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Background. The classic Ph-negative myeloproliferative neoplasms (MPN) are a group of clonal haematopoietic disorders, including polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF), whose shared and diverse phenotypic signatures are caused by a dysregulated JAK/STAT signal transduction because of acquired somatic mutations. It has been demonstrated that autoimmune diseases and MPN can be associated (Kristinsson et al., Haematologica. 2010 Jul;95(7):1216-20.), suggesting a common background of immune dysregulation (Barosi, Curr Hematol Malig Rep. 2014 Dec;9(4):331-9). SARS-CoV-2 infection displays extreme inter-individual clinical variability, ranging from silent infection to lethal disease. It has been described that at least 10% of patients with life-threatening coronavirus disease 2019 (COVID-19) pneumonia have neutralizing autoantibodies (AAbs) against type I IFNs, that precede SARS-CoV-2 infection (Bastard et al., Science. 2020 Oct 23;370(6515):eabd4585). In this study we searched for AAbs against type I IFNs in a cohort of MPN patients to evaluate the prevalence of these AAbs in the MPN population and to check for clinical correlations, including severity of COVID-19. Methods. Plasma samples from consecutively referred MPN patients were prospectively collected between November 2020 and June 2021 and frozen at -30°C immediately after collection. Levels of AAbs against type I IFN subtypes including IFNs alpha, beta and omega were measured using the enzyme-linked immunosorbent assay (ELISA) and a neutralization assay, as previously reported (Bastard et al., Science. 2020 Oct 23;370(6515):eabd4585; Moreews et al., Sci Immunol. 2021 May 25;6(59):eabh1516). Results. We included a total of 219 MPN patients (101 ET, 76 PV, 36 MF and 6 MPN unclassificable). Neutralizing AAbs to type I IFNs were detected in 29 patients (13.2%, 95%CI: 9.1% - 18.5%). Comparing patients with and without AAbs we observed a significant difference in terms of distribution of MPN diagnosis (P = 0.029) and driver mutations (P = 0.019), while we did not observe a difference in terms of age, sex, and treatment (Table 1). Overall, 29 patients (13%) got SARS-CoV-2 infection and 8 of them (28%) required hospitalization due to severe COVID-19. AAbs against type I IFNs were detected in 4 of the 29 SARS-CoV-2 infected patients. A higher rate of hospitalization for severe COVID-19 was observed in patients with AAbs to type I IFNs (2 of 4 patients, 50%) compared to those without these AAbs (6 of 25 patients, 24%), although the difference did not reach a statistical significance (P = 0.300). Conclusions. In this study, we detected a prevalence of AAbs against type I IFNs which is much higher in our MPN cohort (13%) than in the general population (2-3%). We also found a correlation between the presence of AAbs to type I IFNs and both the hematological diagnosis and the driver mutation. Despite a comparable prevalence of SARS-CoV-2 infection between MPN patients with or without AAbs to type I IFNs, we observed a different rate of hospitalization due to severe COVID-19 which is almost twice in those with AAbs against type I IFNs compared to those without these AAbs. However, this difference did not reach a statistical significance, probably because of the low number of SARS-CoV-2 infection in the subgroup of patients with AAbs against type I IFNs. Thus, further studies to analyse the prevalence of AAbs against type I IFNs in patients with MPN, their association with other forms of auto-immunity and severe COVID-19 are warranted. Figure 1 Figure 1. Disclosures Arcaini: Gilead Sciences: Research Funding; Bayer, Celgene, Gilead Sciences, Roche, Sandoz, Janssen-Cilag, VERASTEM: Consultancy; Celgene, Roche, Janssen-Cilag, Gilead: Other: Travel expenses; Celgene: Speakers Bureau. Rumi: Novartis, Abbvie: Consultancy. more...

Introduction: B-cell immunophenotype could be swiftly assessed by flow cytometry on blood samples or bone marrow aspirate specimens. It provides crucial information later refined with histologic, genetic and molecular features to assert accurate diagnosis of chronic B-cell lymphoproliferative disorders (B-CLPD). Besides Matutes score we identified additional useful markers, i.e. CD148 and CD180 to classify mantle cell lymphoma (MCL) and marginal zone lymphoma (MZL), respectively. Furthermore, CD200 is known to be highly expressed in chronic lymphoid leukemia (CLL) while absent in MCL. Hypothesis: The determination of CD148, CD180 and CD200 expression on B-cells by flow cytometry on blood samples and/or bone marrow aspirates could be a potent tool to accurately identify B-CLPD. We postulated the existence of the following specific expression patterns in B-CLPD: CD148 dim/CD180 dim/CD200 bright for CLL, CD148 dim/CD180 dim/CD200 dim for lymphoplasmocytic lymphoma (LPL), CD148 bright/CD180 dim/CD200 neg/dim for MCL and CD148 dim/CD180 bright/CD200 dim for MZL . Methods: In a prospective study we investigated the expression of CD148/CD180/CD200 on B-cells from 673 patients at the time of B-CLPD diagnosis in our hospital from 2014 to 2020. We analyzed 440 blood and 233 bone marrow aspirate specimens using a BD FACSCanto II flow cytometry instrument. Based solely on CD148/CD180/CD200 specific expression patterns we postulated a diagnosis of CLL, LPL, MCL or MZL. These postulated diagnoses were later confronted to the final diagnoses when all histologic, genetic and molecular features were finalized. Sensitivity, specificity, positive and negative predictive values of the expression profiles were determined. In addition, to investigate the relative importance of these three CD markers we then normalized their mean fluorescence intensities (MFI) and applied several supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM). Results: Out of the 673 clinical samples the CD148/CD180/CD200 expression patterns classified 212 specimens as CLL/SLL (30.8%), 160 as LPL (23.8%), 76 as MCL (11.28%) and 169 as MZL (25%). These diagnosis hypotheses were retrospectively compared to the final diagnoses based on all histologic, genetic and molecular features These diagnosis hypotheses of CLL, LPL, MCL and MZL were consistent with the final diagnosis in 583 out of the 617 corresponding cases (94%) with high positive and negative predictive values. The characteristics of the diagnosis accuracy are detailed in the table below. HCL and FL were not further investigated as their immunophenotype usually do not overlap with those of other B-CLPD. Seventeen out of 617 patients (17/617, 5.3%) did not displayed a clear CD148/CD180/CD200 pattern: 9 LPL, 4 CLL and 4 MZL. In sixteen patients (16/617, 5.0%) the diagnosis hypothesis based on this strategy was not confirmed after completion of the exploration including karyotype, MYD88 L265P mutational status, CCND1 overexpression and pathology explorations. We next investigated the relative importance of these 3 markers. We focused on MFI values of CD148, CD180 and CD200 and three categorical "positive or negative" markers (CD5, CD23, FMC7) that were assembled into a composite marker. After Cox-box normalization of CD148, CD180 and CD200 MFIs, a set of supervised machine learning algorithms including Logistic Regression, Random Forest and Light Gradient Boosting Machine (LightGBM) were applied to the cohort of CLL, LPL, MCL and MZL. We established that the highest diagnosis weights were obtained for CD200 in CLL, CD200 and CD148 in MCL (negatively and positively, respectively), CD180 in MZL. In LPL, CD148, CD180 and CD200 had the highest weights using LightGBM and Random Forest algorithms, while Logistic Regression determined that CD5 and CD23 had the highest (negative) weights. In conclusion, the determination of CD148/CD180/CD200 surface expression patterns by flow cytometry, along with morphology, allowed to assert an accurate diagnosis hypothesis in CLL, MCL, LPL and MZL with high positive and negative predictive values. Machine learning algorithms allowed to measure the relative importance of these markers, that could be of great help in case of discordant expression of the main diagnosis markers. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare. more...

