ALBERT

Description: 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.

Description: Introduction Diffuse Large B Cell Lymphoma (DLBCL) is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling (GEP) has identified three main subtypes of DLBCL: Germinal Center B-cell like (GCB), Activated B-Cell like (ABC) and Primary Mediastinal B-cell Lymphoma (PMBL). Recently, Next Generation Sequencing (NGS) has enabled a more detailed characterization of DLBCL mutational profiles. Conventional techniques such as immunohistochemistry (IHC) and FISH are also widely used to describe DLBCL. However, no study has yet performed an integrative analysis of the mutational, gene expression, IHC and FISH profiles of DLBCL, in order to provide a comprehensive view of this disease. Methods 215 patients with de novo DLBCL in the prospective, multicenter and randomized LNH-03B clinical trials led by the LYmphoma Study Association (LYSA) were included in this study. Microarray-based GEP identified 81 ABC, 83 GCB, 18 PMBL and 33 other. Mutational profiles of patients' tumor DNA were established using Lymphopanel NGS, designed to identify mutations in 34 genes important for lymphomagenesis. For each recurrently mutated gene, we applied ROMER (Ritchie, Nucleic Acids Res, 2015) to perform gene set enrichment analysis on differential expression profiles of mutant and wild-type patients, using a multifactorial model accounting for subtype. The gene sets were obtained from the MSIGDB Hallmarks (Subramanian A, PNAS, 2005) and Signaturedb (Schaffer, Immunol Rev, 2006) collections. When possible, IHC was performed for IgM (n=150), MYC (n=140), BCL2 (n=148), BCL6 (n=146), CD10 (n=152), FOXP1 (n=147) and MUM1 (n=152); FISH was performed for MYC (n=131), BCL2 (n=133) and BCL6 (n=131). Results As expected, EZH2 mutations were significantly associated with upregulation of GCB gene expression (p

Description: The molecular heterogeneity of diffuse large B-cell lymphoma (DLBCL) has been highlighted by gene expression profiling (GEP), dividing DLBCL into the following three main molecular subtypes with different clinical outcomes and responses to immunochemotherapy: the germinal center B-cell-like (GCB) subtype, the activated B-cell-like (ABC) subtype and the primary mediastinal B-cell lymphoma (PMBL) subtype. Despite frequent translocations involving the IGH (heavy chain) locus, B-cell receptor (BCR) expression is retained in almost all DLBCL, indicating that BCR signaling is crucial for the survival of malignant cells. Furthermore, DLBCL are characterized by recurrent somatic mutations that are supposed to be oncogenic drivers. Here, with the aim to more accurately define the DLBCL pathogenic subgroups, we report a detailed immunogenetic analysis of IGH rearrangements in a well-defined GEP DLBCL cohort and correlate these features with the somatic mutation status of recurrently mutated genes involved in lymphomagenesis. Methods: 204 DLBCL enrolled in the prospective LYSA LNH03 trial program were classified into molecular subtypes by GEP using Affymetrix arrays [82 ABC (40.2%), 77 GCB (37.7%), 29 (14.2%) unclassified and 16 PMBL (7.8%)]. The amplification of complete VDJ rearrangements was realized using BIOMED-2 protocols. Somatic hypermutation (SHM) characteristics were studied (mutation rate, glycosylation N-X-S/T sites, RGYW hotspots, composition of CDR3) and correlated with the molecular subtypes. To obtain a more comprehensive molecular portrait of the DLBCL cases, GEP and IGH VDJ analyses were correlated with the mutational status of a panel of 34 recurrently mutated genes in DLBCL such as MYD88, EZH2, CD79B, TNFAIP3, CARD11 and GNA13 (Dubois et al. Clinical Cancer Research 2016). Results: A total of 153/204 (75%) clonal VDJ rearrangements were successfully determined. Failures to detect clonotypic sequences were predominantly seen in PMBL (56%) and unclassified (31%) cases. The ABC subtype display a significantly lower SHM rate than the 3 other subtypes, especially in contrast to the PMBL subgroup that was characterized by a higher SHM rate (p

Description: 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.

