ALBERT

Description: Introduction Next generation sequencing (NGS) studies have documented the highly heterogeneous landscape of somatic mutations in CLL. Beside the mere genetic diversity of the disease, frequency and clinical impact of mutations vary between analyzed cohorts since samples may be collected at variable clinical stages. To address this point, we undertook an integrative systematic genomic study of patients included in a prospective clinical trial. Material and methods We performed conventional karyotype (K), fluorescence in situ hybridization (FISH) and investigated 31 genes and 2 non-coding regions, in pretreatment samples obtained from 146 patients enrolled on a phase 3 trial comparing fludarabine (F)-cyclophosphamide (C)-rituximab vs. F-C-alemtuzumab (Lepretre et al, Blood 2012). All patients were Binet stage B (n=115) or C (n=31) and devoid of 17p deletion. All gave their informed consent according to the declaration of Helsinki. We studied by targeted deep sequencing exonic regions of 31 genes including TP53, SF3B1, NOTCH1, ATM, BIRC3, FBXW7, NFKBIE, MED12, MYD88 and POT1. DNA was extracted from total blood mononuclear cells (fraction of tumor cells determined by flow cytometry 〉80%). Targeted regions were PCR-amplified using an AmpliSeq (Life technologies) approach and PCR products sequenced in a MiSeq (Illumina) instrument with a mean depth of 1000X. We used a variant allele frequency (VAF) minimal threshold of 5% to detect mutation. EGR2, PAX5 enhancer and NOTCH1 3'UTR regions were analyzed by Sanger sequencing. For each identified mutation, clinical and biological variables were compared between mutant and wild-type patients using either Wilcoxon or Fisher exact test. For each mutation present in a sufficient number of samples, prognostic impact on overall survival (OS) and progression free survival (PFS) was assessed using log-rank test. For important clinical and molecular prognostic variables, adjusted prognostic effect was assessed using a multivariate Cox model for OS and PFS with a stepwise-forward selection based on Akaike criteria. Results Chromosomal abnormalities were observed by K in 63% of 126 patients, 11% showing complex K and 23% carrying translocations. FISH revealed 78/144 (54%) 13q deletion, 25/144 (17%) trisomy 12 (tri12), 30/146 (21%) 11q deletion. Unmutated (UM) IGHV status was observed in 84/146 (58%) cases. At least one mutation was observed in 118/146 (81%) patients, with 1, 2, 3 and 4 or 5 mutations in, respectively, 34%, 23%, 17% and 7%. The most frequently mutated genes were SF3B1 (23%), NOTCH1 (16%), ATM (14%), NFKBIE (10%), XPO1 (10%), MED12 (7%), EGR2 (6%), TP53 (5%), BIRC3 (5%), FBXW7 (5%), PAX5 enhancer (5%), CHD2 (5%), PCLO (5%), MYD88 (4%), POT1 (4%), KMTD2 (4%) and NOTCH1 3'UTR (4%). MYD88 and SAMHD1 mutations were always observed as clonal (VAF〉40%) whereas BIRC3 and BRAF mutationsalways observed as sub-clonal. NOTCH1 mutations were associated with high CD38 expression (P

Description: Introduction: CLL is a heterogeneous disease in terms of response to treatment, with some patients reaching complete and prolonged remissions, while others relapsing early and requiring several lines of treatments. This highly variable course is partly explained by the existence of a heterogenic panel of genetic alterations (mutations, chromosomal abnormalities) that allow the development of drug-resistant aggressive CLL subclones. Therefore, a functional characterization of the cytogenetic alterations associated to CLL drug resistance may provide new means of improving the current therapeutic strategies. We and others have already reported that the gain of 2p (2p+) is recurrent in CLL. However, the candidate gained gene(s) on the 2p remain to be identified. Previously data: we have observed that the 2p gain is frequent in previously untreated CLL Binet stages B/C (21/132, 