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CD34 positivity has been considered as an adverse prognostic factor in acute myeloid leukemia (AML). Although nucleophosmin 1-mutated (NPM1m) AML is usually CD34 negative, this marker may be expressed at diagnosis or acquired at relapse in a variable number of cases. Our objective was to ascertain if CD34 expression has any influence on the general outcome of this form of acute leukemia. Analysis of clinical outcome (complete remissions, relapses, disease-free survival, and overall survival) was performed depending on the degree of expression of CD34 determined by flow cytometry, in 67 adult patients with NPM1m AML. CD34 expression did not have any influence on the variables analyzed whatever the percentage of blasts expressing this marker. In contrast to other forms of AML, CD34 expression is not an unfavorable prognostic factor in NPM1m AML, neither at diagnosis nor at relapse.

BACKGROUND Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL). METHODS This study analyzed via multiplex ligation–dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL. RESULTS The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)–positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively). CONCLUSIONS Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL. Cancer 2015;121:3809–3817. © 2015 American Cancer Society.

Objective:To characterize the effect of the HIV-1 infection and antiretroviral treatment (ART) in the human memory B (MEB)-cell compartment.Design:A cross-sectional study was designed to analyze MEB cells of HIV-1(+) ART treated and ART-naive study participants, and uninfected individuals.Methods:Frequency and absolute counts of MEB cell subsets in blood were determined by multicolor flow cytometry. Spontaneous cell death and B-cell proliferative capacity was evaluated in vitro by cell culture and flow cytometry. Splenic function was determined by pitted erythrocytes quantification in HIV-1(+) ART-treated study participants.Results:HIV-1(+) ART-treated individuals did not show functional hyposplenism despite the lack of recovery IgM(+)IgD(+)CD27(+) marginal zone-like B cells. Moreover, two germinal center-dependent MEB cells subsets were also dysregulated in HIV-1(+) individuals: IgM(+)IgD(-)CD27(+) (IgM only) cells were increased, whereas the switched subset (IgM(-)IgD(-)) was reduced in viremic individuals. Althought ART restored the numbers of these populations; the switched MEB cells were enriched in CD27(-) cells, which showed the highest susceptibility to spontaneous cell death ex vivo. In addition, B cells from viremic individuals showed a poor response to B-cell receptor and toll-like receptor 9 stimulation that was circumvented when both stimuli were used simultaneously.Conclusion:B cells from HIV-1(+) study participants show a poor stimulation capacity, that may be bypassed by the proper combination of stimuli, and a dysregulated MEB cell pool that suggest an affectation of the germinal center reaction, only partially normalized by ART. Interestingly, hyposplenism does not explain the lack of recovery of the marginal zone-like B cells in ART-treated HIV-1(+) individuals.

Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients does not achieve the optimal response or are resistant to TKI. ABL1 kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Although deletion or insertion of nucleotides in BCR-ABL1 has rarely been described, we identified a CML patient with an already described 35 nucleotides insertion (BCR-ABL1(35INS)) of controversial significance, that confers resistance to imatinib but sensitivity to dasatinib.

Acute myeloid leukemia (AML) is a heterogeneous group of hematological neoplasms with distinct clinical and genetic features. Chromosomal abnormalities are detectable by conventional cytogenetic an...

It is unclear whether higher CD34 + cell doses infused for ASCT have any influence on survival or relapse in patients with lymphoma. We analyzed the correlation of infused CD34 + cell dose with relapse, survival, and hematopoietic recovery in 146 consecutive patients undergoing ASCT for lymphoma. Higher doses (>5 × 106/kg) were significantly correlated with earlier hematopoietic recovery, fewer infectious episodes, lower transfusion needs. No differences were observed in lymphoma outcomes (4-year relapse incidence of 38% [95%CI: 29%–48%] in the lower dose group versus 51% [95%CI: 30%–69%] in the higher dose group, 10-year OS probabilities of 58% [95%CI: 48%–68%] versus 75% [95%CI: 59%–91%], 10-year DFS probabilities of 47% [95%CI: 37%–57%] versus 42% [95%CI: 23%–61%], p = NS for all outcomes). In this series, a higher infused CD34 + cell dose did not correlate with survival or relapse but correlated with earlier hematopoietic recovery and lower resource consumption.

