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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.

Background Recently, copy number variations (CNV) impacting genes involved in oncogenic pathways have attracted an increasing attention to manage disease susceptibility. CNV is one of the most important somatic aberrations in the genome of tumor cells. Oncogene activation and tumor suppressor gene inactivation are often attributed to copy number gain/amplification or deletion, respectively, in many cancer types and stages. Recent advances in next generation sequencing protocols allow for the addition of unique molecular identifiers (UMI) to each read. Each targeted DNA fragment is labeled with a unique random nucleotide sequence added to sequencing primers. UMI are especially useful for CNV detection by making each DNA molecule in a population of reads distinct. Results Here, we present molecular Copy Number Alteration (mCNA), a new methodology allowing the detection of copy number changes using UMI. The algorithm is composed of four main steps: the construction of UMI count matrices, the use of control samples to construct a pseudo-reference, the computation of log-ratios, the segmentation and finally the statistical inference of abnormal segmented breaks. We demonstrate the success of mCNA on a dataset of patients suffering from Diffuse Large B-cell Lymphoma and we highlight that mCNA results have a strong correlation with comparative genomic hybridization. Conclusion We provide mCNA, a new approach for CNV detection, freely available at https://gitlab.com/pierrejulien.viailly/mcna/ under MIT license. mCNA can significantly improve detection accuracy of CNV changes by using UMI.

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.

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.

International audience

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.

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.

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 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.

International audience; Introduction: Primary cutaneous diffuse large B‐cell lymphoma leg‐type (PCLBCL‐LT) is recognized by the World Health Organization (WHO) classification as a rare and aggressive disease accounting for 5 to 10% of primary cutaneous lymphoma. It displays original clinical features occurring mostly in the elderly and preferentially involving the legs but date the genetic specificities of this entities, as compared to other B‐cell aggressive lymphoma is unknown.Methods: To determine whether the mutational profile of primary cutaneous diffuse large B‐cell lymphoma leg‐type (PCLBCL‐LT) is unique by comparison with other diffuse large B‐cell lymphoma (DLBCL) subtypes, we analyzed a total cohort of 28 PCLBCL‐LT cases by next generation sequencing with a Lymphopanel designed for DLBCL. We also analyzed 12 pairs of tumor and control DNA samples by whole exome sequencing which led us to perform resequencing of three selected genes not included in the Lymphopanel: TBL1XR1, KLHL6 and IKZF3. To pinpoint specificities, comparison with a cohort of DLBCL and Primary central nervous System lymphoma (PCNSL) analyzed by the same targeted panel was performed.Results: Our study clearly identifies an original mutational landscape of PCLBCL‐LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1 and CD79B. Other genes involved in B‐cell signaling, NFKB activation or DNA modeling were found altered notably TBL1XR1 (33%), MYC (26%) CREBBP (26%) and IRF4 (21%) or HIST1H1E(41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of cases. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3 and CIITA. The high recurrence of specific gene mutations such as MYD88 and the absence of mutations of KMT2D or FOXO1 are distinct features from nodal DLBCLs of either GC or ABC subtypes. Interestingly PCLBCL‐LT exhibits a mutational pattern that is closer to PCNSL than to ABC‐type nodal DLBCL but also has distinctive features, such as a very high mutational rate for PIM1, a higher rate of CD79B mutations and other original mutations such as CREBBP, MYC and IRF4 mutations (figure 1).Conclusion: This study describes for the first time the genomic landscape of a series of untreated PCLBCL‐LT. Our results obtained by WES and targeted sequencing underscore several similarities with ABC‐DBCL and more specifically with PCNSL subtypes. On the other hand, we pinpoint specificities that may sustain distinctive clinical features and guide therapeutic strategies according to an individual genetic analysis.

