Search

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.

Background The use of combination antiretroviral therapy (CART) has improved the prognosis of HIV-related diffuse large B cell lymphoma (DLBCL). In the pre-CART era, HIV-infected individuals usually presented DLBCL of primary extranodal origin and advanced stage, unlike non-HIV-infected individuals. In the CART era, studies conducted before the introduction of immunochemotherapy pointed outcomes of HIV-infected patients to be approaching those of non-HIV-infected patients. Recently, studies have reported the lack of influence of HIV-infection in the prognosis of Burkitt’s lymphoma treated with immunochemotherapy. Nevertheless, there is scarce information comparing the clinical presentation and outcome of DLBCL treated with immunochemotherapy between non-HIV-infected patients and HIV-infected patients in CART era. Methods We retrospectively studied two series of DLBCL patients treated with RCHOP; 81 HIV-infected patients included in a Spanish multicentre trial and 84 non-HIV-infected patients diagnosed in our institution between 2002 and 2010. Demographic, HIV-infection, and DLBCL data on each case were collected. DLBCL cases were classified in nodal and extranodal according to a previously reported definition (JCO 2005). Continuous and categorical variables are presented using descriptive statistics. Survival analyses were performed using the Kaplan-Meier method, and compared using the log-rank test. P-values of less than 0.05 were considered statistically significant. Results The median follow-up of HIV-infected patients was 6.5 years and of non-HIV-infected patients was 4.6 years. Non-HIV-infected patients were older at the time of DLBCL diagnosis than HIV-infected patients, median age (range) 62 years (24-80) and 44 years (24-74) respectively; (P Conclusions In the CART era, DLBCL of HIV-infected patients still have aggressive features but the incidence of extranodal involvement equalizes that of non-HIV-infected patients. The prognosis of DLBCL treated with RCHOP is worse in HIV-infected than in non-HIV-infected patients. Supported in part by grants EC11-041 and RD12/0036/0029 RTICC from Instituto Carlos III, Spain. Disclosures: No relevant conflicts of interest to declare.

Introduction MYC translocations involving chromosome 8q24 can occur in a wide variety of B-cell lymphomas other than Burkitt’s lymphoma (BL), especially diffuse large B-cell lymphoma (DLBCL) and B-cell lymphoma unclassifiable with intermediate features between DLBCL and BL (BCLU). These lymphomas can harbor additional translocations of BCL2 and/or BCL6, that have been referred to as double and triple-hit lymphomas. MYC positive lymphomas other than BL are not well characterized and the standard treatment has to be established. Aim The objective was to study the clinic-biological characteristics and prognosis of a series of lymphomas with MYC-translocation other than BL. Methods Retrospective study of patients with MYC-translocation other than BL treated in three hospitals of Spain between 2003 and 2012. Cases with diagnosis of BL, Burkitt’s-like lymphoma and any other with MYC-translocation were reviewed and classified according to the WHO 2008 criteria. The status of MYC, BCL2 and BCL6 genes were evaluated in all cases by fluorescent in situ hybridization (FISH) using dual-colour break-apart commercial probes (LSI MYC DC BA, LSI BCL6 DC BA and LSI BCL2 DC BA; Abbot Molecular, Abbot Park, IL, USA) on whole tissue sections of formalin-fixed paraffin-embedded tissue. Main clinical and biological data were collected from the records. Results Between 2003 and 2012, 34 patients with a median follow-up of 1.9 years (range 0.7-9.7) were included. Median age was 59.5 years (range 36-83) and 21 (62%) were male. ECOG score at diagnosis was ≥2 in 14 patients (42%), 16 (49%) had ≥2 extranodal sites involved, serum LDH was elevated in 24 out of 32 (75%), Ann Arbor stage III/IV in 24 (73%) and B symptoms were present in 18 (56%). IPI was high or intermediate/high in 19 out of 32 (59%). Eighteen patients (53%) presented with a mass (13 abdominal location). Twenty-four cases were diagnosed with DLBCL and 10 with BCLU. Conventional cytogenetics showed a complex karyotype in the 9 studied cases. MYC rearrangement alone without BCL2 or BCL6 rearrangements was observed in 13 (38%) cases, 15 were double-hit, and 6 triple-hit lymphomas. There were no differences between patients with MYC translocation alone and patients with double or triple-hit, regarding the clinical and biological characteristics. Moreover, there were no clinical and biological differences between patients with DLBCL and those with BCLU. Twenty-one cases were treated with R-CHOP (19 DLBCL and 2 BCLU), and 13 with a specific treatment for BL (Burkimab) (8 BCLU and 5 DLBCL) (P=0.002). Complete response (CR) was achieved in 14 out of 32 (44%) cases (9 out of 21 [45%] treated with R-CHOP and 5 out of 13 [42%] with Burkimab). Two patients died during chemotherapy. Six out of the 9 patients in CR to R-CHOP relapsed, but none of the 5 patients in CR to Burkimab did it. The overall survival (OS) probability and progression free survival (PFS) at 2 years, (95% CI) for the whole series were 32% (14%, 50%) and 21% (6%, 36%) respectively. The two-year OS and PFS probabilities were not significantly different between patients treated with Burkimab and those treated with R-CHOP: 62% (95% CI: 36%, 88%) versus 28% (95% CI: 8%, 48%) for OS (P=0.67); and 46% (95% CI: 19%, 73%) versus 15% (95% CI: 0%, 31%) for PFS (P=0.549), respectively. Conclusions Lymphomas with MYC translocation other than BL present aggressive characteristics at diagnosis and have poor response to immunochemotherapy. Supported in part by grants EC11-041 and RD12/0036/0029 RTICC from Instituto Carlos III, Spain Disclosures: Lopez-Guillermo: Roche: Membership on an entity’s Board of Directors or advisory committees.

