ALBERT

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: PAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Description: The nucleoporin gene NUP98 is known to be rearranged in several recurrent translocations occurring in de novo and therapy-related myelodysplastic syndrome and acute leukemia, in children or adults. All these abnormalities seem to have bad prognosis, thus, it is important to identify NUP98 rearrangements. As other genes (MLL, ETV6, RUNX1, ...), NUP98 may fuse to different partners, often homeobox genes such as HOXA, HOXC, HOXD, PMX1, but also non homeobox genes such as RAP1GSD1, NSD1, NSD3, LEDGF, ADD3, DDX10, and TOP1. The fusion transcript always juxtapose the N-terminal FG repeats of NUP98, required for its docking function, to the C-terminus of the partner gene, which contains the homeodomain in the case of a homeobox partner gene. We identified a t(9;11)(q34.1;p15.5) by conventional cytogenetic analysis, in a 65 years old Caucasian female who developed a t-AML four years after lymphoma treatment. The involvement of NUP98 gene was confirmed by FISH analysis using BAC RP11-120E20 and PAC RP11-1173K1. 3′RACE analysis allowed to identified a novel partner gene, the class II homeobox gene PRRX2 (Paired Related homeobox 2), located on 9q34. The breakpoint occurred in an infrequent breaking region in exon 11 of NUP98, and in exon 2 of PRRX2. The NUP98-PRRX2 fusion transcript was cloned and sequenced, and confirmed by RT-PCR. As its homologue PRRX1 (PMX1), PRRX2 is a DNA-binding transcription factor that is essential for fetal development. PRRX1 has been described to be fused with NUP98 in t(1;11)(q23;p15), but it is the first time PRRX2 is implicated in leukemia, and even in malignancy. Further studies are necessary to confirm the recurrence of this translocation and its prognosis. Furthermore, transformation assays in cells lines and transgenic mice studies would be interesting to understand the leukemogenicity induced by the NUP98-PRRX2 fusion transcript.

Description: Acute basophilic leukemia (ABL) is a rare subtype of acute leukemia with clinical features and symptoms related to hyperhistaminemia because of excessive growth of basophils. No known recurrent cytogenetic abnormality is associated with this leukemia. Rare cases of t(X;6)(p11;q23) translocation have been described but these were sporadic. We report here 4 cases of ABL with a t(X;6)(p11;q23) translocation occurring in male infants. Because of its location on chromosome 6q23, MYB was a good candidate gene. Our molecular investigations, based on fluorescence in situ hybridization and rapid amplification of cDNA ends, revealed that the translocation generated a MYB-GATA1 fusion gene. Expression of MYB-GATA1 in mouse lineage-negative cells committed them to the granulocyte lineage and blocked at an early stage of differentiation. Taken together, these results establish, for the first time, a link between a recurrent chromosomal translocation and the development of this particular subtype of infant leukemia.

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: Abstract 2879 Introduction: Whole genome and exome sequencing studies recently revealed that CLL genome harbors frequent recurrent mutations as NOTCH1, TP53 and SF3B1 genes. Mutational status of these three genes strongly impacted on treatment-free survival (TFS) and overall survival (OS) in pioneer reports. In this retrospective series, we evaluated the relative role of these mutations along with the karyotype in untreated CLL patients, and after immunochemotherapy given first-line. Patients and methods: 164 patients were included (all naïve from chemotherapy), including 108 who further necessitated therapy according to NCI2008 guidelines. Mutational studies (IgVH sequence, NOTCH1, SF3B1, and TP53 genes) were available in 164/164, FISH (del17p and 11q) in 156/164, and conventional karyotype in 140/164. TFS and OS were calculated from diagnosis and first-line treatment or death respectively, progression-free survival (PFS) was calculated in 108 patients who received rituximab-based immunochemotherapy frontline (94 FCR, 14 R-alkylators), from end of therapy to relapse (according to NCI2008 definition). In 60/94 patients, minimal residual disease (MRD) was assessed three months after completion of FCR by 4-color flow cytometry, according to published guidelines for MRD monitoring. Results: Thirty-seven patients (22.6%) have at least one somatic mutation (TP53, SF3B1 or NOTCH1 genes), 22 NOTCH1 mutations (13.4%), 10 SF3B1 mutations (6.1%) and 8 TP53 mutations (4.5%). SF3B1 mutations are located mainly on the exon 14. TP53 mutations are located mainly on the exon 8 (4/9) and double mutation of TP53 was not detected. Three patients had a mutation of NOTCH1 or SF3B1 combined to a TP53 mutation. Conventional karyotype was obtained in 135/140 CLL (96.4%, 5 culture failures) and 116/135 (85.9%) were associated with chromosomal aberrations. Complex karyotype (CK) and balanced translocations involving IgH locus were shown in 23.9% and 8.1% of patients, respectively. NOTCH1 mutations were found significantly associated with unmutated IgVH and trisomy 12, SF3B1 mutations with male gender and advanced Binet stage, both somatic mutations being mutually exclusive. As expected, TP53 mutations associated with del17p. After a median follow-up of 5 years, 108/164 patients had received frontline immunochemotherapy, for a median treatment-free survival (TFS) of 41.6 months from diagnosis of CLL. Only 8/164 had died, for a median overall survival (OS) not reached, but a 10y probability of survival of 92%. On univariate analysis, shorter TFS was correlated with age