Strategies to improve the efficiency of bronchoalveolar lavage (BAL) are needed. We conducted a study to establish the diagnostic value of BAL in patients with hematological malignancies and pulmonary infiltrates.The correlation of cytologic and flow cytometric study of BAL with the microbiological findings and the clinical evolution was determined.Seventy BAL were performed and flow cytometric study was analyzed in 23 of them. Fifty-three patients did not present any adverse event attributable to BAL. Anti-infectious therapy was modified in 64 (91%) patients. T lymphocyte count0.3×10Our study confirms the clinical value of BAL for treatment decision making in patients with hematological malignancies and acute respiratory failure.

// Laura Palomo 1, 2 , Olga Garcia 3 , Montse Arnan 4 , Blanca Xicoy 3 , Francisco Fuster 1 , Marta Cabezon 3 , Rosa Coll 5 , Vera Adema 1 , Javier Grau 3 , Maria-Jose Jimenez 3 , Helena Pomares 4 , Silvia Marce 3 , Mar Mallo 1 , Fuensanta Milla 3 , Esther Alonso 4 , Anna Sureda 4 , David Gallardo 5 , Evarist Feliu 3 , Josep-Maria Ribera 3 , Francesc Sole 1 , Lurdes Zamora 3 1 MDS Research Group, Josep Carreras Leukaemia Research Institute, ICO-Hospital Germans Trias i Pujol, Universitat Autonoma de Barcelona, Badalona, Spain 2 Departament de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, Badalona, Spain 3 Hematology Service, ICO-Hospital Germans Trias i Pujol, Josep Carreras Leukaemia Research Institute, Universitat Autonoma de Barcelona, Badalona, Spain 4 Hematology Service, ICO-Hospital Duran i Reynals, Barcelona, Spain 5 Hematology Service, ICO-Hospital Josep Trueta, Girona, Spain Correspondence to: Lurdes Zamora, email: lzamora@iconcologia.net Keywords: chronic myelomonocytic leukemia, normal karyotype, gene mutations, targeted deep sequencing, prognostic factors Received: May 20, 2016 Accepted: July 01, 2016 Published: July 29, 2016 ABSTRACT Clonal cytogenetic abnormalities are found in 20-30% of patients with chronic myelomonocytic leukemia (CMML), while gene mutations are present in >90% of cases. Patients with low risk cytogenetic features account for 80% of CMML cases and often fall into the low risk categories of CMML prognostic scoring systems, but the outcome differs considerably among them. We performed targeted deep sequencing of 83 myeloid-related genes in 56 CMML patients with low risk cytogenetic features or uninformative conventional cytogenetics (CC) at diagnosis, with the aim to identify the genetic characteristics of patients with a more aggressive disease. Targeted sequencing was also performed in a subset of these patients at time of acute myeloid leukemia (AML) transformation. Overall, 98% of patients harbored at least one mutation. Mutations in cell signaling genes were acquired at time of AML progression. Mutations in ASXL1 , EZH2 and NRAS correlated with higher risk features and shorter overall survival (OS) and progression free survival (PFS). Patients with SRSF2 mutations associated with poorer OS, while absence of TET2 mutations ( TET2 wt) was predictive of shorter PFS. A decrease in OS and PFS was observed as the number of adverse risk gene mutations ( ASXL1 , EZH2, NRAS and SRSF2 ) increased. On multivariate analyses, CMML-specific scoring system (CPSS) and presence of adverse risk gene mutations remained significant for OS, while CPSS and TET2wt were predictive of PFS. These results confirm that mutation analysis can add prognostic value to patients with CMML and low risk cytogenetic features or uninformative CC.

