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Myelodysplastic syndromes (MDS) are hematological malignancies classically defined by the presence of cytopenia(s) and dysmorphic myeloid cells. It is now known that MDS can be preceded by a pre-malignant condition called clonal cytopenia of unknown significance (CCUS), which associates a clonality marker with cytopenia in the absence of criteria of dysplasia. However, to date, it is not clear whether chromosomal abnormalities should be considered in the definition of CCUS or if they carry a prognostic impact in CCUS patients. In this study, we analyzed the clinico-biological features and outcomes of 34 patients who presented with one or more cytopenias, an absence of significant dysplasia, and a presence of a chromosomal abnormality (CA). We named this entity chromosomal abnormality with cytopenia of undetermined significance (CACtUS). We show that these patients are slightly older than MDS patients and that they more frequently presented with normocytic anemia. Most CACtUS patients exhibited only one unbalanced CA. The number and type of mutations were comparable between CACtUS patients and MDS patients. Regardless of the cytogenetic abnormality, the clinicobiological characteristics, overall survival, and risk of progression to high-risk (HR) MDS were similar between CACtUS patients and low-risk MDS patients. Thus, we suggest that CACtUS patients can be considered as HR-CCUS and should receive the follow-up regimen recommended for MDS patients.

Key Points We detail at clinical, cytological, cytogenetic, and molecular levels 113 cases of MDS and MDS/MPN with del(11q), a rare recurrent event.CADM1, a tumor suppressor gene identified initially in solid tumors, ATM, CBL, and KMT2A are deleted and/or mutated in del(11q)., Visual Abstract, Myelodysplastic syndromes (MDS) represent a heterogeneous group of clonal hematopoietic stem cell disorders characterized by ineffective hematopoiesis leading to peripheral cytopenias and in a substantial proportion of cases to acute myeloid leukemia. The deletion of the long arm of chromosome 11, del(11q), is a rare but recurrent clonal event in MDS. Here, we detail the largest series of 113 cases of MDS and myelodysplastic syndromes/myeloproliferative neoplasms (MDS/MPN) harboring a del(11q) analyzed at clinical, cytological, cytogenetic, and molecular levels. Female predominance, a survival prognosis similar to other MDS, a low monocyte count, and dysmegakaryopoiesis were the specific clinical and cytological features of del(11q) MDS. In most cases, del(11q) was isolated, primary and interstitial encompassing the 11q22-23 region containing ATM, KMT2A, and CBL genes. The common deleted region at 11q23.2 is centered on an intergenic region between CADM1 (also known as Tumor Suppressor in Lung Cancer 1) and NXPE2. CADM1 was expressed in all myeloid cells analyzed in contrast to NXPE2. At the functional level, the deletion of Cadm1 in murine Lineage-Sca1+Kit+ cells modifies the lymphoid-to-myeloid ratio in bone marrow, although not altering their multilineage hematopoietic reconstitution potential after syngenic transplantation. Together with the frequent simultaneous deletions of KMT2A, ATM, and CBL and mutations of ASXL1, SF3B1, and CBL, we show that CADM1 may be important in the physiopathology of the del(11q) MDS, extending its role as tumor-suppressor gene from solid tumors to hematopoietic malignancies.

Diagnosis of B-cell chronic lymphocytic leukemia (B-CLL) is usually straightforward, involving clinical, immunophenotypic (Matutes score), and (immuno)genetic analyses (to refine patient prognosis for treatment). CLL cases with atypical presentation (e.g., Matutes ≤ 3) are also encountered, and for these diseases, biology and prognostic impact are less clear. Here we report the genomic characterization of a case of atypical B-CLL in a 70-yr-old male patient; B-CLL cells showed a Matutes score of 3, chromosomal translocation t(14;18)(q32;q21) (BCL2/IGH), mutated IGHV, deletion 17p, and mutations in BCL2, NOTCH1 (subclonal), and TP53 (subclonal). Quite strikingly, a novel PAX5 mutation that was predicted to be loss of function was also seen. Exome sequencing identified, in addition, a potentially actionable BRAF mutation, together with novel somatic mutations affecting the homeobox transcription factor NKX2-3, known to control B-lymphocyte development and homing, and the epigenetic regulator LRIF1, which is implicated in chromatin compaction and gene silencing. Neither NKX2-3 nor LRIF1 mutations, predicted to be loss of function, have previously been reported in B-CLL. Sequencing confirmed the presence of these mutations together with BCL2, NOTCH1, and BRAF mutations, with the t(14;18)(q32;q21) translocation, in the initial diagnostic sample obtained 12 yr prior. This is suggestive of a role for these novel mutations in B-CLL initiation and stable clonal evolution, including upon treatment withdrawal. This case extends the spectrum of atypical B-CLL with t(14;18)(q32;q21) and highlights the value of more global precision genomics for patient follow-up and treatment in these patients.