Background : APL is, in the vast majority of cases, driven by t(15 ;17) translocation, which leads to PML/RARA rearrangement. Remarkably, APL is an uncommon genetically simple disease and only few additional alterations, cooperating with PML/RAR, have been described at diagnostic (Welch et al, Cell 2012). Most APL can be cured with targeted therapy combining all-trans retinoic acid (ATRA) and chemotherapy (CT). However, genetic mechanisms underlying the 10-15% relapses observed with this regimen remain unclear. The goal of the present study was to identify mutations that cooperate with PML/RAR and those responsible for acquired resistance to ATRA-CT treatment in APL patients by whole-exome sequencing of diagnostic/ remission/relapse trios. Methods: Newly diagnosed APL patients included in clinical trials of the French Swiss Belgian APL group between 1994 and 2008, treated with ATRA-CT, before the introduction of first-line ATO, who experienced at least one relapse and had adequate material, were studied. We collected retrospectively 64 samples from 23 patients, including 23 diagnostic samples, 18 at first complete remission (CR) and 23 at relapse (22 first relapse and 1 second relapse). Whole exome-sequencing was performed on all samples. DNA libraries were prepared with the SureSelect human v5 kit (Agilent) and sequenced on Hiseq1000 (Illumina). The bioinformatic analysis was performed by GECO/integragen using CASAVA variant calling (Illumina) and dedicated pipeline. 18 trios and 5 duos passed the stringent quality control and were analyzed for somatic variants and copy number variations (CNV). Results : After elimination of polymorphisms, the median number of somatic variants corresponding to de novo mutation at diagnosis was 14, while only 3 new somatic variants appeared at relapse (figure 1). Notably, we failed to detect oncogene alterations other than PML/RARA in 7/23 (30%) patients. At diagnostic, 39% of patients (9/23) presented the common FLT3 alterations and at relapse 22% (5/23) of patients presented the known RARA mutations. Moreover, recurrent alterations were observed in activators of the MAPK signaling (22%): NRAS (2 patients), BRAF (1 patient), KRAS (1 patient), SPRY1 (1 patient). Mutations in the NT5C2 gene (3 patients), coding a 5'nucleotidase implicated in resistance to nucleoside-analog therapy, were solely observed at relapse, as in acute lymphoblastic leukemia (ALL). Abnormalities of epigenetic regulators were also detected at diagnostic and/or relapse: WT1 (7 patients, 30%), NSD1 (2 patients), TET2 (1 patient), ASXL1 (1 patient) and MED12 (2 patients). Homozygote WT1 inactivation by mutation plus neutral copy LOH occurred in 3 patients at relapse. The genetic markers identified allowed us to construct several evolution models. In 8 patients (35%), the diagnostic and relapse clones were clearly distinct, supporting the fact that they independently derived from pre-leukemic cells that survived ATRA/chemotherapy. In contrast, other relapses appeared to derive from the diagnostic clone. Conclusion: Our data highlight the genetic simplicity of APL with very few alterations detected and 30% patients without identified mutations in addition to PML/RARa. Our results support the existence of two prototypic mechanisms of relapse: re-emergence of a new APL from persisting pre-leukemic cells and relapse from APLs often expressing strong oncogenes at diagnosis, impeding therapy response and favoring the acquisition of resistance mutations at relapse, including PML/RARA or NT5C2. It will be interesting to assess the prevalence of those two mechanisms in the exceptional cases of relapse in patients treated with more recent frontline regimens that combine ATRA and arsenic in APL. Disclosures Ades: Celgene, Takeda, Novartis, Astex: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Fenaux:Celgene, Janssen,Novartis, Astex, Teva: Honoraria, Research Funding. more...

Polycythemia vera (PV) is an acquired myeloproliferative disorder with primary expansion of the red cell mass leading to an increased risk of thrombosis and less frequently to myelofibrosis and secondary acute leukemia. Standard therapies include cytoreduction with either phlebotomy or chemotherapeutic agents and antithrombotic drugs. Because long-term exposure to cytotoxic chemotherapy may increase the risk of acute transformation, new therapeutic options are needed. Tipifarnib is a nonpeptidomimetic inhibitor of farnesyl transferase that was developed as a potential inhibitor of RAS signaling. In the present study we report that tipifarnib used at pharmacologically achievable concentrations strongly inhibits the erythroid burst-forming unit (BFU-E) autonomous growth that characterizes patients with PV. Moreover, at low tipifarnib concentrations (0.15 μM), the inhibitory effect was preferentially observed in PV BFU-E progenitors and not in normal BFU-E progenitors and was not rescued by erythropoietin (EPO). Thus tipifarnib may specifically target PV stem cells and may be of clinical interest in the treatment of patients with PV. more...