Description: We have identified a novel recurrent translocation and the genes involved therein, which is associated with both lymphoid and myeloid acute leukemia. Fluorescence in situ hybridization (FISH) and molecular analyses revealed an in-frame fusion of ETV6 (alias TEL) and NCOA2 (alias TIF2) in six cases of childhood acute leukemia. ETV6 encodes an ets transcription repressor and is frequently involved in chromosome rearrangements with a multitude of partners in lymphoid and myeloid leukemia, and to a lesser extent also in solid tumors. NCOA2 is a member of the p160 family of nuclear receptor transcriptional coactivators (NRCoAs), which have a common domain structure with a conserved N-terminal bHLH-PAS domain, a centrally located (nuclear) receptor interaction domain (RID/NID), and a C-terminal transcriptional activation domain (AD2). NRCoAs typically display HAT activity and/or directly interact with CBP. In this context, NCOA2 also contains a C-terminal CBP-interaction domain (CID), and thus is a transcription factor that mediates transcriptional activation in a ligand-dependent fashion by a mechanism involving chromatin remodeling. As a result of the translocation the pointed (PNT) protein interaction domain of ETV6 is fused to the C-terminus of NCOA2 including the CBP and AD2 motifs. The same C-terminal domains of NCOA2 are also involved in the previously identified MOZ-NCOA2 fusion, which is generated by a inv(8)(p11q13). The absence of the reciprocal NCOA2-ETV6 transcript in one of the cases, and the facts that MOZ-NCOA2 is transforming and that the reciprocal NCOA2-MOZ is not expressed, strongly suggest that the ETV6-NCOA2 chimeric protein and not the reciprocal NCOA2-ETV6 is responsible for leukemogenesis. Unlike in cases with a ETV6-RUNX1 fusion, which is frequently associated with a concurrent secondary deletion of the non-rearranged ETV6 allele, in all cases the second ETV6 and also one NCOA2 allele were retained and both genes were readily expressed. With respect to the immunophenotype, according to the European Group for the Immunological Characterization of Leukemias (EGIL), three of the cases were classified as T-ALL (one as T-I My+), two as biphenotypic leukemia (BAL), and one as AML with M1 phenotype. Nevertheless, all leukemias displayed aberrant co-expression of various T-cell and myeloid/NK markers, and were double-negative for CD4/CD8, suggesting transformation of early progenitors. In contrast to the MOZ-NCOA2 fusion, which is exclusively associated with AML, mostly of M4 and M5 subtype, the ETV6-NCOA2 fusion seems to define a new entity of acute leukemia with aberrant co-presence of T lymphoid and myeloid antigens.

