Abstract: Introduction Aggressive B-cell lymphomas are heterogeneous in their clinical course and biological characteristics. They include diffuse large B-cell lymphoma not otherwise specified (DLBCL-NOS), high grade-B-cell lymphoma with double/triple-hit or NOS (HGBL), primary mediastinal B-cell lymphoma (PMBL). In order to better differentiate these entities, the WHO classification recommends using immunohistochemistry (IHC), FISH, targeted sequencing and gene expression profiles (GEP). However, these techniques are most often performed retrospectively in clinical trials, which is not representative of real life. In order to use these information to improve the current standard of treatment with targeted therapies adapted to lymphoma biology, we have set up a national network on behalf of the LYSA called RT3 (for Real-time tailored therapy) to demonstrate that we are able to comprehensively characterize aggressive B cell lymphomas in a clinically relevant timeline. Materials and methods Patients & gt; 18 years of age with untreated aggressive B-cell lymphoma were included prospectively from 21 LYSA centers. FFPE specimens were analyzed and classified according to WHO classification, benefiting from an IHC profile (Hans algorithm, BCL2, MYC), FISH analysis (BCL6, BCL2, MYC break apart and MYC-IGH/IGL/IGK fusion probes), targeted NGS analysis and a targeted GEP using RT-MLPA (Bobée et al. J Mol diagn 2017). A first part of the study phase was carried out in an oligocentric manner with only 2 referral platforms for pathological analysis and molecular characterization. The second phase was the implementation of 7 RT3 platforms spread over France. The main objective was to evaluate the capacity of the network to provide an exhaustive histopathological and molecular characterization 4 days before theoretical R-CHOP21 C3 administration (within 38 days after D1 cycle 1). Results The oligocentric cohort 1 prospectively included 72 patients in 6 months: 19 had insufficient material or inappropriate diagnosis to be qualified and 53 benefited from the complete analysis on referral platforms. A complete characterization 4 days before RCHOP-C3 was provided to the clinician in 47 cases (88.7%), allowing to further implement the network with 7 platforms and 23 clinical investigation centers. 183 patients were included in the second phase in 9 months, 35 were excluded for inadequate diagnosis/material. On this population, 143 (96.6%) complete patient-reports were provided with a median time of 32 days (1-50). Finally, 201 cases were retained in the Full Analysis Set for which tumor samples fulfilled all prerequisites and diagnosis of DLBCL was confirmed by RT3 platforms. The clinical characteristics were as follows: median age of 61 y (20-92), with 45.2% of pts with aaIPI 2-3and 67.1% with Ann Arbor stage III-IV; 1L treatment consisted mainly of RCHOP (RCHOP14/21 = 136, 72.3%). 7% of patients were treated with experimental drugs. 76% presented with extranodal involvement. After pathological review, 139 (69%) patients were classified as DLBCL-NOS (41% GCB; 58% nGCB), 11 (5%) as HGBL-NOS, 8 (4%) as HGBL-DH and 18 (9%) as PMBL, 3 (1%) as EBV+ DLBCL-NOS, 9 (4%) as FL-3B, 7 (3%) transformed DLBCL, 2 (1%) plasmablastic, 1 (0.5%) unclassified, 1 (0.5%) DLBCL with IRF4 rearrangement. By immunohistochemistry, 22 cases were CD5+, 74% were BCL2+, and 53% MYC+ and 49% as double expressors. BCL2/18q21, BCL6/3q27 and MYC/8q24 breaks were observed in 14% (29/201), 25% (50/201) and 12% (25/201) of the cases respectively. MYC partner gene was available in 10/29 cases (5 MYC-IG, 5 non-IG). At the molecular level, PIM1 was the most frequently mutated gene in the entire cohort (36%). In the ABC subtype, MYD88 was mutated in 48% of cases. In the GCB cohort, SOCS1 was the most frequently mutated gene (30%). In the HGBL cohort, MYC was the most mutated gene (43%). Conclusion This study demonstrates that this RT3 LYSA network allowed a real time pathological and molecular characterization of aggressive B-cell lymphomas according to the WHO recommendations. The RT3 network provides relevant informations to the clinician before R-CHOP21 C3, that might contribute to modify the treatment eventually adding targeted therapies or tailor a second line treatment. This network should be used as a basis for future clinical trials. Inter platforms reproducibility and prognostic relevance of the RT3 project will be presented. Disclosures Haioun: Gilead: Honoraria; Janssen: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Takeda: Honoraria; Miltenyi: Honoraria; Servier: Honoraria; Roche: Honoraria; Celgene: Honoraria. Le Gouill:Roche Genentech, Janssen-Cilag and Abbvie, Celgene, Jazz pharmaceutical, Gilead-kite, Loxo, Daiichi-Sankyo and Servier: Honoraria; Loxo Oncology at Lilly: Consultancy. Ribrag:arGEN-X-BVBA: Research Funding; BAY1000394 studies on MCL: Patents & Royalties; Institut Gustave Roussy: Current Employment; argenX: Current equity holder in publicly-traded company, Research Funding; Epizyme: Consultancy, Current equity holder in publicly-traded company, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees; Infinity: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; MSD: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; BMS: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; Nanostring: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; F. Hoffmann-La Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel Expenses; AstraZeneca: Honoraria, Membership on an entity's Board of Directors or advisory committees; Gilead: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Servier: Consultancy, Honoraria; Pharmamar: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; AZD: Honoraria, Other; Eisai: Honoraria; Incyte: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Immune Design: Consultancy, Membership on an entity's Board of Directors or advisory committees; Roche/Genentech: Consultancy, Membership on an entity's Board of Directors or advisory committees. Salles:Takeda: Honoraria; Karyopharm: Honoraria; Genmab: Honoraria, Other; Debiopharm: Consultancy, Honoraria, Other: consultancy or advisory role; Autolos: Other: consultancy or advisory role; Abbvie: Other: consultancy or advisory role; Roche: Honoraria, Other: consultancy or advisory role; Novartis: Honoraria, Other: consultancy or advisory role; MorphoSys: Honoraria, Other: consultancy or advisory role; Janssen: Honoraria, Other: consultancy or advisory role; Epizyme: Honoraria, Other: consultancy or advisory role; Kite, a Gilead Company: Honoraria, Other: consultancy or advisory role ; BMS/Celgene: Honoraria, Other: consultancy or advisory role. Sujobert:Sunesis: Research Funding; Janssen: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Gilead/Kyte: Membership on an entity's Board of Directors or advisory committees. Bouabdallah:Gilead Sciences: Consultancy, Honoraria; Takeda: Consultancy, Honoraria; Roche: Consultancy, Honoraria. Sibon:takeda france: Consultancy.

