VAF_P2mutA) / maxVAF_P1mutA. If the mutation corresponding to the maximal VAF at P1 was not present at P2 and if one or more mutations was present at P2, the common mutations between the 2 time points were selected and the mutation with the highest VAF at P2 was used to calculate the ratio as follow: (VAF_P1mutA-VAF_P2mutA)/ VAF_P1mutA (this type of calculation was used in 2 cases). If no mutation was present at P2 the ratio was calculated as follow: maxVAF_P1mutA/maxVAF_P1mutA leading to a ratio at 1 (this type of calculation was used in 11 cases). If no mutation was present at P1 and one or more mutation was present at P2 the ratio was calculated at follow: (0-maxVAF_P2mutA)/ VAF_P2mutA and lead to a ratio of -1 (this type of calculation was used in 4 cases). In the cases with 2 different unique mutations present at the 2 time points, the ratio was calculated as follow VAF_P1mutA-VAF_P2mutB/VAF_P1mutA (this type of calculation was used in 1 case). Based on this ratio, we defined 3 groups of patients: patients with no detectable ctDNA at P1 and P2 were classified in a "negative ctDNA" group; patients with ctDNA at least P1 or P2 were divided in 2 groups according to the ratio median value. For both arms combined, prognostic value of defined groups was assessed according to RECIST criteria (ORR and disease control rate), then on PFS and OS.

Finally, we evaluated the effect of ctDNA at baseline on efficacy of treatment by assessing an interaction between the presence of ctDNA and survival (OS and PFS) using a univariate and multivariate analyses. A significant p-value was defined as a p-value <0.01 at the exception of interaction analyses. A significant interaction was defined by a p-value <0.05, and an interesting interaction by a p-value <0.1. All statistical analyses were performed with R survival package.

Results

Patients' characteristics

At least one plasma sample was available in 122 of the 141 patients (87%). Plasma samples were available at baseline in 117 patients and at one month (day 1 of 2 nd cycle) in 88 patients.

Characteristics at baseline of the 122 patients with at least one plasma sample available were not different of those of the 141 patients included in the phase IIb trial (see supplementary Data File S1). OS and PFS of the patients with or without available plasma sample were not different (see supplementary Data File S2). Similar to the results in the ITT population (N=141), eryaspase was associated with a significant improvement of both OS and PFS in this subgroup of 122 patients (see supplementary Data File S2).

ctDNA analysis at baseline

For baseline time point, failure of sequencing was observed in 4 patients either due to too low amount of DNA or non-interpretable sequencing. CtDNA was detectable by the presence of at least one mutated gene in 77 of 113 patients (68.1%). Among those, the most frequent mutated genes were KRAS (n=64, 83%) and TP53 (n=38, 49%). The number of mutated genes identified was 4 in one patient (1%), 3 in 9 patients (12%), two in 33 patients (43%) and one in 34 patients (44%). The most frequent combination of mutations, KRAS and TP53, was observed in 34 patients (44%).

No difference in baseline characteristics was observed between patients with or without detectable ctDNA (see supplementary Data File S3).

Prognostic value

Among the 113 patients for whom NGS analysis was available at baseline, the presence of ctDNA was associated with significant shorter OS (median 4.6 vs 8.8 months; p=0.0025) and PFS (median 1.6 vs 3.3 months; p= 0.00043). The hazard ratio (HR) adjusted to gender, CA19.9 at baseline, treatment arm, number of metastatic site and delay before the inclusion were 1.92 (95% CI: 1.18-3.1) and 2.13 (95% CI: 1.3-3.5) for OS and PFS, respectively (Figure 1A and1B). ORR was not significantly different among patients with or without detectable ctDNA: 10.4% (8/77) vs 13.9% (5/36), respectively.

The mean of the maximum of the variant allelic fraction (VAF) at baseline was 0.097 (median: 0.025; range [0-0.74]). The maximal VAF frequency observed at baseline was significantly negatively correlated with OS (HR=23.9; 95% CI: 6.7-84.6; p<0.0001) and PFS (HR=8.27; 95% CI: 2.4-28.3; p=0.0008) in univariate analysis. In multivariate analysis, a significant correlation was observed with OS (HR=16.8; 95% CI: 3.83-73.58; p=0.00018) and a clear trend with PFS (HR= 5.66; 95% CI: 1.43-22.39; p= 0.0135) (see supplementary Data File S4). Prognostic value of ctDNA at baseline was then analyzed by tertiles according to VAF frequency (cut points: 0.007 and 0.0683). Characteristics of patients of three tertiles are described in supplementary Data File S5. Higher tertiles of VAF frequency was prognostic of OS and PFS (Figure 2A and2B).

