Abstract: 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.

Abstract: Beside the rare true leukemic forms of follicular lymphoma (FL), low levels of circulating tumor cells (CTC) are detected by PCR in the vast majority of FL patients at diagnosis. By a regular recirculating process, those CTC could reflect the total solid tumor mass. Alternatively, they could represent a subpopulation of tumor cells with distinct molecular profile including adhesion molecules and, as such, could add to the prognostic value of solid tumoral mass previously reported in FL (Dupuis, JCO 2013). In order to address these issues, we retrospectively selected FL patients treated in our institution between 2007 and 2014 who were simultaneously evaluated for both t(14;18) cells in peripheral blood (PB) and FDG-PET/CT tumor mass at diagnosis or at relapse. Absolute quantification of t(14;18) positive cells was performed using quantitative droplet digital PCR (ddPCR). Total metabolic tumor volume (TMTV) and total lesion glycolysis (TLG) were calculated from FDG-PET/CT, and baseline clinical characteristics and outcomes were collected. One hundred fourteen patients fulfilled the inclusion criteria. Using a routine 10-4 sensitive biomed 2 t(14;18) PCR assay, 75 had a positive PCR, either in peripheral blood alone (n=37), in peripheral blood and tumor biopsy (n=27) or in tumor biopsy but not PB (n= 11). Absolute quantitative ddPCR was performed for the 56 t(14;18) MBR+ patients. Clinical characteristics are given in Table 1. Median CTC value (number of t(14;18) positive cells out of total peripheral blood nucleated cells) was 1.6 10-3 (range 0-0.96), with only 5 CTC (-) patients and 13 patients with 〉 10% CTC . Median TMTV was 267 cm3 (range 4.61-1900) and median TLG was 1473 (range 10.24-5912). A positive correlation was found between number of CTC and TMTV (R2 = 0.49; P 〈 0.001; Fig.1) and to a lesser extend with TLG (R2 = 0.38; P 〈 0.001). With a median follow-up of 32 months, OS of FLIPI 0-2 patients was 100% vs 87% for FLIPI 3-4 patients (p=0.003). A number of CTC 〉 6%, TMTV 〉 432 cm3 and TLG 〉 2717 tend to be associated with poorer OS (Fig. 2). The combined presence of 〉 6% CTC and TLG 〉 2717 allowed to identify a group of patients with 3-year OS of 71%, compared with 100% when both criteria were negative or dissociated (P= 0.01) (Fig. 2). In the subset of 42 patients with an untreated and untransformed FL, incremental prognostic value of circulating mass and metabolic tumor burden remained significant (P=0.03). Disclosures Dupuis: ABBVIE: Membership on an entity's Board of Directors or advisory committees; ROCHE: Speakers Bureau.

Abstract: Abstract 1559 Follicular lymphoma (FL) is the most common indolent subtype of non-Hodgkin's lymphoma in the western world. The genetic hallmark of FL is the t(14;18)(q32;q21) translocation leading to the deregulation of BCL2 expression which occurs in up to 90% of the grade 1–2 FL. However, a minority of FL without BCL2 gene rearrangement harbour genetic abnormalities involving the BCL6 gene (5–15% of the cases) and show distinct pathological features. The activation of Signal Transducer and Activator of Transcription 6 (STAT-6) has been observed in Primary Mediastinal B-cell Lymphoma (PMBL) and Hodgkin Lymphoma but also in FL. Because missense mutations of STAT6 DNA binding domain have been described in PMBL, we searched for such mutations in FL. Using a PCR HRM (High Resolution Melting) assay as a screening tool and conventional Sanger sequencing in all cases with abnormal denaturation curves, we analyzed the frequency of STAT6 mutations in FL samples. We focused our screening on exon 12, which encodes part of the DNA binding domain and which had been shown to be a hotspot mutation in PMBL. A series of 40 FL lymphomas samples diagnosed at the University Hospital of Créteil (Henri Mondor) and Centre Henri Becquerel in Rouen were retrieved. These tumors showed characteristic histopathological features of FL according to the World Health Organization (WHO) classification. DNA analysis was performed on DNA extracted from fresh/frozen samples (Rouen) or FFPE (formalin-fixed and paraffin-embedded) tissues (Créteil) with standard procedures. We detected 5 (12%) mutated tumors in this series of FL. These 5 mutations were single missense mutations targeting amino acids 419–421. All mutations were observed in histological grade 1or 2 lymphomas, and no mutations were found in the 9 cases classified as 3A or 3B. Only one classical FL out of 15 cases (6%) with BCL2 rearrangement showed STAT6 mutation. Strikingly, the 4 other mutated cases showed specific features. There were 3 female and 1 male, with a mean age of 52 years, and all presented with inguinal involvement and stage III or IV Ann Arbor disease. Morphologically, these cases displayed a follicular growth pattern. The immunophenotype was CD20+, CD5-, CD10+ (3/4), BCL6+, CD23+ (3/4) but BCL2 was negative (4/4). Cytogenetically, these 4 cases were characterized by BCL6 gene rearrangement without BCL2 gene rearrangement by interphase FISH on FFPE tissue sections. Thus, STAT6 mutations were observed in 4/11 (36%) grade 1–2, and 0/6 grade 3A/3B BCL2 negative FL with BCL6 rearrangement. No case was found to be mutated in the FL group without BCL2 and BCL6 rearrangement (8 cases). In conclusion, this is the first time that mutations of STAT6 are found in FL and interestingly, they target a rare group of FL with distinct pathological and cytogenetical features. Further investigations are required to identify the mutational mechanisms involved and the oncogenic function associated with these mutations. Disclosures: No relevant conflicts of interest to declare.

