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Supplementary methods, figures, and tables Figure S1: Variant filters applied to Lymphopanel NGS data. Figure S2: Validation of variants according to SIFT and CADD scores. Figure S3: PMBL samples harbor more mutations than other DLBCL subtypes. Figure S4: Role of AID-induced SHM in DLBCL. Figure S5: Mutation frequency by subtype. Figure S6: Clustering of gene mutations according to subtype. Figure S7: Protein representations of observed mutations. Figure S8: Correlations between age and specific gene mutations. Figure S9: Prognosis according to IPI and subtype. Figure S10: Prognostic impact of gene mutations among the Lymphopanel. Table S1: Clinical characteristics of patients in our cohort. Table S2: Overview of the Lymphopanel used for NGS analysis. Table S3: Overview of pathway selection Table S4: Validation of variant filtering by an independent study of seven non-tumoral samples from DLBCL patients Table S5: Validated variants. Table S6: Lymphopanel NGS detection of genes with high AID target frequencies. Table S7: Prognostic impact of mutations in Lymphopanel genes

Supplementary Figures 1-10 Figure S1. MYD88 mutation VAF according to L265P or non-L265P variants. Figure S2. Age according to MYD88 mutation status and variant among ABC patients. Figure S3. Genomic profiles of DLBCL according to the presence of MYD88 L265P or non-L265P variants. Figure S4. Relative L265P allele expression level according to L265P VAF. Figure S5. Gene expression according to MYD88 status. Figure S6. NFkB pathway gene expression according to MYD88 mutational status. Figure S7. LPS score according to subtype and downstream NFkB alterations. Figure S8. Clustering according to expression levels of 27 genes involved in LPS response and differentially expressed according to MYD88 L265P. Figure S9. TNFAIP3 and CARD11 expression according to their copy number status. Figure S10. Additional survival analyses.

Clinical characteristics of the 17 patients with CNS relapse are provided, included time to CNS relapse, first-line treatment and response after first-line treatment. CR, Complete Response; PR, Partial Response; PD, Progressive Disease.

Purpose: Next-generation sequencing (NGS) has detailed the genomic characterization of diffuse large B-cell lymphoma (DLBCL) by identifying recurrent somatic mutations. We set out to design a clinically feasible NGS panel focusing on genes whose mutations hold potential therapeutic impact. Furthermore, for the first time, we evaluated the prognostic value of these mutations in prospective clinical trials.Experimental Design: A Lymphopanel was designed to identify mutations in 34 genes, selected according to literature and a whole exome sequencing study of relapsed/refractory DLBCL patients. The tumor DNA of 215 patients with CD20+de novo DLBCL in the prospective, multicenter, and randomized LNH-03B LYSA clinical trials was sequenced to deep, uniform coverage with the Lymphopanel. Cell-of-origin molecular classification was obtained through gene expression profiling with HGU133+2.0 Affymetrix GeneChip arrays.Results: The Lymphopanel was informative for 96% of patients. A clear depiction of DLBCL subtype molecular heterogeneity was uncovered with the Lymphopanel, confirming that activated B-cell–like (ABC), germinal center B-cell like (GCB), and primary mediastinal B-cell lymphoma (PMBL) are frequently affected by mutations in NF-κB, epigenetic, and JAK–STAT pathways, respectively. Novel truncating immunity pathway, ITPKB, MFHAS1, and XPO1 mutations were identified as highly enriched in PMBL. Notably, TNFAIP3 and GNA13 mutations in ABC patients treated with R-CHOP were associated with significantly less favorable prognoses.Conclusions: This study demonstrates the contribution of NGS with a consensus gene panel to personalized therapy in DLBCL, highlighting the molecular heterogeneity of subtypes and identifying somatic mutations with therapeutic and prognostic impact. Clin Cancer Res; 22(12); 2919–28. ©2016 AACR.See related commentary by Lim and Elenitoba-Johnson, p. 2829

All relevant clinical data is included. Immunohistochemical results for BCL2, CD10, BCL6, MUM1, FOXP1, IgM and MYC are transcribed: 0 indicates negativity and 1 indicates positivity. For BCL2 and MYC, positivity is according to a threshold of 50% or 40% respectively.

Purpose: MYD88 mutations, notably the recurrent gain-of-function L265P variant, are a distinguishing feature of activated B-cell like (ABC) diffuse large B-cell lymphoma (DLBCL), leading to constitutive NFκB pathway activation. The aim of this study was to examine the distinct genomic profiles of MYD88-mutant DLBCL, notably according to the presence of the L265P or other non-L265P MYD88 variants.Experimental Design: A cohort of 361 DLBCL cases (94 MYD88 mutant and 267 MYD88 wild-type) was submitted to next-generation sequencing (NGS) focusing on 34 genes to analyze associated mutations and copy number variations, as well as gene expression profiling, and clinical and prognostic analyses.Results: Importantly, we highlighted different genomic profiles for MYD88 L265P and MYD88 non-L265P–mutant DLBCL, shedding light on their divergent backgrounds. Clustering analysis also segregated subgroups according to associated genetic alterations among patients with the same MYD88 mutation. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFκB pathway activation, although the majority of MYD88 L265P–mutant cases harbors downstream NFκB alterations, which can predict BTK inhibitor resistance. Finally, although the MYD88 L265P variant was not an independent prognostic factor in ABC DLBCL, associated CD79B mutations significantly improved the survival of MYD88 L265P–mutant ABC DLBCL in our cohort.Conclusions: This study highlights the relative heterogeneity of MYD88-mutant DLBCL, adding to the field's knowledge of the theranostic importance of MYD88 mutations, but also of associated alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. Clin Cancer Res; 23(9); 2232–44. ©2016 AACR.

Supplementary methods, supplementary figure & table legends, and supplementary tables 3, 4, 8 & 9. Table S3: Overview of the Lymphopanel used for NGS analysis. Table S4: Overview of pathway selection Table S8. Genes involved in the LPS score. Table S9. Expression levels of genes involved in response to LPS stimulation according to MYD88 L265P.