15.9%), and is associated with bad prognostic factors, such as 11q deletion (p=0.0008) and unmutated IGHV (p=0.02). Using a SNP-array approach, we have identified a minimally gained region of 1.28Mb on 2p16.1-15. This region included the gene CRM1/XPO1 (Chromosome Region Maintenance 1/Exportin-1), a gene also recurrently mutated in CLL. A qPCR assessment confirmed that XPO1 was overexpressed in the 2p+/CLL patients (1.4-fold increase compared to 2p-/CLL; p=0.02). The objective of our work was to identify the potential role of XPO1 in CLL drug resistance by using the selective XPO1 inhibitor Selinexor (KPT-330, provided by Karyopharm Therapeutics), which is currently in Phase II human clinical trials in hematological and solid cancers. Methods: We have analyzed 36 2p+/CLL and we have searched for XPO1 mutations in 436 CLL samples. CLL drug resistance associated to XPO1 overexpression/mutation was assessed by measuring the rate of programmed cell death (PCD) on cells from 2p- and wildtype (wt) XPO1/CLL (n=20), 2p+/XPO1 wt/CLL (n=8) and on XPO1 mut/CLL (n=6). After 24 hours treatment with Fludarabin + Cyclophosphamid + Rituximab (FCR), Ibrutinib (Ibru), Idelalisib + Rituximab (Ide+R) and Selinexor, cells were stained with Annexin-V and propidium iodide and PCD was assessed by flow cytometry. KPT-301 was used as a negative control. For the inhibition assay, the inhibitor Q-VD-Oph was added 30 min before inducing cell death. Mitochondrial membrane depolarisation was assessed using tetramethyllrhodamine ethyl ester probe and flow cytometry analysis. Results: (i) Using a FISH approach, we fully confirmed the gain of XPO1 in 2p+/CLL samples. Additionally, we found that the XPO1 gain was often subclonal, suggesting that it tends to arise late in leukemic development. Longitudinal FISH analyses, performed on 8 2p+/CLL-treated patients, showed a similar or increasing percentage of cells carrying XPO1 gain at relapse, when compared to diagnosis; (ii) XPO1 was mutated in 23/436 (5.3%) CLL and in 2/30 (6.7%) 2p+/CLL; (iii) Selinexor induced PCD in 2p-/XPO1 wt/CLL (35% of PCD). The results were similar in all tested CLL, independently of prognostic factors (del13q, tri12, del11q, del17p, IGHV status), while sparing the non leukemic cells from patients or B cells from healthy donors; (iv) Selinexor induced CLL PCD through a caspase-dependant apoptotic pathway, as evidenced by inhibition of cell death by Q-VD-Oph, and cleavage of the caspase-3. Selinexor also induced mitochondrial depolarization and was associated with upregulation and activation of the pro-apopototic Bax protein; (v) XPO1 mut/CLL were significantly resistant to PCD induced by Selinexor (p=0.003). In contrast, the mutations in XPO1 had no effect in FCR and Ibru PCD induction; (vi) 2p+/CLL cells were resistant to PCD induced by all tested drugs: FCR (p=0.01), Ibru (p=0.003), Ide+R (p=0.004) and Selinexor (p=0.0001). Conclusion: Our data show that 2p+/CLL is associated to FCR, Ibru and Ide+R drug resistance. Strikingly, Selinexor, a new XPO1 inhibitor, is unable to induce PCD in 2p+ and/or XPO1 mut CLL, which strongly suggests a key role for XPO1 in the CLL drug resistance associated to the 2p gain. Altogether, our work provide substantial progress in the understanding of the role of XPO1 in CLL drug resistance and suggests that the assessment of the 2p gain and the mutations in XPO1 will be considered before to decide a CLL therapy. As 2p gain could be observed in other B malignancies, it is tempting to extend these recommendations to all Selinexor treatments. Disclosures Choquet: Janssen: Consultancy; Roche: Consultancy. Leblond:Janssen: Consultancy, Honoraria, Speakers Bureau; GSK: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Honoraria, Speakers Bureau; Roche: Consultancy, Honoraria, Other: Travel, Accommodations, Expenses, Speakers Bureau; Mundipharma: Honoraria.