Ibrutinib, obinutuzumab, and venetoclax demonstrate synergy in preclinical models of mantle cell lymphoma (MCL). OAsIs (NCT02558816), a single-arm multicenter prospective phase 1/2 trial, aimed to determine the maximum tolerated dose of venetoclax in combination with fixed doses of ibrutinib and obinutuzumab, in relapsed MCL patients. At the venetoclax MTD, extension cohorts were opened for relapsed and untreated patients. Safety and efficacy were secondary objectives. Minimal residual disease (MRD) was assessed by allele-specific oligonucleotide quantitative polymerase chain reaction. Between 14 October 2015 and 29 May 2018, 48 patients were enrolled. No dose-limiting toxicity was reported, and venetoclax at 400 mg per day was chosen for extension. Eighteen (75%) relapsed and 8 (53%) untreated patients experienced grade 3/4 adverse events. The complete response rate assessed by positron emission tomography at the end of cycle 6 was 67% in relapsed and 86.6% in untreated patients. MRD clearance for evaluable patients was seen in 71.5% of relapsed (10/14 patients) and 100% of untreated MRD-evaluable patients (n = 12) at the end of 3 cycles. The median follow-up for relapsed patients was 17 months (range, 10-35 months). The 2-year progression-free survival (PFS) was 69.5% (95% confidence interval [CI], 52.9%-91.4%) and 68.6% (95% CI, 49.5%-95.1%) for overall survival. The median follow-up was 14 months (range, 5-19) for untreated patients, the 1-year PFS was 93.3% (95% CI, 81.5%-100%). The combination of obinutuzumab, ibrutinib, and venetoclax is well tolerated and provides high response rates, including at the molecular level, in relapsed and untreated MCL patients. This trial was registered at www.clinicaltrials.gov as #NCT02558816.

TP53 aberrations, including somatic mutations of TP53 gene or 17p deletion leading to the loss of the TP53 locus, are a major predictive factor of resistance to fludarabin based chemotherapy in chronic lymphocytic leukemia (CLL) and remain an adverse prognostic factor in the chemofree era. Therefore, detection of TP53 alteration before each new line of treatment is required for theranostic stratification. In order to better characterize the distribution and combination of the TP53 variants in CLL, we collected the TP53 sequencing data of 343 patients harboring TP53 mutations from centers of the French Innovative Leukemia Organization-CLL (FILO) and established a large data base of 573 TP53 mutations. Mutations were identified through NGS sequencing (covering exon 2 to 11) allowing the detection of low frequency variants down to 1% VAF. Several distinct low VAF mutations were orthogonally confirmed by digital PCR. TP53 variants were analyzed through UMD_TP53 data gathering 90 000 TP53 mutations from all type of cancers. IGHV mutational status and FISH analysis were available for 224 and 176 patients respectively. Using ACMG criteria from the UMD_TP53 database, we confirmed that 523 could be classified as pathogenic, 42 were likely pathogenic and 8 were VUS (Variants of Unknown Significance). As expected, the mutation distribution along the p53 protein exhibited a clustering of variants in the DNA binding domain of the protein. We also confirmed the presence of a specific hotspot at codon 234 (6%) which is noticeable in other CLL cohorts but absent in solid tumors. 431 TP53 variants led to the expression of a mutant protein whereas the remaining 142 led a TP53 null phenotype. For 8 patients without 17p deletion and a mutation VAF larger than 50%, SNP analysis indicate that these tumors had a copy number neutral loss of heterozygosis at 17p with a duplication of the mutant allele leading to homozygous mutations of TP53. When focusing on the allele burden of TP53 mutations, 264/573 (46%) variants had an allele frequency Among the 343 patients, 113 (33%) were poly-mutated and harbored more than one pathogenic TP53 variants (2 to 11 variants per patient): 57 (16,7 %) had 2 variants, 32 (9,3%) had 3, 10 had 4 (3%) and 14 patients (4%) had 5 to 11 variants. Using both long range sequencing and in silico analysis, we could show that all these variants were distributed in different alleles supporting an important intratumoral heterogeneity and a strong selection for TP53 loss of function during tumor progression in these patients. Null variants were rarely found as single alteration: only 46 patients (13,4%) patients harbored a single null mutation. Null mutations were predominantly found in patients with multiclonal mutations (87% with 4 or more). Median size of variants significantly decreased with the number of mutations and most of low VAF (less than 10%) variants were found in multiclonal combinations. Multiclonal mutations were predominantly found in previously treated patients (41% treated versus 10 % untreated) but whether all these variants preceded treatment and were further selected is currently unknown. We observed that 71,5 % of patients were IGHV unmutated and multiclonal mutations were surprisingly more frequent in mutated IGHV cases than in unmutated ones. Only 50% of cases carried a 17p deletion, highlighting again the importance of testing for TP53 mutations in addition to FISH analysis. Presence or absence of 17p deletion was unrelated to the number of TP53 mutations. Taken together these observations suggest that the TP53 mutational landscape in CLL is very complex and can involve multiple mechanisms, converging to a total loss of TP53 function and tumor progression. NGS provides a powerful tool for detecting all these alterations including variants with low VAF and should become a standard for CLL screening prior to each line of treatment. Disclosures Leblond: Amgen: Honoraria, Speakers Bureau; Abbvie: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Janssen: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Gilead: Honoraria, Speakers Bureau; Astra Zeneca: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Roche: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Letestu:Abbvie: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Janssen: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Roche: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts; Alexion: Membership on an entity's Board of Directors or advisory committees, Other: speaker fee, expert contracts. Cymbalista:Abbvie: Honoraria; Roche: Research Funding; Sunesis: Research Funding; Gilead: Honoraria; Janssen: Honoraria; AstraZeneca: Honoraria.

Background Expression of SRY [sex-determining region Y]-box11 (SOX11) is specific to mantle cell lymphoma (MCL) and contributes, in conjunction with immunoglobulin variable heavy chain gene mutation status, to the identification of two forms of this disease. Methods The aim of this report was firstly, to design an easy and suitable RT-qPCR method to quantify SOX11 mRNA expression in mantle cell lymphoma and other B cell malignancies with the proper reference gene; secondly, to define the best threshold of relative quantity of SOX11 mRNA in order to reach the best compromise between sensitivity and specificity. Results For best discrimination of MCL and non-MCL groups we determined an area under the curve (AUC) of 0.9750 and a threshold of 1.76 with 100% sensitivity and 88% specificity. AUC and threshold values of respectively 0.91/1.346 [87% sensitivity, 80% specificity] and 0.9525/1.7120 [100% sensitivity, 88% specificity] for GAPDH and RPLP0 respectively denote that the RPLP0 reference gene alone is sufficient for PCR housekeeping gene. Conclusion This work describes an RT-qPCR assay for SOX11 expression in order to better characterize MCL at diagnosis. Further studies on larger cohorts are needed to evaluate this molecular tool, especially for the follow-up of minimal residual disease. Electronic supplementary material The online version of this article (10.1186/s40164-018-0097-6) contains supplementary material, which is available to authorized users.