Quantitative monitoring of ctDNA under treatment

The mean of the VAF ratio between P1 and P2 was 0.12 (IQR: 1.06) and the median 0.28 (range [-5.33-1]). Twenty-five patients were classified in the "negative ctDNA" group (no ctDNA at P1 and P2); 23 in the "ctDNA responders" group and 23 in the "ctDNA nonresponders" group.

ORR was correlated with the groups defined by ctDNA variation: 20 % (5/25), 26% (6/23) and 0% (0/23) in "negative ctDNA", "ctDNA responders" and "ctDNA non-responders" groups (p<0.04), respectively. Significant correlation was observed for disease control rate: 68 % (n=17/25), 61% (14/23) and 22% (5/23) in "negative ctDNA", "ctDNA responders" and "ctDNA non-responders" groups (p=0.002), respectively.

For both arms combined, the groups defined by ctDNA variation were significantly correlated with OS and PFS (Figure 3A and3B).

Plasma samples were available before the first day of the three cycles in 40 patients. Patients who had a disease control had more frequently a negative ctDNA or a decrease of the maximal VAF after treatment initiation (supplementary Data File S6).

ctDNA and treatment arm

For OS, an interesting interaction between the presence of ctDNA at baseline and eryaspase efficacy was observed in univariate (HR=0.39; 95% CI: 0.14-1.09; p=0.073) and multivariate (HR= 0.35; 95% CI: 0.12-1.05; p= 0.060) analyses. For PFS, there was a trend for an interesting interaction in univariate analysis (HR=0.47; 95% CI: 0.18-1.22; p=0.122) and in multivariate (HR= 0.42; 95% CI: 0.15-1.20; p= 0.105) analysis (see supplementary Data File S7).

Among patients with detectable ctDNA at baseline, eryaspase was associated with a not significant increase in disease control rate (47% (n= 15/32) vs 29% (n= 4/14); p= 0.3) and a significant increase of OS (HR=0.52; 95% CI: 0.29-0.91: p<0.001) and PFS (HR=0.53; 95% CI: 0.3-0.94; p<0.001). No significant difference of treatment efficacy was observed in the subgroup of patients with unknown ctDNA status (Figure 4A and4B).

No correlation was observed between asparagine synthetase (ASNS) expression level and ctDNA status. Among patients with negative and positive ctDNA at baseline, null-low (0-1) ASNS expression rates were 72% (n=26/36) and 68% (n=52/77) (p=0.776), respectively.

Discussion

The aim of this exploratory translational study was to evaluate the prognostic and predictive value of ctDNA from plasma samples collected during the randomized phase 2 trial which has assessed eryaspase efficacy in second-line in patients with advanced PAC. For this, we used a NGS method adapted to plasma analysis and allowing to search the main mutated genes in PAC [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF]. The prognostic value of ctDNA at diagnosis (baseline) is now well established in several cancer types, and more specifically in PAC, irrespective of the stage of the disease [START_REF] Bettegowda | Detection of Circulating Tumor DNA in Early-and Late-Stage Human Malignancies[END_REF][START_REF] Wan | Liquid biopsies come of age: towards implementation of circulating tumour DNA[END_REF][START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Lee | Circulating tumor DNA as a prognostic indicator in resectable pancreatic ductal adenocarcinoma: A systematic review and meta-analysis[END_REF][START_REF] Buscail | High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery[END_REF]. The rate of detectable ctDNA at diagnosis accounts for approximately two thirds of patients with metastatic disease [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][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][START_REF] Buscail | High Clinical Value of Liquid Biopsy to Detect Circulating Tumor Cells and Tumor Exosomes in Pancreatic Ductal Adenocarcinoma Patients Eligible for Up-Front Surgery[END_REF][START_REF] Pietrasz | Methylated circulating tumor DNA (Met-DNA) as an independent prognostic factor in metastatic pancreatic adenocarcinoma (mPAC) patients[END_REF][START_REF] Takai | Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer[END_REF]. The current study represents the largest cohort investigating the prognostic and predictive value of quantitative ctDNA in second-line setting.