Statistically significant differences (p

This table presents all variants identified among our cohort by Lymphopanel NGS. Variant type and variant allele frequency (VAF) are indicated.

This table presents all CNVs identified among our cohort using the ONCOCNV software. Duplication was considered when copy number was superior to 2 and deletion was considered when copy number was inferior to 2. False discovery rate (FDR) cutoff was set at 0.01.

COO information according to the three techniques used in this study is given according to the various MYD88 variants present in our cohort.

Few data exist concerning circulating tumor DNA (ctDNA) relevance in primary mediastinal B-cell lymphoma (PMBL). To explore this topic, we applied a 9-gene next-generation sequencing pipeline to samples from forty-four PMBL patients (median age 36.5 years). The primary endpoint was a similarity between paired biopsy/plasma mutational profiles. We detected at least one variant in 32 plasma samples (80%). The similarity between the biopsy and ctDNA genetic profiles for the 30 patients with paired mutated biopsy/plasma samples was greater than or equal to 80% in 19 patients (63.3%). We then compared PMBL ctDNA features with those of a cohort of Hodgkin lymphoma patients (

The relevance of circulating tumor DNA (ctDNA) analysis as a liquid biopsy and minimal residual disease tool in the management of classical Hodgkin lymphoma (cHL) patients was demonstrated in retrospective settings and remains to be confirmed in a prospective setting. We developed a targeted Next-Generation sequencing (NGS) panel for fast analysis (AmpliSeq® technology) of nine commonly mutated genes in biopies and ctDNA of cHL patients. We then conducted a prospective trial to assess ctDNA follow-up at diagnosis and after two cycles (C2) of chemotherapy. Sixty cHL patients treated by first line conventional chemotherapy (BEACOPPescalated [21.3%], ABVD/ABVD-like [73.5%] and other regimens [5.2%, for elderly patients]) were assessed in this noninterventional study. The median age of the patients was 33.5 years (range: 20-86). Variants were identified in 42 (70%) patients. Mutations of NFKBIE, TNFAIP3, STAT6, PTPN1, B2M, XPO1, ITPKB, GNA13 and SOCS1 were found in 13.3%, 31.7%, 23.3%, 5%, 33.3%, 10%, 23.3%, 13.3% and 50% of patients, respectively. ctDNA concentration and genotype were correlated with clinical characteristics and presentation. Regarding early therapeutic response, 45 patients (83%, not available [NA] =6) had a negative positron emission tomography (PET) after C2 (Deauville Score 1-3). The mean of DeltaSUVmax after C2 was -78.8%. ctDNA after C2 was analysed in 54 patients (90%). ctDNA became rapidly undetectable in all cases after C2. Variant detection in ctDNA is suitable to depict the genetic features of cHL at diagnosis and may help to assess early treatment response, in association with PET. Clinical Trial reference: NCT02815137.

The mutational status of the variable region of the immunoglobulin heavy chain gene (IGHV) is a very important biomarker for chronic lymphocytic leukemia (CLL) patients. However, the routine detection of IGHV mutational status is time-consuming and costly. Therefore, we performed 5' Rapid amplification of cDNA ends (5' RACE) in 81 CLL patients who previously underwent detection using Biomed-2. The agreement rate of these two methods was 93.8 %. Regarding the discordant cases, 5' RACE was more sensitive to identify unproductive and multiple rearrangements. Furthermore, 5' RACE can also be used to simultaneously sequence light chains. In most CLL cases, the mutational statuses of heavy and light chains are concordant, except in IGLV3-21. Most IGLV3-21 (24/25) rearrangement shared a similar LCDR3 (QVWDSSSDHPWV) and harbored a single point mutation, namely, IGLV3-21

Synchronous Hodgkin Lymphoma and Primary Mediastinal B‐cell Lymphoma is possible, with molecular analyses proving the absence of clonal filiation between both entities. This suggests a common etiology but the existence of two divergent clones.

International audience; In the era of precision medicine, it is crucial to identify molecular alterations that will guide the therapeutic management of patients. In this context, circulating tumoral DNA (ctDNA) released by the tumor in body fluids, like blood, and carrying its molecular characteristics is becoming a powerful biomarker for non-invasive detection and monitoring of cancer. Major recent technological advances, especially in terms of sequencing, have made possible its analysis, the challenge still being its reliable early detection. Different parameters, from the pre-analytical phase to the choice of sequencing technology and bioinformatic tools can influence the sensitivity of ctDNA detection.

Background: Gene expression profiling (GEP), next-generation sequencing (NGS) and copy number variation (CNV) analysis have led to an increasingly detailed characterization of the genomic profiles of DLBCL. The aim of this study was to perform a fully integrated analysis of mutational, genomic, and expression profiles to refine DLBCL subtypes. A comparison of our model with two recently published integrative DLBCL classifiers was carried out, in order to best reflect the current state of genomic subtypes. Methods: 223 patients with de novo DLBCL from the prospective, multicenter and randomized LNH-03B LYSA clinical trials were included. GEP data was obtained using Affymetrix GeneChip arrays, mutational profiles were established by Lymphopanel NGS targeting 34 key genes, CNV analysis was obtained by array CGH, and FISH and IHC were performed. Unsupervised independent component analysis (ICA) was applied to GEP data and integrated analysis of multi-level molecular data associated with each component (gene signature) was performed. Findings: ICA identified 38 components reflecting transcriptomic variability across our DLBCL cohort. Many of the components were closely related to well-known DLBCL features such as cell-of-origin, stromal and MYC signatures. A component linked to gain of 19q13 locus, among other genomic alterations, was significantly correlated with poor OS and PFS. Through this integrated analysis, a high degree of heterogeneity was highlighted among previously described DLBCL subtypes. Interpretation: The results of this integrated analysis enable a global and multi-level view of DLBCL, as well as improve our understanding of DLBCL subgroups. Keywords: Diffuse large B-cell lymphoma, Independent component analysis, Transcriptomic variability, Gene signatures, prognosis