International audience; B-cell prolymphocytic leukemia (B-PLL) is a rare hematological disorder whose underlying oncogenic mechanisms are poorly understood. Our cytogenetic and molecular assessment of 34 patients with B-PLL revealed several disease-specific features and potential therapeutic targets. The karyotype was complex ({greater than or equal to}3 abnormalities) in 73% of the patients and highly complex (>5 abnormalities) in 45%. The most frequent chromosomal aberrations were translocations involving MYC [t(MYC)] (62%), deletion (del)17p (38%), trisomy (tri)18 (30%), del13q (29%), tri3 (24%), tri12 (24%), and del8p (23%). Twenty-six of the 34 patients (76%) exhibit MYC aberration, resulting from mutually exclusive translocations or gains. Whole-exome sequencing revealed frequent mutations in TP53, MYD88, BCOR, MYC, SF3B1, SETD2, CHD2, CXCR4, and BCLAF1 The majority of B-PLL used the IGHV3 or IGHV4 subgroups (89%), and displayed significantly mutated IGHV genes (79%). We identified three distinct cytogenetic risk groups: low-risk (no MYC aberration), intermediate-risk (MYC aberration but no del17p), and high-risk (MYC aberration and del17p) (p=.0006). In vitro drug response profiling revealed that the combination of a B-cell receptor or BCL2 inhibitor with OTX015 (a bromodomain and extra-terminal motif (BET) inhibitor targeting MYC) was associated with significantly lower viability of B-PLL cells harboring a t(MYC). We conclude that cytogenetic analysis is a useful diagnostic and prognostic tool in B-PLL. Targeting MYC may be a useful treatment option in this disease.

International audience

Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS.

Non-Hodgkin's lymphomas and lymphoproliferative disorders include a high number of heterogeneous entities, described in the 2008 WHO classification. This classification reflects the crucial role of a multidisciplinary approach which integrates cytogenetic results both for the notion of clonality and for differential diagnosis between these entities. The prognostic impact of some cytogenetic abnormalities or genome complexity is also confirmed for many of these entities. Novel provisional entities have been described, such as BCLU (B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma) for which karyotype is critical to distinguish BCLU from Burkitt's lymphoma. The karyotype can be established from any tumour or liquid infiltrated by lymphoma cells. Recent adaptations of technics for cellular cultures according to the subtype of known (or suspected) lymphoma have significantly improved the percentage of informative karyotypes. Conventional karyotypes remain the best technical approach recommended for most of these subtypes. Interphase and/or metaphase FISH also represents a solid and rapid approach, because of the significant number of recurrent (sometimes specific) rearrangements of these entities. Next generation sequencing technologies contribute to enrich genomic data and substantially improve the understanding of oncogenic mechanisms underlying these lymphoid malignancies. Some molecular biomarkers are already part of the diagnostic process (for example, somatic mutation of MYD88 in Waldenstrom disease) thus reinforcing the essential principle of a multidisciplinary approach for the diagnosis of all the mature lymphoid malignancies.

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 The median overall survival (OS) for the entire cohort was 126 months with a median follow-up time of 47 months [ 0.2-141]. We found MYC activation (translocation or gain) to be associated with a shorter OS (p=0.03). Regarding MYC and del17p, we identified 3 distinct cytogenetic prognostic groups, with significant differences in OS (p=0.0006) (Fig 2). The patients without MYC activation had the lower risk (n=8, median not reached). Patients with a MYC activation without del17p had an intermediate risk (n=18, 125 months). The highest risk group corresponded to patients with both MYC and TP53 aberrations (n=7, 11 months). We performed drug response profiling on primary B-PLL cells using the ATP-based CellTiter Glo kit (Promega) (n=5). We observed that after 48h of exposure to increased doses, response was heterogeneous, with a majority of samples resistant to fludarabine (n=3), ibrutinib (n=3), idelalisib (n=4), venetoclax (n=3) and OTX015 (n=4). Annexin/PI assays using flow cytometry showed that the induced cell death could be increased by combination of ibrutinib or venetoclax with OTX015 or JQ1, two BET protein inhibitors that target MYC signaling (n=1/2). In summary, B-PLL have complex and highly complex K, a high frequency of MYC activation by translocation or gain, frequent 17p deletion, and frequent mutations in MYC, TP53, BCOR, and MYD88 genes. We identified 3 prognostic subgroups according to MYC and 17p status. Patients with MYC activation + 17p deletion have the shorter OS, and should be considered as a high-risk "double-hit" subgroup. Our results show that cytogenetic analysis is a useful diagnostic tool in B-PLL that improves prognostic stratification. We recommend to perform K and FISH (MYC and TP53) analyses systematically when a B-PLL is suspected. Our in vitro data suggest that drugs targeting the BCR and BCL2 in combination with MYC inhibition may be a therapeutic option in some patients. Disclosures Baseggio: Takeda Oncology: Honoraria.