The prevalence rate of 68% is similar to that found in other studies of metastatic PAC. To our knowledge, the features of metastatic PAC patients with undetectable ctDNA are not well described, unlike metastatic colorectal cancer for which metastatic spread to the liver is clearly correlated to detectable ctDNA [START_REF] Jones | Core signaling pathways in human pancreatic cancers revealed by global genomic analyses[END_REF]. The detection of ctDNA seems correlated with differentiation grade and CA 19-9 level but not clearly with tumor burden [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF]. Given the strong prognostic value of ctDNA at baseline, future studies should aim to better define the subset of patients with undetectable ctDNA, with the goal of stratifying future trials based on ctDNA content.

In addition to its prognostic value at diagnosis, the value of the dynamic change of ctDNA during follow-up is very promising [START_REF] Siravegna | How liquid biopsies can change clinical practice in oncology[END_REF][START_REF] Bettegowda | Detection of Circulating Tumor DNA in Early-and Late-Stage Human Malignancies[END_REF][START_REF] Wan | Liquid biopsies come of age: towards implementation of circulating tumour DNA[END_REF]. Indeed, the effect of chemotherapy on ctDNA levels could inform on treatment efficacy [START_REF] Pietrasz | Plasma Circulating Tumor DNA in Pancreatic Cancer Patients Is a Prognostic Marker[END_REF][START_REF] Takai | Clinical utility of circulating tumor DNA for molecular assessment in pancreatic cancer[END_REF]. In a prospective cohort of patients with advanced PAC, a significant correlation between the kinetics of ctDNA, from day 0 to day 14, and objective response rate was reported [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]. Moreover, an increase in ctDNA level under treatment indicated progression with a sensitivity of 83% and specificity of 100%. Changes in ctDNA levels appeared more informative than changes in more "classical" biomarkers such as CA 19-9 and CEA [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]. In our study, we assessed the value of early changes, between days 0 and 28, in ctDNA levels and identified three distinct groups that were strongly correlated with ORR, PFS and OS. Generally, only half of PAC patients are able to receive second line therapy [START_REF] Thierry | FOLFIRINOX versus Gemcitabine for Metastatic Pancreatic Cancer[END_REF][START_REF] Hoff | Increased Survival in Pancreatic Cancer with nab-Paclitaxel plus Gemcitabine[END_REF][START_REF] Ducreux | ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up[END_REF], therefore, it is prudent to argue that identifying early disease progression based on increasing levels of ctDNA and subsequently instating salvage therapy before objective disease progression may have a positive effect on OS and improving the patients outcome.

The main strengths of this exploratory study are that approximately 85% of the patients in this trial had analyzable ctDNA samples, confirming the utility of our approach and the correlation with efficacy indicators in the ITT population. While our results are promising, there are still several outstanding issues that should be addressed in a prospective clinical trial, such as the optimal timing for collecting ctDNA samples, the ctDNA threshold and the different methods available to evaluate ctDNA. Furthermore, the clinical utility of using early evolution of ctDNA during treatment follow-up should also be addressed in future trials.

The eryaspase benefit appeared most significant for OS and PFS in the subgroup of patients with detectable ctDNA, which is also the subgroup of patients with the worst prognosis. This result could be explained either by an efficacy of eryaspase on circulating tumor cells (potentially correlated to ctDNA), or by an increase anticancer drug delivery in the subgroup of pancreatic adenocarcinomas with ctDNA positivity. Given this correlation, these results provide further support for the role of targeting metabolic pathways as a novel therapeutic modality in PAC. Interestingly, to our knowledge no specific therapy has demonstrated efficacy in a subgroup defined by the presence of ctDNA. PAC is characterized by extensive reprogramming of cellular metabolism, specifically a robust glycolytic activity and glutamine addiction [START_REF] Jones | Core signaling pathways in human pancreatic cancers revealed by global genomic analyses[END_REF], which plays a role in controlling proliferation of cancer cells and enabling invasion and metastasis in a nutrient-poor, hypoxic microenvironment. These metabolic switches are driven by the acquisition of activating KRAS mutations [START_REF] Seo | Autophagy is required for PDAC glutamine metabolism[END_REF]. Therefore, eryaspase provides a novel therapeutic target for the treatment of PAC [START_REF] Bachet | Asparagine Synthetase Expression and Phase I Study With L-Asparaginase Encapsulated in Red Blood Cells in Patients With Pancreatic Adenocarcinoma[END_REF][START_REF] Hammel | Erythrocyte-encapsulated asparaginase (eryaspase) combined with chemotherapy in secondline treatment of advanced pancreatic cancer: an open-label, randomized Phase IIb trial[END_REF]. The current results provide a rationale for utilizing ctDNA as a potential marker of response. At the opposite, no benefit of eryaspase was observed in the subgroup of patients with undetectable ctDNA at baseline. This observation could be due to the small number of patients of this subgroup (n=36; 8 vs 28). It is therefore important to confirm these findings in the ongoing phase 3 trial. ASNS expression was not predictive of eryaspase efficacy in the phase 2 trial, and no correlation was found between ASNS expression and ctDNA status.