Description: Abstract 2900 Poster Board II-876 Ph-negative myeloproliferative neoplasms: polycythemia vera (PV), essential thrombocythemia (ET) and primitive myelofibrosis (PMF) carry an acquired somatic mutation JAK2V617F in 95% (PV), and in 50 to 60% (ET or PMF) of the patients. Mutations of the TET2 gene have been observed with roughly similar frequencies in the three MPN, irrespective of the presence of JAK2V617F. Evolution to myelofibrosis or acute leukemia may occur with time in MPN patients. Although its molecular bases are poorly understood, the evolution is likely due to the acquisition of additional mutations. To investigate whether cytogenetic abnormalities are distributed differently according to type of transformation and to the JAK2 and TET2 statuses, the Groupe Francophone de Cytogénétique Hématologique has collected 82 patients with transformation of MPN. There were 66 (80%) acute myeloid leukemia or myelodysplastic syndromes (AML/MDS) and 16 (20%) myelofibroses (MF). Of note pipobroman (Pi) treatment seems to be associated with MF, and hydroxyuera (Hu) with AML/MDS evolution in our series. Statistical analyses of clinical, cytogenetic and molecular data are shown Table 1. On the cytogenetical point of view, several points are noteworthy. Some abnormalities were unevenly distributed: there were significantly more -7/del7q and -5/del5q in AML/MDS and tri1q and tri9 in MF. MF and PMF cytogenetic profile looked similar, suggesting a potential link between cytogenetic markers and the phenotype. Although the derivative chromosome der(1;7), observed in 9 patients, is responsible for a loss of 7q, it seemed different from patients with -7/del7q [excluding der(1;7)]. In the -7/del7q group, AML/MDS patients were more numerous than MF patients and the overall survival was shorter compared with the der(1;7) group (22/22 (100%) vs 6/9 (67%) AML/MDS, p=0.02; median: 4 vs 41 months, p=0.0007 respectively). Some specific associations could be observed, such as 17p deletions with 5q deletion (12/30, 40% vs 4/48, 8%, p=0.0007) and 20q deletion with der(1;7) (4/9 (44%) vs11/69 (16%), p=0.03). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in transformed MPN, with all possible combinations between the wildtype and mutated forms of both genes. For one post-ET AML patient, JAK2V617F had been observed in a fraction of the granulocytes at the chronic phase. Analysis of blood cDNA obtained at chronic phase showed the same TET2 mutation as observed at acute phase. Because the blast cells were JAK2wt-TET2mut and carried a t(10;16)(q22;q23) affecting the CBFB gene, it is likely that the resulting non-MYH11 CBFB fusion gene transformed a JAK2wt-TET2 mutated progenitor that predominated in the chronic phase. In conclusion, no specific chromosomal abnormality was associated with TET2 or JAK2 mutations. Chromosomal abnormalities were associated with a type of transformation (AML/MDS or MF), suggesting a specific role in the process. In addition, association between some chromosomal abnormalities suggest a specific oncogenic cooperation.Table 1.n=82AML/MDS n=66 MF n=16 p univariate p multivariate Sex F39 (59%)5 (31%)nsnsPV/ET/PMF30/26/1013/3/0nsnsAge at diagnosis of MPN54 [20-82]55.5 [31-69]nsnsChronic Phase (duration, years)12 [2-34]14.5 [3-28]nsnsPrior treatments (n=73*)57*16..No treatment (n=6)60nsnsOne treatment (n=40)33 (58%)7 (44%)nsnsTreatments with Hu (n=57)48 (73%)9 (56%)0.03Treatments with Pi (n=41)26 (46%)15 (93%)0.00060.05Age