Chromosomal translocations involving the immunoglobulin heavy chain locus (IGH@) are recurrent but rare in B-cell precursor acute lymphoblastic leukemia (BCP-ALL), and various partner genes have been described. Here, we report a new series of 29 cases of BCP-ALL with IGH@ translocations. The partner gene was identified by fluorescence in situ hybridization and/or molecular cloning in 20 patients. Members of the CEBP gene family (n = 11), BCL2 (n = 3), ID4 (n = 3), EPOR (n = 2), and TRA/D@ (n = 1) were identified and demonstrated by quantitative real-time reverse transcriptase-polymerase chain reaction to be markedly up-regulated. The present cases, added to those already reported, confirm the diversity of the partner genes, which, apart from BCL2, are specific to BCP-ALL. Collectively, patients with IGH@ translocations may represent a novel sub-group of BCP-ALL occurring in adolescents and young adults.

Pediatric acute myeloid leukemia (AML) is a rare disease whose prognosis is highly variable according to factors such as chromosomal abnormalities. Recurrent genomic rearrangements are detected in half of pediatric AML by karyotype. NUcleoPorin 98 (NUP98) gene is rearranged with 31 different fusion partner genes. These rearrangements are frequently undetected by conventional cytogenetics, as the NUP98 gene is located at the end of the chromosome 11 short arm (11p15). By screening a series of 574 pediatric AML, we detected a NUP98 rearrangement in 22 cases (3.8%), a frequency similar to CBFB-MYH11 fusion gene (4.0%). The most frequent NUP98 fusion gene partner is NSD1. These cases are homogeneous regarding their biological and clinical characteristics, and associated with bad prognosis only improved by bone marrow transplantation. We detailed the biological characteristics of these AML by exome sequencing which demonstrated few recurrent mutations (FLT3 ITD, WT1, CEBPA, NBPF14, BCR and ODF1). The analysis of the clonal structure in these cases suggests that the mutation order in the NUP98-rearranged pediatric AML begins with the NUP98 rearrangement leading to epigenetic dysregulations then followed by mutations of critical hematopoietic transcription factors and finally, activation of the FLT3 signaling pathway.

We previously showed that complex karyotypes (CK) and chromosome 13q abnormalities have an adverse prognostic impact in childhood Burkitt lymphomas/leukemias (BL) and diffuse large B-cell lymphomas (DLBCL). The aim of our study was to identify recurrent alterations associated with MYC rearrangements in aggressive B-cell lymphomas with CK. Multicolor fluorescence in situ hybridization (M-FISH) was performed in 84 patient samples (59 adults and 25 children), including 37 BL (13 lymphomas and 24 acute leukemias), 12 DLBCL, 28 B-cell lymphomas with intermediate features (DLBCL/BL), 4 B-cell precursor acute lymphoblastic leukemias (BCP-ALL), and 3 unclassifiable B-cell lymphomas. New (cytogenetically undetected) abnormalities were identified in 80% of patients. We also refined one-third of the chromosomal aberrations detected by karyotyping. M-FISH proved to be more useful in identifying chromosomal partners involved in unbalanced translocations and in revealing greater complexity of 13q rearrangements. Most of the newly identified or refined recurrent alterations involved 1q, 13q and 3q (gains/losses), 7q and 18q (gains), or 6q (losses), suggesting that these secondary aberrations may play a role in lymphomagenesis. Several patterns of genomic aberrations were identified: 1q gains in BL, trisomies 7 in DLBCL, and 18q-translocations in adult non-BL. BCP-ALL usually displayed an 18q21 rearrangement. BL karyotypes were less complex and aneuploid than those of other MYC-rearranged lymphomas. BCP-ALL and DLBCL/BL were associated with a higher rate of early death than BL and DLBCL. These findings support the categorization of DLBCL/BL as a distinct entity and suggest that BL with CK are indeed different from other aggressive MYC-rearranged lymphomas, which usually show greater genetic complexity. © 2012 Wiley Periodicals, Inc.

// Pascale Flandrin-Gresta 1,2 , Francoise Solly 1,2 , Carmen Mariana Aanei 1 , Jerome Cornillon 3 , Emmanuelle Tavernier 2,3 , Nathalie Nadal 1 , Franck Morteux 2 , Denis Guyotat 2,3 , Eric Wattel 2 , Lydia Campos 1,2 , 1 Laboratoire d’Hematologie, University Hospital of Saint-Etienne, University Hospital of Saint-Etienne, 42055 Saint-Etienne Cedex 2, France. 2 UMR5239 CNRS, Universite Claude Bernard Lyon 1, Faculte deMedecine J Lisfranc Saint-Etienne, 42023 Saint-Etienne Cedex 2, France. 3 Institut de Cancerologie de la Loire, University Hospital of Saint-Etienne, 42271 Saint Priesten Jarez cedex, France. Correspondence: Pascale Flandrin-Gresta, email: // Keywords : HSP90, MDS, FAK, 17-AAG Received : July 18, 2012, Accepted : September 25, 2012, Published : September 28, 2012 Abstract Myelodysplastic syndromes are characterized by a high risk of evolution into acute myeloid leukaemia which can involve activation of signalling pathways. As the chaperone heat shock protein 90 (HSP90) has a key role in signal transduction, we investigated its role in the pathogenesis and evolution of myelodysplastic syndromes. Expressions of HSP90 and signalling proteins clients (phosphorylated-AKT (pAKT), Focal Adhesion Kinase (FAK) and phosphorylated-FAK (pFAK)), were assessed in bone marrow mononuclear and CD34-positive (CD34 + ) cells from 177 patients with myelodysplasia. Effects of HSP90 inhibition were also evaluated in 39 samples. The levels of all proteins studied were significantly higher in patients with high grade disease, than those with low grade myelodysplastic syndrome or chronic myelomonocytic leukaemia. High levels of HSP90, FAK, pFAK and pAKT were associated with shorter survival and increased risk of progression into acute leukaemia. A down regulation of pFAK and pAKT and increased apoptosis was observed in mononuclear and CD34 + cells after 12 hours of incubation with 17-AAG. In conclusion, our data suggest the implication of HSP90 and FAK and AKT activation in the pathogenesis of myelodysplastic syndromes with excess of blasts and evolution to leukaemia. Moreover this signalling network could be a therapeutic target through HSP90 inhibition.