Introduction: 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 NFkB pathway activation. The frequency of MYD88 mutations in DLBCL and other hematologic malignancies is well described; however, there has not yet been a large-scale study of a MYD88 mutated patient cohort with additional Next Generation Sequencing (NGS), copy number variation (CNV), and gene expression data, in order to thoroughly characterize the associated genomic profiles of these patients. The aims of our study were to compare the L265P and non-L265P mutations in terms of pathological and genetic features, to better detail the genomic background associated with MYD88 mutations in order to delineate patients potentially sensitive to targeted therapies, and to define the prognostic value of MYD88 mutations according to different genomic contexts. Methods: A cohort of 361 DLBCL patients (94 MYD88 mutant and 267 MYD88 wild-type) was selected among the prospective, multicenter and randomized LNH-03B and LNH09-7B (NCT01195714) LYSA trials, as well as among patients sequenced at our institution as part of routine procedure. Cell of origin (COO) classification was obtained with HGU133+2.0 Affymetrix GeneChip arrays for 214 patients, with RT-MLPA for 77 patients1 and with Hans immunohistochemistry (IHC) method for 49 patients. All cases were submitted to next generation sequencing (NGS) focusing on 34 genes (Lymphopanel2) in order to analyze associated mutations and copy number variations (CNVs), as well as IHC, FISH, 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 segregated subgroups according to associated genetic alterations among patients with either MYD88 L265P or non-L265P mutations. As such, clustering separated MYD88 L265P mutated DLBCL with associated PIM1 (52%), CD79B (52%), KMT2D (42%), and PRDM1 (32%) mutations, as well as MYD88 L265P mutated DLBCL with CDKN2A/B (67%/50%), PRDM1 (57%) and TNFAIP3 (52%) CNVs. We showed that associated CD79B and MYD88 L265P mutations act synergistically to increase NFkB pathway activation, although the majority of ABC MYD88 L265P mutant cases harbor downstream NFkB alterations, which can potentially predict BTK inhibitor resistance. Of note, 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 both in OS (p=0.02) and PFS (p=0.01), whereas the association of CARD11 or TNFAIP3 alterations did not impact survival. Interestingly, MYD88 mutant DLBCL cases were significantly more likely to experience central nervous system (CNS) relapse than MYD88 WT cases (p=0.02), as were MYD88 L265P mutant cases specifically (p=0.03). This result still tended toward statistical significance when considering only ABC patients (7 of 11 ABC CNS-relapsing cases were MYD88 mutant, p=0.1) but would have to be confirmed in a larger cohort. Conclusions: This study highlights the relative heterogeneity of MYD88 mutant DLBCBL, adding to the field's knowledge of the distinct genetic backgrounds of these subgroups. Our data highlights the theranostic and prognostic relevance of examining MYD88 and associated genomic alterations, emphasizing the usefulness of genomic profiling to best stratify patients for targeted therapy. 1. Mareschal S, Ruminy P, Bagacean C, et al. Accurate Classification of Germinal Center B-Cell-Like/Activated B-Cell-Like Diffuse Large B-Cell Lymphoma Using a Simple and Rapid Reverse Transcriptase-Multiplex Ligation-Dependent Probe Amplification Assay: A CALYM Study. The Journal of molecular diagnostics : JMD. 2015;17(3):273-283. 2. Dubois S, Viailly P-J, Mareschal S, et al. Next Generation Sequencing in Diffuse Large B Cell Lymphoma Highlights Molecular Divergence and Therapeutic Opportunities: a LYSA Study. Clinical cancer research : an official journal of the American Association for Cancer Research. 2016;22(12):2919-2928. Disclosures Salles: Novartis: Consultancy, Honoraria; Mundipharma: Honoraria; Amgen: Consultancy, Honoraria; Celgene: Consultancy, Honoraria; Gilead: Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Roche/Genentech: Consultancy, Honoraria, Research Funding.