at transformation66.5[37-92]68 [45-80]nsnsAbnormal karyotype62 (94%)16 (100%)nsnsComplex karyotype45 (68%)7 (44%)nsns-7/del7q28 (42%)3 (18%)0.07ns-7/del7q[without der(1;7)]22 (33%)00.0040.04-5/del5q28 (42%)2 (12%)0.03ns-13/del13q5 (8%)3 (19%)nsns-20/del20q11 (17%)4 (25%)nsns-17/del17p15 (23%)1(6%)nsns+1q14 (22%)9 (56%)0.01ns+95 (8%)4 (25%)0.04ns+811 (17%)3 (19%)nsnsdic17 (26%)3 (19%)nsnsder(1;7)6 (9%)3 (19%)nsnsAmplification MLL0 (0%)nsnsJAK2mut17/31 (55%)7/9 (78%)nsnsTET2mut6/19 (32%)2/6 (33%)nsnsMedian overall survival (months)448

Description: B-PLL is defined by the presence of prolymphocytes in peripheral blood exceeding 55% of lymphoid cells. The diagnosis, mainly based on clinical and morphological data, can be difficult because of overlap with other B-cell malignancies. Because of the rarity of the disease, only case reports and small series describe its cytogenetic features. Few prognostic markers have been identified in this aggressive leukemia usually resistant to standard chemo-immuno therapy. We report here the cytogenetic and molecular findings in a large series of B-PLL. We also studied the in vitro response to novel targeted drugs on primary B-PLL cells. The study included 34 cases with a diagnosis of B-PLL validated by morphological review performed by three independent expert cytologists. The diagnosis of mantle cell lymphoma was excluded by karyotype (K) and FISH using CCND1, CCND2 and CCND3 probes. Median age at diagnosis was 72 years [46-88]. K was complex (≥3 abnormalities) in 73%, and highly complex (HCK≥5) in 45%. Combining K and FISH data, the most frequent chromosomal aberrations were: translocation targeting the MYC gene [t(MYC)] (21/34, 62%), 17p deletion including TP53 gene (13/34, 38%), trisomy 18/18q (10/33, 30%), 13q14 deletion (10/34, 29%), trisomy 3 (8/33, 24%), trisomy 12 (8/34, 24%) and 8p deletion (7/31, 23%). Whole-Exome Sequencing analysis of paired tumor-control DNA was performed in 16 patients. The most frequently mutated genes were TP53(6/16, 38%), associated with del17p in all, MYD88 (n=4), BCOR (n=4), MYC (n=3), SF3B1 (n=3), FAT1 (n=3), SETD2 (n=2), CHD2 (n=2), CXCR4 (n=2), BCLAF1 (n=2) and NFASC (n=2). Distribution of the chromosomal aberrations is shown in Fig 1. The main group of patients (21/34, 62%) had a t(MYC) that was associated with a higher % of prolymphocytes (86 vs 76, p=0.03), CD38 expression (90% vs 15%,p

Description: Hepatitis C virus (HCV) infection is a well-established risk factor for the development of B cell non-Hodgkin lymphoma (B-NHL). Two main pathways have been proposed to explain the role of the virus in lymphomagenesis: 1) transformation of B cells either directly by oncogenic viral proteins, or by a hit-and-run mechanism inducing a mutator phenotype, and 2) chronic antigenic stimulation through the B cell receptor (BcR) in conjunction to the binding of the viral E2 protein to its receptor CD81. There are however conflicting data regarding whether the BcR of the lymphomatous cells are capable of directly recognizing viral proteins or, rather, they possess a rheumatoid factor (RF) activity, binding instead polyclonal immunoglobulin (IG) within immune complexes trapping the virus. To further elucidate the role of antigenic stimulation in the natural history of HCV-associated B-NHL, we analyzed the IG gene repertoire of both heavy and light chains expressed by neoplastic cells in 41 cases of HCV-associated NHL, including: 29 marginal zone lymphoma (MZL); 7 diffuse