Non-Hodgkin B-cell lymphomas (B-NHLs) are a highly heterogeneous group of mature B-cell malignancies. Their classification thus requires skillful evaluation by expert hematopathologists, but the risk of error remains higher in these tumors than in many other areas of pathology. To facilitate diagnosis, we have thus developed a gene expression assay able to discriminate the seven most frequent B-cell NHL categories. This assay relies on the combination of ligation-dependent RT-PCR and next-generation sequencing, and addresses the expression of more than 130 genetic markers. It was designed to retrieve the main gene expression signatures of B-NHL cells and their microenvironment. The classification is handled by a random forest algorithm which we trained and validated on a large cohort of more than 400 annotated cases of different histology. Its clinical relevance was verified through its capacity to prevent important misclassification in low grade lymphomas and to retrieve clinically important characteristics in high grade lymphomas including the cell-of-origin signatures and the MYC and BCL2 expression levels. This accurate pan-B-NHL predictor, which allows a systematic evaluation of numerous diagnostic and prognostic markers, could thus be proposed as a complement to conventional histology to guide the management of patients and facilitate their stratification into clinical trials.

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The XPO1 gene encodes exportin 1 (XPO1) that controls the nuclear export of cargo proteins and RNAs. Almost 25% of primary mediastinal B-cell lymphoma (PMBL) and classical Hodgkin lymphoma (cHL) cases harboured a recurrent XPO1 point mutation (NM_003400, chr2:g61718472C&gt, T) resulting in the E571K substitution within the hydrophobic groove of the protein, the site of cargo binding. We investigated the impact of the XPO1E571K mutation using PMBL/cHL cells having various XPO1 statuses and CRISPR&ndash, Cas9-edited cells in which the E571K mutation was either introduced or knocked-out. We first confirmed that the mutation was present in both XPO1 mRNA and protein. We observed that the mutation did not modify the export capacity but rather the subcellular localisation of XPO1 itself. In particular, mutant XPO1 bound to importin &beta, 1 modified the nuclear export/import dynamics of relevant cargoes.

Introduction Non-Hodgkin B-cell lymphomas (B-NHLs) are a highly heterogeneous group of mature B-cell malignancies associated with very diverse clinical behaviors. They rely on the activation of different signaling pathways for proliferation and survival which might be amenable to targeted therapies, increasing the need for precision diagnosis. Unfortunately, their accurate classification can be challenging, even for expert hemato-pathologists, and secondary reviews recurrently differ from initial diagnosis. To address this issue we have developed a pan-B-NHL classifier based on a middle throughput gene expression assay coupled with a random forest algorithm. Material and Methods Five hundred ten B-NHL diagnosed according to the WHO criteria were studied, with 325 diffuse large B-cell lymphomas (DLBCL), 43 primary mediastinal B-cell lymphomas (PMBL), 55 follicular lymphomas (FL), 31 mantle cell lymphomas (MCL), 17 small lymphocytic lymphomas (SLL), 20 marginal zone lymphomas (MZL), 11 marginal zone lymphomas of mucosa-associated lymphoid tissue (MALT) and 8 lymphoplasmacytic lymphomas (LPL). To train and validate the predictor the samples were randomly split into a training (2/3) and an independent validation cohort (1/3). A panel of 137 genes was designed by purposely selecting the differentiation markers identified in the WHO classification for their capacity to provide diagnostic and prognostic information in NHLs. Gene expression profiles were generated by ligation dependent RT-PCR applied to RNA extracted from frozen or FFPE tissue and analyzed on a MiSeq sequencer. For analysis, the sequencing reads were de-multiplexed, aligned with the sequences of the LD-RTPCR probes and counted. Results were normalized using unique molecular indexes counts to correct PCR amplification biases. Results In DLBCL, unsupervised gene expression analysis retrieved the expected GCB, ABC and PMBL signatures (Fig A). These tumors also showed higher expressions of the KI67 (proliferation), CD68 and CD163 (tumor associated macrophages), and PD-L1/2 (immune response) markers. We also observed that the dual expression of MYC and BCL2 at the mRNA level significantly associates with inferior PFS and OS, independent from the International Prognostic Index and from the GCB/ABC cell-of-origin signature, validating the capacity of the assay to identify these highly aggressive lymphomas (Fig C). Overall, low-grade lymphomas were characterized by a significant T cell component. FLs associated with the GCB (BCL6, MYBL1, CD10 and LMO2) and Tfh (CD3, CD5, CD28, ICOS, CD40L, CXCL13) signatures. Other small B-cell lymphomas tended to overexpress activated B-cell markers (LIMD1, TACI, IRF4,FOXP1...), and the expected CD5, CD10, CD23 and CCND1 differential expressions in SLL, MCL and MZL were correctly retrieved (Fig B). Surprisingly, our analysis revealed that the Ie-Ce sterile transcript, expressed from the IGH locus during IgE isotype switching, is almost exclusively expressed by FLs, constituting one of the most discriminant markers for this pathology. We next trained a random forest classifier to discriminate the 7 principal subtypes of B-NHLs. The training cohort comprised 162 DLBCLs (ABC or GCB), 28 PMBL, 35 FLs (grade 1-3A), 21 MCLs, 12 SLLs, and 25 NHLs grouped into the MZL category (13 MZLs, 8 MALT and 4 LPLs). The independent validation series comprised 90 DLBCLs classified as GCB or ABC DLBCLs by the Lymph2Cx assay, 15 PMBLs, 12 FLs (grade 1-3A), 10 MCLs, 5 SLLs and 14 MZLs (7 MZL, 3 MALT and 4 LPL). The RF algorithm classified all cases of the training series into the expected subtype, as well as 94.5% samples of the independent validation cohort (Fig D). For ABC and GCB DLBCLs, the concordance with the Lymph2Cx assay in the validation cohort was 94.3%. Conclusion We have developed a comprehensive gene expression based solution which allows a systematic evaluation of multiple diagnostic and prognostic markers expressed by the tumor and by the microenvironment in B-NHLs. This assay, which does not require any specific platform, could be implemented in complement to histology in many diagnostic laboratories and, with the current development of targeted therapies, enable a more accurate and standardized B-NHL diagnosis. Together, our data illustrate how the integration of gene expression profiling and artificial intelligence can increase precision diagnosis in cancers. Figure Disclosures Oberic: Takeda: Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria; Roche: Membership on an entity's Board of Directors or advisory committees. Haioun:Miltenyi: Honoraria; Takeda: Honoraria; Servier: Honoraria; F. Hoffmann-La Roche Ltd: Honoraria; Novartis: Honoraria; Amgen: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Janssen: Honoraria. Salles:Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Amgen: Honoraria, Other: Educational events; BMS: Honoraria; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria. Tilly:roche: Membership on an entity's Board of Directors or advisory committees; servier: Honoraria; merck: Honoraria; Roche: Consultancy; Celgene: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Karyopharm: Consultancy; BMS: Honoraria; Janssen: Honoraria; Gilead: Honoraria. Jardin:celgene: Honoraria; roche: Honoraria; amgen: Honoraria; Servier: Honoraria; janssen: Honoraria.