In conclusion, we confirm that the presence of ctDNA at baseline is a prognostic factor in patients with advanced PAC. The feasibility of this approach and its potential prognostic value provides a rationale for stratifying patients in future clinical trials. Our results suggest that presence of ctDNA could be a predictive biomarker of eryaspase efficacy. Taken together, the detection of ctDNA in approximately two thirds of patients with metastatic PAC and eryaspase being associated with a good safety profile, argue for future first-line development of this combination in the event of a positive phase 3 results.

Figure 1A .

 1A Figure 1A. Overall survival curves according to the presence or absence of ctDNA at

Figure 1B .

 1B Figure 1B. Progression free survival curves according to the presence or absence of ctDNA at

Figure 2A .

 2A Figure 2A. Overall survival curves according to the tertiles defined by the VAF frequency of

Figure 2B .

 2B Figure 2B. Progression free survival curves according to the tertiles defined by the VAF

Figure 3A .

 3A Figure 3A. Overall survival according to the groups defined by ctDNA variation between first

Figure 3B .

 3B Figure 3B. Progression free survival according to the groups defined by ctDNA variation

Figure 4A .

 4A Figure 4A. Overall survival curves according to ctDNA at baseline and treatment arm.

Figure 4B .

 4B Figure 4B. Progression free survival curves according to ctDNA at baseline and treatment

5 HR: 2 .

 52 Figure 1B

  Figure 3 A/

  Supplementary Data File S4: Multivariate analysis of prognostic factors at baseline Factors estimate std.error statistic p.value conf.low conf.high