The term « double » or « triple hit lymphoma » is commonly used to describe mature B cell neoplasms with either BCL2 and/or MYC and/or BCL6 gene rearrangements. These rare and aggressive lymphomas with high Ki67 expression are categorized as « B-cell lymphomas unclassified, with features intermediate between diffuse B-cell lymphoma and Burkitt lymphoma » category in the WHO 2008 classification. To our knowledge, only 2 cases of lymphoma have been described with four specific genetic events (quadruple hit) involving MYC, BCL2, BCL6 and CCND1 genes (Bacher U. et al., Genes Chromosomes Cancer 2011). We describe here the third observation. A patient, a 79 years old man, suffering from paraesthesias for 4 months, was admitted for polyneuritis in a context of poor general condition. Clinical examination showed the presence of numerous axillary, supraclavicular, mediastinal and inguinal lymphadenopathy, neuro-meningeal invasion and skin infiltration. The biopsy of a skin nodule of left arm revealed an infiltration consisting in large proliferating cells (Ki67 80%) stained by anti-CD20, BCL2 and BCL6 antibodies, but not with CD10 nor CD23, consistent with a diffuse large B-cell lymphoma (DLBCL), NOS diagnosis. Blood cell count showed 8.1 G/L leukocytes, 13.2 g/dL hemoglobin, 166 G/L platelets. LDH and β-2 microglobulin were elevated (989 U/I, and 9.14 mg/L respectively). Blood cell film examination showed the presence of 28% of pathological basophil lymphocytes (medium sized, with slightly clumped chromatin and frequent prominent nucleoli). Flow cytometry revealed that these cells expressed a monotypic lambda immunoglobulin light chain and were CD19, CD20, FMC7, CD22 positive, with a dim CD5 positivity and no CD10 staining. The negativity of CD23 associated to strong CD148 staining (Miguet et al, J Proteome Res2009) were in favour of the diagnosis of variant pleomorphic mantle cell lymphoma (MCL). Cytogenetic study performed in the WBC found a complex hyperdiploid karyotype (47 chromosomes) with a t(3;22) translocation involving BCL6 and IGL genes, structural abnormality of a chromosome 8 resulting in juxtaposition of 5’MYC and BCL2 in FISH (with break of the MYC probe in FISH), a derivative chromosome 18 from a t(14;18) translocation with fusion of 5’IGH and BCL2, and a t(11;14) complex translocation involving IGH and CCND1 (54% of the nuclei). Overexpression of cyclin D1 was detected in the WBC by RQ-PCR, as well as in the skin lesion using immunochemistry. Others numeral (trisomy 12) and structural abnormalities (involving the 1, 7, 14 and 21 chromosomes) were also detected. The patient was treated with a chemotherapy combining rituximab, ifosfamide, cytosine arabinoside and intrathecal methotrexate. He is still alive 5 months after the diagnosis. This third case of quadruple hit lymphoma confirms the complexity of the classification of such aggressive malignancies. Initial rearrangement of the CCND1 gene characterizes MCL that may harbour in very rare cases additional rearrangements of MYC or BCL6. Conversely, cyclin D1 overexpression is considered a rare feature in DLBCL. Recently, Ok CY et al. (Cancer 2014) proposed to reclassify DLBCL with expression of cyclin D1 and CCND1 chromosomal rearrangement and CD5 positivity as aggressive pleomorphic MCL variant. But no case with rearrangement of 2 (or more) genes (BCL2 and/or MYC and/or BCL6) was described in this study. Juskevicius D et al. (Am J Surg Pathol 2014) suggest the existence of a “gray zone” in which morphologic, clinical and genetic features are insufficient to segregate CD5- and SOX11-negative lymphomas with overexpression of cyclin D1/ translocation involving CCND1between blastoid MCL from cyclin D1-positive DLBCL. In our case, immunophenotyping of circulating cells (CD5+, CD148++) as well as genetic and molecular features (translocation involving CCND1 and IGH, and overexpression of cyclin D1) allow us to diagnose a probable genetically unstable aggressive pleomorphic MCL variant with rearrangement of several extra genetic hits. Disclosures No relevant conflicts of interest to declare. more...