From a liquid biopsy, cell-free DNA (cfDNA) can provide information regarding basal tumoral genetic patterns and changes upon treatment. In a prospective cohort of 30 diffuse large B-cell lymphomas (DLBCL), we determined the clinical relevance of cfDNA using targeted next-generation sequencing and its correlation with PET scan imaging at the time of diagnosis and during treatment. Using a dedicated DLBCL panel, mutations were identified at baseline for 19 cfDNAs and profiles were consistent with expected DLBCL patterns. Tumor burden-related clinical and PET scan features (LDH, IPI, and metabolic tumor volume) were significantly correlated with the quantity of tumoral cfDNA. Among the four patients presenting additional mutations in their cfDNAs, three had high metabolic tumor volumes, suggesting that cfDNA more accurately reflects tumor heterogeneity than tissues biopsy itself. Mid-treatment, four patients still had basal mutations in their cfDNAs, including three in partial response according to their Deauville scores. Our study highlights the major interests in liquid biopsy, in particular in the context of bulky tumors where cfDNA allows capturing the entire tumoral mutation profile. Therefore, cfDNA analysis in DLBCL represents a complementary approach to PET scan imaging.

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Introduction: The presence of circulating tumor DNA (ctDNA) was demonstrated years ago in solid tumors and represents a promising technology for the non-invasive management of cancers. In the case ...

Le lymphome cutane primitif B diffus a grandes cellules type jambe (LBC-TJ), le plus agressif des lymphomes B cutanes, presente des caracteristiques proches des lymphomes B diffus a grandes cellules (LBDGC) de type « Activated B- Cell-like » (ABC) avec une activation constitutive de la voie NF-κB (nuclear factor-kappa B). Initialement, notre equipe a demontre la haute prevalence de la mutation MYD88L265P dans les LBC-TJ, puis associee a l’equipe du Pr Jardin (INSERM UMR1245, Rouen), nous avons confirme par exome sequencing la tres haute prevalence des mutations MYD88, PIM1 et CD79B [1] . La prise en charge de ces patients demeure un enjeu therapeutique malgre l’amelioration de la survie a 5 ans de 38 % a 74 % par l’association du Rituximab (anti-CD20) avec une polychimiotherapie adaptee a l’âge mais environ 50 % de non reponse sont observees sans facteur predictif connu. Nous souhaitons identifier un profil moleculaire predictif de reponse therapeutique permettant une meilleure prise en charge de ces patients. Nous avons analyse 14 patients repondeurs et 18 non repondeurs a la R-polychimiotherapie par NGS via un panel de 36 genes importants dans la lymphomagenese. Pour 14 patients, nous avons egalement analyse la biopsie a la progression/rechute afin d’identifier les alterations genetiques selectionnees lors de l’echappement therapeutique. Les patients presentant une mutation de la voie du BCR (CD79A/B ou CARD11) ont une survie sans progression et une survie specifique diminuee (p = 0,002 et p = 0,03 respectivement) [2] . L’analyse des echantillons au diagnostic et a la rechute montre que les mutations des genes MYD88 et CD79B sont les plus conservees et que la frequence allelique de ces mutations est superieure a la progression/rechute. La caracterisation d’une mutation dans la voie du BCR chez les patients atteints d’un LBTJ laisse presager une resistance a la polychimiotherapie conventionnelle. Ces patients pourraient beneficier d’un traitement adjuvant ou d’une seconde ligne d’emblee afin d’ameliorer leur prise en charge.

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 > 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.

Little is known on the phylogenetic relationship between diagnostic and relapse clones of diffuse large B-cell lymphoma (DLBCL). We applied high throughput sequencing (HTS) of the VDJ locus of Immunoglobulin heavy chain (IGHV) on 14 DLBCL patients with serial samples, including tumor biopsies and/or peripheral blood mononuclear cells (PBMC). Phylogenetic data were consolidated with targeted sequencing and cytogenetics. Phylogeny clearly showed that DLBCL relapse could occur according either an early or a late divergent mode. These two modes of divergence were independent from the elapsed time between diagnosis and relapse. We found no significant features for antigen selection pressure in complementary determining region both at diagnosis and relapse for 9/12 pairs and a conserved negative selection pressure for the three remaining cases. Targeted HTS and conventional cytogenetics revealed a branched vs. linear evolution for 5/5 IGHV early divergent cases, but unexpected such "oncogenetic" branched evolution could be found in at least 2/7 IGHV late divergent cases. Thus, if BCR signaling is mandatory for DLBCL emergence, oncogenetic events under chemotherapy selection pressure may be the main driving forces at relapse. Finally, circulating subclones with divergent IGHV somatic hypermutations patterns from initial biopsy could be detected in PBMC at diagnosis for 4/6 patients and, for two of them, at least one was similar to the ones found at relapse. This study highlights that oncogenetic intraclonal diversity of DLBCL should be evaluated beyond the scope a single biopsy and represents a rationale for future investigations using peripheral blood for lymphoid malignancies genotyping. Am. J. Hematol. 92:68-76, 2017. © 2016 Wiley Periodicals, Inc.