Introduction Monoclonal B-Cell Lymphocytosis (MBL) is a frequent condition characterized by a clonal B-cell population detected in the blood or bone marrow, in otherwise asymptomatic individuals.A small proportion of patients with MBL need clinical attention. Its prevalence is around 3-5% in healthy adults over age 40 and rises with age. The largest studies of MBL features are focused on healthy population, therefore additional data regarding the population referred to hospital are necessary. Genotype and immunophenotype data are well described in the literature so far, but a comprehensive framework of the condition is still required. Data of features not well described heretofore like bone marrow pattern of infiltration would help in the differential diagnosis. The aim of this work was to analyze the clinical and laboratory findings of MBL reclassified from CLL or other B-cell lymphoproliferative disorders (LPD) with peripheral involvement, referred to a centre. Patients and methods Three hundred seventy-one LPD patients with peripheral blood involvement diagnosed between years 2000 and 2009 in a single centre were revised to reclassify them according to the World Health Organization consensus cut-off of 5x109/L circulating B cells. From those reclassified as MBL, blood count, biochemistry, immunophenotype, cytogenetics, bone marrow trepine biopsy and results of CT screening were registered, as well as treatment and outcome data. Only those patients who were followed in our centre were included. Results Seventy-three LPD (20%) were reclassified as MBL, with a median age of 72 years (range: 42-94) and a 52% of males. Proportions of CLL-like LPD, CD5 negative non CLL-like and CD5 positive non CLL-like switched from 65%, 21% and 14% to 84%, 7% and 9% after the reclassification. In only seven out of 41 patients the bone marrow was not infiltrated, while the majority had an interstitial and a mixed nodular and interstitial infiltration (44% and 36%, respectively). Only 2 out of 49 patients had a concomitant infection with hepatitis C virus. Fifty-nine % of patients had a FISH alteration, predominantly chromosome 13 deletion. Thirty-seven out of 69 patients (54%) fulfilled criteria of LPD progression, with 10 patients (15%) requiring treatment (7 out of them because of progression of CLL). The mean (SD) B-lymphocyte count at diagnosis of patients with LPD progression was 3.58x10^9/L (0.9x10^9/L) versus 3.03x10^9/L (1.1x10^9/L) in those who did not progress, p=0.023. The 12-year progression free survival of the whole series was 38% (CI 95% 23-53) with a median of 4.08 years (range: 2.3-5.8). The 12-year overall survival was 54% (29-79), with a median follow-up of 7.73 years (1.7 – 12.9). Conclusions An important proportion of LPD with peripheral involvement can be reclassified to MBL, with a higher prevalence of CLL-like LPD than the global set of patients. The pattern of bone marrow infiltration was predominantly interstitial, followed by a mixed pattern of infiltration. A high percentage of patients in this series evolved to LPD, but a small subset required specific treatment. The patients with LPD progression had a significantly higher B-lymphocyte count than those who did not. Disclosures: No relevant conflicts of interest to declare.