There are a number of small B-cell proliferations that do not fall into any of the types of B-cell neoplasms recognized in the WHO classification using classical immunophenotypic markers. This situation is notably encountered in the case of the differential diagnosis of marginal zone lymphoma (MZL), atypical chronic lymphocytic leukemia (aCLL) mantle cell lymphoma (MCL) or lymphoplasmacytic lymphoma (LPL). This is mostly due to the lack of immunological specific markers especially when histological samples are not available or during leukemic phases of atypical B-cell neoplasms. In order to find new markers to discriminate between these different malignancies, we have previously developed a proteomic strategy based on the analyses of plasma membrane microparticles and proposed two new specific markers: CD148 and CD1801, 2 for MCL and MZL respectively. The simultaneous use of these two markers, together with the CD200 that is positive in most cases of CLL and negative in MCL3 could be of great interest to better assess the differential diagnosis. In the present study, we report the results obtained by the combination of the three markers studied in addition to the routine flow cytometry panel: CD148 (Clone 143-41 FITC); CD180 (Clone G28.8 PE) and CD200 (Clone OX104 APC). An expression profile of these proteins have been established on a well characterized set of patients: CLL with a Matutes score > 3 (N=28); MCL harboring t(11;14) translocation or CCND1 overexpression (N=20); LPL (N=16) classified following cytological morphology, IgM peak and positivity of CD38, and MZL (N=27), displaying a CD5- CD23-immunophenotype associated to a splenomegaly. For each group the mean of fluorescence intensity and Standard Error have been determined. MCL patients exhibited a strong expression of CD148 (MFI = 1480) combined with a weak expression of CD180 and CD200 (MFI = 888 and 426 respectively). A weak expression of CD148 and CD180 (MFI = 495 and 754) coupled to a strong expression of CD200 (MFI = 3750) was typical of the CLL group and a weak expression of CD148 and CD200 (MFI = 640 and 1200) coupled to a strong expression of CD180 (MFI = 5300) was observed in the MZL group. A moderate expression of these three markers was observed in the LPL group. A threshold corresponding to MFI +/- 4 standard error was then calculated for each group (see table 1), and patients were categorized following the expression profile of these 3 markers. Table 1: threshold calculated from the average MFI and the associated standard error for each studied pathologies. CD148 CD180 CD200 MCL >980 500 MZL 2700 In this cohort, the above described profiles correctly identified MCL cases with a specificity of 96% and a sensitivity of 65%, CLL cases with a specificity of 95% and a sensitivity of 79%, LPL cases with a specificity of 95% and a sensitivity of 31% and MZL cases with a specificity of 99% and a sensitivity of 52%. These results strongly suggest that the incorporation of these three markers CD148 CD180 and CD200 in addition of the routinely used flow cytometry panel can be helpful in a number of cases for which the diagnosis remains difficult. References: 1) Miguet et al. Leukemia 2013 2) Miguet et al. Journal of Proteome Research 2009 3) Palumbo et al. Leukemia Research 2009 Disclosures No relevant conflicts of interest to declare. more...

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. more...

TET1 genomic breakpoints and clinical features of MLL-TET1 rearrangements have been described in 13 acute leukemia cases, 11 in AML, 2 in B cell-precursor ALL. The incidence of this rare translocation was evaluated to 0.3% of MLL rearranged AML cases (5 out of 1590 MLL [Meyer C. Leukemia 2013]). Although those cases are very uncommon, their study can improve our current understanding of leukemogenesis. We report here the first t(10;11) MLL-TET1 positive case of lymphoblastic T lymphoma occurring in a 31 years old male patient, with a subsequent transformation to AML. The patient was referred for a large mediastinal mass and right pleural effusion. Mediastinal and bronchus biopsies led to the diagnosis of a precursor-T cell lymphoblastic lymphoma, expressing CD3, CD5, CD4, CD8, CD10 antigens, without any expression of CD34 or CD79. Molecular analyses of the malignant T-cells showed a clonal TCR gamma-chain gene rearrangement together with HOXA10 overexpression. FISH analysis showed a MLL breakage. The partner gene, TET1, was identified using RP11-9E13 and RP11-314J18 BACs, corresponding to the recurrent translocation t(10;11)(q22;q23). MLL-TET1 fusion transcript was detected (intron 8 of TET1 fused to exon 8 of MLL), as well as its reciprocal transcript. The patient was treated following the (GELA) LL03 protocol, and was considered in complete remission after induction and consolidation phases. Fourteen months after the diagnosis, a bone marrow examination was performed for thrombopenia (6 G/L) which revealed a myelomonocytic acute leukemia with trilineage dysplasia. At this time, The MLL-TET1 fusion transcript as well as HOXA10 overexpression was still present but the TCR rearrangement was not detected. A non-familial donor allogeneic bone marrow transplant was performed in CR after intensive chemotherapy, that was complicated by a grade IV acute graft-versus-host disease. The patient died 54 days after the transplant of bacterial sepsis leading to multi-organ failure. MLL-TET1 fusions have been described in 13 cases in the literature, mainly in AMLs (11/13 cases, mostly AML M4 or M5 cases) and in B-ALLs (2/13 cases). The case reported here presents two interesting features. Firstly, this patient harbors the first MLL-TET1 fusion reported to date in T-ALL, and secondly this case presented a lymphoid to myeloid phenotype switch during the time course of the disease. This strongly suggests that the translocation occurred very early during hematopoietic differentiation, prior to the lymphoid/myeloid commitment. As described for the Ph1 chromosome rearrangement, the t(10;11) translocation may arise in hematopoietic stem cell rather than in committed progenitors. These features are also described in 8p11 stem cell syndrome that involves FGFR1. In both cases, genetic rearrangements arise in myeloid or lymphoid neoplasms with possible subsequent transformation. TET family enzymes convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) and play a key role in active DNA demethylation. TET1 and TET2 are also the key enzymes responsible for the presence of 5hmC in mouse embryonic stem cells (ESCs) (Koh KP., Cell Stem Cell 2011), and TET1 functions to regulate the lineage differentiation potential of ESCs. In addition to its role in DNA demethylation, TET1 interacts physically with NANOG and NANOG/TET1 co-occupy genomic loci of genes associated with both maintenance of pluripotency and lineage commitment in embryonic stem cells (Costa Y., Nature 2013). Taken together, these observations enlighten possible mechanism of the lineage switch observed in this case, and may be a rationale for using demethylating agents in MLL-TET1 neoplasms. Disclosures: No relevant conflicts of interest to declare. more...

STK, a new member of the hepatocyte growth factor receptor family, is the receptor for macrophage-stimulating protein (MSP), which acts on murine resident peritoneal macrophages. We established polyclonal and monoclonal antibodies against STK and characterized the structure of STK protein and STK expression on cells of the mononuclear phagocyte system. Western blotting showed that the STK transcript is translated into a single-chain precursor and then cleaved into a 165-kD disulfide- linked heterodimer composed of a 35-kD alpha-chain and a 144-kD beta- chain. Western blotting detected STK protein on resident peritoneal macrophages, a target of MSP, and showed that it was autophosphorylated in cells stimulated by MSP. By flow cytometric analysis using a monoclonal anti-STK antibody, we showed that STK protein is expressed on restricted macrophage populations such as resident peritoneal macrophages, but not on exudate peritoneal macrophages or mononuclear phagocytes of the bone marrow, peripheral blood, spleen, or alveoli. Resident peritoneal macrophages were classified into two fractions according to their reactivity with an anti-STK antibody and a marker antibody for macrophages: STKhigh-F4/80high cells and STKnegative- F4/80low cells. Acute exudative macrophages were all STKnegative- F4/80low, but they gradually became predominantly STKhigh-F4/80high several days after entrance into the peritoneal cavity. These results showed that after monocytes migrate into the peritoneal cavity, they undergo terminal differentiation in the peritoneal microenvironment. This is the first evidence of tissue-specific terminal differentiation of peritoneal macrophages, and this terminal differentiation can be characterized by the expression of STK receptor tyrosine kinase. more...