Motivation Next-generation sequencing has become the go-to standard method for the detection of single-nucleotide variants in tumor cells. The use of such technologies requires a PCR amplification step and a sequencing step, steps in which artifacts are introduced at very low frequencies. These artifacts are often confused with true low-frequency variants that can be found in tumor cells and cell-free DNA. The recent use of unique molecular identifiers (UMI) in targeted sequencing protocols has offered a trustworthy approach to filter out artefactual variants and accurately call low-frequency variants. However, the integration of UMI analysis in the variant calling process led to developing tools that are significantly slower and more memory consuming than raw-reads-based variant callers. Results We present UMI-VarCal, a UMI-based variant caller for targeted sequencing data with better sensitivity compared to other variant callers. Being developed with performance in mind, UMI-VarCal stands out from the crowd by being one of the few variant callers that do not rely on SAMtools to do their pileup. Instead, at its core runs an innovative homemade pileup algorithm specifically designed to treat the UMI tags in the reads. After the pileup, a Poisson statistical test is applied at every position to determine if the frequency of the variant is significantly higher than the background error noise. Finally, an analysis of UMI tags is performed, a strand bias and a homopolymer length filter are applied to achieve better accuracy. We illustrate the results obtained using UMI-VarCal through the sequencing of tumor samples and we show how UMI-VarCal is both faster and more sensitive than other publicly available solutions. Availability and implementation The entire pipeline is available at https://gitlab.com/vincent-sater/umi-varcal-master under MIT license. Supplementary information Supplementary data are available at Bioinformatics online.

Next Generation Sequencing (NGS) has become the go-to standard method for the detection of Single Nucleotide Variants (SNV) in tumor cells. The use of such technologies requires a PCR amplification step and a sequencing step, steps in which artifacts are introduced at very low frequencies. These artifacts are often confused with true low-frequency variants that can be found in tumor cells and cell-free DNA. The recent use of Unique Molecular Identifiers (UMI) in targeted sequencing protocols has offered a trustworthy approach to filter out artifactual variants and accurately call low frequency variants. However, the integration of UMI analysis in the variant calling process led to developing tools that are significantly slower and more memory consuming than raw-reads-based variant callers. We present UMI-VarCal, a UMI-based variant caller for targeted sequencing data with better sensitivity compared to other variant callers. Being developed with performance in mind, UMI-VarCal stands out from the crowd by being one of the few variant callers that don9t rely on SAMtools to do their pileup. Instead, at its core runs an innovative homemade pileup algorithm specifically designed to treat the UMI tags in the reads. After the pileup, a Poisson statistical test is applied at every position to determine if the frequency of the variant is significantly higher than the background error noise. Finally, an analysis of UMI tags is performed, a strand bias and a homopolymer length filter are applied to achieve better accuracy. We illustrate the results obtained using UMI-VarCal through the sequencing of tumor samples and we show how UMI-VarCal is both faster and more sensitive than other publicly available solutions.

International audience; About Related Information ePDFPDF Request permission Export citation Add to favorites Track citationShare a linkShare on Email Facebook Twitter LinkedIn RedditIntroduction: The relevance of circulating tumor DNA (ctDNA) analysis as a liquid biopsy and minimal residual disease tool in the management of classical Hodgkin Lymphoma (cHL) patients was demonstrated in retrospective settings and remains to be confirmed.Methods: We developed a targeted Next‐Generation sequencing (NGS) panel for fast analysis (AmpliSeq® technology) of nine commonly mutated genes in biopsy and ctDNA of cHL patients. We then conducted a prospective trial to assess ctDNA follow up at diagnosis and after 2 cycles of chemotherapy (C2). A dedicated bioinformatics pipeline to optimize detections of variants with low rates and minimize artefactual misinterpretations was built. Sixty cHL patients treated by first line conventional chemotherapy (BEACOPPescalated [21.3%], ABVD/ABVD‐like [73.5%] and other regimens [5.2%, for elderly patients] were included in this non‐interventional study (NCT02815137).Results: Median age of the patients was 33.5 years (range 20‐86) with a predominance of male patients, scleronodular subtype and ECOG 0‐1 (53.3%, 70% and 88.3%, respectively). Variants were identified in 33 (55%) patients, precisely in 16/30 (53.3%) and 30/60 (50%) of available biopsy and ctDNA samples respectively. Concordance between genetic profiles of biopsy and ctDNA was accurate for 22/30 patients (73.3%). Mutations of NFKBIE, TNFAIP3, STAT6, PTPN1, B2M, XPO1, ITPKB, GNA13 and SOCS1were found in 11.7% (mean number of variants by sample [range]: 1 [0‐1]), 25% (1.1 [0‐2]), 21.7% (1.4 [0‐2]), 1.7% (1 [0‐1]), 25% (1.3 [0‐3]), 6.7% (1 [0‐1]), 15% (1.4 [0‐3]), 5% (1.3 [0‐2]) and 31.7% (1.8 [0‐7]) of all patients, respectively. Unsupervised hierarchical clustering was performed among the 9 genes to represent the association of alterations (See Figure 1).Higher level of [ctDNA] at diagnosis was associated with adverse characteristics: age ≥45 years, presence of anemia (hemoglobin

Primary low-grade dural marginal zone lymphoma is an indolent low grade lymphoma occurring especially among middle-aged immunocompetent women, and is not associated to an infectious process, contrary to gastric or intestinal marginal zone lymphomas. Dural location is rare since only 105 cases have been reported so far. We report herein on two additional cases, a 72-year-old woman and a 36-year-old man whose lymphoma was revealed by partial seizures and headaches. Morphological analysis of surgical specimens displayed a tumoral proliferation made of small lymphocytes arranged in sheets or in nodules with CD20, CD79a and BCL2-immunopositivity, but CD5 and CD10 negativity. Molecular analysis using a panel of 34 genes involved in lymphomagenesis disclosed a deletion of SOCS1 and TNFAIP3 genes, implicated in the JAK/STAT and NFκB pathways respectively in the first patient that could explain unfavourable prognosis despite complementary radiotherapy. No anomaly was identified in the second patient who is alive with no recurrence or progression seven years after the diagnosis. Currently, there are no standardized treatment schedules, but the vast majority of patients are treated by surgery, then radiotherapy followed by adjuvant chemotherapy using methotrexate alone or in combination with rituximab. Literature review indicates that five-year survival has been estimated at 96.7%, suggesting a better prognosis compared to other locations.

Primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) preferentially involves the lower limb in elderly subjects. A combination of polychemotherapy and rituximab has improved prognosis. However, about 50% of patients will experience progression or relapse without any predictive biologic marker of therapeutic response. The mutational profile of PCLBCL-LT has highlighted mutations contributing to constitutive NF-κB and B-cell receptor (BCR) signaling pathways but has not demonstrated clinical utility. Therefore, the mutational status of 32 patients with PCLBCL-LT (14 patients with complete durable response and 18 patients with relapsing or refractory disease) was determined with a dedicated lymphopanel. Tumor pairs at diagnosis and relapse or progression were analyzed in 14 relapsing or refractory patients. Patients with PCLBCL-LT harboring one mutation that targets one of the BCR signaling genes, CD79A/B or CARD11, displayed a reduced progression-free survival and specific survival (median 18 months, P = 0.002 and 51 months, P = 0.03, respectively, whereas median duration in the wild-type group was not reached) and were associated with therapeutic resistance (P = 0.0006). Longitudinal analyses revealed that MYD88 and CD79B were the earliest and among the most mutated genes. Our data suggest that evaluating BCR mutations in patients with PCLBCL-LT may help to predict first-line therapeutic response and to select targeted therapies.

Primary mediastinal B-cell lymphoma (PMBL) is an entity of B-cell lymphoma distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We investigated the prevalence, specificity, and clinical relevance of mutations of XPO1, which encodes a member of the karyopherin-β nuclear transporters, in a large cohort of PMBL. PMBL cases defined histologically or by gene expression profiling (GEP) were sequenced and the XPO1 mutational status was correlated to genetic and clinical characteristics. The XPO1 mutational status was also assessed in DLBCL, Hodgkin lymphoma (HL) and mediastinal gray-zone lymphoma (MGZL).The biological impact of the mutation on Selective Inhibitor of Nuclear Export (SINE) compounds (KPT-185/330) sensitivity was investigated in vitro. XPO1 mutations were present in 28/117 (24%) PMBL cases and in 5/19 (26%) HL cases but absent/rare in MGZL (0/20) or DLBCL (3/197). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in GEP-defined PMBL and was associated with shorter PFS. Age, International Prognostic Index and bulky mass were similar in XPO1 mutant and wild-type cases. KPT-185 induced a dose-dependent decrease in cell proliferation and increased cell-death in PMBL cell lines harboring wild type or XPO1 E571K mutant alleles. Experiments in transfected U2OS cells further confirmed that the XPO1 E571K mutation does not have a drastic impact on KPT-330 binding. To conclude the XPO1 E571K mutation represents a genetic hallmark of the PMBL subtype and serves as a new relevant PMBL biomarker. SINE compounds appear active for both mutated and wild-type protein.

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in many human tumors. Here, we report HACE1 as a candidate tumor suppressor gene down-regulated by a combination of deletion and epigenetic mechanisms. HACE1 deletions were observed in 40% of B-cell lymphoma tumors. Hypermethylation of the HACE1 promoter CpG177 island was found in 60% (68/111) of cases and in all tested B-cell lymphoma lines. Using HDAC inhibitors, we observed predominantly inactive chromatin conformation (methylated H3 histones H3K9me2) in HACE1 gene promoter region. We demonstrated in Ramos and Raji cells that down-regulation of HACE1 expression was associated with a significant decrease in apoptosis and an accumulation of cells in the S and G2/M phases. Our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and can be downregulated by deacetylation of its promoter region chromatin, which makes HACE1 a potential target for HDAC inhibitors.

Despite the many efforts already spent to enumerate somatic mutations in diffuse large B-cell lymphoma (DLBCL), previous whole-genome and whole-exome studies conducted on patients of mixed outcomes failed at characterizing the 30% of patients who will relapse or resist current immunochemotherapies. To address this issue, we performed whole-exome sequencing of normal/tumoral DNA pairs in 14 relapsed/refractory (R/R) patients subclassified by full-transcriptome arrays (six activated B-cell like, three germinal center B-cell like, and five primary mediastinal B-cell lymphomas), from the LNH-03 LYSA clinical trial program. Aside from well-known DLBCL features, gene and pathway level recurrence analyses proposed several interesting leads including TBL1XR1 and activating mutations in IRF4 or in the insulin regulation pathway. Sequencing-based copy number analysis defined 23 short recurrently altered regions involving genes such as REL, CDKN2A, HYAL2, and TP53. Moreover, it highlighted mutations in genes such as GNA13, CARD11, MFHAS1, and PCLO as associated with secondary variant allele amplification events. The five primary mediastinal B-cell lymphomas (PMBL), while unexpected in a R/R cohort, showed a significantly higher mutation rate (P = 0.003) and provided many insights on this classical Hodgkin lymphoma related subtype. Novel genes such as XPO1, MFHAS1, and ITPKB were found particularly mutated, along with various cytokine-based signaling pathways. Among these analyses, somatic events in the NF-κB pathway were found preponderant in the three DLBCL subtypes, confirming its major implication in DLBCL aggressiveness and pinpointing several new candidate genes.