4809 Introduction: Chronic myelomonocytic leukemia (CMML) is a clonal haematopoietic malignancy characterized by features from both myelodisplastic syndromes and myeloproloferative neoplasms. CMML median overall survival is 20 months and 15–30% of cases progress to acute myeloid leukemia (AML). Molecular biology of CMML is poorly understood. Clonal cytogenetic abnormalities are found in 20–30% of cases. Recently, some recurrent gene mutations have been reported. The most frequent mutated genes are TET2 (30–50%), ASXL1 (35–56%), SRSF2 (28–47%), RUNX1 (8–37%), CBL (10–22%) and K/NRAS (2–22%). We sought to characterize type, frequency and prognostic implication of cytogenetic alterations and genetic mutations ( ASXL1 , CBL , JAK2 and SRSF2 ) in a cohort of 20 patients with CMML. Methods: A retrospective study was performed on 20 CMML cases (11 CMML-1, 9 CMML-2) for whom clinical and biological data were available. Median age at diagnosis was 66 years (range: 49 – 84 years), with a male predominance of 1.9:1 and 42% frequency of progression to AML. Conventional cytogenetic analysis was performed in bone marrow (BM) samples obtained at diagnosis (n=19) and at progression to AML (n=8). For mutation analysis, DNA was extracted from BM samples at diagnosis (n= 20). Sanger sequencing was performed to study mutations in ASXL1 (exon 12), SRSF2 (exon 1) and CBL (exons 8 and 9) genes, while JAK2 V617F mutation was analyzed by endpoint genotyping. Survival analysis was performed using Kaplan-Meier estimate and log-rank tests were used for comparisons. The χ2 and Fisher's exact tests were used to analyze differences in the distribution of variables among patient subsets. Results: Aberrant karyotypes were seen in 5/19 (26%) cases at diagnosis and 6/8 (83%) cases at progression to AML. Alterations observed at diagnosis were typical from CMML (trisomy 8, 7q deletions and 12p structural alterations), while those observed at progression were more common in AML ([Table 1][1]). Heterozygous somatic ASXL1 mutations were detected in 8/19 (42%) cases. A total of 6 different mutations were seen in these 8 cases, being the recurrent mutation p.G646WfsX12 (g.76303dupG) the only one seen in more than one patient (4/8, 50%). In addition, 4 cases harboured two point mutations located at 3' of exon 12 (g.79017A>G and g.78128C>T). Somatic SRSF2 mutations were detected in 4/20 (20%) cases. All of them were heterozygous, missense mutations located at hotspot P95. In 3 cases P95 changed to P95H (g.535 C>A), while in 1 case it changed to P95L (g.535 C>T). In addition, 1 silence mutation was detected in 1 patient at the beginning of exon 1 (g.395 C>T) of SRSF2 . No mutations were seen in CBL and JAK2 genes. ASXL1 and SRSF2 mutations did not correlate with either CMML-1/CMML-2 subtypes or myelodysplastic/myeloproliferative variants. Survival analysis revealed that the only adverse prognostic factor was CMML-2 subtype. Compared to CMML-1, patients with CMML-2 had worse overall survival (median OS at 2 years: 18% vs 21%, p=0.05) and progression-free survival (median PFS at 2 years: 21% vs 57%, p=0.002). Mutations in ASXL1 had no prognostic impact, while a trend towards higher PFS was seen in cases with SRSF2 mutations, although it was not statistically significant. Conclusions: Cytogenetic analysis revealed that the percentage of aberrant karyotypes in CMML increases considerably at progression to AML. Mutational analysis showed that ASXL1 and SRSF2 are frequently mutated in CMML. There are multiple ASXL1 mutations throughout the exon 12, while missense SRSF2 mutations located at the hotspot P95. CMML-2 is still the only prognostic factor associated to worse OS and PFS. | CMML (n=19) | AML (n=8) | |:-------------------------------------:| ------------------------------------------------------------------------------------------------------------------ | | Patients that did not progress (n=10) | | | 46,XY \[20\] (n=4) | | | 46,XX \[20\] (n=5) | | | 46,XY,del(11)(q23) [18]/46,XY [2] | | | 47,XY,+8,del(12)(p13) [20] | | | Patients that progressed to AML (n=8) | | | 46,XX [20] | No data | | 46,XY [20] | No data | | 46,XY [20] | 46,XY [20] | | 46,XX,t(9;11)(p22;q23) [13]/46,XX [7] | 46, XX, t(9;11)(p22;q23) [16]/46,XX [4] | | 46,XY [20] | 44,XY,del(3)(p21),del(5)(q22q23,del(7)(q11.2),-9,-13,-14,+mar1,+mar2 ![Formula][2] /46,XY ![Formula][3] | | 46,XY [20] | 46,XY,t(8;16)(p11;p13) [18]/46,XY [2] | | 46,XX,t(1;3)(p36;q21) [20] | 46,XX,t(1;3)(p36;q21)[19]/46,XX,t(1;3)(p36;q21),der(1)[1] | | 46,XY,del(7q) [3]/46,XY [17] | 46,XY,t(2;3)(p23;q26.2) [30] | Table 1. Cytogenetic alterations of CMML at diagnosis (n=19) and at progression to AML (n=8) Disclosures: No relevant conflicts of interest to declare. [1]: #T1 [2]: T1/embed/tex-math-1.gif [3]: T1/embed/tex-math-2.gif