Background: Congenital acute leukemia is a rare event, presenting usually as an aggressive disease with a poor prognosis. A differential diagnosis is the transient myeloproliferative disorder observed in Down syndrome. We describe the case of an apparently healthy newborn male child presenting with normal peripheral blood (PB) counts but with a blast population on differentials. The child's condition and the blast population remained unchanged during the first year of life. Methods: Bone marrow and PB were morphologically analyzed. Multiparametric flow cytometry was performed at the time of diagnosis and repeated at 1 year. These studies were completed by cytogenetic and molecular analyses. Results: Bone marrow contained 40% of undifferentiated blasts. Multiparametric flow cytometry showed low expression of CD38, HLA-DR, and CD33 markers. All other markers were negative. Constitutional and blast cell karyotypes were normal. Fluorescence in situ hybridization analysis showed no rearrangement. Molecular studies were negative. The blast percentage remained stable during several months. After 1 year, the PB counts showed thrombocytopenia, with an increase of blast cells exhibiting the same phenotype. Clinically, an enlarged spleen was found. The child did not respond to chemotherapy and only partially to gemtuzumab ozogamicin. A cord blood cell transplantation was finally performed. With a follow-up of 12 months, the child is doing well. Conclusions: To our knowledge, this is the first case of congenital acute leukemia with initially indolent course in a newborn without Down syndrome. This observation emphasizes the importance of a careful follow-up. © 2010 International Clinical Cytometry Society

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more -7/del(7q) (P = 0.004) and -5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example -5/del(5q) and -17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and -5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of -5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild-type to mutated forms of both genes. The mutated and wild-type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process.

We analysed, by flow cytometry, the “adhesive” phenotype of acute myelogenous leukemia (AML) cells from 36 patients treated in our institution. In univariate analysis, the main prognostic factor for CR achievement was lower CXCR4 expression (p = 0.03). Overall survival (OS) was negatively influenced by higher CXC chemokine receptor 4 (CXCR4) (p = 0.01), very late antigen-4 (VLA-4) (p = 0.01), and focal adhesion kinase (FAK) expression (p = 0.04). Combination of these markers allowed to distinguish two prognostic groups: patients overexpressing 2 or 3 factors had a significantly shorter OS (p = 0.015). CXCR4, VLA-4 and FAK are new phenotypic markers which could be helpful to establish risk-stratified therapeutic strategies.

A series of 38 patients with acute myeloblastic leukemia (AML) with 49 or more chromosomes and without structural abnormalities was selected within the Groupe Francophone de Cytogénétique Hématologique (GFCH) to better define their characteristics. The median age of the patients was 65 years, and all FAB subtypes were represented. Although all chromosomes were gained, some seems to prevail: chromosome 8 (68%), 21 (47%), 19 (37%), and 13 and 14 (34% each). Since MLL rearrangement leads patients in a group with an unfavorable prognosis, search for cryptic rearrangements of MLL was performed in 34 patients and showed abnormalities in 5 (15%). When we applied the most frequent definition of complex karyotypes (three or more abnormalities), all patients with high hyperdiploid AML fall in the unfavorable category. Among the 18 patients without MLL rearrangement receiving an induction therapy, 16 (89%) reached CR and 6 (33%) were still alive after a 31-month median follow-up (14-61 months). Although this study was retrospective, these results suggest that high hyperdiploid AML without chromosome rearrangement seems to be a subgroup of uncommon AML (less than 1%), and may be better classified in the intermediate prognostic group.

Deletions of the long arm of chromosome 14 [del(14q)] are rare but recurrently observed in mature B-cell neoplasms, particularly in chronic lymphocytic leukemia (CLL). To further characterize this aberration, we studied 81 cases with del(14q): 54 of CLL and 27 of small lymphocytic lymphoma (SLL), the largest reported series to date. Using karyotype and fluorescence in situ hybridization (FISH), the most frequent additional abnormality was trisomy 12 (tri12), observed in 28/79 (35%) cases, followed by del13q14 (12/79, 15%), delTP53 (11/80, 14%) delATM (5/79, 6%), and del6q21 (3/76, 4%). IGHV genes were unmutated in 41/53 (77%) patients, with a high frequency of IGHV1-69 (21/52, 40%). NOTCH1 gene was mutated in 14/45 (31%) patients. There was no significant difference in cytogenetic and molecular abnormalities between CLL and SLL. Investigations using FISH and SNP-array demonstrated the heterogeneous size of the 14q deletions. However, a group with the same del(14)(q24.1q32.33) was identified in 48% of cases. In this group, tri12 (P = 0.004) and NOTCH1 mutations (P = 0.02) were significantly more frequent than in the other patients. In CLL patients with del(14q), median treatment-free survival (TFS) was 27 months. In conclusion, del(14q) is associated with tri12 and with pejorative prognostic factors: unmutated IGHV genes (with over-representation of the IGHV1-69 repertoire), NOTCH1 mutations, and a short TFS.

There was an error in the name of the 13th author. The correct name is Florence Nguyen-Khac.