International audience; To determine whether the mutational profile of primary cutaneous diffuse large B-cell lymphoma, leg type (PCLBCL-LT) is unique by comparison with other diffuse large B-cell lymphoma subtypes, we analyzed a total cohort of 20 PCLBCL-LT patients by using next-generation sequencing with a lymphoma panel designed for diffuse large B-cell lymphoma. We also analyzed 12 pairs of tumor and control DNA samples by whole-exome sequencing, which led us to perform resequencing of three selected genes not included in the lymphoma panel: TBL1XR1, KLHL6, and IKZF3. Our study clearly identifies an original mutational landscape of PCLBCL-LT with a very restricted set of highly recurrent mutations (>40%) involving MYD88 (p.L265P variant), PIM1, and CD79B. Other genes involved in B-cell signaling, NF-κB activation, or DNA modeling were found altered, notably TBL1XR1 (33%), MYC (26%) CREBBP (26%), and IRF4 (21%) or HIST1H1E (41%). MYD88L265P variant was associated with copy number variations or copy neutral loss of heterozygosity in 60% of patients. The most frequent genetic losses involved CDKN2A/2B, TNFAIP3/A20, PRDM1, TCF3, and CIITA. Together, these results show that PCLBCL-LT exhibits a unique mutational landscape, combining highly recurrent hotspot mutations in genes involved in NF-kB and B-cell signaling pathways, which provides a rationale for using selective inhibitors of the B-cell receptor.

International audience; Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma. It includes three major subtypes termed germinal center B-cell-like, activated B-cell-like, and primary mediastinal B-cell lymphoma. With the emergence of novel targeted therapies, accurate methods capable of interrogating this cell-of-origin classification should soon become essential in the clinics. To address this issue, we developed a novel gene expression profiling DLBCL classifier based on reverse transcriptase multiplex ligation-dependent probe amplification. This assay simultaneously evaluates the expression of 21 markers, to differentiate primary mediastinal B-cell lymphoma, activated B-cell-like, germinal center B-cell-like, and also Epstein-Barr virus-positive DLBCLs. It was trained using 70 paraffin-embedded biopsies and validated using >160 independent samples. Compared with a reference classification established from Affymetrix U133 + 2 data, reverse transcriptase multiplex ligation-dependent probe amplification classified 85.0% samples into the expected subtype, comparing favorably with current diagnostic methods. This assay also proved to be highly efficient in detecting the MYD88 L265P mutation, even in archival paraffin-embedded tissues. This reliable, rapid, and cost-effective method uses common instruments and reagents and could thus easily be implemented into routine diagnosis workflows, to improve the management of these aggressive tumors.

International audience; Classical Hodgkin lymphoma is one of the most common lymphomas and shares clinical and genetic features with primary mediastinal B-cell lymphoma. In this retrospective study, we analyzed the recurrent hotspot mutation of the exportin 1 (XPO1, p.E571K) gene, previously identified in primary mediastinal B-cell lymphoma, in biopsies and plasma circulating cell-free DNA from patients with classical Hodgkin lymphoma using a highly sensitive digital PCR technique. A total of 94 patients were included in the present study. This widely expressed XPO1 E571K mutation is present in one quarter of classical Hodgkin lymphoma patients (24.2%). Mutated and wild-type classical Hodgkin lymphomas were similar regarding the main clinical features. Patients with a detectable XPO1 mutation at the end of treatment displayed a tendency toward shorter progression-free survival, as compared to patients with undetectable mutation in plasma cell-free DNA (2-year progression-free survival: 57.1%, 95% confidence interval: 30.1-100% versus 2-year progression-free survival: 90.5%, 95% confidence interval: 78.8-100%, respectively, P=0.0601). To conclude, the detection of the XPO1 E571K mutation in biopsy and plasma cell-free DNA by digital PCR may be used as a novel biomarker in classical Hodgkin lymphoma for both diagnosis and minimal residual disease, and pinpoints a crucial role of XPO1 in classical Hodgkin lymphoma pathogenesis. The detection of somatic mutation in the plasma cell-free DNA of patients represents a major technological advance in the context of liquid biopsies and noninvasive management of classical Hodgkin lymphoma.

Diffuse large B-cell lymphoma (DLBCL) is an aggressive and heterogeneous malignancy harboring frequent targetable activating somatic mutations. Emerging evidence suggests that circulating cell-free DNA (cfDNA) can be used to detect somatic variants in DLBCL using Next-Generation Sequencing (NGS) experiments. In this proof-of-concept study, we chose to develop simple and valuable digital PCR (dPCR) assays for the detection of recurrent exportin-1 (XPO1) E571K, EZH2 Y641N, and MYD88 L265P mutations in DLBCL patients, thereby identifying patients most likely to potentially benefit from targeted therapies. We demonstrated that our dPCR assays were sufficiently sensitive to detect rare XPO1, EZH2, and MYD88 mutations in plasma cfDNA, with a sensitivity of 0.05%. cfDNA somatic mutation detection by dPCR seems to be a promising technique in the management of DLBCL, in addition to NGS experiments.