Abstract 1437 JAK2 mutation testing and karyotye are routinely used for diagnosis of myeloproliferative neoplasms (MPNs) but they have not been incorporated into risk stratification. Although JAK2V617F mutation in MPNs has been one of the most seminal medical discoveries in recent years, it is not clear the importance of the amount of JAK2V617F allele. Some studies correlate the JAK2 allele burden with a higher hemoglobin level, leukocyte count, splenomegaly and thrombosis and more probability of transformation to MF or AML. The aim of this study was to determine whether cytogenetic data, JAK2 mutation status and allele burden correlates with cytological subtypes, clinical complications or provide prognostic information. Methods A retrospective study was conducted with samples centralized in a unique laboratory since 2005. A total of 526 patients were included (median age 63; 243 males) with classic MPNs (348 ET, 135 PV, 43 PMF) fulfilling 2008 WHO criteria and in accordance with the Declaration of Helsinki. Conventional cytogenetic was performed in bone marrow samples obtained at diagnosis (n=205) and at progression to MF or AML (n=46). DNA was extracted from peripheral blood using QIAamp DNA mini kit (Qiagen). All samples were coded and assayed blindly in duplicate to detect JAK2V617F mutation with an allele-specific PCR using TaqMan allelic discrimination, with 2 specific probes to measure the respective fluorescence of each allele. Then, JAK2 MutaQuant assay (Ipsogen, Luminy Biotech) was used to detect the JAK2V617F quantity by real-time PCR, detecting fluorescent signals using double-dye hyrolysis oligonucleotide probes with calibration standards at 4 different concentrations. Homozygous (HOZ) ratio was considered when percentage was higher than 50. Laboratory (hemoglobin, WBC and platelet counts) and clinical data (constitutional symptoms, splenomegaly, complications, OS and DFS) were collected. For continuous variables parametric and non parametric statistics were used. Survival analysis was performed using Kaplan-Meier estimate and log-rank tests were used for comparisons. The χ2 and Fisher's exact tests were used to analyze differences in the distribution of variables among patient subsets. p-value less than 0.05 were considered statistically significant. Results Aberrant karyotypes were seen in 15/205 (7%) cases at diagnosis (4% in ET and PV and 40% in PMF). At progression to MF or AML we have cytogenetic studies in 22 patients, and 10 (45%) harbor alterations. A total of 283 patients (64%) were JAK2V617F, 61% ET (4% HOZ), 75% PV (28% HOZ) and 55% PMF (16% HOZ). The median value of JAK2V617F was 26% (range, 1–99.9%). No correlation was seen between JAK2 and karyotype at diagnosis, but 7/9 patients with aberrant karyotype at progression had JAK2V617F mutation. JAK2 correlations with laboratory and clinical data are summarized in Table 1 and 2. Conclusions JAK2V617F is associated with a more pronounced myeloproliferative phenotype (higher hemoglobin level, platelet or leukocyte count). Patients with JAK2V617F HOZ have a higher probability of splenomegaly. Hematological complications do not depend on JAK2 ratio but mutated patients have more probability of thrombosis or hemorrhage. OS and PFS do not depend on JAK2 status, but patients with JAK2V617F heterozygous seem to have a slightly better outcome. Disclosures: No relevant conflicts of interest to declare.

Abstract 2551 Background: The presence of multilineage dysplasia (MLD) is the hallmark to define acute myeloid leukemia (AML) with myelodysplasia-related changes. The recognition by the WHO classification of this category as a separate entity implies the presence of differential biological and clinical features. While this seems to be confirmed for patients with AML and MLD associated with high risk cytogenetic abnormalities, the prognostic significance of MLD in cases with intermediate risk karyotype is unclear. Recent studies have shown that MLD has no prognostic impact in patients with mutated NPM1, but the prognosis of patients with intermediate-risk cytogenetics and MLD harboring a wild-type configuration of NPM1 is uncertain. Objective: The aim of this study was to analyze the presence of MLD and its prognostic value in AML patients with intermediate risk cytogenetics (IR-AML) and wild-type NPM1. Patients and methods: One-hundred eighty-two patients diagnosed with de novo IR-AML and wild-type NPM1 treated with the same chemotherapy protocol (CETLAM-2003) were included (M/F:101/81; median age: 55.5, 18–73). Bone marrow aspirate smears performed at diagnosis were reviewed by a panel of expert cytologists, and the cases were categorized as having MLD by applying strictly the WHO criteria (Harris et al, WHO 2008, MLD-1). Additionally, cases with dysplasia in ≥50% of the cells in one cell line, and between 30% and 50% in another cell line, were classified as having significant dysplasia (MLD-2). Results: The degree of dysplasia was evaluable in 139 of the 182 cases, observing strictly WHO-defined multilineage dysplasia (MLD-1) in 45 (25%), significant dysplasia (MLD-2) in 17 additional patients (9%), and absence of MLD in 80 (44%). In 43 (30%) cases dysplasia could not be thoroughly quantified in all hematopoietic lines, mostly due to the lack of enough evaluable precursors in a context of massive blast infiltration. No major differences concerning main characteristics were observed between patients with or without MLD, including age, leucocyte count at presentation, degree of BM involvement, proportion of normal karyotype, and frequency of mutations of FLT3 (internal tandem duplication), CEBPA, and partial tandem duplication of MLL gene. Since the outcome of patients with MLD-2 was similar to that of patients with stringent WHO MLD, and markedly inferior to patients lacking MLD (table 1), these two groups were merged in a unique MLD group (MLD-1+MLD-2). Thus, MLD was associated with a lower probability to achieve complete remission (CR) after frontline induction chemotherapy (66% vs. 80%, p=0.033). In addition, patients with MLD showed a trend to an inferior overall survival (OS at 5 years: 39.2±5% vs. 22.9±8.1%, p=0.064). Of note, a multivariate analysis identified increasing age, higher leucocyte count at presentation, and presence of MLD (hazard ratio, 95% CI: 1.569,1.056–2.331; p=0.026) as the only variables associated with a shortened survival; this analysis also included presence of FLT3-ITD and cytogenetics (normal karyotype vs. other intermediate-risk abnormalities). Age was the only variable with independent prognostic value on leukemia-free survival in this cohort of patients, whereas MLD did not reach statistical significance. Interestingly, the outcome of patients with MLD-AML with a minimum CR duration of 3 months was significantly better after allogeneic stem cell transplantation in first CR compared to other post-remission treatments (5-year OS: 82±12% vs. 31±9%, p=0.006), suggesting that alloHSCT could partially overcome the adverse prognosis associated with this AML category. Conclusions: The presence of MLD confers adverse prognosis to patients with IR-AML and wild-type NPM1. Presence of significant dysplasia in two hematopoietic cell lines without fulfilling stringent WHO criteria was associated to an outcome similar to that of patients with WHO-defined criteria, an observation that leads to a wider interpretation of significant MLD in AML. These results have an important clinical relevance and should lead to the search of new genetic and epigenetic markers associated with MLD in this setting. This study has been partially supported by grant n° 03/0423 from Instituto de Salud Carlos III/FIS0. Disclosures: No relevant conflicts of interest to declare.