large B-cell lymphoma (DLBCL) of which 3 originated from transformed MZL ; and 5 other low-grade B-cell NHL. Tumor cells were obtained from blood in 39 cases, bone marrow and a lymph node biopsy in 1 case each. Forty-three productive IGHV-IGHD-IGHJ gene rearrangements were obtained, which displayed a clear biased gene composition as 3 IGHV genes contributed to almost half of the repertoire: IGHV1-69 (11/43, 25,6%), IGHV3-7 (5/43, 11.6%), IGHV3-21 (4/43, 9.3%). This was also true for both IGHD genes (IGHD3-22: 12/43, 27.9%), and IGHJ genes (IGHJ4: 17/43, 39.5%; IGHJ3: 17/43, 25.6%). All but 3 sequences carried somatic hypermutations (SHM) in their IGHV genes with a median identity to the germline of 97.6% (range 86.5-100%). Thirty-eight productive IG light chain rearrangement sequences were obtained from 36 cases, of which 30 (78.9%) were IGK. Similarly, they exhibited strong gene usage bias with an over-representation of IGKV3-20 (9/30, 30%), as well as IGKJ1 (11/30, 33.3%) and IGKJ2 (6/30, 20%). IG light chain sequences were found to harbor similar SHM load with a 97% median identity to germline (range 91-100%). As previously described, we observed preferential pairing of heavy and light chain genes, with 6 of the 9 IGHV1-69 cases (with available light chain sequence data) being associated with IGKV3-20. Using established criteria, we found 5 cases (11.6%) carrying stereotyped BcR i.e. IGHV-IGHD-IGHJ gene rearrangements with quasi-identical amino-acid (AA) sequences, including the highly variable complementary determining region 3 (CDR3). Three cases concerned IGHV3-7/IGHD3-22/IGHJ3 rearrangements with an 18 AA-long CDR3, and 2 concerned IGHV1-69/IGHD3-22/IGHJ4 rearrangements with a 13 AA-long CDR3. Identity within the CDR3 extended to AA encoded by randomly inserted N-region nucleotides. We failed to establish correlations between histological categories and IG repertoire, probably due to the uneven distribution of lymphoma subtypes within our cohort. In contrast, most if not all sequences with biased IG gene usage, including the 5 stereotyped BcR ones, were found amongst the 28/41 cases (68.3%) with mixed cryoglobulinemia type II and/or positive for RF (MC/RF+). These two categories of patients differed also regarding the SHM load of their IGHV genes since MC/RF+ cases were signlificantly less mutated than MC/RF- cases (median identity to germline: 97.9% vs 95.9%, p= 0.048). We then searched for similar sequences in public databases and collaborative studies. Stereotyped BcR sequences similar to those of our cases were detected in HCV-associated lymphoma, but also in other HCV-negative B-cell maligancies e.g. MALT lymphoma (some associated with RF) and chronic lymphocytic leukemia, non malignant B cells with RF activity, and non malignant marginal zone splenic B cells. Sequence similarity extended to some shared AA replacements, e.g. identical AA introduced by HSM at the same positions. In conclusion we confirm the highly biased IG repertoire of HCV-associated lymphoma. However this feature seems to be linked essentially to the presence of a MC and/or RF. As quasi-identical sequences are found in HCV-negative malignant and normal B cells, our data support the hypothesis that HCV-associated lymphomatous cells originate from precursors endowed with auto-immune properties rather than B cells expressing an anti-virus BcR. Disclosures Stamatopoulos: Gilead Sciences: Research Funding; Janssen Pharmaceuticals: Research Funding.