The PRDM16 (1p36) gene is rearranged in acute myeloid leukaemia (AML) and myelodysplastic syndrome (MDS) with t(1;3)(p36;q21), sharing characteristics with AML and MDS with MECOM (3q26.2) translocations. We used fluorescence in situ hybridization to study 39 haematological malignancies with translocations involving PRDM16 to assess the precise breakpoint on 1p36 and the identity of the partner locus. Reverse-transcription polymerase chain reaction (PCR) was performed in selected cases in order to confirm the partner locus. PRDM16 expression studies were performed on bone marrow samples of patients, normal controls and CD34(+) cells using TaqMan real-time quantitative PCR. PRDM16 was rearranged with the RPN1 (3q21) locus in 30 cases and with other loci in nine cases. The diagnosis was AML or MDS in most cases, except for two cases of lymphoid proliferation. We identified novel translocation partners of PRDM16, including the transcription factors ETV6 and IKZF1. Translocations involving PRDM16 lead to its overexpression irrespective of the consequence of the rearrangement (fusion gene or promoter swap). Survival data suggest that patients with AML/MDS and PRDM16 translocations have a poor prognosis despite a simple karyotype and a median age of 65 years. There seems to be an over-representation of late-onset therapy-related myeloid malignancies.

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.

International audience

International audience; The t(8;16)(p11;p13) is a rare translocation involved in de novo and therapy-related myelomonocytic and monocytic acute leukemia. It fuses two genes encoding histone acetyltransferases (HATs), MYST3 located at 8p11 to CREBBP located at 16p13. Variant translocations involve other HAT-encoding genes such as EP300, MYST4, NCOA2 or NCOA3. MYST3-linked acute myeloid leukemias (AMLs) share specific clinical and biological features and a poor prognosis. Because of its rarity, the molecular biology of MYST3-linked AMLs remains poorly understood. We have established the genome and gene expression profiles of a multicentric series of 61 M4/M5 AMLs including 18 MYST3-linked AMLs by using array comparative genome hybridization (aCGH) (n=52) and DNA microarrays (n=44), respectively. We show that M4/5 AMLs have a variety of rare genomic alterations. One alteration, a gain of the MYB locus, was found recurrently and only in the MYST3-linked AMLs (7/18 vs 0/34). MYST3-AMLs have also a specific a gene expression profile, which includes overexpression of MYB, CD4 and HOXA genes. These features, reminiscent of T-cell acute lymphoid leukemia (ALL), suggest the targeting of a common T-myeloid progenitor.

Background: The knowledge of normal marrow is mandatory to assess the malignant counterpart of normal cells and define leukemia-associated immunophenotypes (LAIPs). In this study, the expression of a variety of antigens expressed in normal and postchemotherapy bone marrow (BM) was analyzed to provide a frame of reference for the identification of myeloid LAIPs. Methods: Multiparameter four- and six-color flow cytometry was used to define antigen combinations totally absent or present at very minimal levels in marrow cells of normal individuals (n = 20) and patients receiving chemotherapy for acute lymphoblastic leukemia (n = 20). Immature (blast) cells were gated according to CD45/SSC properties. Fifty-three acute myeloid leukemia (AML) samples were studied in six-color combinations. Results: In six-color flow cytometry, 47 phenotypes were totally absent from blast gate in all normal samples. Forty-one other phenotypes were identified in less than 0.05% of blast cells. There was no difference between normal and postchemotherapy BMs. The four-color panel allowed to identify only 30 phenotypes present at a frequency

The 90-kDa heat shock protein (HSP90) is implicated in the conformational maturation and stabilization of a variety of client proteins with receptor and signal transduction functions. The objective of this study was to assess its expression in primary acute myeloid leukemia (AML) cells and to evaluate its biological and clinical significance. The in vitro effects of 17-AAG, a selective inhibitor of HSP90, was also evaluated. Cells from 65 patients with newly diagnosed AML were studied. The expression of HSP90 correlated with that of CD34, p170, and bcl-2 proteins but not with white cell counts, FAB or WHO subtype, or cytogenetics. HSP90 levels were also higher in samples exhibiting an autonomous growth in liquid culture or forming spontaneous colonies. A concomitant constitutive activation of the extracellular signal-regulated kinase and phosphatidylinositol 3-kinase/AKT pathways was observed in a majority of samples and was significantly correlated with HSP90 expression. All patients received induction chemotherapy. The percentages of HSP90-, CD34-, bcl-2-, and p170-positive cells were higher in patients who did not attain complete remission. Survival was also shorter in patients with high levels of HSP90. In vitro exposure of leukemic cells to 17-allylamino-demethoxy geldanamycin (17-AAG) resulted in inhibition of growth in liquid and clonogeneic cultures and in apoptosis, at concentrations which in most cases were not toxic for normal CD34-positive or progenitor cells. The concentration inhibiting 50% growth at 72 h in liquid culture correlated with HSP90 expression. Our study suggests that HSP90 is overexpressed in poor-prognosis AML cells and plays a role in cell survival and resistance to chemotherapy. Targeted therapy with 17-AAG represents a promising antileukemic strategy in adult AML.

Using flow cytometry, we investigated the clinical and hematologic relevance of expression of heat-shock proteins (HSP) HSP27, HSP60, HSP70, HSP90 and HSP110 in bone marrow of 142 patients with newly diagnosed myelodysplastic syndromes, together with that of the membrane differentiation antigen CD34 and the drug-resistance related protein, P170 (Pgp).