Background Primary Central Nervous System Lymphoma (PCNSL) are rare and aggressive primary brain tumors. Histological diagnosis can be difficult at initial stages or at relapse due to deep brain structure involvement. Finding a minimally invasive biomarker aiding the diagnosis remains an unsolved question. Plasma Cell-Free DNA (cfDNA) seems to have shown its diagnostic and prognostic value in nodal Diffuse Large B Cell Lymphomas (DLBCL) [Roschewski et al, Lancet Oncology, 2015; Kurtz et al, Blood, 2015]. Our main objective was to demonstrate that targeted sequencing of cfDNA in plasma at time of diagnosis could identify PCNSL somatic mutations. Methods 30 immuno-competent patients suffering from newly diagnosed PCNSL, without any extranevraxic lesions, were enrolled from 2008 to 2014. Tumor tissues and plasma samples were collected at the time of diagnosis and frozen until use. High throughput sequencing was performed on primitive tumors using a panel of 34 genes relevant to lymphomagenesis, as previously reported [Dubois et al, Oncotarget, 2015; Bohers et al, Haematologica, 2015]. We next performed patient-specific targeted sequencing of identified somatic mutations in cfDNA. The detection sensitivity threshold was set at 1% for all SNVs, except for MYD88 L265P, which was set at 0.1%. The primary endpoint was the proportion of patients having at least one somatic mutation found in the plasma. Results Among 24 available plasmas, 15 patients (63%) had at least one detected somatic mutation in cfDNA. All plasmas had detectable cfDNA (mean concentration 1.6 ng/µL). No correlation was found between tumor volume and cfDNA concentration (R squared coefficient 0.01). Regarding the whole sequenced cohort (n=30) 21 (70%) were classified as nonGC subtype, 8 (27%) as GC subtype and 1 patient (3%) as unclassifiable, according to the Hans algorithm. 29 tumors had at least one somatic mutation, mainly nonsynonymous single nucleotide variants (SNV). The NF-kB pathway was the most affected by mutations: MYD88 (n=23, 77%), PIM1 (n=11, 37%), TNFAIP3 (n=6, 20%), IRF4 (n=3, 10%), CARD11 (n=3, 10%) and PRDM1 (n=3, 10%). Among the 23 tumors harboring a MYD88 mutation, the L265P variant was the most frequent (20 patients, 67%); mean tumor variant allele frequency was 46% [min 8%, max 91%]. One tumor harbored a single MYD88 L265P mutation with no other detectable abnormality. Among patients with both available plasma and a somatic MYD88 L265P mutation in the tumor, 15 patients (88%) had an identifiable L265P variant in cfDNA, with a mean variant allele frequency of 4% [min 0.1%, max 28%]. PIM1 and TNFAIP3 SNVs were also detected in cfDNA for respectively two and one patient. The second most affected pathway was the apoptotic pathway: genes affected by mutations included GNA13 (n=7, 23%), TP53 (n=2, 7%), MYC (n=1, 3%), CDKN2A (n=2, 7%) and BCL2 (n=1, 3%). The B Cell Receptor (BCR) pathway was also affected, mainly due to mutations targeting CD79B (n=10, 33%) and ITPKB (n=3, 10%) mutations. 8 tumors (27%) harbored a dual alteration affecting MYD88 and CD79B. One tumor of the GC subtype had one EZH2 SNV (Y646H), but the mutation was not found in cfDNA. There was no significant difference in overall survival (OS) between patients with and without mutations detected in cfDNA: mean OS 27 months versus 18 months (HR 0.6; IC95% [0.2 - 1.6], p value 0.3). Tumor gene copy number variations were detected in 29/30 patients with either homo or heterozygous deletions or copy gains. CDKN2A/2B deletions were detected in 23 cases (77%). Conclusion To our knowledge this is the first study that provides evidence that somatic mutations can be detected in cfDNA in patients suffering from PCNSL, therefore constituting a minimally invasive tool helping for diagnosis. Further studies are now required to improve prognosis and predictive values of this new promising procedure for PCNSL patient care. Disclosures No relevant conflicts of interest to declare.