Some copy number alterations (CNAs) have independent prognostic significance for adults with acute lymphoblastic leukemia (ALL).This study analyzed via multiplex ligation-dependent probe amplification the frequency and prognostic impact of CNAs of 12 genetic regions in 142 adolescents and adults with de novo precursor B-cell ALL.The cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) deletion (59 of 142 or 42%) was the most frequent CNA, and it was followed by Ikaros family zinc finger 1 (IKZF1) losses (49 of 142 or 35%). IKZF1 deletions were more prevalent in Philadelphia chromosome (Ph)-positive ALL and were associated with advanced age and high white blood cell (WBC) counts. The multivariate analysis showed that advanced age and early B-cell factor 1 (EBF1) deletions were associated with chemotherapy resistance in both the whole series (hazard ratios, 0.949 and 0.135, respectively) and the Ph-negative subgroup (hazard ratios, 0.946 and 0.118, respectively). High WBC counts and focal IKZF1 deletions correlated with disease recurrence (hazard ratios, 1.005 and 1.869, respectively), whereas advanced age and CDKN2A/B losses influenced overall survival in both the whole series (hazard ratios, 1.038 and 2.545, respectively) and the Ph-negative subgroup (hazard ratios, 1.044 and 2.105, respectively).Deletions of EBF1, IKZF1, and CDKN2A/B have an independent adverse prognosis for adolescents and adults with B-precursor ALL, and this suggests that these CNAs should be included in the initial risk assessment of ALL.

In December 2013 mutations were described in the calreticulin (CALR) gene in 67–71% and 56–88% of patients with JAK2 V617F and MPL negative essential thrombocythemia (ET) and primary myelofibrosis ...

Objectives: To compare, from a biological and clinical perspective, a significant group of patients with AML with inv(3)(q21q26.2) or t(3;3)(q21;q26.2) with another group of AML carrying different abnormalities of 3q at q21 or q26, the latter named as the AML abn(3q) group. Methods: We developed a national survey with the participation of 13 Spanish hospitals, and retrospectively reviewed (from 1990 to 2010) these subtypes of AML. Fifty-five patients were collected: 35 with AML inv(3)/t(3;3) and 20 with AML abn(3q). A data collecting page that included main features at diagnosis, therapeutic approach and response, and survival variables, was distributed and completed. Results: We did not find significant differences in sex, age, history of myelodysplastic syndrome or chemo-/radiotherapy, clinical presentation, WBC and platelet counts, hemoglobin level, blasts immunophenotype, serum lactatedehydrogenase, peripheral blood and bone marrow cellular dysplasia, and bone marrow biopsy findings. Although the association with monosomy 7 was significantly more frequent in AML inv(3)/t(3;3), this did not seem to influence outcome. The lack of response to the different modalities of treatment and the aggressive course of the disease were the standard in both cohorts of patients. Discussion: Although not yet recognized by the World Health Organization classification, our results are in agreement with the findings of other authors, who include both subsets of AML together in the same group of adverse prognosis. Conclusion: In an attempt to simplify and bound entities with similar genetic background and clinical behavior, it would be desirable to bring together both subgroups of AML in a single section.