Abstract: 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.

Abstract: Skin biopsies of 41 angioimmunoblastic T-cell lymphoma patients were retrospectively analyzed for the expression of follicular helper T-cell (TFH) markers, Epstein-Barr virus (EBV), and the presence of RHOA (p.G17V) and IDH2 (p.R172K/S) mutations using allele-specific polymerase chain reaction. We categorized cases into 4 distinctive patterns: (1) low-density lymphocytic perivascular infiltrates (n=11), (2) dense perivascular infiltrates with atypical cells and occasional inflammatory cells (n=13), (3) diffuse infiltrates reminiscent of angioimmunoblastic T-cell lymphoma (n=4), or (4) other aspects (n=13). Two EBV + and 2 plasmacytoid lymphoproliferative disorders were seen. We observed variable expression of TFH markers (CD10 [50%], BCLB6 [84%] , PD1 [94%], CXCL13 [84%] , and ICOS [97.5%]), and EBV + B-blasts (26%). A TFH phenotype was identified in 82% and 73%, respectively, of cases with the most challenging patterns 1 and 2. TFH markers and EBV can thus help for diagnosis and are detected in samples with low-density infiltrates. We found RHOA G17V and IDH2 R172K/S mutations in the skin in 14/18 (78%) and 3/16 (19%) cases, respectively. The RHOA G17V mutation was identified in a proportion of biopsies with patterns 1 and 2, which represent a diagnostic challenge. The RHOA G17V mutation was detected both in the skin and lymph node (LN) biopsies in 7/9 (64%) cases, and in only the skin or the LN of 1 sample each. The frequency of RHOA G17V mutation was similar to that reported in LNs. It may represent a sensitive diagnostic marker in the skin, helpful in cases with low-density infiltrates.

Abstract: Abstract 2632 Hans algorithm using immunohistochemistry correlates well with gene expression data in Diffuse large B-cell lymphomas (DLBCL) (Meyer PN, 2011) and has demonstrated in some studies clear survival differences in favor of germinal-centre (GC) vs non-germinal centre (n-GC) B-cell among DLBCL treated with R-CHOP. We undertook an immunohistochemical study among patients aged 18 to 59 years with aaIPI 1 included in the GELA trial LNH 03-2B that compared R-ACVBP intensified immunochemotherapy to standard R-CHOP. This trial demonstrated an improvement of EFS, PFS and overall survival (OS) of patients treated with R-ACVBP (C Recher et al, in press). Our goal was to evaluate survival of patients with GC and n-GC DLBCL according to treatment regimens. We analyzed by immunohistochemistry the expression of CD10, BCL6 and MUM1 and classified patients as GC or n-GC according to the Hans algorithm. Among the 380 patients enrolled in this study, 229 patients were available for Hans algorithm classification. There was no differences considering clinical characteristics of these 229 patients (age, sex, B symptoms, PS, Stage, LDH, number of extranodal sites, bulky mass, bone marrow involvement) compared to the whole LNH03-2B population. 175 DLBCL cases were present on a tissue microarray (TMA) and 54 other cases were analyzed using unstained slides. 101 patients were classified as GC and 128 as n-GC. 107 patients were treated by R-ACVBP and 122 by R-CHOP. EFS, PFS and OS were not different between the GC and n-GC profile among the whole population (P=.82, P=.90, P=.68, respectively). There was no statistical difference in EFS, PFS and OS between R-ACVBP and R-CHOP in GC patients (P=.78; P=.84, P=.33, respectively). Interestingly, EFS, PFS and OS were significantly much longer among n-GC patients treated by R-ACVBP compared to R-CHOP (P=.02; P=.007, P=.007, respectively). Results were similar considering only TMA population (P=.02, P=.001, P=.001, respectively). This subgroup analysis suggests that the survival benefit related to R-ACVBP over R-CHOP in the LNH 03-2B is in large part linked to a survival improvement in the n-GC population. This algorithm, easy to apply on routine paraffin-embedded tissue, might be useful in the future to select patients that can primarily benefit from this intensive regimen. Disclosures: No relevant conflicts of interest to declare.

Abstract: 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. Am. J. Hematol. 91:923–930, 2016. © 2016 Wiley Periodicals, Inc.

Abstract: Ten cases of an indolent T-cell lymphoproliferative disease of the gastrointestinal tract are reported. It is important to recognize this condition because it can be mistaken for aggressive T-cell lymphoma, which may lead to unnecessary therapy.