Abstract: Background: Recent advances in next-generation sequencing (NGS) techniques enable the quantitation of circulating tumor DNA (ctDNA) encoding the clonal rearranged V(D)J immunoglobulin (IG) receptor gene sequence. The aims of this study were 1) To evaluate the clonal heterogeneity of follicular lymphoma (FL) and its variations between tumor and plasma at diagnosis in patients (pts) included in PRIMA trial, and 2) To assess the prognostic value of the level of ctDNA at diagnosis on PFS (median follow up of 6 years). Methods: Using the NGS-based immunosequencing method (Adaptive Biotechnologies, South San Francisco, CA), the lymphoma clonotype was first established in the tumor biopsy using locus-specific primer sets for IGH-V, IGH-D and IGK rearrangements. Clonotypes regarded as originating from the lymphoma clone were present at a frequency greater than 5%. The lymphoma-derived sequences identified in the biopsy sample were then used as targets to assess the presence of ctDNA in plasma samples. The quantity and frequency of the ctDNA in plasma is calculated relative to the total number of reads in the sample and defined as lymphoma clonotype molecules per million diploid genomes. For 34 FL pts from the PRIMA trial, both tumor biopsy and plasma samples at diagnosis were available. Results: One tumor clonotype or more could be detected in 29 pts (85%) in the tumor diagnostic sample (DNA isolated from fresh frozen tissue). Indeed, with the IGH-V assay, between 1 to 3 different clonotypes were identified in 23 pts. With the IGH-D assay, 1 clonotype was detected in 2 pts. With the IGK assay, 1 to 2 different clonotypes were detected in 17 pts. The tumor clonotypes were detected in diagnostic plasma samples in 25 out of the 29 pts (83%). Moreover, in 14 pts we detected clones in the plasma samples that are related (with point mutations in the V(D)J sequence) to the highest frequency index clone identified in the tumor sample. In half of the cases, we observed a different repartition of the subclonal populations between tumor and plasma samples at diagnosis: for these cases the highest frequency clone in the tumor was not the highest frequency clone in the plasma. Among the 24 pts with an IGH clonotype (IGH-V or IGH-D assay), ctDNA could be detected in 19 cases. We could not find a correlation between peripheral blood dissemination and the presence of a stereotyped sequence or an N-glycosylation motif within the CDR3 region of IGH clonotypes. Interestingly, in one case, two different related clones were detected at high frequency in tumor sample and the putative parental clone, not detected in the tumor sample, was detected in plasma sample as the highest frequency clone. The level of ctDNA ranged from 0 to 345,000/million diploid genomes in the 29 plasma samples (median 39,720). The presence of ctDNA was correlated with bone marrow involvement (p=.005), but not with FLIPI score, LDH level, anemia, bulky disease, presence of circulating lymphoma cells (detected by morphology or flow examination), Ann Arbor stage or β2microglobulin level. The absolute level of ctDNA in diagnostic samples did not correlate with any of the clinical characteristics. We assessed the prognostic value of the level of ctDNA at diagnosis. Pts (14/29) with higher levels of ctDNA at diagnosis ( 〉 40,000/million diploid genomes) had shorter PFS than patients with lower levels of ctDNA (median 15.7 m vs NR, p=.027, Fig 1). In an exploratory multivariate Cox regression model including FLIPI score, bulky disease and β2-microglobuline level, high ctDNA level was the only factor significantly associated with a worse PFS (HR 4.2, p=.039). In the observation arm, pts with high ctDNA levels had a median PFS of only 11.6 months vs NR (p=.01). Whereas in the maintenance arm, the prognostic value of elevated ctDNA levels was erased (p=.6). Conclusion: Using the NGS-based immunosequencing method, we were able to show a variation in clonal heterogeneity between tumor biopsy and plasma samples obtained at diagnosis. The poor prognostic value of elevated ctDNA levels in plasma at diagnosis appeared higher than other clinical parameters in a multivariate cox regression model. These findings need to be validated in larger cohorts. Figure 1. PFS according to the level of circulating tumor DNA Figure 1. PFS according to the level of circulating tumor DNA Disclosures Carlton: Adaptive Biotechnologies Corp.: Employment, Equity Ownership. Faham:adaptive biotech: Employment, Other: stockholders. Salles:Roche: Honoraria, Research Funding.