Background & Objective: Acute Lymphoblastic Leukemia (ALL) is an aggressive neoplasia characterized by a high genetic heterogeneity both at diagnosis and at relapse. Due to the high incidence of relapse in adults and the dismal prognosis beyond recurrence, diagnosis and relapse samples of adult ALL patients were carefully analyzed in order to identify genetic alterations related with drug resistance and disease progression. Patients & Methods: Paired diagnosis-relapse bone marrow samples from 5 adult B-cell precursor ALL (B-ALL) patients were analyzed (Ph+ ALL [n=2], normal karyotype [n=1], t(1;19)(q23;p13) [n=1] and t(8;13)(p21-22;q12) [n=1]). Copy Number Alterations (CNA) were studied with Multiplex Ligation-dependent Probe Amplification (MLPA, kits P-335 and P-202 from MRC-Holland, Amsterdam, Netherlands) and Affymetrix CytoScan HD arrays (Affymetrix, Santa Clara, USA). In the array analyses, only the CNA that encompassed at least 25 markers were considered significant. Results: Regarding karyotype, 2 patients (1 Ph+ and 1 t(1;19) at diagnosis) showed the same chromosomal translocations within a complex karyotype at relapse. On the contrary, the other Ph+ patient showed a normal karyotype at relapse, while 2 patients did not experience any karyotypic change. Regarding immunophenotype, 3/5 patients showed changes on antigen expression from diagnosis to relapse such as expression of markers of immaturity (CD34, TdT positivity and CD38 negativity), loss of lymphoid markers (CD20 and CD22) and/or acquisition of myeloid markers (CD33 and CD66c). Concerning CNA, all relapse samples were genetically related to the diagnosis clone (common clonal origin). All relapsed populations lost CNA detected at diagnosis and/or acquired new CNA but retained some of the CNA showed at diagnosis revealing clonal evolution from ancestral clones. CNA in B-ALL key genes involved in lymphoid development (IKZF1, PAX5, EBF1,VPREB1 and BLNK), proliferation (CDKN2A/B, RB1, CRLF2, C-MYC and ERG), apoptosis (BTG1, TP53 and ATM), hematopoiesis transcription factors (ETV6 and MLL) and histone modifications (KDM6A) were detected, among others. Losses in 9p were the most recurrent event both at diagnosis and at relapse. CDKN2A/B deletions were observed in all relapse samples (3/5 in homozygosis) while PAX5 deletions were present in 4/5 relapsed cases. Interestingly, all relapse samples showed CNA favoring the activation and/or the transcription of proteins involved in the Akt/C-MYC signaling pathway. Another common feature (4/5 patients) were CNA affecting genes involved in drug transport such as several ABC transporter genes and genes related to drug resistance such as PRKDC and RUNX1T1 (in 3/4 of the cases, the CNA appeared exclusively at relapse or were already present at diagnosis and increased their frequency at relapse). CNA in genes that may confer stem cell characteristics (EGR1 and USP16) were another recurrent event at relapse (3/5 samples, 2 of them were not present at diagnosis). CNA affecting the X/Y PAR1 region (CRLF2, CSF2RA and IL3RA) or VPREB1 at 22q11.22 were detected in 3/5 relapse samples, respectively. An important apoptosis cluster at 11q21q24.2 (BIRC2/3, CASP1/4/5/12, hsa-miR-34b/c, ATM and BTG4) was lost in 2/5 relapse samples (one of them was not detected at diagnosis and the other increased its frequency at relapse). Finally, ETV6 deletion (12p13.2) and duplication of Xq26.2q28 (containing ABCD1, BCAP31 and genes coding for several cancer/testis antigens) were observed in 2 relapse samples. Conclusions: SNP arrays analysis of paired B-cell precursor ALL samples at diagnosis and at relapse allows the identification of genetic alterations potentially related with ALL progression. The systematic analysis of relapse samples could contribute to the identification of specific genetic targets with potential therapeutic impact for each patient (personalized medicine). Disclosures Martínez-López: Novartis: Honoraria, Speakers Bureau. Sole:Celgene: Membership on an entity's Board of Directors or advisory committees.