Background and aim of the study Primary mediastinal B-cell lymphoma (PMBL) is an entity of aggressive B-cell lymphoma that is clinically and biologically distinct from the other molecular subtypes of diffuse large B-cell lymphoma (DLBCL). We recently detected by Whole exome sequencing a recurrent point mutation in the XPO1 (exportin 1) gene (also referred to as chromosome region maintenance 1; CRM1), which resulted in the Glu571Lys (p.E571K) missense substitution in 2 refractory/relapsed PMBL (Dubois et al., ICML 2015; Mareschal et al. AACR 2015). XPO1 is a member of the Karyopherin-b superfamily of nuclear transport proteins. XPO1 mediates the nuclear export of numerous RNAs and cellular regulatory proteins, including tumor suppressor proteins. This mutation is in the hydrophobic groove of XPO1 that binds to the leucine-rich nuclear export signal (NES) of cargo proteins. In this study, we investigated the prevalence, specificity, and biological / clinical relevance of XPO1 mutations in PMBL. Patients and methods High-throughput targeted or Sanger sequencing of 117 PMBL patients and 3 PMBL cell lines were performed. PMBL cases were defined either molecularly by gene expression profile (mPMBL cohort) or by standard histological method (hPMBL cohort) and enrolled in various LYSA (LYmphoma Study Association) clinical trials. To assess the frequency and specificity of XPO1 mutations, cases of classical Hodgkin lymphoma (cHL) and primary mediastinal grey zone lymphoma (MGZL) were analysed. Cell experiments were performed to assess the impact of the E571 mutation on the activity of selective inhibitor of nuclear export (SINE) molecules. Results XPO1 mutations were present in 28/117 (24%) PMBL cases but were rare in cHL cases (1/19, 5%) and absent from MGZL cases (0/20). A higher prevalence (50%) of the recurrent codon 571 variant (p.E571K) was observed in PMBL cases defined by gene expression profiling (n = 32), as compared to hPMBL cases (n = 85, 13%). No difference in age, International Prognostic Index (IPI) or bulky mass was observed between the PMBL patients harboring mutant and wild-type XPO1 in the overall cohort whereas a female predominance was noticed in the mPMBL cohort. Based on a median follow-up duration of 42 months, XPO1 mutant patients exhibited significantly decreased PFS (3y PFS = 74% [CI95% 55-100]) compared to wild-type patients (3y PFS = 94% [CI95% 83-100], p=0.049) in the mPMBL cohort. In 4/4 tested cases, the E571K variant was also detected in cell-free circulating plasmatic DNA, suggesting that the mutation can be used as a biomarker at the time of diagnosis and during follow-up. Importantly, the E571K variant was detected as a heterozygous mutation in MedB-1, a PMBL-derived cell line, whereas the two other PMBL cell lines tested, Karpas1106 and U-2940, did not display any variants in XPO1 exon 15. KPT-185, the SINE compound that blocks XPO1-dependent nuclear export, induced a dose-dependent decrease in cell proliferation and increased cell death in the PMBL cell lines harbouring wild type or mutated alleles. To test directly if XPO1 mutation from E571 to E571K alters XPO1 inhibition by SINE compounds, the mutated protein was tested in vitro. The E571XPO1 mutated allele was transiently transfected into osteosarcoma U2OS cells which stably express the fluorescently labelled XPO1 cargo REV. Cells were treated with the clinical SINE compound selinexor, which is currently in phase I/II clinical trials and nuclear localization of REV-GFP was analysed in red transfected cells. The results showed that the nuclear export of the mutated XPO1 protein was inhibited by selinexor similarly to the wild-type XPO1 protein (Figure 1). Conclusion Although the oncogenic properties of XPO1 mutations remain to be determined, their recurrent selection in PMBL strongly supports their involvement in the pathogenesis of this curable aggressive B-cell lymphoma. XPO1 mutations were primarily observed in young female patients who displayed a typical PMBL molecular signature. The E571K XPO1 mutation represents a novel hallmark of PMBL but does not seem to interfere with SINE activity. Rev-GFP (green fluorescent) expressing U2OS cells were transfected with wild type XPO1-RFP (red fluorescent protein), XPO1-C528S-RFP, XPO1-E571K-mCherry, and XPO1-E571G-mCherry. The cells were then treated with 1µM KPT-330 for 8 hours. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Figure 1. Rev-GFP expressing U2OS cells transfected with XPO1 variants. Disclosures Landesman: Karyopharm Therapeutics: Employment. Senapedis:Karyopharm Therapeutics, Inc.: Employment, Patents & Royalties. Argueta:Karyopharm Therapeutics: Employment. Milpied:Celgene: Honoraria, Research Funding.