	Figure 4B
	Supplementary Data File S1: Description of the studied population Supplementary Data File S5: Description of patients' characteristics according to tertiles of
	ctDNA VAF frequency at baseline
		Patients included in	Patients included in the
	0.25 0.50 0.75 1.00 0.25 0.50 0.75 1.00 maximal VAF frequency observed at Overall survival Disease Free survival Age Mean (SD) Median [Min, Max] Gender F M Treatment Arm intent to treat Control arm Eryaspase arm CA19.9 Normal vs Elevated Normal Overall Survival Age Mean (SD) Median [Min, Max] Age baseline* Treatment Arm: Eryaspase vs Control Mean (SD) Age* Median [Min, Max] Gender: Male vs Female Gender Female Gender F CA19.9 levels at baseline: Normal vs Elevated Male M Delay before inclusion* Treatment Arm intent to treat Number of metastatic sites* Control Treatment Arm intend to treat Control Eryaspase Eryaspase Progression Free Survival CA19.9 Normal vs Elevated maximal VAF frequency observed at Normal baseline* 0.00 0.00 Elevated CA19.9 Normal vs Elevated Treatment Arm: Eryaspase vs Control Elevated	+ Strata + p = 2e-04 + + + + Log-rank p = 0.00015 Log-rank Control Arm & Presence of ctDNA median: 1.4 m + Control Arm & Presence of ctDNA median: 3.1 m + Eryaspase Arm & Presence of ctDNA median: 5.8 m Eryaspase Arm & Absence of ctDNA median: 7.3 m Control Arm & Absence of ctDNA median: 11.7 m + + Control Arm and Absence of ctDNA at baseline Control Arm and Presence of ctDNA at baseline Eryaspase Arm and + + HR* (Eryaspase vs Control): 0.52 CI95%: 0.29-0.91 HR* (Eryaspase vs Control): 0.93 CI95%: 0.32-2.67 *Adjusted for Age, Gender, CA19.9 level at baseline, Number of metastatic sites, delay before inclusion + Absence of ctDNA at baseline Eryaspase Arm and Presence of ctDNA at baseline Eryaspase Arm & Presence of ctDNA median: 1.7 m Eryaspase Arm & Absence of ctDNA median: 3.2 m Control Arm & Absence of ctDNA median: 4.5 m HR* (Eryaspase vs Control): 0.53 CI95%: 0.3-0.94 HR* (Eryaspase vs Control): 0.99 CI95%: 0.38-2.57 *Adjusted for Age, Gender, CA19.9 level at baseline, Number of metastatic sites, delay before inclusion Strata Control Arm and Absence of ctDNA at baseline Control Arm and Presence of ctDNA at baseline Eryaspase Arm and Absence of ctDNA at baseline Eryaspase Arm and Presence of ctDNA at baseline translational study (n=122) phase IIb trial (n=141) 62.4 (9.48) 62.6 (9.72) 63.0 [37.0, 84.0] 63.0 [37.0, 84.0] 52 (42.6%) 58 (41.1%) 70 (57.4%) 83 (58.9%) 36 (29.5%) 46 (32.6%) 86 (70.5%) 95 (67.4%) 22 (18.0%) 25 (17.7%) No detectable Detectable ctDNA Overall 1st tertile 2nd tertile 3rd tertile (n=38) (n=37) (n=38) ctDNA (n=36) (n=77) (n=113) 61.4 (9.70) 62.3 (8.93) 62.6 (9.72) 62.5 [42.0, 80.0] 63.0 [37.0, 84.0] 63.0 [37.0, 84.0] 16.80 0.75 3.74 0.00018 3.83 73.58 0.97 0.26 -0.10 0.92 0.58 61.6 (9.57) 61.8 (9.56) 62.6 (8.51) 1.63 0.98 0.01 -1.31 0.20 0.96 62.5 [42.0, 80.0] 64.0 [37.0, 78.0] 62.5 [42.0, 84.0] 1.01 0.87 0.23 -0.62 0.53 0.56 1.35 17 (44.7%) 16 (43.2%) 16 (42.1%) 15 (41.7%) 34 (44.2%) 0.86 0.30 -0.50 0.61 0.48 1.55 21 (55.3%) 21 (56.8%) 22 (57.9%) 58 (41.1%) 21 (58.3%) 43 (55.8%) 1.00 0.01 -0.44 0.66 0.97 1.02 83 (58.9%) 0.98 0.17 -0.09 0.93 0.71 8 (21.1%) 9 (24.3%) 17 (44.7%) 1.37 8 (22.2%) 26 (33.8%) 46 (32.6%) 28 (77.8%) 51 (66.2%) 95 (67.4%) 30 (78.9%) 28 (75.7%) 21 (55.3%) 5.66 0.70 2.47 0.0135 1.43 22.39 6 (15.8%) 6 (16.2%) 7 (18.4%) + + 88 (72.1%) 97 (68.8%) 0.87 0.25 -0.57 0.56 0.53 1.42 27 (71.1%) 31 (83.8%) 25 (65.8%)
	0 8 26 28 51 0 Number at risk 2 7 19 28 43 2 0 5 (13.9%) 4 6 10 23 34 4 2 1.00 5 (13.2%) 8 5 25 5 26 17 43 19 0 2 Number at risk Time from initial diagnosis to randomization ----Strata Missing Normal Age* Missing ----Strata Mean (SD) Elevated 26 (72.2%) Gender: Male vs Female 0.93 Time from initial diagnosis to randomization Median [Min, Max] 8.10 [2.50, 86.9] 6 8 10 5 4 4 6 2 1 14 14 11 23 9 7 6 8 10 4 6 12 (9.8%) 14 (18.2%) 12 3 0 9 4 12 8 0.01 -0.37 0.71 14 0 0 6 4 14 0 (0%) 4 3 2 2 0 0 8 8 2 5 4 1 4 6 8 11.0 (10.1) 57 (74.0%) 0.22 -0.30 0.76 Number of metastatic sites Mean (SD) 1.37 (0.645) Median [Min, Max] 1.00 [0.00, 5.00] Missing 5 (13.9%) 6 (7.8%) CA19.9 levels at baseline: Normal vs 0.82 0.29 -0.69 0.48 Mean (SD) 12.8 (14.0) 10.1 (8.33) Elevated* Time from initial diagnosis to Delay before inclusion* 1.00 0.01 0.10 0.92 Median [Min, Max] 9.92 [2.76, 86.9] 7.98 [2.50, 47.9] randomization Mean (SD) 12.9 (14.2) 9.96 (7.40) Number of metastatic sites* 1.07 0.19 0.35 0.73 Number of metastatic sites Mean (SD) 1.45 (0.555) 1.35 (0.588) Median [Min, Max] 9.92 [2.76, 86.9] 7.98 [2.50, 47.9] Median [Min, Max] 1.00 [1.00, 3.00] 1.00 [1.00, 3.00] Number of metastatic sites 1 or 2 sites 117 (95.9%) Number of metastatic sites Abbreviation: VAF: variant allelic fraction Number of metastatic sites more than 2 sites 5 (4.1%) Mean (SD) 1.44 (0.558) 1.38 (0.689) * Continuous variables 1 or 2 sites 37 (97.4%) 35 (94.6%)	16 0 0 5 3 16 19 (13.5%) 18 0 0 5 3 18 12 25 (17.7%) 20 0 0 2 2 20 0.97 6 (15.8%) 1.02 14 0 0 0 0 2 1 1 0 12 14 97 (68.8%) 0.60 1.45 10.7 (9.71) 8.08 [2.50, 86.9] Months Months 10 Months 1 0 2 1 10 Months 1.39 (0.674) 19 (13.5%) 0.46 1.44 9.69 (6.33) 0.98 1.03 7.90 [2.99, 31.4] 0.74 1.54 10.7 (9.71) 1.39 (0.790) 1.00 [0.00, 5.00] 8.08 [2.50, 86.9] 1.00 [1.00, 5.00] 134 (95.0%) 7 (5.0%) 1.39 (0.674) 36 (94.7%)	22 0 0 2 2 22	24 0 0 2 2 24 16 0 0 1 0 16	26 0 0 1 0 26	18 0 0 1 0 18	28 0 0 1 0 28	30 0 0 1 0 30 20 0 0 0 0 20
	Median [Min, Max] more than 2 sites	1.00 [1.00, 3.00] 1 (2.6%)	1.00 [1.00, 5.00] 2 (5.4%)	1.00 [0.00, 5.00] 2 (5.3%)
	Number of metastatic sites
	1 or 2 sites	35 (97.2%)	73 (94.8%)		134 (95.0%)
	more than 2 sites	1 (2.8%)	4 (5.2%)		7 (5.0%)