Description: In acute leukemia, recurrent chromosomal translocations which result in the fusion of two genes are frequent. Some of these markers have a well established prognostic and therapeutic impact and are routinely screened at diagnosis by cytogenetics and RT-PCR. Yet, due to the limitations of these methods, only a few among the dozens of known rearrangements are systematically tested. Many abnormalities which could provide important clinical information thus remain ignored, mainly due to the impossibility of performing a cost effective multi-targeted screening. To overcome this obstacle, we have developed a simple and parsimonious assay which allows a reliable detection of dozens of fusion genes in only a few hours. Our assay was designed to screen simultaneously more than 50 translocations involving 70 genes, recurrent in acute myeloid (AML), acute lymphoid (ALL) and chronic myeloid (CML) leukemia. cDNA samples obtained from leukemic cells are first incubated with a mix of oligonucleotides probes which are complementary to the ends of the exons, at the abnormal junctions on the fusion mRNAs (Figure). For most genes, different probes were designed to detect different transcripts resulting from alternative genomic breakpoints (for example on exons 1, 13, 14 and 19 for BCR, and on exons 2 and 3 for ABL). The mix we created thus regroups more than 150 probes and targets more than 400 different fusion transcripts. All left probes have a common tail (T1) at their 5' ends, all right probes a common tail (T2) at their 3' ends. Additional probes were also included to detect the most frequent mutations of NPM1 (A, B, D). If one translocation is present in the sample, two probes hybridize side to side on the fusion cDNA. A DNA ligase is next used to create a covalent link between these probes, allowing their amplification by PCR with the T1 and T2 primers. If a PCR product is amplified, the two partners are identified by sequencing analysis. To test this method, we applied it to a retrospective series of 430 patients, (252 AML and 178 childhood ALL). In B-ALL (147 cases), all 33 ETV6-RUNX1, 6 BCR-ABL and 5 TCF3-PBX1 as well as 6 MLL rearrangements (3 AF4; 1 ENL, 1 AF9 and 1 AFF4) identified at diagnosis by conventional methods were detected, as well as 5 previously unnoticed P2RY8-CRLF2 junctions. We only failed to detect one MLL-AF4 fusion detected by cytogenetics but not by RT-PCR, probably due to the poor quality of the RNA. Further analysis of 10 MLL-AF4 positive adult ALL patients allowed the correct identification of this fusion in all cases. In T-ALL (31 cases), we detected 6 known (4 SIL-TAL, 2 CALM-AF10) and 5 previously undetected fusions (2 NUP214-ABL, 1 MLL-ENL, 1 ETV6-ABL, and a new PLZF-ABL junction). In AML, we detected 86 fusions: 23 PML-RARA, 2 PLZF-RARA, 18 CBFB-MYH11, 12 RUNX1-RUNX1T1, 4 NUP98-NSD1, 2 BCR-ABL, 1 DEK-NUP214, 1 CALM-AF10, 1 MOZ-CBP, 22 MLL rearrangements (13 PTD, 3 AF9, 2 AF6, 1 AF10, 1 ENL, 1 AF1Q and 1 MAPRE) and 44 NPM1 mutations. Two PML-RARA cases, one with a BCR2 breakpoint (not included in our design), one weakly positive by RT-PCR but negative by cytogenetics, two t(11;17) validated by FISH but negative by RT-PCR, and one NPM1J mutation (not included in our assay) were not detected. Importantly, 20 translocations in this series, including 14 MLL fusions and a cytogenetically cryptic t(8;21) had not been identified at diagnosis. Furthermore, all these new abnormalities (in AML and ALL) could be confirmed by conventional RT-PCR and sequencing analysis, demonstrating the specificity of the method. In the whole cohort of 430 patients, the three methods thus detected 157 fusions. 85 (54.1%) and 112 (71.3%) had been detected at diagnosis by cytogenetics and RT-PCR respectively, and 152 (96.8%) by our assay. Two were revealed only by cytogenetics, one only by RT-PCR and 30 only by our method. In conclusion, we have developed a simple multiplexed assay which can reveal a very large number of recurrent gene fusions in leukemia. Its short turn-around time (we repeatedly tested up to 40 patients in parallel and the results can be obtained in less than one day) and low cost (only one PCR module, one pyrosequencer and basic molecular biology reagents are needed) make it particularly suitable for a daily practice. Its capacity to detect many abnormalities which are almost never tested in daily practice could provide many important diagnosis and prognosis information, and enable the stratification of patients in prospective clinical trials. Figure 1 Figure 1. Disclosures Ruminy: Centre Henri Becquerel: Patents & Royalties. Marchand:Centre Henri Becquerel: Patents & Royalties.

Description: 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.

Description: DLBCL is the most common lymphoid malignancy, accounting for 30-40% of all Non Hodgkin Lymphomas. Gene expression profiling has identified two main subtypes: Germinal Center B-Cell like (GCB) and Activated B-Cell like (ABC). EZH2 plays an essential role in epigenetic regulation of DLBCL by specifically mono-, bi- and tri-methylating histone H3 lysine 27 (H3K27me1/-me2/-me3). Recurrent somatic heterozygous gain-of-function mutations of EZH2 have been identified in DLBCL, mostly affecting tyrosine 641 (Y641), inducing increased H3K27me3. Novel EZH2 inhibitors are currently being tested in phase 1 and 2 clinical trials in patients with and without EZH2 Y641 mutations, but no study has examined which patients would most benefit from this treatment. We studied a cohort of 100 patients with DLBCL with available biopsies (96 at diagnosis and 4 at relapse) and developed an immunohistochemical (IHC) assay based on antibodies specifically targeting EZH2, H3K27me3 or H3K27me2. Eighty-six biopsies (12 EZH2 Y641 mutant and 74 WT EZH2) were usable for IHC analysis. Biopsies were scored based on staining intensity and percentage of tumor cells stained, and a me3/me2 score (log of me3 to me2 ratio) was calculated for each patient. Sanger sequencing of EZH2was performed on all patients, GCB/ABC signature was determined by DASL technology based on the expression of 19 genes, and t(14;18) translocation was detected by karyotyping and FISH. The cohort was also extended to 15 patients with EZH2 Y641 mutations and 17 WT EZH2 patients for Next Generation Sequencing (NGS) analysis of a panel of 34 genes involved in lymphomagenesis. Among our cohort, 45 patients were ABC, 50 were GCB, and 5 were unclassified. Sanger sequencing identified 14 patients with EZH2 Y641 mutations (12 GCB, 1 ABC, 1 unclassified). The t(14;18) translocation was more frequent in patients with EZH2 Y641 mutations (9/14, 64%) (p0, n=12/86), a me3-low/me2-high profile (me3/me2 score0 profiles (n=7/12), whereas patients with WT EZH2 are split between intermediate (n=29/74) and me3/me2 score