// Sydney Dubois 1 , Sylvain Mareschal 1 , Jean-Michel Picquenot 1, 2 , Pierre-Julien Viailly 1 , Elodie Bohers 1 , Marie Cornic 2 , Philippe Bertrand 1 , Elena Liana Veresezan 1, 2 , Philippe Ruminy 1 , Catherine Maingonnat 1 , Vinciane Marchand 1 , Helene Lanic 1, 3 , Dominique Penther 1 , Christian Bastard 1 , Herve Tilly 1, 3 , Fabrice Jardin 1, 3 1 INSERM U918, Centre Henri Becquerel, Universite de Rouen, IRIB, Rouen, France 2 Department of Pathology, Centre Henri Becquerel, Rouen, France 3 Department of Clinical Hematology, Centre Henri Becquerel, Rouen, France Correspondence to: Fabrice Jardin, e-mail: fabrice.jardin@chb.unicancer.fr Keywords: DLBCL, EZH2, Methylation, Immunohistochemistry, NGS Received: October 20, 2014 Accepted: January 15, 2015 Published: February 05, 2015 ABSTRACT Enhancer of Zeste Homolog 2 (EZH2) plays an essential epigenetic role in Diffuse Large B Cell Lymphoma (DLBCL) development. Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, most notably affecting tyrosine 641 (Y641), inducing hyper-trimethylation of H3K27 (H3K27me3). Novel EZH2 inhibitors are being tested in phase 1 and 2 clinical trials but no study has examined which patients would most benefit from this treatment. We evaluated the immunohistochemical (IHC) methylation profiles of 82 patients with DLBCL, as well as the mutational profiles of 32 patients with DLBCL using NGS analysis of a panel of 34 genes involved in lymphomagenesis. A novel IHC score based on H3K27me2 and H3K27me3 expression was developed, capable of distinguishing patients with wild-type (WT) EZH2 and patients with EZH2 Y641 mutations ( p = 10 −5 ). NGS analysis revealed a subclonal EZH2 mutation pattern in EZH2 mutant patients with WT-like IHC methylation profiles, while associated mutations capable of upregulating EZH2 were detected in WT EZH2 patients with mutant-like IHC methylation profiles. IHC and mutational profiles highlight in vivo hyper-H3K27me3 and hypo-H3K27me2 status, pinpoint associated activating mutations and determine EZH2 mutation clonality, maximizing EZH2 inhibitor potential by identifying patients most likely to benefit from treatment.

Diffuse large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30–40% of newly diagnosed cases of non-Hodgkin lymphomas. The molecular heterogeneity of DLBCL has been deciphered by gene expression profiling, and DLBCL have been divided into three main molecular subtypes: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype, and the primary mediastinal B-cell lymphoma subtype with distinct clinical outcomes and responses to immunochemotherapy. Next-generation sequencing (NGS) technologies, which allow for massive, parallel, high-throughput DNA sequencing, have emerged over the past decade and have provided new insights into the genomic characterization of DLBCL. Recurrent single nucleotide variants (SNV) are now well defined and provide new therapeutic opportunities for the three molecular subtypes. The SNV target genes play a crucial role in several pathways, including B-cell receptor signaling (CD79A/CD79B), NFκ-B (CARD11), Toll-like receptor signaling (MYD88), immunity (CD58, TNFSRF14, B2M), cell cycle/apoptosis (TP53, BCL2) and epigenetic regulation (EZH2,CREBBP, MLL2).1,2