Acknowledgements

The authors would like to thank all patients and their families participating in the trial and the SIRIC CARPEM for facilitating the access to the sequencing platform.

work. P. Hammel has received personal fees from Amgen, AstraZeneca, Celgene, Sanofi. J.P. Metges has received personal fees Bristol-Myers Squibb, Lilly, Novartis, Sanofi, and Syncore. J. Cain is employed by Erytech Pharma. J. Cros has received personal fees from Novartis. P. Laurent-Puig has received grants from AGEO during the conduct of the study; personal fees from Amgen, Merck Serono, Boehringer Ingelheim, Astrazeneca, Pfizer, Bristol-Myers Squibb, outside the submitted work.

Funding

This work was funded by ERYTECH Pharma. The funding source was involved in the design, interpretation or conduct of this evaluation, manuscript writing, and the decision to submit or publish the data. However, the authors are fully responsible for the content of this manuscript, and the views and opinions described in the publication reflect solely those of the authors.

Authors' Contributions

Conception and design: J.B. Bachet, I. El Hariry, J. Cain, J. Cros, P. Laurent-Puig Development and methodology: J.B. Bachet, H. Blons, I. El Hariry, J. Cain, J. Cros, P.

Laurent-Puig.

Acquisition of data (provided animals, acquired and managed patients, provided facilities, etc..): J. B. Bachet S2a: Overall survival of patients with or without at least one available plasma sample.

Median overall survivals were of 5.6 and 4.7 months for patients with and without at least one available plasma sample (p=0.52), respectively. Median progression free survivals were of 1.8 and 2.1 months for patients with and without at least one available plasma sample (p=0.61), respectively. Supplementary Data File S3: Patients characteristics at baseline according the presence or not of ctDNA Supplementary Data File S6: Evolution of the maximal variant allelic fraction during the first three cycles in the 40 patients with all three available points.

For each graph, the number on the bottom line corresponds to the number of patients with nondetectable (negative) ctDNA at each point.

Supplementary Data File S7: Interaction between the presence of ctDNA at baseline and eryaspase efficacy on PFS and OS