Abstract 4943 Background: The use of highly active antiretroviral therapy (HAART) has dramatically improved the prognosis of AIDS-related Non-Hodgkin lymphoma (NHL), causing a remarkable increase in patients' long-term survival. On the other hand, with the introduction of HAART, the incidence of non-AIDS-related malignancies is increasing among patients with HIV infection. The incidence of second neoplasms after treatment of aggressive NHL have been reported to be higher than in the general population. Nevertheless, there is scarce information regarding second malignancies occurring in HIV-infected patients after NHL treatment. We sought for second neoplasms among patients treated of AIDS-related NHL, hence suffering from 2 conditions which may predispose them to develop malignancies: therapy for lymphoma and HIV infection. Methods: We conducted a retrospective study of patient data on AIDS-related NHL individuals diagnosed between 1989 and 2010 in our institution. Demographic, HIV infection, and lymphoma data on each case were collected. Two groups were further considered: patients who did not take HAART at all, and those who started HAART at any stage of their HIV infection. Continuous and categorical variables are presented using descriptive statistics. Survival analyses were performed using the Kaplan-Meier method and compared using the log-rank test. P-values of less than 0.05 were considered statistically significant. Results: Out of a series of 146 patients diagnosed with AIDS-related NHL, 138 patients were eligible for the study with a median follow-up of 8.24 years: 70 patients belonged to the no-HAART group, and 68 to the HAART group. Most interesting data regarding both groups are listed in table 1. Briefly, patients in the HAART group were older at the time of lymphoma diagnosis than those who did not receive it (p=0.024); in addition, there were more male patients in the no-HAART group (p=0.011). These differences show the demographic changes among HIV-infected patients in the HAART era. Furthermore, a higher number of complete responses (CR) to lymphoma treatment was observed in the HAART group (p Four out of the 40 patients at risk (10%), developed a second malignancy in the HAART group, and none in the no-HAART one. However, no statistically significant differences were found, probably due to the low number of cases. The most important features of the 4 patients with a second malignancy are listed in table 2. All 4 patients were diagnosed with diffuse large B-cell lymphoma (DLBCL), were treated with CHOP, and had a prior diagnosis of AIDS. Two of the 4 were on HAART at lymphoma diagnosis (one of them in both virological and immunological response). The patient with hepatocarcinoma was HCV positive. As shown in table 1, three of the patients had responded to HAART when the second neoplasm (carcinoma) was diagnosed; and the reminder, who did not respond to HAART, developed a Kaposi sarcoma. All four patients died because of neoplasm progression. Conclusions: As expected, a higher frequence of second neoplasms was observed after treatment of AIDS-related NHL among patients who receive HAART. Second malignancies were non-HIV related neoplasms in patients with response to HAART. Abbreviations: CT, chemotherapy; RT, radiotherapy; IVDU: intravenous drug user. Disclosures: No relevant conflicts of interest to declare.

Abstract 4897 Objective. To study the frequency, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML and MDS. Patients and methods. One hundred thirty patients were studied between 2000 and 2010. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2009. Results. Clonal abnormalities were observed in 36/130 patients (28%) with a median follow-up was 11 months (range 3–131). Initial diagnosis (OMS 2008): AML with maduration (8 patients), acute erythroid leukaemia (4), acute monocytic leukaemia (3), AML with multilineage dysplasia (3), AML with myelodysplasia-related changes (3), AML with inv(16)(p13q22) (2), acute monoblastic leukaemia (2), AML with minimal differentation (2), AML without maduration (1), acute myelomonocytic leukaemia (1), refractory anaemia with excess blasts (4), chronic myelomonocytic leukaemia (2) and refractory cytopenia with multilineage dysplasia (1). Treatment before SCT: idarubicin, cytarabine, etoposide and mitoxantrone (25), idarubicin, cytarabine and etoposide (6), idarubicin, cytarabine, etoposide and gemtuzumab (2), FLAG-ida (2) and azacitidine (1). SCT type: autologous (23 patients), allogeneic of reduced intensity (7), allogenic (4), umbilical cord blood (1) and syngeneic transplant (1). Conditioning regimen: TBI and cyclophosphamide (24 cases), fludarabine and busulfan (7), busulfan and cyclophosphamide (4) and thymoglobulin, thiotepa, fludarabine and busulfan (1). The median time between SCT and the appearance of clonal abnormalities was 6.5 months (range 2–51). At the time of clonal abnormality detection, 32 patients were in cytological and/or clinical relapse and 4 in cytological remission. In 15 cases the initial clone reappeared, 2 showed the initial abnormalities with an acquired abnormality and 19 presented de novo clonal abnormalities (53%). The most frequent clonal chromosomal abnormalities observed were: complex karyotype (22%), estructural abnormalities with afection of chromosomes 1, 4, 6, 7, 10, 11, 12, 16 and 17 (22%), +21 (5%) and +11 (3%). The median survival from the appearance of clonal abnormalities was 2.5 months (range 1–100). There were no differences when we compared the survival of patients with de novo clonal abnormalities with that of patients with initial abnormalities (including those with acquired abnormality). Furtheremore there were no differences when we compared the median survival of relapsed patients with normal Karyotype with that of patients with clonal chromosomal abnormalities following SCT (2.5 months [range 1–25] and 2.5 months [range 1–100] respectively). At the time of the analysis only 1 patients are alive and in complete remission (100 months). Conclusions. 1. The appearance of clonal abnormalities following SCT in patients with AML and MDS is frequent and the majority of cases are detected at the time of relapse. Half of cases presented de novo clonal abnormalities and the majority were not related with prior irradiation exposition, alkylating agents neither topoisomerase II inhibitors. The prognosis of patients with acquired clonal abnormalities after SCT is poor. Disclosures: No relevant conflicts of interest to declare.