INTRODUCTION: The myelodysplastic syndromes (MDS) are a group of hematologic disorders characterized by ineffective hematopoiesis and increased risk of transformation to acute myeloid leukemia (AML). Aberrant DNA methylation is the dominant and most well-studied epigenetic alteration in MDS. Various genes, including cell cycle regulators, apoptotic genes, and DNA repair genes, are epigenetically silenced and play a role in pathogenesis and transformation to leukemia. The clinical response of MDS and AML to drugs that revert the aberrant hypermethylation, such as 5-aza-2x-deoxicitidine and 5-azacitidine (AZA), suggests that this hypermethylation could have an important role in the disease and it is not a secondary effect to other mechanisms. The aim of this study was to define the methylation pattern of DNA at diagnosis in patients with high-risk MDS and secondary AML to determine if there are some pattern predictive for response to AZA treatment or relapse. MATERIAL AND METHODS: Genomic DNA was obtained from bone marrow (n=100): 39 patients at diagnosis prior AZA treatment (24 MDS, 10 AML and 5 CMML), 10 control samples (peripheral blood of donors of bone marrow stimulated with hematologic growth factors) and 51 samples at different moments of follow-up (3 months, 6 months, 12 months and/or 18 months). Genome-wide DNA methylation profiling was performed using the Illumina Infinium 450K methylation array. We have used the IWG-2006 criteria for classifying the responders versus non responders. RESULTS: We have focused on MDS patients treated with AZA comparing methylation profiles at diagnosis and at the time of first detected response to treatment (mostly at 3 months). We performed different experiments of DNA methylation assays using RnBeads tool. The global methylation dendogram (Fig.1) clearly demonstrate that the subgroup of responders (7 patients) show strong demethylation after the treatment, while in non-responders methylation profiles has not changed that dramatically even after 6 months from the beginning of the treatment. We also have found several hundreds differentially methylated regions (DMR) at gene promoters, gene bodies and other genomic locations, which are significantly hypomethylated after the treatment in responders, while remain almost unchanged after the treatment in non responders. CONCLUSIONS: Illumina Infinium 450K methylation array serves as an informative methodology to study the methylation changes in bone marrow samples. A more detailed investigation of obtained DMR might shed light on the mechanisms of the response to AZA treatment and as result to allow detection of methylation markers of response to treatment or relapse. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Josep Carreras, Obra Social "La Caixa" and Celgene Spain. Diana Domínguez for her excellence technical assistance. Figure 1. Bi-clustering of the methylation levels of gene promoters in responders (green: diagnosis and orange: response) Figure 1. Bi-clustering of the methylation levels of gene promoters in responders (green: diagnosis and orange: response) Disclosures Valcárcel: Celgene Corporation: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; GlaxoSmithKline: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau.

INTRODUCTION: Several mutations have been described in patients with BCR-ABL1 –negative chronic myeloproliferative neoplasms, including primary myelofibrosis (PMF). The most frequent mutation is JAK2 V617F, followed by calreticulin exon 9 (CALR), MPL exon 10 and ASXL1 exon 12. Currently, less than 10% of patients lack molecular marker. CALR and ASXL1 mutations have been consistently found to have favorable and unfavorable prognostic implications, respectively. The aim of this study was to describe the frequency and prognostic impact of JAK2 V617F, CALR, MPL and ASXL1 mutations in patients diagnosed with PMF in 6 Spanish hospitals. METHODS: To detect the presence of JAK2 V617F mutation, an allele-specific PCR using TaqMan probes was used. Screening for insertions and deletions in CALR gene was performed with 6-FAM labeled primers spanning exon 9 and CALR mutations were described by Sanger sequencing. Sanger sequencing was also used to detect MPL exon 10 and ASXL1 exon 12 mutations. RESULTS: Sixty-eight patients were included in the study. All of them were screened for JAK2 and CALR mutations. Forty-five of them (66%) were positive for the JAK2 V617F mutation, while 11/68 (16%) were positive for CALR mutations. Of the 11 CALR mutations, 10 were JAK2 wild-type. MPL exon 10 mutation analysis was only performed in JAK2 wild-type patients and was positive in 4/23 patients (17%), and all of them were CALR wild-type. At the time of submission, ASXL1 exon 12 has been assessed in 18 patients (analysis in the rest of them is currently ongoing). ASXL1 mutations have been found in 3/18 (17%) patients, two of them also with a CALR mutation and the other one with the JAK2 V617F mutation. All three cases were indel mutations. Overall, no mutation was detected in 9/68 (13%) patients. JAK2 V617F, CALR and MPL mutations had no prognostic impact on overall survival. The effect of ASXL1 mutation on prognosis (with and without CALR mutation) will be assessed once all samples have been sequenced. CONCLUSION: JAK2 V617F, CALR and MPL mutations were found in our series of PMF patients in the same proportion found in larger series. ASXL1 has so far been found in a smaller percentage but the entire series of patients will need to be sequenced before reaching definitive conclusions. Studying these genes, only 13% of patients with PMF did not have a clonal marker. None of the studied mutations had prognostic significance. ACKNOWLEDGMENTS: The authors would like to thank Diana Dominguez for her excellent technical assistance and to the grant 2014 SGR225 (GRE), Generalitat de Catalunya. Disclosures No relevant conflicts of interest to declare.

