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: Introduction We (Bobée et al, 2020) have recently described a new molecular classifier combining gene expression profiling of 137 genes and machine learning able to diagnose B-cell lymphomas. As this tool can be easily performed from formalin fixed paraffin embedded tissue, we proposed to evaluate how it may be useful after the process of central review by expert pathologists in a LYSA diffuse large B-cell lymphoma (DLBCL) clinical trial, the GAINED trial (Le Gouill et al, 2021). Methods The GAINED trial was a multicenter randomized phase 3 trial comparing obinutuzumab to rituximab followed by a PET-driven consolidation in patients with DLBCL. Histological inclusion criteria were : DLBCL, high grade B-cell lymphoma double/triple hit (DH/TH) or high grade NOS. 670 patients were included and 646 underwent a central review of the biopsy by at least two LYSA pathologists. The central review performed immunostaining of full slides by anti-CD20, CD5, CD10, BCL6, MUM1, MYC, BCL2 if sufficient material was available as well as RNA extraction for the molecular classifier, the nanostring Lymph2CX and DNA extraction for targeted NGS. The molecular classifier was able to deliver 8 different signatures: DLBCL GCB, DLBCL ABC, primary mediastinal large B-cell lymphoma (PMBL), follicular lymphoma (FL), mantle cell lymphoma (MCL), marginal zone lymphoma/lymphoplasmacytic lymphoma, CLL/lymphocytic Lymphoma, T-cell rich signature. We compared classification by the histopathology review and by the molecular classifier. Discordant cases were reviewed both by two histopathologists (JB, TJM) and the molecular biologist (PR) and a reconciliation diagnosis was proposed Results The central histopathological review alone identified 580 patients with DLBCL or high grade B-cell lymphoma and 40 patients with a lymphoma subtype not eligible for the trial. Among the 470 patients evaluated by the molecular classifier, the reconciliation diagnosis identified 292 DLBCL NOS with 40 associated with a small B-cell component, 13 High grade DH/TH, 9 high grade NOS, 109 PMBL, 16 T-cell rich large B-cell Lymphoma (TCRBCL), 5 DLBCL EBV+ NOS. 29 cases did not fit with inclusion criteria and were mainly FL (3A or 3B), MCL, Nodular lymphocyte Predominant Hodgkin Lymphoma (NLPHL). Among them, the classifier helped to identify one case of CD5- MCL initially diagnosed as DLBCL with CD5- marginal zone lymphoma as well as one case of CD10+ follicular T-cell lymphoma (T-cell rich signature with Rho A mutation) initally diagnosed as FL 3B. Among 235 patients with GCB/ ABC subtyping, the correlation with Nanostring was 97.4% for the molecular classifier and 86,4% for the Hans algorithm. Although the main aim of the pathology review was not dedicated to differentiate DLBCL NOS from PMBL but to validate histological inclusion criteria, after reconciliation 22 DLBCL NOS were reclassified as PMBL. Overall, among the 109 PMBL , PET-CT at baseline in 108 cases evaluable found in 93 cases (86%) a mediastinal mass unique (37) or with satellite lymph nodes(56), in 12 cases (11%) a mediastinal mass with extramediastinal localization and in only 3 cases (3%) the absence of mediastinal mass. This observation fits well with the known anatomical distribution of PMBL and with the rare non mediastinal lymphomas with a PMBL signature. However, the molecular classifier was able to identify 8 signatures and could not recognize other lymphoma subtypes such as High grade B-cell Lymphomas NOS or DH/TH. In addition, 14 follicular lymphomas in our series comprising 5 grade 3A and 6 grade 3B were diagnosed molecularly mainly as DLBCL GC or rarely ABC . Although the TCRBCL were all included in the T-cell signature lymphoma, this signature included high grade B-cell lymphoma, DLBCL NOS, PMBL, DLBCL EBV+, NLPHL. Conclusion Overall, the molecular classifier in addition to the expert pathologic review increased accuracy of diagnostic by the pathologists particularly for difficult or rare cases, as well as it strongly helped to identify PMBL and GC/ABC DLCBL. However, the pathologist should be aware of the limit of the tool when dealing with DLBCL differential diagnosis, particularly for the identification of FL3A, FL3B or for cases with a T-cell rich microenvironment such as TCRBCL. Disclosures Bobée: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Drieux: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties.. Casasnovas: ROCHE: Consultancy, Research Funding; TAKEDA: Consultancy, Research Funding; Gilead/Kite: Consultancy, Research Funding; BMS: Consultancy; Janssen: Consultancy; Amgen: Consultancy. Ghesquieres: Mundipharma Research Limited: Consultancy, Honoraria; Gilead Sciences: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Celgene: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Roche: Consultancy, Honoraria, Other: Travel, accommodation, expenses; Takeda: Other: Travel, accommodation, expenses. Morschhauser: abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; epizyme: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; gilead: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; jannssen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation. Thieblemont: Cellectis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; BMS: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; roche: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Abbvie: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Hospira: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation; Incyte: Membership on an entity's Board of Directors or advisory committees, Other: travel compensation, Research Funding; Kite: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: travel compensation. Ribrag: Bristol Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Epizyme: Honoraria, Research Funding; Servier: Consultancy, Membership on an entity's Board of Directors or advisory committees; GSK: Research Funding; AstraZeneca: Membership on an entity's Board of Directors or advisory committees; PharmaMar: Honoraria, Membership on an entity's Board of Directors or advisory committees; Nanostring: Membership on an entity's Board of Directors or advisory committees; Argen-X: Research Funding; Astex Pharmaceuticals: Research Funding; Infinity Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; MSD Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Roche: Membership on an entity's Board of Directors or advisory committees; Incyte: Membership on an entity's Board of Directors or advisory committees; Gilead: Membership on an entity's Board of Directors or advisory committees. Haioun: Takeda: Honoraria, Research Funding; Servier: Honoraria, Research Funding; F. Hoffmann-La Roche Ltd: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Janssen: Honoraria, Research Funding; Gilead: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Miltenyi: Honoraria, Research Funding. Jardin: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. . Ruminy: Genexpath: Patents & Royalties: The author is a potential inventor on a patent application for the LymphoSign, which has been licensed for by Genexpath Patents & Royalties. .