As the most common lymphoid malignancy, Diffuse Large B-Cell Lymphoma (DLBCL) has largely benefited from immunochemotherapy combinations developed in the past decade. However 30% to 40% of patients still do not respond to treatment, or relapse in a few months. The mechanisms involved in short term treatment failure are poorly understood, and biomarkers to predict refractoriness in newly diagnosed patients are still lacking. To address this issue, 14 normal / tumoral pairs of exomes from patients who progressed or relapsed within 12 months were sequenced on a HiSeq2500 platform. Somatic events were called using VarScan2, and significantly mutated pathways were highlighted by PathScan (False Discovery Rate, FDR < 0.001). Genes from 175 pathways were found to be more mutated than expected according to the background mutation rate observed throughout the exomes (2.68 non synonymous mutations per megabase, ranging from 0.69 to 4.66), including genes encoding histone monomers (HIST1H1/2) and transcription regulators (CREBBP, EP300, TP53), members and relatives of the NFKB signaling pathway (MYD88, TNFAIP3, NFKBIA/E), or other signaling cascades (IRF4, CIITA, SOCS1). To pinpoint genes and pathways unusually enriched in such refractory patients, a published series of 39 DLBCL genome pairs was used (Morin et al, Blood 2013). Variant annotation and filtering was reapplied to enforce target region consistency, and Fisher tests were conducted independently in the two series. 22 of the 175 pathways selected by PathScan were found to be enriched in refractory patients (FDR < 0.01) but not in the comparative series (FDR > 0.5). One of the most relevant was “IL4 mediated signaling events”, which includes genes such as STAT6, IRF4 or SOCS1. These 3 genes, along with 31 other genes frequently mutated in DLBCL, were sequenced in a series of 216 DLBCLs with an orthogonal method (PGM sequencer). Although the survival impact of IL4 related gene mutations could not be confirmed, their prevalence (17.5%) was far above what was previously described in DLBCL. Molecular profiling by Affymetrix U133+2 arrays revealed a strong association with the Primary Mediastinal B-Cell lymphoma (PMBL) subtype (Fisher p = 5e-10), a molecular subtype observed in 4/14 and 22/216 patients of the refractory and extended series respectively. We propose here an exhaustive description of mutations found in 14 exomes from refractory DLBCL patients, compared to 39 previously published genomes. The extension to 216 patients enrolled in the LNH03 LYSA (LYmphoma Study Association) clinical trial program using targeted resequencing and transcriptomic arrays allowed us to refine these results, and highlight the high prevalence of somatic mutations in IL4 signaling related genes in PMBLs. Citation Format: Sylvain Mareschal, Pierre-Julien Viailly, Philippe Bertrand, Elodie Bohers, Jean-Philippe Jais, Martin Figeac, Thierry J. Molina, Fabienne Desmots, Thierry Fest, Gilles Salles, Corinne Haioun, Hervé Tilly, Karen Leroy, Fabrice Jardin. Exome sequencing of refractory diffuse large B-cell lymphomas highlights candidate genes for targeted resequencing. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4810. doi:10.1158/1538-7445.AM2015-4810

Diff use large B-cell lymphoma (DLBCL) is the most common form of lymphoma, accounting for 30 - 40% of newly diagnosed non-Hodgkin lymphomas. Historically, DLBCL has been thought to involve recurrent translocations of the immunoglobulin heavy (IGH) locus and the deregulation of rearranged oncogenes. Whole exome sequencing (WES) of more than 200 DLBCLs has completely redefi ned the genetic landscape of the disease by identifying recurrent single nucleotide variants and providing new therapeutic opportunities in DLBCL molecular subtypes. Some of these somatic mutations target genes that play a crucial role in B-cell function (B cell receptor (BCR) signaling, nuclear factor κ B (NF- κ B) pathway, Toll-like receptor (TLR) signaling and phosphatidylinositol 3-kinase (PI3K) pathway), immunity, cell cycle/apoptosis or chromatin modifi cation. In this review, following an overview of the somatic mutations reported in DLBCL, we focus on activating and clustered mutations targeting genes including MYD88 , CD79A/B , EZH2 and CARD11 and discuss their clinical and therapeutic relevance in the precision medicine era.