Introduction Recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of ALL have been identified in genes involved in B-cell development, cell cycle regulation, proliferation, apoptosis and drug resistance. Their independent prognostic significance in adult ALL patients is controversial. The aim of this study was to analyze the prognostic significance of CNA in a series of 96 high-risk, Ph-negative, B-precursor adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods MLPA assays (MRC-Holland) were performed for the following genes: IKZF1, IKZF2, IKZF3, EBF1, CDKN2A/B, PAX5, ETV6, BTG1, RB1, hsa-miR-31, X/Y PAR1 region genes (CRLF2, CSF2RA, IL3RA) and 14q32.33 region genes (IGH D, MTA1, KIAA0284) . Fragment analysis was made by Genescan in an ABI-3130 sequencer (Applied Biosystems). Data normalization provided a value indicative of the presence or absence of CNA: 0-0.20 homozygous deletion, 0.21-0.70 heterozygous deletion, 0.71-1.30 normal, 1.31-1.70 heterozygous duplication and 1.71-2.20 homozygous duplication. Univariable and multivariable analyses including the most relevant clinical parameters (age, WBC count, phenotype, cytogenetics, CNS involvement) were performed for CR attainment, CR duration and OS. Results The median age [range] of the 96 patients was 39 [15-72] years, 50 (52%) patients were males, with a median WBC count 14.3 x109/L [0.4-388]. Phenotype: early pre-B 19 (20%), common 51 (54%), pre-B 22 (24%), unknown 2 (2%). Cytogenetics: normal 18 (19%), hyperdiploid 5 (5%), hypodiploid 2 (2%), near haploid 6 (6%), t(1;19) 7 (8%), 11q23/ MLL 11 (12%), complex 1 (1%), other 27 (29%), no growth 17 (18%). The most frequent CNA deletions involved CDKN2A /B (43/96, 45%), PAX5 (34/94, 36%), IKZF1 (32/95, 34%), hsa-miR-31 (25/96, 26%), 14q32.33 region (18/96, 19%), RB1 (17/96, 18%), EBF1 (12/91, 13%) and X/Y PAR (10/96, 10%). The most frequent duplications involved X/Y PAR (11/96, 12%) and 14q32.33 region (7/96, 7%). The CR rate was 83% (80/96), the median (95%CI) of CR duration was 2.7 years (0-5.9) and the median (95%CI) of OS was 2.1 (1.0-3.2), being the median (range) follow-up of the series of 3.8 (0.6-8.0) years. Table 1 shows the results of univariable and multivariable analyses. By multivariable analyses advanced age and EBF1 deletions were significantly associated with less CR rate, WBC count and X/Y PAR duplication were associated with shorter CR duration, and advanced age and CDKN2A /B deletion were associated with shorter OS. Conclusions The CNA of EBF1 , X/Y PAR1 genes and CDKN2A/B have independent prognostic significance in adult patients with high-risk, Ph-negative, B-precursor ALL. This study suggests that these genetic studies should be added to the initial work-up of these patients for more accurate prognostic assessment Supported by grants PI10/01417, RD12-0036-0029 from Instituto Carlos III, 2014 SGR225 (GRE) from Generalitat de Catalunya and a grant from the Spanish Society of Hematology and Hemotherapy (2012). | Variable | CR rate | CR duration | OS | | ---------- | -------- | ------------------ | -------- | ------------------- | | | P (univ) | OR (95%CI) | P (univ) | HR (95%CI) | P (univ) | HR (95%CI) | | Age | 0.011 | 0.93 (0.89 - 0.98) | NS | - | 0.005 | 1.03 (1.01 - 1.05) | | WBC | NS | -