Description: Abstract 801 The genetic bases of Waldenström Macroglobulinemia (WM) are poorly understood. Because of the difficulty in obtaining tumor metaphases for karyotype studies, few recurrent chromosomal abnormalities have been reported in WM. We have studied a cohort of 171 untreated WM patients, enrolled in a prospective randomized trial from the French Cooperative Group on Chronic Lymphocytic Leukemia and Waldenstrom Macroglobulinemia (FCG-CLL/WM) comparing the efficacy of fludarabine to that of chlorambucil, by conventional cytogenetic (CC) and Fluorescence in situ hybridization (FISH). CC was systematically performed on bone marrow or peripheral blood, and FISH analysis carried out using 8 probes CEP4, CEP12, 13q14, 11q22 (ATM), 17p13 (TP53), IGH, BCL2 Abbott, 6q21 Q-Biogene, on metaphases and interphase nuclei. The sex ratio was 2.1M/1F, the average age at inclusion was 66.9 years [40-89]. The mean percentage of lymphoplasmacytic cells was 53% [8-97]. Out of 140/171 successful CC, 65 (46.4%) showed clonal abnormalities. Out of 65 abnormal CC, 19 (29.2%) were complex, with at least 3 chromosomal changes, and 22 (33.8%) showed translocations (balanced and unbalanced). Using CC and FISH, we observed 29/132 (22%) 6q deletion, 18/141 (12.8%) 13q14 deletion, 9/80 (11.2%) trisomy 18, 11/135 (8.1%) TP53 deletion, 10/133 (7.5%) trisomy 4, 10/132 (7.6%) ATM deletion, 4/118(3.4%) IGH rearrangement, and 4/136 (2,9%)trisomy 12. Chromosomal abnormalities were compared to adverse characteristics described by Morel et al (ISSWM, Blood 2009,113(8),4163-70): advanced age (〉 65 years), hemoglobin 〈 11.5g/dl, platelet count 〈 100×109/l, b2-microglobulin 〉 3 mg/l, and serum monoclonal protein concentration 〉 7g/dl. Patients with 6q deletion had significantly more frequently albumin 〈 3.5g/dl (15/29 (51.7%) vs 24/103 (23.3%), p=0.005), b2microglobuline 〉 3 mg/l (20/29 (68.9%) vs 48/103 (46.6%), p=0.04). Similarly, patients with trisomy 4 had significantly more frequently b2microglobuline 〉 3 mg/l (9/10 (90%) vs 59/123 (47.9%), p=0.02). Of note, all patients with trisomy 4 had M-protein concentration 〉 2 g/dl (10/10 (100%) vs 73/123 (59.3%), p=0.02). Finally, there were significantly more patients with age 〉 65 years, when ATM deletion was observed (9/10 (90%) vs 65/122 (53.2%), p=0.04). Cytogenetic abnormalities did not influence the response rate but in multivariate analysis, TP53 deletion was associated with a shorter time to progression (15months (m) versus 35m, p=0.0007) and 6q deletion with a longer time to progression (55m versus 24m, p=0.04) in responding patients. Cytogenetic abnormalities in WM differ from those commonly reported in other B-cell neoplasms and confirm the originality of this disease. The 6q deletion is frequent compared to chronic lymphocytic leukaemia (CLL) or marginal zone lymphoma (MZL) and 13q14 deletion is rare compared to CLL. In our series trisomy 12 is rare compared to atypical-CLL and MZL. Involvement of the IGH locus, which is frequent in multiple myeloma or lymphoplasmocytic lymphoma, is rare in WM. Finally trisomy 4 is present in WM but not reported in other B-cell malignancies. The 6q deletion is the most frequent reported cytogenetic abnormality in WM. We found 22% cases with deletions of 6q21, a lower percentage compared with the literature [38-54%].Conversely to previous publications, 6q deletion was associated with a longer response duration to treatment and did not influence the overall survival. These discrepancies could be explained by the heterogeneity of the previous published series, mixing untreated patients and treated patients in various ways. In our trial, all patients were analyzed before randomization. As in CLL patients, TP53 deletion seems to be associated with a shorter response duration. However, regarding the small numbers of patients with cytogenetic abnormalities, these results must be confirmed in larger series. Disclosures: Leblond: ROCHE: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; MUNDIPHARMA: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; CELGENE: Honoraria, Membership on an entity's Board of Directors or advisory committees; Janssen: Honoraria, Membership on an entity's Board of Directors or advisory committees.

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.