Description: Abstract 582 Chromosomal translocations (t) are usually analyzed as one group, and are associated with poor prognosis in chronic lymphocytic leukemia (CLL). Translocations involving immunoglobulin (IG) genes are recurrent, but uncommon (〈 5%) in CLL. The two most frequent IG-partners are BCL2 (18q21) and BCL3 (19q13). On the behalf of the Groupe Francophone de Cytogenetique Hematologique (GFCH), we report an extensive analysis of 75 t(14;18)-CLLs, and a comparison to our previously published series of 29 t(14;19)-CLLs (Chapiro et al, Leukemia 2008). The 75 t(14;18)-CLLs or variant BCL2-t have been collected between 1985 and 2009. The morphological and immunological reviews were performed by KM, CS, and HM-B. All karyotypes were reviewed by the GFCH. Fluorescence in situ hybridization analyses were performed to detect IG and BCL2 rearrangements, trisomy 12, and deletions of 11q22 (ATM), 17p13 (TP53), 6q21, 13q14 (D13S319). IGHV mutation analyses were performed by referring laboratories. Statistical analyses were carried out using the Fisher's exact test, and continuous data using the Mann-Whitney test. Overall survival (OS) and treatment free survival (TFS) calculated from diagnosis were estimated using the Kaplan-Meier method, and the statistic significance was determined using log-rank test. Among BCL2-CLL, the sex ratio was 57M/18F, the median age at diagnosis was 66 years; of 68 patients with available data, 63 (93%) presented with Binet stage A; median lymphocytosis was 14.6×109/l. There were 47/75 (63%) “classical” CLL and 28/75 (37%) “atypical” CLL, with more than 10% of lymphoplasmacytoid cells and/or large cells. All tested cases (58/58) were CD10-, 69/73 (94%) were CD5+, and 44/63 (70%) were CD38-; 57/68 (84%) had a Matutes score 〉 4, 7/68 (10%) a score = 3, 4/68 (6%) a score 〈 3. We observed 62 t(14;18) and 13 variant translocations [11 t(18;22), 2 t(2;18)]. The karyotype was complex (〉 3 abnormalities) in 15/74 (20%) cases, and the BCL2-t was isolated in 25/74 (34%) cases. There were 33/75 (44%) tri12, 32/68 (47%) del13q14, 1/72 (1%) delTP53, 0/72 (0%) delATM, 0/59 (0%) del6q21. Of 42 analyzed cases, 33 (78%) were mutated. Finally, the median time from diagnosis to first therapy was 24 months (m). Comparisons with the BCL3-CLL showed no difference in sex ratio, age, and Binet stages. The lymphocytosis was lower in BCL2-CLL (14.6 vs 24.4 x109/l, p

Description: Key Points B-PLL is tightly linked to MYC aberrations (translocation or gain) and 17p (TP53) deletion. Cases of B-PLL with MYC aberration and 17p (TP53) deletion have the worst prognosis.

Description: Deletions of the long arm of chromosome 14 are rare (

Description: Numerous cytogenetic and Fluorescence In Situ Hybridization (F) data have been previously published with the aim of defining prognosis subgroups of B-CLL patients. These studies included heterogeneous series of patients (treated or not, and at various stages of the disease). We have studied with conventional cytogenetic and F analyses an homogeneous cohort of 125 untreated Binet stages B or C B-CLL patients enrolled in a prospective trial on the basis of biological and morphological criteria, with a Matutes score 4/5. Karyotype (K) was systematically performed and F analysis carried out using four probes CEP12, 13q14, 11q22 (ATM), 17p13 (TP53) on metaphases and interphase nuclei. Out of 104 successful K, 65 (63%) showed abnormalities. Among them, 32% exhibited complex K, 25% balanced translocations and 35% unbalanced translocations. The most frequent abnormalities were 11q– (13%), +12 (13%), 13q– (10%), 6q– (8%), 17p– (5%), 14q32 rearrangement (5%), X or Y loss (4%), +19 (4%). Two translocations not previously reported involving the 14q32 locus were observed. F analyses were performed on 116 patients, 108 with the four probes, 8 with only 1–3 probes. 82 out of the 108 patients (76%) analyzed with 4 probes showed one or more abnormalities. Deletions of 13q were observed in 52%, ATM in 26%, TP53 in 10%, and trisomy 12 was present in 13%. Abnormal F patterns were observed in 51% of patients with normal K with at least one of the 4 probes, versus 92% of patients with abnormal K. Among abnormal K, F analyses detected additional cryptic deletions in 6/19 cases for ATM (31%), 29/39 for 13q14 (74%), and 4/9 for TP53 (45%). TP53 deletion was never detected among normal K, but in 15% of abnormal K. Among them, there were more TP53 deletion in complex K (24% vs 2%, p=0.005). Additional karyotypic abnormalities were found with a single F anomaly in 6/7 (86%) cases with del ATM, 12/17 (71%) with del13q, 4/6 (67%) with +12, 1/2 with del TP53. In the group of patients with normal F pattern, 81% had a normal K whereas 19% had an abnormal one (p