Background: Clinical, morphological and genetic characteristics of chronic myelomonocytic leukemia (CMML) are heterogeneous and vary from a myelodysplastic predominant profile to a myeloproliferative one. CMML has a highly variable course, with a median overall survival (OS) of 20 months and 15-30% of progression to acute myeloid leukemia (AML). Cytogenetic abnormalities are present in only 20-40% of cases. CMML-specific cytogenetic risk classification stratifies karyotypes into three groups: low risk (normal karyotype and isolated loss of Y chromosome, -Y), poor risk (trisomy 8, monosomy 7, 7q deletion and complex karyotype) and intermediate risk (all other chromosomal abnormalities). According to this model, 65-85% of patients fall into the low risk cytogenetic category. The aim of this study was to characterize type, frequency and prognostic impact of cytogenetic alterations detected by SNP arrays (SNP-A) in a series of 128 patients with CMML and low risk cytogenetic features or no metaphases. Methods: A retrospective study was performed on 128 patients with CMML. Cases with normal karyotype (n=120), isolated -Y (n=4) and no metaphases (n=4) were selected. Median age at diagnosis was 73 years (range 39-98), there was a 2.4:1 male predominance and 22.4% (28/125) of cases progressed to AML. Median follow up of patients was 26 months (range 1-115). Morphological WHO subtypes were CMML-1 in 104 (81%) cases and CMML-2 in 24 (19%). According to the FAB criteria 82 (64%) cases were included in the myelodysplastic variant (CMML-MD) and 46 (36%) in the myeloproliferative one (CMML-MP). High density SNP-A (Cytoscan HD, Affymetrix) were performed using DNA extracted from bone marrow (n=124) or peripheral blood (n=4) samples at diagnosis. The statistical analysis was performed with SPSS. Kaplan-Meier method was used for OS and progression-free survival (PFS) analysis and log-rank test was used for comparisons between groups. Results: SNP-A revealed novel chromosomal alterations (copy number alterations, CNA, and loss of heterozygosity, LOH) in 66% (85/128) of cases. Among the abnormal cases (CNA plus LOH), 1 alteration was detected in 65% (55/85) of cases, 2 in 20% (17/85) and ≥3 in 15% (13/85). The median size of the affected genome for CNA and LOH was 759 Kb (range 0-142Mb). CNA were detected in 38% (48/128) of cases, most of them being gains and losses smaller than 10Mb. Only 7 CNA were larger than 10Mb, four of them corresponded to patients with isolated -Y and three were novel alterations that had not been detected by conventional G-banding cytogenetics. Most affected regions (detected in 5 cases) were gains in chromosomes 3q, 8p and 21q as well as losses in chromosomes 10q and 12p. LOH were detected in 39% (50/128) of patients. Interstitial LOH larger than >25Mb were detected in 30% (39/128) of cases. Recurrent interstitial LOH were detected in: 4q24-4q35 region (12 cases), involving TET2 gene, which is mutated in 40-50% of CMML; and in chromosome 11 (9 cases), 7 of which included 11q13.3-11q25 region, involving CBLgene, mutated in 5-20% of CMML. Although the number of total alterations (1, 2 or ≥3) do not have a survival impact, we found a significant correlation between the size of the affected genome (≥30 Mb, including CNA and LOH) and a poorer OS (OS at 3 years 26% vs. 46%, P=0.039). Regarding type of alterations, cases with interstitial LOH had lower OS than cases without LOH (OS at 3 years 19% vs. 45%, P=0.049). Cases with high risk alterations as defined by CMML-specific cytogenetic risk classification (losses in 7q, gains in 8q and ≥3 CNA) had lower PFS than patients with other aberrations (PFS at 1 year 76% vs. 93%, P=0.036), although statistical significance was not reached for OS (OS at 3 years, 27% vs.49%, P=0.096). Conclusions: SNP-A in CMML patients with low risk cytogenetic features or no metaphases has led to the detection of chromosomal alterations in 66% of cases. Using this technique the prognosis can be better defined, and we can detect a group of patients with worse outcome who could be considered for more intensive treatment. Acknowledgments: Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02519; PI 11/02010); RTICC, FEDER (RD12/0036/0044; RD12/0036/0014); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras, Obra Social “La Caixa” and Celgene Spain; NHRI-EX103-10003NI, Taiwan. Footnote: Francesc Solé and Lurdes Zamora contributed equally Disclosures Xicoy: Celgene: Honoraria. Sole:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.