Primary mediastinal large B-cell lymphoma (PMBL) is a mature large B-cell lymphoma of putative thymic B-cell origin involving the mediastinum with younger age distribution and better prognosis than diffuse large B-cell lymphoma (DLBCL), not otherwise specified. Recently, based on gene expression profile analysis and morphologic findings, cases of PMBL without mediastinal involvement have been reported. In this study, we analyzed 3 cases of nodal DLBCL with morphologic features of PMBL presenting in submandibular or supraclavicular lymph nodes, in middle-aged to elderly patients, 2 of them without clinical or radiologic evidence of mediastinal involvement. The 3 patients presented with stage I/II disease and had excellent response to R-CHOP/R-EPOCH therapy. The 3 cases showed MAL expression and were positive for CD23 and/or CD30. All 3 cases expressed cyclin D1 with copy number gains of CCND1 gene but without rearrangement. There was no rearrangement of CIITA or PDL1/PDL2. Reverse transcriptase-multiplex ligation-dependent probe amplification, a mRNA-based gene expression profile analysis revealed high probability of PMBL (87.6%, 98.7%, and 99%) in these 3 cases. Targeted next-generation sequencing analysis showed SOCS1 mutations in the 3 cases, and TNFAIP3 and XPO1 mutations in one, further supporting the diagnosis of PMBL. In conclusion, we report 3 cases of nodal PMBL, 2 of them without mediastinal mass, and expression of cyclin D1 due to copy number gains of CCND1 gene, a diagnostic pitfall with mantle cell lymphoma and DLBCL, not otherwise specified.

Introduction The outcome of very elderly patients with newly diagnosed diffuse large B cell lymphoma (DLBCL) remains poorer than that of younger patients, often because of co-morbidities and physiological organ function impairment. However, the biological specificities of DLBCL in very elderly patients are unknown. The LYSA conducted a multicentric, phase III, open-label, randomized trial in newly diagnosed DLBCL > 80y evaluating the efficacy of the combination of lenalidomide with R-miniCHOP (R2-miniCHOP) in comparison to the standard R-miniCHOP (SENIOR trial, NCT02128061). Based on targeted NGS sequencing and gene expression profile (GEP) analysis, the BIOSENIOR project aimed to provide a molecular characterisation of the cohort and detect clinically relevant biomarkers in according to treatment arm. Methods The SENIOR study enrolled patients aged of 80 years or more with non-previously treated CD20+ DLBCL, age-adjusted IPI= 0 to 3, and Ann Arbor stage II to IV. 249 patients were randomized (127 in R-miniCHOP arm and 122 in R2-miniCHOP arm). Median age was 83y (range 80-96. Patients with available tumor DNA /RNA from FFPE biopsies were included in the BIOSENIOR substudy. GEP was performed using Lymph2CX assay (Nanostring®) and a new RTMLPA assay (Bobée et al. J Mol Diagn 2017) able to quantify the expression of 137 relevant genes involved in B-cell biology, microenvironment or therapeutic response (cereblon). Genotyping (QiaSeq ®) was performed using a dedicated 36 genes panel. GEP features were compared to DLBCL patients < 60y enrolled in the LYSA GAINED trial (NCT01659099). Results Tumor DNA/RNA was available for 164/249 patients (66%). GEP was performed in 154 cases by RTMLPA and in 103 cases by Lymph2CX. The concordance to classify GCB/ABC was of 94% between the two technologies. DLBCL were mainly classified in the ABC subtype [77 = ABC (50%); 53 = GCB (34.4%); 17 unclassified (11%)]. Four cases (2.6%) were classified as EBV+ DLBCL and 1 as PMBL. 81 cases (52.5%) were considered as BCL2+, 23 (15%) as MYC+ and 16 (10.4%) as double-expressors (DE) by GEP. Comparison of GEP with younger patients highlighted distinct features and geriatric specificities (figure 1A). Genotypes were obtained in 159 patients. MYD88, PIM1, CREBBP, CD79B and TP53 were the five most frequently mutated genes (Figure 1B). Correlations between biomarkers and outcomes (OS and PFS) were performed in the overall population and according to treatment arms. The GCB/ABC subtyping, as determined by Lymph2CX (n = 97) was related to OS [HR = 0.38 (0.18 - 0.81) Log-rank: 0.009] and PFS in the overall population. This result was not confirmed in the extended cohort (n = 154) by RTMLPA. The MYC / BCL2 DE status assessed by GEP was a strong predictor of outcome (OS, HR = 2.89 (1.39 - 5.97) Log-rank: 0.003] in both arms. Importantly MYC/BCL2 DE was independent of the IPI score and albuminemia in multivariate analysis. In an exploratory analysis, cereblon (HR = 2.360; CI-95% 1.157-4.812, Log-rank: 0.015) and PD1 [HR = 0.33 (0.16 - 0.66) Log-rank: 0.001] expressions correlate exclusively with PFS in the R2-miniCHOP arm. MYD88 mutations (L265P and others variants) were associated with a worse outcome in the overall population (OS, HR= 2.05, CI-95% 1.190-3.554, Log-rank: 0.008; PFS, HR =1.731, CI-95% 1.059-2.830 Log-rank: 0.027). FOXO1 mutations were detected in 15 patients (9%). Its impact was only observed in the R-miniCHOP arm (OS, HR = 4.07 (1.52 - 10.87) Log-rank: 0.0024; PFS HR = 4.139 (1.59 - 10.79) Log-rank: 0.0016)] and was erased in the R2-miniCHOP arm. TP53 mutations, reported in 24% of cases were predictive of the PFS in the overall population [HR = 1.811 (1.09 - 3.01) Log-rank: 0.0199](prognostic factors are summarized in Table 1). Conclusion BIOSENIOR is an ancillary study of the first prospective phase III trial in ³80 years old patients with newly diagnosed DLBCL. Some biomarkers, known to be predictive in younger patients, are relevant in this cohort, including MYC expression, BCL2/MYC DE, mutations of MYD88, TP53 or FOXO1. However, GEP characteristics of DLBCL occurring in this very old population are highly different of younger patients, suggesting distinct underlying oncogenic mechanisms. BIOSENIOR indicates that, beyond geriatric frailty, molecular features of DLBCL patients > 80y play also a crucial role that should be considered in the design of future trials. Markers that may predict R2-miniCHOP efficacy remain to be confirmed. Disclosures Thieblemont: Janssen: Honoraria; Celgene: Honoraria; Gilead: Honoraria; Novartis: Honoraria; Kyte: Honoraria; Roche: Honoraria, Research Funding; Cellectis: Membership on an entity's Board of Directors or advisory committees. Feugier:gilead: Honoraria, Research Funding, Speakers Bureau; roche: Honoraria, Research Funding, Speakers Bureau; janssen: Honoraria, Research Funding, Speakers Bureau; abbvie: Honoraria, Research Funding, Speakers Bureau. Salles:Autolus: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merck: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis, Servier, AbbVie, Karyopharm, Kite, MorphoSys: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Roche, Janssen, Gilead, Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Amgen: Honoraria, Other: Educational events; BMS: Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Educational events; Epizyme: Consultancy, Honoraria. Tilly:Roche: Consultancy; Celgene: Consultancy, Research Funding; Astra-Zeneca: Consultancy; Karyopharm: Consultancy; BMS: Honoraria; Janssen: Honoraria; Gilead: Honoraria; merck: Honoraria; servier: Honoraria; roche: Membership on an entity's Board of Directors or advisory committees. Haioun:celgene: Honoraria; novartis: Honoraria; servier: Honoraria; amgen: Honoraria; janssen cilag: Consultancy; takeda: Consultancy; gilead: Consultancy; celgene: Consultancy; roche: Consultancy. Le Gouill:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Roche-Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support. Casasnovas:Takeda: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Roche: 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, Research Funding; Gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel expenses; Merck Sharp and Dohme: Membership on an entity's Board of Directors or advisory committees, Other: Travel support, Research Funding; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Travel support; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees. Jardin:janssen: Honoraria; roche: Honoraria; amgen: Honoraria; celgene: Honoraria; Servier: Honoraria. OffLabel Disclosure: lenalidomide is evaluatd in combination with RminiHOP