Abstract 4442 Introduction: Chronic myeloid leukemia (CML) is a model of disease in the development of targeted therapies. Tyrosine kinase inhibitors (TKIs) have transformed the approach to management of CML and have dramatically improved patients' outcome. Clinical response is obtained in the majority of patients. However, a significant proportion of patients do not achieve the optimal desirable outcome or are completely resistant to this treatment. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKIs has produced high rates of hematologic and cytogenetic response in mutated ABL patients. The aim of this study was analyzed the presence of ABL mutations in imatinib resistant patients and determine the importance of changing to second-generation TKIs treatment as soon as failure or suboptimal response is recognized. Patients and methods: From 420 CML patients diagnosed in 6 centers between 2004 and 2010, we have amplified and sequenced the ABL1 domain from BCR-ABL1 amplicon of 45 imatinib resistant patients (23 patients with suboptimal response, 14 with treatment failure, 4 who lost the molecular response and 4 patients who progressed to blast phase). The obtained sequences were compared with the published ABL1 sequence, GenBank U07563, using BLAST 2 software. Results: We have detected mutations in 15 of 45 patients (33%), some of them with more than one mutation (Table 1). Seven of these patients were treated with second-generation TKIs as a single treatment. Three of them achieve a major molecular response (MMR), one patient is in complete cytogenetic response (CCyR) and the other two patients are in major (MCyR) and partial (PCyR) cytogenetic response. Another patient received nilotinib followed by hematological stem cell transplantation (HSCT) and is in MMR. Two patients were submitted to a HSCT and achieve MMR. Only one patient treated with nilotinib as second option has not reach a cytogenetic response one year after detection of the mutation. Two of the patients with the T315I mutation were treated with IFN and nilotinib achieving PCyR and MCyR, respectively, and are still alive. The other T315I patient, and two patients in blast-crisis (BC) disease with the F317L mutation who received dasatinib prior to the study of ABL mutations, died before a change of treatment could have been performed. Conclusions: Disclosures: No relevant conflicts of interest to declare.

Abstract 4838 Introduction: Chronic myeloid leukemia (CML) is a clonal hematopoietic malignancy characterized by the presence of BCR/ABL fusion gene. The resulting protein has a high tyrosine kinase (TK) activity. The first-line treatment for CML is Imatinib, which allow the achievement of cytogenetic and molecular response in most of patients with CML in chronic phase. However, some patients do not respond to this treatment or lose their initial response. Imatinib has been reported to be incorporated into the cell through hOCT1 transporter (human organic cation transporter). The aim of this study was to determine whether the expression of hOCT1 at diagnosis of CML influenced the achievement of molecular response. Patients and Methods: We analyzed hOCT1 gene expression by quantitative PCR in 42 patients at diagnosis and 18 months after treatment with Imatinib. We compared the expression with the presence of compleat molecular response (CMR) at 18 months. We consider CMR when the Ratio (BCR-ABL/ABL)×100 was Results: Of the 42 patients, 2 were in hematological response, 22 were in cytogenetic response and 18 in CMR at 18 months. We found a higher hOCT1 gene expression at 18 month than at diagnosis (53.3 versus 29.6, p Conclusions: Partially funded by FICJ-P-EF-09, RD06/0020/1056 de RTICC and Novartis. We want to thank Dr. David Marin for providing us plasmid for quantitative analysis. Disclosures: No relevant conflicts of interest to declare.