Abstract 4890 Gene amplification is a mechanism whereby a tumor cell can increase the copy number of specific gene sequences and gain a proliferative advantage. Although amplifications are common in solid tumors, they are relatively rare in hematological neoplasms. We report here on a patient with an acute erythroid leukemia and a jumping translocation of a hsr(11q) in a complex karyotype, without MLL amplification. The patient was 40 years old when he was admitted for asthenia, peripheral blood showing pancytopenia (Hb=111G/L; neutrophils= 0.23 G/L; platelets 92 G/L), without circulating blast cells. Bone marrow was hypercellular with the presence of more than 50% of erythroid precursors and more than 20% of myeloid blast cells in non-erythroid cell population, leading to the diagnosis of erythroblastic acute leukemia. A marked dysgranulopoiesis was also detected. Conventional cytogenetics showed a complex karyotype with a del(5q), a unbalanced t(7;17) leading to partial 7q deletion and homogeneously staining regions (hsr). Fluorescence in situ hybridization confirmed the del(5q), and showed that TP53 was not deleted in the t(7;17). Multi-FISH (M-FISH) pointed out that the hsr consisted of chromosome 11 and was implicated in 3 different translocations with 3 different partner chromosomes in 3 different clones. To further characterize the amplicon and determine which bands were implicated, we used a chromosome 11 m-band probe. It revealed that the bands implicated are the same on the der(3), der(12) and der(20) and are localized between 11q23.3 and 11q25. A series of BAC probes showed that different genes, present in these regions, were amplified: ETS1, FLI1, KCNJ5, NFRKB, SNX19, HNT, OPCM, but not MLL. Amplifications of chromosome 11q usually includes MLL, but more telomeric amplicons have also been reported in AML and myelodysplatic syndromes (MDS). A very close 11q amplification was identified in 3 AML/MDS cases (Crossen et al, 1999; Tyybäkinoja et al, 2006). Also, the ETS1 oncogene was found to be rearranged and 30-fold amplified in a case of acute myelomonocytic leukaemia, in which an hsr occurred on 11q23 (Rovigatti et al, 1986). The role of chromosomal amplifications in leukemia is unclear, but it has been suggested that they are associated with rapid disease progression and short survival. Jumping translocations are an unusual phenomenon and have been rarely reported in hematological malignancies. Whether jumping translocations play a role in tumor genesis or confer a selective growth advantage on tumor cells is unknown. The combination of hsr and jumping translocations as described here are very rare in hematological malignancies. The cases of the literature (Yoshida et al, 1999) and the one presented here suggest that the 11q24-q25 region may harbor new candidate oncogenes, together with unusual chromosomal mechanisms. Disclosures: No relevant conflicts of interest to declare. more...

Abstract 565FN2 Hyperdiploidy >50 chromosomes (HD>50) has long been recognized as favorable group in childhood B-ALL but there is still debate on the factors contributing to heterogeneity of prognosis observed within this entity. Better outcome has been reported for patients (pts) presenting DNA index (DI) >1.16 (Blood 1985;10:213), ≥56 chromosomes (Leukemia 1996;10:213), triple trisomies (TT) +4,+10,+17, double trisomies +4,+10 (Leukemia 2005;19:734) and trisomy 18 (Blood 2003;102:2756) but there is no consensus between the reports and these factors are differently applied in current protocols. We studied these factors in the pts with HD>50 enrolled in the ALL 58951 trial, BFM related. HD>50 were detected by cytogenetics (karyotype/FISH) and/or flow cytometry (DI). In order to analyze the outcome of HD>50 itself, pts with recurrent unfavorable translocations t(9;22), 11q23/MLL+, with t(1;19), t(12;21) or Down Syndrome were excluded, as well as near-triploidies/duplication of hypodiploidies 30–39 chromosomes. Pts were stratified into 4 risk groups (VLR/AR1/AR2/VHR) according to DI, Modal Number of Chromosomes (MNC), WBC, CNS/gonadal involvement, presence of VHR features (unfavorable translocations, poor response to prephase, residual disease (MRD) at the end of induction >10-2). VLR was defined as: DI>1.16 or MNC>50, WBC Overall Results: Out of 1651 B-lineage pts registered in the 58951 study over a 10-year period (1998-2008), a total of 541 pts had HD>50. Median age was 3 years and median WBC was 5.6×109/L. After prephase, 3% (N=17) were poor responders; initial risk group distribution into VLR/AR1/AR2/VHR was 45%/47%/5%/3%. After induction, 540 (99.8%) reached complete remission, 455 of whom had an MRD evaluation: MRD10-3 (N=26;6%) or MRD≥10-2 (N=13;2%). At median follow-up of 6 years, 48 pts (9%) relapsed, 22 pts (4%) died and 6 (1%) died without relapse. The 6-yr EFS was 89% (SE=1.5%). MNC was assessed in 446 pts (82%) with successful karyotypes, MNC ranged from 51 to 66 chromosomes and peaked at 55–56; 87 pts had 51 to 53 chromosomes (HD51-53), 258 pts had 54 to 57 (HD54-57) and 101 pts had ≥58 chromosomes (HD≥58). In these 3 groups, VLR regimen was given to 16%, 50% and 64% respectively. Structural abnormalities were detected in 46% of pts and associated with all MNCs. DI, assessed in 460 pts was Prognostic Factors: The only significant prognostic factors for EFS were MNC, DI, TT, DT and MRD. EFS was clearly improved (p10-2 and 2 pts: MRD≥10-3) and only 1 pt (MNC=58) relapsed. No specific profile of chromosome gains was identified in HD ≥58 since all chromosomes contributed to tri/tetrasomies, except chromosome 1. Likewise, the higher the DI the better the outcome (p=0.01): 6-yr EFS was 83% (DI TT were detected in 168 out of 468 pts, and DT in 242 out of 466 pts. There was no overlap with HD ≥58 since TT and DT were distributed from 52 to 66 chromosomes. One third of TT and of DT had MNC≥58. TT and DT were also of prognostic importance for outcome: the 6-yr EFS rate was 96% (TT) vs 86% (non-TT) (p=0.005) and 94% (DT) vs 84% (non-DT) (p=0.003). A hierarchical variable based on presence of HD≥58, TT or DT showed that HD ≥58 (N=101; 6-yr EFS: 99%) group had a better outcome than TT without HD≥58 (N=115; 6-yr EFS: 93%) and DT without HD≥58 and without +17 (N=55; 6-yr EFS: 84%) groups (p=0.04). We can infer from our results that the good outcome observed for all of the TT and DT was partially due to their association with HD '58. Consequently, the best indicator for excellent outcome was a high MNC (≥58 chromosomes). From our 58951 trial, we can assume that among children with B-ALL and HD>50, those with ≥58 chromosomes, stand every chance of being cured. Our results stress the necessity of karyotype for identifying them since this is the only way to assess MNC. They can also be detected (less accurately) by DI (DI ≥1.24). Therefore, both MNC and DI should be used for stratifying pts in the very low risk groups. Disclosures: No relevant conflicts of interest to declare. more...