Classical Hodgkin lymphoma (cHL) accounts for 30% of all lymphomas [1]. Currently, 20–25% of cHL cases relapse or are refractory after first-line standard treatments, leading to the need to better ...

Acquired estrogen receptor gene (ESR1) mutations have been recently reported as a marker of resistance to aromatase inhibitors in hormone receptor positive metastatic breast cancer. We retrospectively considered seven patients treated for metastatic breast cancer with available samples from the primary tumor before any treatment, cryopreserved metastasis removed during progression and concomitant plasmas. All these seven patients were in disease progression after previous exposure to aromatase inhibitors for at least 6 months, and were assessed for ESR1 mutations detection in tumor and circulating DNA. For these patients, Sanger sequencing identified four metastases with clear ESR1 mutation and one possible, whereas digital PCR identified six mutated metastases. Then, under blind conditions and using digital PCR, corresponding circulating ESR1 mutations were successfully detected in four of these six metastatic breast cancer patients. Moreover, in two patients with serial blood samples following treatments exposure, the monitoring of circulating ESR1 mutations clearly predicted disease evolution. In the context of high interest for ESR1 mutations, our results highlight that these acquired recurrent mutations may be tracked in circulating tumor DNA and may be of clinical relevance for metastatic breast cancer patient monitoring.

// Maxime Fontanilles 1, 2 , Florent Marguet 3, 4 , Elodie Bohers 2 , Pierre-Julien Viailly 2 , Sydney Dubois 2 , Philippe Bertrand 2 , Vincent Camus 1, 2 , Sylvain Mareschal 2 , Philippe Ruminy 2 , Catherine Maingonnat 2 , Stephane Lepretre 1 , Elena-Liana Veresezan 5 , Stephane Derrey 6 , Herve Tilly 1, 2 , Jean-Michel Picquenot 5 , Annie Laquerriere 3, 4 and Fabrice Jardin 1, 2 1 Department of Hematology, Cancer Center Henri Becquerel, 76000 Rouen, France 2 INSERM U1245, Cancer Center Henri Becquerel, Institute of Research and Innovation in Biomedicine (IRIB), University of Normandy, UNIVROUEN, 76000 Rouen, France 3 INSERM U1245 and Hopital Charles Nicolle, NeoVasc Team, University of Normandy, UNIVROUEN, CHU-Hopitaux de Rouen, 76031 Rouen, France 4 Department of Neuropathology, Hopital Charles Nicolle, Normandy Center for Genomic and Personalized Medicine, CHU-Hopitaux de Rouen, 76031 Rouen, France 5 Department of Pathology, Cancer Center Henri Becquerel, 76000 Rouen, France 6 Department of Neurosurgery, Hopital Charles Nicolle, CHU-Hopitaux de Rouen, 76031 Rouen, France Correspondence to: Fabrice Jardin, email: fabrice.jardin@chb.unicancer.fr Keywords: primary central nervous system lymphoma, circulating cell-free tumor DNA, somatic mutation, liquid biopsy, next-generation sequencing Received: March 08, 2017 Accepted: May 03, 2017 Published: June 01, 2017 ABSTRACT Purpose: Primary central nervous system lymphomas (PCNSL) have recurrent genomic alterations. The main objective of our study was to demonstrate that targeted sequencing of circulating cell-free DNA (cfDNA) released by PCNSL at the time of diagnosis could identify somatic mutations by next-generation sequencing (NGS). Patients and Methods: PlasmacfDNA and matched tumor DNA (tDNA) from 25 PCNSL patients were sequenced using an Ion Torrent Personal Genome Machine (Life Technologies ® ). First, patient-specific targeted sequencing of identified somatic mutations in tDNA was performed. Then, a second sequencing targeting MYD88 c.T778C was performed and compared to plasma samples from 25 age-matched control patients suffering from other types of cancer. Results: According to the patient-specific targeted sequencing, eight patients (32% [95% CI 15-54%]) had detectable somatic mutations in cfDNA. Considering MYD88 sequencing, six patients had the specific c.T778C alteration detected in plasma. Using a control group, the sensitivity was 24% [9-45%] and the specificity was 100%. Tumor volume or deep brain structure involvement did not influence the detection of somatic mutations in plasma. Conclusion: This pilot study provided evidence that somatic mutations can be detected by NGS in the cfDNA of a subset of patients suffering from PCNSL.