Abstract 1003 Poster Board I-25 Introduction: Recently, the cooperative group HOVON-SAKK has refined the prognostic impact of cytogenetic abnormalities in acute myeloid leukemia (AML) by introducing the concept of monosomal karyotype (MK). This consists of ≥ 2 autosomal monosomies or one autosomal monosomy in addition to a structural alteration. In their experience, MK would explain the poor prognosis of AML with a complex karyotype. Objective: To investigate the prognostic impact of MK in patients with primary (de novo) AML enrolled in the Spanish CETLAM group protocols (AML 94/99/03). Also, to determine whether considering MK added predictive value to the cytogenetic classification of the Medical Research Council (MRC). Methods: Retrospective analysis of data from 1149 AML patients. Chromosomal formula was centrally reviewed with karyotypes being classified by the presence of MK and allocated into the MRC risk categories. Complete remission (CR) rate, disease-free survival (DFS) and overall survival (OS) were calculated. Results: The karyotype was assessable in 904 (79%) of the 1149 cases. In 145 of the 904 cases (16%), abnormalities involving CBF gene were detected and in 437 (48%) the karyotype was normal (NK). In 253 (28%) additional patients the karyotype was not monosomal; of them, 61 (24%) belonged to the unfavorable MRC with 17 cases harboring a complex karyotype ≥ 5 abnormalities, 7 cases with rearrangements 3q, 13 cases with -7, 9 cases with 5q abnormalities and 16 cases with t(6;9)). The remaining 69 (7.7%) patients had a MK; of them, 59 (85.5%) were from the unfavorable MRC category and included 43 cases with complex karyotype ≥ 5 abnormalities, 6 cases with rearrangements 3q, 5 cases with -7, 5 cases with alterations of 5q). The following table summarizes the results in terms of CR rate, DFS and OS: Conclusions: The addition of MK to the MRC cytogenetic classification refines the prognostic prediction. In our series, the dismal outcome of patients with MK is confirmed; these patients had worse prognosis than those with adverse cytogenetics without MK. Alternative treatment strategies are mandatory for MK+ patients. Supported in part by grants: GR1-01075, ECO07/90065, PI080672 and RD06/0020/0101. Disclosures: No relevant conflicts of interest to declare.

Objective. To study the incidence, type and prognostic significance of clonal chromosomal abnormalities following SCT in patients with AML. Patients and methods. From February 2000 to March 2008, 74 patients were studied. Karyotypes were analysed by G-banded chromosomes obtained from 24 hours bone marrow cultures, and were described according to ISCN 2005. Results. Clonal abnormalities were observed in 17/74 patients (23%). Median follow-up was 11 months (range 5–47). Initial diagnosis: AML2 (6 cases), AML5a (3), AML6 (3), AML with inv(16)(p13;q22) (1), AML with multilineage dysplasia (1), AML5b (1), AML1 (1) and AML0 (1). Treatment before SCT: idarubicin, cytarabine and etoposide (16), FLAG-ida (1). All patients presented a normal karyotype at the time of SCT. SCT type: autologous (12 patients), allogeneic (3), allogeneic of reduced intensity (2). Conditioning regimen: TBI and cyclophosphamide (14 cases), busulfan and cyclophosphamide (1), fludarabine and busulfan (2). The median time between SCT and the appearance of clonal abnormalities was 8 months (range 3–36). At the time of clonal abnormality detection, 16 patients were in cytological and/or clinical relapse and 1 in complete remission. In 4 cases the initial clone reappeared, 1 showed the initial abnormalities with an acquired abnormality and 12 presented de novo clonal abnormalities. The median survival from the appearance of clonal abnormalities was 2 months (range 1–40). At the time of the analysis all patients had died. Conclusions. The appearance of clonal abnormalities following SCT in patients with AML is frequent. Time between SCT and the appearance of clonal abnormalities is short. The majority of patients presented de novo clonal abnormalities with cytological and/or clinical relapse and poor prognosis.

INTRODUCTION. Mantle cell lymphoma (MCL) is a well defined mature B-cell neoplasm, usually with an aggressive behaviour and a median survival of 3–5 years. It is characterized by the presence of t(11;14)(q13;q32), that leads to overexpression of CCND1. This translocation is detected by conventional cytogenetics (CC) in more than 65% of the cases. Nevertheless, cyclin D1 overexpression alone is not sufficient to give rise to a MCL. Several secondary genetic abnormalities with a potential role in the oncogenic process have been described using different molecular and cytogenetic techniques but no data from large series of MCL cases studied by CC have been correlated with survival and proliferation information. AIMS. 1. To correlate secondary chromosomal abnormalities, histological subtype and proliferative index with survival. 2. To describe the frequency of secondary chromosomal aberrations using CC and a large series of MCL patients. PATIENTS AND METHODS. We selected 145 MCL cases (94M/51F; mean age 65.4 yrs; mean survival 33 mo.), all of them with an abnormal karyotype. Histological subtype, proliferative index (Ki-67) and survival data were collected. All patients were studied by conventional GTG-banding cytogenetics, and FISH with a dual colour dual fusion CCND1/IGH probe was applied in those cases with an abnormal karyotype but without t(11;14) detected by CC. RESULTS. Translocation t(11;14) was detected as a single anomaly in 36% of patients, whereas 57% of cases displayed t(11;14) plus other additional aberrations and 6% of patients showed an abnormal karyotype without t(11;14) which was detected by FISH. We observed a total of 550 abnormalities (147 numerical and 403 structural), being the most frequent deletions of 1p (12%), 13q and 17p (10% each) and gains of 3q (9%). Recurrent breakpoints were identified at 1p31–p32, 1p21–p22, 17p13 and 1p36 (in eight, six, five and four cases, respectively). Blastoid variants of MCL displayed more karyotypic complexity with high number of structural alterations, higher number of 1p and 17p deletions, higher proliferation index and poor survival. CONCLUSIONS. 1. MCL cases associated with 17p losses (overall survival at 4 yrs: 15% vs 50%; P=0.01) and high proliferative index (15% vs 55%; P=0.005), generally corresponding to blastoid variants, display a worse outcome. 2. Our results confirm, by means of a routine technique as CC, previous observations made using more sophisticated molecular techniques in shorter series of patients.