Background: Numerous recurrent chromosomal abnormalities have been described in adult Ph-negative ALL, often observed in small patient cohorts. In the largest MRC/ECOG study (Moorman, Blood 2007), t(4;11)(q21;q23), 14q32 involvement, complex karyotype (≥5 abnormalities), and low hypodiploidy/near triploidy (Ho-Tr) were associated with shorter event-free survival (EFS), while patients with high hyperdiploidy or del(9p) had a better outcome. We aimed to confirm these observations in 955 adult patients (15-60y; median, 35y) with Ph-negative ALL treated in the pediatric-inspired GRAALL-2003/2005 trials. Patients and Methods: Overall, a karyotype was performed for 946 (611 BCP-ALL, and 335 T-ALL), successful for 811 (523 BCP-ALL and 288 T-ALL) and abnormal in 590 patients (387 BCP-ALL and 203 T-ALL). FISH and/or PCR screening for relevant abnormalities and DNA index were also performed, finally allowing for the identification of cytogenetic abnormalities in 677/955 patients (71%). All were centrally reviewed. Ultimately, 857/955 patients (90%; 542 BCP-ALL and 315 T-ALL) could be classified in 18 exclusive primary cytogenetic subsets as detailed below. Endpoints were cumulative incidence of failure (CIF, including primary refractoriness and relapse) and EFS. With a median follow-up of 4 years, 5-year CIF and EFS were estimated in these patients at 31% and 51%, respectively. As some abnormalities, including MLL rearrangements, Ho/Tr, t(1;19)(q23;p13)/TCF3 and complex karyotypes were used to stratify allogeneic stem cell transplantation (SCT) in GRAALL trials, some comparisons were repeated after censoring patients transplanted in first CR at SCT time. Results: The 542 informative BCP-ALL patients were classified as: t(4;11)(q21;q23)/MLL-AFF1 (n=72; 13%); other MLL+ 11q23 abnormalities (n=11; 2%); t(1;19)(q23;p13)/TCF3-PBX1 (#28; 5%); Ho/Tr (n=33; 6%); high hyperdiploidy (n=36; 7%); abnormal 14q32/IGH translocation (n=27; 5%); t(12;21)(p13;q22)/ETV6-RUNX1 (n=2; 0.4%); iAMP21 (n=3; 0.6%); other abnormalities (n=210; 39%); and no abnormality (n=120; 22%). The 315 informative T-ALL patients were classified as: t(10;14)(q24;q11)/TLX1 (n=64; 20%); other 14q11 or 7q34/TCR (n=31; 10%); t(5;14)(q35;q32)/TLX3 (n=29; 9%); t(10;11)(p12;q14)/PICALM-MLLT10 (n=14; 4%); deletion 1p32/SIL-TAL (n=18; 6%); MLL+ 11q23 abnormalities (n=6; 2%); other abnormalities (n=93; 30%); and no abnormalities (n=60; 19%). A complex karyotype was observed in 27/527 (5%) BCP-ALL and 21/298 (7%) T-ALL patients and a monosomal karyotype (as per Breems, JCO 2008) in 82/518 (16%) BCP-ALL and 26/286 (9%) T-ALL patients. In BCP-ALL, trends towards higher CIF and shorter EFS were observed in t(4;11) patients, with or without SCT censoring (HRs, 1.34 to 1.64; p values Conclusion: These results show that, in the context of an intensified pediatric-inspired protocol designed for adult Ph-negative ALL patients, few cytogenetic subsets remained reliably predictive of response to therapy. Differences observed in EFS might partly be due to treatment-related mortality. Combining cytogenetics, molecular genetics and minimal residual disease monitoring could allow for better individual risk assessment (Beldjord, Blood 2014). Disclosures No relevant conflicts of interest to declare.

To the editor: A family history of chronic lymphocytic leukemia (CLL) is one of the best characterized risk factors for the development of CLL. First-degree relatives of individuals with CLL have a 3- to 8-fold increased risk for CLL.[1][1],[2][2] CLL often has an indolent behavior, but some

Adult Refsum disease (ARD) is a rare autosomal recessive neurologic disorder associated with the accumulation in blood and tissues of phytanic acid, a natural compound of exogenous origin whose catabolism is impaired in patients. We present here genome wide linkage analysis of an atypical Refsum disease family where L-pipecolic acid level in blood was also increased, suggesting that the patients suffer from a new peroxisomal disorder intermediate between ARD and Infantile Refsum Disease (IRD, a peroxisomal deficiency disease). We were able to demonstrate significant linkage (lod score = 3.6) between Refsum Disease with increased Pipecolic Acidaemia (RDPA) and the interval defined by D10S249 and D10S466 on 10p in this single consanguineous family by combining lod score values obtained from analysis of the multiple affected sibs, haplotype homozygosity and from discrimination between healthy carriers and non carriers based on phytanate oxidase measurements. This illustrates the power of homozygosity mapping with a dense map of microsatellite markers. A similar strategy will allow testing for homogeneity/heterogeneity between RDPA and ARD or the rare complementation groups of IRD.

6531 Background: The PRDM16 gene on chromosome 1p36 is reported to be rearranged in sparse cases of acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS) with t(1;3)(p36;q21). Methods: We...