Abstract 2708 RUNX1 is implicated in numerous chromosomal abnormalities acquired in acute myeloid leukemia (AML). The most frequent one, the t(8;21) is associated with a particular morphology together with a favorable prognosis. This is not the case for other 21q abnormalities, that are much less frequent and for which the prognosis is quite different. Moreover, beside point mutations, conventional cytogenetics failed to detect some of chromosomal alterations involving RUNX1. Recently 3 cases of the rare and semi-cryptic t(7;21)(p22;q22) translocation expressing the RUNX1-USP42 fusion transcripts have been reported, demonstrating the recurrence of this abnormality in AML. We describe here 3 additional cases with the same translocation and fusion transcripts, associated to 5q alterations leading to EGR1 and CSF1R heterozygous losses. In all our patients, the t(7;21)(p22.1;q22.3) was initially detected by the systematic FISH evaluation of the blastic populations using ETO-AML1 Dual Fusion probe. Patient#1 bone marrow karyotype was characterized by a tetraploid clone (89,XXYY) with loss of chromosomes 15, 17 and 18 in addition to the t(7;21), and a unbalanced translocation der(5)t(5;13)(q23;q?) between long arms of chromosomes 5 and 13, resulting in a heterozygous loss of EGR1 and CSF1R. Patient #2 blood and bone marrow karyotypes revealed a diploid clone with a del(5)(q31q33) associated with the t(7;21). The FISH analysis confirmed EGR1 and CSF1R deletions. In patient #3, the bone marrow karyotype showed diploid/tetraploïd clones, both harboring the t(7;21)(p22;q22), confirmed by FISH experiments (WCP7, AML1 probes). In addition, a der(5)t(1;5)(q3?2;q21-23) was identified within the tetraploïd clone, resulting in the loss of EGR1 and CSF1R, confirmed by FISH. In all three cases a RUNX1-USP42 fusion transcript was detected using RT-PCR, as well as the reciprocal transcript. Sequence analysis of RT-PCR products showed that the breakpoints occurred exactly in the same introns of USP42 and RUNX1 as in the previously described cases. For patient #1 and #3 a chimeric transcript was found formed of the RUNX1 exon 7 fused to the USP42 exon 3. In patient #2, a shorter chimeric transcript arised from the fusion of the RUNX1 exon 5 to the exon 3 of USP42. As already noticed in the previous reports, an alternative splicing of the RUNX1 exon 6 has been detected in these three cases. The description of these 3 novel t(7;21) confirm the recurrence of this balanced translocation in AML, and shows that this chromosomal abnormality is often associated with diploid/tetraploid clones and/or 5q alterations. Special attention should be paid in karyotype analysis of AML with diploid or tetraploid clones harboring 5q alterations. In such cases RUNX1 rearrangements should be explored using FISH analysis, and RUNX1-USP42 fusion transcript should be searched by RT-PCR in positive cases. Prospective and retrospective studies of AML have now to be settled in order to assess the incidence and clinical relevance of this cryptic translocation. Disclosures: No relevant conflicts of interest to declare. more...

Abstract 1121 Introduction: D variant is a common phenotype in Africans. However, the allele multiplicity challenges the serological methods which often fail to detect D variant patients in such populations. Recently, two cases of strong anti-D allo-immunization occurred in our blood bank in ccDee patients who received transfusions with Rh positive red blood cells. The D phenotype of these patients was determined using two different monoclonal and one polyclonal anti-D antibodies, with normal agglutination (4+) observed. Considering these results, we decided to investigate the prevalence of D variant for the ccDee patients of our cosmopolitan population. The aim of this study was to determine whether ccDee blood recipients should systematically undergo molecular screening for D variant prior to transfusion in order to avoid anti-D allo-immunization. Materiel and methods: Fresh EDTA blood from 35 ccDee patients (22 Africans and 13 Europeans) was analyzed using both serological and molecular methods. Serological characterization was performed using two monoclonal and one human polyclonal anti-D antibodies. In addition, each sample was tested using an extended partial RhD typing set from Diamed (consisting of 12 monoclonal antisera). Molecular determination was performed using two polymerase chain reactions with sequence-specific priming (PCR-SSP) kits intended to diagnose either partial D or weak D phenotypes (BAGene, BAG, Lich, Germany). Result: Among the 35 patients screened, discordant results between serological and molecular methods were found in 12. While serological tests revealed a normal D phenotype for these patients with all anti-D antibodies tested, molecular tests diagnosed them as D variant (nine were DAU; two were weak D types 4.0 and 4.1; one was DAR). As expected, the vast majority (9/12) of these patients were of African descent. No discordance between serological and molecular screening was observed for the rest of the screened population (23 patients). Conclusion: Our study reveals that approximately one-third (12/35) of our ccDee patients present a D variant genotype despite normal serological results. It emphasizes the fact that transfusion of Rh positive red blood cells to ccDee patients (mainly of African origin) requires precautionary measures. Molecular D variant screening in ccDee patients should be performed in order to improve the handling of transfusions in ccDee patients. Disclosures: No relevant conflicts of interest to declare. more...