[(18)F]-fluorodeoxyglucose positron emission tomography (FDG-PET) imaging is essential to optimize the initial staging and to predict the prognosis of diffuse large B-cell lymphoma (DLBCL). To assess the relationship between the germinal center B cell-like/activated B cell-like (GCB/ABC) classification and PET scan features in DLBCL, 57 cases treated with rituximab and a cyclophosphamide, doxorubicin, vincristine and prednisone (CHOP)/CHOP-like regimen were analyzed. The expression profile of 18 GCB/ABC related genes and five genes coding for glucose transporters (GLUTs) was determined from frozen tissues using DASL (cDNA-mediated Annealing, Selection, Ligation and extension) technology. According to the gene expression profile (GEP), 30 cases of DLBCL were classified as GCB subtype (2-year progression-free survival [PFS] 76%) and 27 cases as ABC subtype (2-year PFS 51%, p = 0.03). Using a semiquantitative assessment of the decrease in standard uptake value (SUV) at interim PET performed after 3-4 cycles of chemotherapy, we defined fast (n = 36) and slow (n = 9) metabolic responders. In multivariate analysis, GCB/ABC subtype, age-adjusted international prognostic index (aaIPI) and slow/fast metabolic response were independent variables that predicted outcome. A score incorporating aaIPI, fast/slow metabolic response and GCB/ABC classification was used to define two groups with highly significantly distinct outcomes. Our study suggests that the combination of GEP, aaIPI and interim PET more accurately predicts DLBCL prognosis and is therefore suitable for tailoring therapeutic strategies.

HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. We show here, by RT-qPCR, that HACE1 gene is constitutively expressed in Normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of diffuse large B-cell lymphoma (DLBCL) cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases. A HACE1 heterozygous deletion was observed in 38.1% and an homozygous deletion in 2.4% of cases. These deletions were associated with a significant gene expression decrease. The molecular epigenetic mechanisms underlying HACE1 downregulation were also investigated. Using pyrosequencing assays, as compared to normal B-cells, we observed an hypermethylation of HACE1 promoter CpG177 island in 60% (68/111) of DLBCL cases and in all tested B-Lymphoma cell lines. However, no significant correlation between promoter methylation status and gene expression level was demonstrated. Furthermore, RT-qPCR assays revealed that the demethylating agent 5′azacytidine (5′AZA) did not induce a HACE1 gene expression increase in the different cell lines. By contrast, the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and LBH589 strongly reactivated the expression of HACE1 in Ramos, Raji and RL cells in which the CpG 177 island was fully methylated. We next performed ChIP experiments to determine whether HACE1 locus chromatin was in an active or inactive conformation in Ramos cell line, the most sensitive cell line to TSA effect. We found that the chromatin of HACE1 gene promoter region was predominantly in the inactive conformation (methylated H3 histones). TSA treatment was able to reverse this pattern, switching the conformation of HACE1 promoter chromatin to an active one predominantly associated with acetylated H3 histones. The putative role of HACE1 in B-cell lymphomagenesis was further investigated using lentiviral transduction (shHACE1). We demonstrated in Ramos and Raji cells that a down-regulation of HACE1 expression was associated with a significant decrease of apoptosis level and cell cycle arrest in G2/M phase. To conclude, our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and be downregulated by deacetylation and methylation of its promoter region chromatin constituting a potential target for HDAC inhibitors. Citation Format: Abdelilah Bouzelfen, Marion Alcantara, Hafid Kora, Philippe Bertrand, Sylvain Mareschal, Elodie Bohers, Catherine Maingonnat, Philippe Ruminy, Sahil Adriouch, Gaetan Riou, Martin Figeac, Thierry Fest, Christian Bastard, Hervé Tilly, Jean-Baptiste Latouche, Fabrice Jardin. HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4947. doi:10.1158/1538-7445.AM2015-4947