Abstract 1721 Introduction: CD133 is a transmembrane protein expressed by hematopoietic stem cells and other cell types, whose physiological functions remain unknown. To determine its potential prognostic value in AML, we analyzed its expression on blasts from 76 patients at diagnosis. Moreover, since leukemic stem cells play a critical role in the resistance to chemotherapeutic drugs and may be responsible for relapse, we compared the growth potential and activated cellular pathways of CD133+ and CD133- cells from 20 AML patients. Methods: First, bone marrow samples were collected at diagnosis from 76 AML patients (median age: 65, range: 1–84). CD133 expression was evaluated on blasts cells and CD34+ cells using 6-color flow cytometry (FC) and the monoclonal antibody AC133. The mean fluorescence intensity ratio (MFIR) was calculated by dividing the MFI of CD133 by the MFI of its isotypic control. Second, purified CD34+, CD34+CD133+ and CD34+CD133- cells were isolated from 20 AML bone marrows using the magnetic separation system Minimacs (Miltenyi Biotec). These fractions were submitted to an in vitro colony forming assay during 14 days. Finally, the level of expression of survival proteins involved in the PI3K/AKT, Mitogen-Activated Protein Kinase (MAPK) and Bcl-2 pathways, as well as CXCR4 and Focal Adhesion Kinase (FAK) migration proteins, was analyzed by FC in these fractions. Results: The level of CD133 expression on blasts cells was significantly different between AML patients with favorable (n=12), intermediate (n=38) and unfavorable karyotypes (n=23) (p=0.007). In particular, it was significantly higher in patients with unfavorable karyotype than in patients with intermediate karyotype (p=0.032) or favorable karyotype (p=0.005). Moreover, when considering NPM1 and FLT3 mutation status in patients with intermediate karyotype, the level of CD133 expression was significantly higher in patients with an intermediate molecular profile than in patients with a favorable molecular profile (p=0.004). Most interestingly, we showed that the level of CD133 expression has an impact on overall survival in multivariate analysis, independently of leukocytosis, karyotype and achievement of complete remission after induction therapy (p=0.04). Second, in 20 patients, we found that AML progenitor cells (AML CFU) were present in all sorted fractions, but the CD34+CD133+ fraction gave rise to larger and more numerous colonies (74.9±39.6) than the CD34+CD133- fraction (11,25±6). Moreover, the CD34+CD133+ fraction was the only one able to form secondary colonies (52.1±31.8). Finally, we found that the expression of PI3K (mean percentage of positive cells: 78±20.1), pAKT (80±19.8), pERK (80.1±19), CXCR4 (71.6±22.7), pFAK (81.2±19.8), Bcl-2 (72.9±26.1), Bcl-xL (45.7±24.8), and pBad (63.9±36.1) was significantly upregulated in CD34+CD133+ cells compared to CD34+CD133- cells, whereas Bax was downregulated. Discussion: CD133 is known to be expressed in hematopoietic progenitors, but its role in the resistance to chemotherapy is not well studied. In our work, the level of expression of CD133 on AML blasts appeared as an independent immunophenotypic prognostic factor, encouraging its further evaluation in a larger number of cases. We also showed that CD133+ cells have greater replicative properties than CD133- cells, and exhibit activation of various signaling pathways. Since CD133+ cells might escape from apoptosis through activation of PI3K/AKT and ERK, these proteins could represent potential therapeutic targets. In conclusion, our results revealed that CD133+ cells exhibit more aggressive behavior than CD133- ones, and that CD133+ AML progenitors might contribute to resistance to chemotherapy through preferential activation of PI3K/AKT, MAPK and Bcl-2 survival response. Disclosures: No relevant conflicts of interest to declare.

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 Disclosures: No relevant conflicts of interest to declare.

Deregulation of signal transduction pathways (STPs) including JAK/STAT, RAS/Raf/MEK/ERK and PI3K/AKT may promote leukemogenesis by conferring cells proliferation and survival advantages in acute myelogenous leukemia (AML). The activation of these pathways had an adverse prognosis in AML and development of targeted therapies seems to be promising. Heat-shock proteins (HSP) are involved in the conformational maturation of a number of signaling proteins, and HSPs expression in AML is associated with other adverse prognostic factors (Bcl2, MRP). The aim of this work was to study STPs expression in AML, and there correlation to other adverse prognostic factors and complete remission rate. Sixty five patients with primary AML were analyzed by flow cytometry for constitutive ERK, PI3K and AKT activation, HSP90, Bcl2 and P170 expression. FAB subtypes were M0=3, M1=19, M2=16, M4=10, M5=16, M6=1. All patients received an induction treatment, 44 patients reached complete remission. Cytogenetics was available in 63 cases (Intermediate = 36, favorable = 3, unfavorable= 24). We performed a 3 color flow cytometry protocol using CD45 and CD34 to identify blast cells, and used a third antibody to the following transduction proteins (ERK, pERK, AKT, pAKT, PI3K) or intracytoplasmic proteins (Bcl2, HSP90, p170). Spontaneous growth of leukemic progenitor cells (CFU-L) was investigated in 44 samples. In AML, activated proteins were found in CD34+ cells. ERK and PI3K/AKT were frequently co-activated. Flow cytometry results showed that levels of STPs were significantly higher (p

The recently described episomal amplification of the NUP214-ABL1 fusion in 6% of T-ALL leads to the expression of a constitutively activated chimeric tyrosine kinase sensitive to imatinib. We collected additional cases in order to better characterize this new entity with respect to genetic presentations and clinical course. We collected 14 new NUP214-ABL1 positive cases by FISH (LSI-BCR/ABL1 ES, Vysis and ABL1 break-apart home-made probes) or RT-PCR (fusion transcript) screening. FISH analysis detected episomal amplification of NUP214-ABL1 in 11 patients with a highly variable number of nuclei with amplification ( These observations emphasize the interest of combining both (quantitative) RT-PCR and FISH for the screening and characterisation of NUP214-ABL1 fusion and amplification, demonstrate the coexistence of different NUP214-ABL1 genomic presentations in one patient (episomal amplification, 9q34 insertions, HSR) compatible with the model in which genomic amplification start with episome formation in order to create the NUP214-ABL1 fusion followed by their amplification and optional secondary reintegration, confirm occurrence of NUP214-ABL1 in T-ALL with HOX11 and HOX11L2 involvement, raise the question of the rather worse prognosis for cases with intrachromosomal amplification as previously suggested, raise the signification of minor NUP214-ABL1 clones and variable genomic presentations in the leukemogenesis of this subgroup of T-ALL that could potentially benefit from imatimib. ° on behalf of the GFCH (Groupe Francophone de Cytogénétique Hématologique) and the BCGHO (Belgian Cytogenetic Group for Hematology and Oncology).