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 4 (84% vs 5/20 (25%), p Comparison to common CLL showed that BCL2-CLLs had more tri12 (p Compared to BCL3-CLLs, BCL2-CLLs have a much less aggressive behavior, indicating that distinguishing the individual translocations and the cytogenetic partners would allow a better patients' stratification. Disclosures: No relevant conflicts of interest to declare. more...

Most chromosomal translocations in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) involve oncogenes which are either up-regulated or form part of new chimeric genes. The t(2;11)(p21;q23) translocation has been cloned in 19 cases of MDS and AML. In addition to this, we have shown that this translocation is responsible for a strong up-regulation of miR-125b (6 to 90 fold). In vitro experiments revealed that miR-125b was able to block monocytic and granulocytic differentiation of leukemic cells and primary CD34+ human blasts. Therefore, miR-125b up-regulation may represent a new mechanism of myeloid cell transformation and myeloid neoplasms carrying the t(2;11) translocation define a new clinico-pathological entity. more...

The diagnosis of mature B-cell neoplasms remains difficult in a number of cases, especially leukemic phases of non Hodgkin lymphomas, for which discriminating criteria or marker are often lacking. In order to identify new surface markers, we developed an original proteomic approach based on mass spectrometry analysis of plasma membrane microparticles derived from chronic B-cell lymphoproliferations: chronic lymphocytic leukemia/ small cell lymphoma (CLL/SLL) and mantle cell lymphoma (MCL). The approach consisted of protein extraction from plasma membrane microparticles (MPs), generated following actinomycin D stimulation and recovered using differential centrifugation. The complex protein mixture was further separated using 1D-gel electrophoresis separation and gel bands were systematically cutted at 2 mm intervals, trypsin digested and the resulting peptide mixtures was subsequently analysed by tandem mass spectrometry prior to protein identification. The lists of the proteins obtained for each pathology were then examined with respect to the membrane localization of the proteins in order to fulfill biological requirements needed for its further clinical use. Comparison of the lists of the proteins obtained for each pathology identified CD148, a membrane receptor with phosphatase activity, as a candidate for a discriminating marker detected in MCL but not in CLL in this approach. Flow cytometry studies, performed on 55 patients and 10 controls, showed that an anti-CD148 antibody stained significantly higher MCL (n=22) than CLL/SLL (n=33) circulating cells (p Figure Figure more...

Conventional cytogenetics (CC) of B-cell lymphoproliferations remains difficult because of low mitotic in vitro activity of the leukemic cells. Therefore, mitogen stimulation of B-cells is required to analyze an adequate number of metaphases. Chromosome abnormalities using CC with TPA can be detected in up to 50% of chronic lymphocytic leukemia (CLL), but the development of FISH techniques has allowed the detection of selected chromosome abnormalities in more than 80% of CLL. However, FISH is restricted, since information is only available for the genes/loci for which probes are used. So, for a comprehensive genetic analysis, CC is essential because it provides an overview of all microscopically visible chromosome abnormalities important as prognostic factors. The use of the immunostimulatory CpG-oligonucleotide DSP30 to effectively induce cell cycle progression of CLL cells in vitro has been reported. This proliferation is markedly enhanced upon addition of Interleukine-2 to cultures. To our knowledge and to date, there has been no direct comparison of classical TPA versus DSP30+IL-2. DSP30+IL-2 stimulation has been successfully tested in CLL but no data are available for other lymphoproliferations. We cultured 132 B-cell lymphoproliferations (80 CLL and 52 other B-cell lymphoid neoplasms (BCLN)) in parallel, in presence of TPA or DSP30+IL-2. The objective of this study was to evaluate the suitability of DSP30+IL-2 as a routine B-cell mitogen for metaphase cytogenetics. CC successfully analyzed 94.9% of CLL and 98.1% of BCNL with more than 80% abnormal karyotypes. For CLL, failures of karyotypes were more frequent in cultures with DSP30+IL-2 (14%) than in those with TPA (4%). The rate of failures were similar for BCLN (6% versus 4%). For CLL, there were significantly fewer metaphases in DSP30+IL-2 than in TPA spreads (mean of 50 versus 72 metaphases per slide respectively, p=0.0007), as well as for BCLN (mean of 50 versus 71 metaphases per slide respectively, p=0.009). However, the proportion of abnormal metaphases was significantly higher in DSP30+IL-2 (mean of 59%) compared to TPA cultures (mean of 26%, p=0.00265) for CLL and for BCLN (mean of 57% versus 33%, p=0.0065). Stimulation with DSP30+IL-2 allowed to detect more abnormalities, more abnormal subclones and more complex karyotypes in CLL and in the majority of BCLN. Though FISH exploration using a large probe panel has yielded valuable results in lymphoproliferative diseases, it underestimates the heterogeneity of chromosomal aberrations. Complexity of chromosomal changes, recently associated to unfavorable outcomes, can only be assessed with CC. In conclusion, our results in both CLL and BCLN indicate that the immunostimulatory oligonucleotide DSP30 in combination with IL-2 is an easy and efficient stimulus in metaphase generation for routine chromosomal banding. more...

Loss of the Y chromosome (-Y) in bone marrow cells frequently occurs in elderly patients. It is thus unclear whether loss of the Y chromosome, when found in Philadelphiapositive cells, should be overlooked, or whether it represents a clonal evolution (a criterion of cytogenetic acceleration of the disease) or an additional cytogenetic abnormality (ACA), considered of little importance in patients treated with imatinib mesylate (IM). We have collected diagnostic characteristics, clinical, cytogenetic and molecular evolution data from 29 patients diagnosed with CML from 1991 to April 2007 in 6 participating centres, who presented a loss of the Y chromosome at diagnosis (n=20) or during evolution (n=9) and who were treated with imatinib in first (n=19) or second (n=10) line. They were compared to patients with a Philadelphia chromosome as sole anomaly, diagnosed at similar dates in the same centres, thus benefitting from the same management. Age and Sokal score at diagnosis were similar. In both -Y and control groups, two thirds of patients had received prior treatment (mostly interferon, hydroxyurea and aracytine). Median follow-ups were respectively 54 months (range: 7–146) and 58 months (range: 13–149), censored if therapy was changed for a second generation tyrosine-kinase inhibitor or bone marrow transplantation. The time to achieve complete cytogenetic remission (CCR) (22 months vs 7, p=0.02) and major molecular response (MMR) (not reached vs 30 months, p=0.0015) were significantly higher in -Y patients. Acceleration (8/29 vs 1/29, p=0.003), acutisation (4/29 vs 1/29, p=0.1) and death (3/29 vs 1/29) were more frequent in the -Y group, but owing to the rare occurrence of these events, statistical significance was clear for acceleration only. Interestingly, prognosis (time to CCR, MMR and occurrence of acceleration) was worse if loss of the Y chromosome was present in a subclone (suggesting a clonal evolution) rather than all mitoses (ACA) and also when it was observed at diagnosis rather than during the evolution of the disease. Consequently, survival was significantly reduced (55 months vs not reached, p=0.009) in those patients for whom -Y was present in a subclone at diagnosis. In conclusion, loss of the Y chromosome in Philadelphia-positive cells is an important finding since it affects the prognosis of CML patients, especially, but not exclusively, when present at diagnosis in a subclone. In this case, loss of the Y chromosome even predicts a decreased survival after imatinib treatment. more...

T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive malignancy of thymocytes that accounts for about 15 percent of ALL cases. Leukemic transformation of immature thymocytes is caused by a multistep pathogenesis involving numerous genetic abnormalities providing uncontrolled cell growth. Accumulating evidence suggests the presence of at least 5 different molecular-cytogenetic subgroups in T-ALL, ie. TAL/LMO , TLX1 , TLX3 , HOXA and MYB . Recently, non coding microRNAs were discovered as important regulators of gene and/or protein expression and subsequently shown to be directly implicated in cancer. Nevertheless, it is currently unclear in which way deregulated miRNA expression may contribute to the pathogenesis of T-cell acute leukemia. In this study, we investigated whether different genetic subgroups in T-ALL are characterized by distinct miRNA expression patterns. Therefore, we profiled a total of 360 miRNAs through automated qRT-PCR using high-throughput quantitative stem-loop RT-PCR in a genetically well characterized T-ALL patient cohort (n=52), including 11 HOXA (3 MLL rearranged, 5 inv(7)(p15q35) and 3 CALM - AF10 ), 16 TAL/LMO (7 LMO2 rearranged, 8 TAL1 rearranged, 1 LMO2/TAL1 rearranged), 11 TLX3 and 5 TLX1 rearranged patient samples. Since T-ALL blasts originate from maturating T lymphocytes, we also profiled different subsets of sorted T-cell populations (CD34 + , CD4 + /CD8 + /CD3 − , CD4 + /CD8 + /CD3 + , CD4 + SP and CD8 + SP). These miRNA profiles of normal T-cells served as a negative control for the identification of deregulated miRNA expression that may be truly leukemia associated. SAM analysis (t-test and wilcoxon, FDR=0) identified significant and differentially expressed miRNAs between the HOXA, TLX3 and TAL/LMO subgroups. No significant and differentially expressed miRNAs were obtained for the TLX1 subgroup, probably due to the limited number of patient samples. The HOXA subgroup showed specific up-regulation of miR-196a and miR-196b , which are encoded at the HOXB and HOXA cluster, respectively, but no significantly down-regulated miRNAs could be identified in this subgroup. The TLX3 subgroup was uniquely characterized by the up-regulation of miR-99a , miR-125b , let-7c , miR-508 and miR-509 , and down-regulation of miR-127 and miR-182 . Finally, specific up-regulation of miR-424 , miR-148a , miR-422 , miR-362 , miR-148a , miR-502 , miR-10a , miR-200c , miR-31 , miR-660 and miR-15b , was identified in the TAL/LMO rearranged subgroup, which was also characterized by the specific down-regulation of miR-99b , miR-155 , miR-125a , miR-153 , miR-135a , miR-34a and miR-193b . Next, we evaluated the expression pattern of all significant and differentially expressed miRNAs in the different subsets of sorted T-cell populations. The expression patterns of these miRNAs could be classified into consistently active, completely absent or temporally regulated during T-cell development. For the miRNAs showing a temporal regulation during T-cell maturation, their differential expression in T-ALL subtypes may reflect differences in the maturation arrest of the T-cell of origin, rather than pointing to an oncogenic event. Nevertheless, their constitutive (in)activation in primary T-ALL patients could still be of oncogenic relevance, similar to transcription factors like TAL1 or LMO2 which also show a temporal regulation during T-cell maturation. In contrast, some other miRNAs showed no expression in any of the T-cell populations, providing stronger evidence that their activation in specific T-ALL subtypes may contribute to T-ALL pathogenesis. In conclusion, this study shows that molecular-cytogenetic subgroups in T-ALL are characterized by a specific miRNA expression signature. In addition, correlation of our findings to the expression of these miRNAs in normal T-cell subsets may guide us to the miRNAs with true oncogenic potential. This report paves the way for further investigation directed at the role of these miRNAs in the pathogenesis of T-ALL, which may provide us with further insight in the oncogenic pathways that are (in)activated in different T-ALL subgroups. Ultimately, these deregulated miRNAs may offer new targets for therapeutic intervention. more...

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. more...