Background. Several studies have reported cases of Ph-positive CML treated with imatinib developing chromosomal abnormalities in Ph-negative cells. Trisomy 8 is the most frequent of these abnormalities, but monosomy 7 has also been detected. The association of these cytogenetic alterations with severe myelodysplasia has been scarcely reported. We present 2 cases of monosomy 7 with severe myelodysplasia arising during imatinib treatment of Ph-positive CML, one evolving as a transient clonal abnormality, and the other with a fatal evolution to AML. Case 1. A 55 year-old man was diagnosed with Ph-positive chronic phase CML in July 2000. He started treatment with IFN-α without cytogenetic response, and in October 2002 he began imatinib-mesylate (400 mg/day). After 6 months on imatinib he developed anemia and neutropenia, with severe dysplastic features on peripheral blood. Dysplastic features in the 3 series with 70% of ring syderoblasts were observed in the bone marrow. The ultrastructural study showed numerous erythroblasts with iron-loaded mitochondria and some “ring forms”. The cytogenetic study was: 46,XY,t(9;22)(q34;q11)[4]/ 45,XY,-7[12]/46,XY[6]. The dose of imatinib was reduced (300 mg/day). Three months later, peripheral blood cell counts were normal, the myelogram showed mild dyserythropoiesis without ring syderoblasts and the cytogenetic study was: 46,XY,t(9;22)(q34;q11)[7]/47,XY,+8[15]. Monosomy 7 was not detected by FISH. From January 2004 he has been treated with imatinib (300 mg/day) and IFN-α and the karyotype is: 46,XY,t(9;22)(q34;q11)[15]/ 46,XY[6]. Monosomy 7 and trisomy 8 have not been detected. Case 2. A 47 year-old man was diagnosed with Ph-positive chronic phase CML in October 2001. He was initially treated with hydroxyurea and in February 2002 a peripheral blood allogeneic stem cell transplant from a HLA-identical sibling, conditioned with busulfan and cyclophosphamide, was performed. Seven months later Ph chromosome was detected in 100% of metaphases and the patient was treated with 2 donor lymphocyte infusions without response. Imatinib at a dose of 400 mg/day plus G-CSF was started. After 14 months on imatinib, a complete cytogenetic response was obtained and 9 months later, molecular response was achieved too. Three months later (March, 2005), pancytopenia was detected. In the bone marrow, severe dysplasia in the 3 series with predominance of erythroid precursors and 41% of ring syderoblasts, were observed. The cytogenetic study showed a complex karyotype harboring monosomy 7: 45,XY,-7[9]/45,X,add(Y)(q12),-7[4]/46,XY,-7,+21[2]/46,XY[5]. Imatinib was stopped and 2 months later the patient was diagnosed with refractory anemia with excess of blasts. He rapidly progressed to AML and has been treated with idarubicine, cytarabine and etoposide as induction therapy without response. Monosomy 7 (2 cases) and trisomy 8 (case 1) were not retrospectively detected by FISH, prior to the introduction of imatinib. Conclusions. Severe myelodysplasia with monosomy 7 can develop in Ph-negative metaphases of patients with CML treated with imatinib. Although in some cases these alterations are transient, in others the evolution can be fatal to AML. Imatinib may play a role in the appearence of these clonal abnormalities.

Introduction and objectives. Flow-cytometry based automated blood cell counters measure the volume (V), conductivity (C) and light scatter (S) of cells and make available these values for each subpopulation of white blood cells. Although differences in V, C and S among several lymphoproliferative disorders, reactive lymphocytes and normal lymphocytes have been described, the ability to use this this information to detect lymphoproliferative disorders has not been defined. Methods. We registered VCS data of all consecutive cases of lymphocytosis (lymphocytes > 3.5x10e9/l) detected in routine laboratory testing during 50 non-consecutive days. Lymphocytes were evaluated morphologically, immunophenotypically and by conventional cytogenetics if necessary and the patients were evaluated clinically to document diagnosis adequately. Discriminant analisys modelling was used to assess the ability to predict diagnosis of VCS data. Results. 202 cases of lymphocytosis were detected in 120 males and 82 females, mean age 53 years (SD 16). Patients were considered to have normal lymphocytes (N=102, 50%), to present a reactive lymphocytosis (N=43, 21%, 20 of them HIV infected patients) or achieved a final diagnosis of lymphoproliferative disorder (N=57, 28%, 43 of them CLL). The mean volume of CLL lymphocytes was significantly lower than that of normal and reactive lymphocytes (p=0.009) and so was light scatter (p=0.018). When mean VCS values and their standard deviations were included in a discriminant analysis model, a unique function adequately classified 166/189 cases (87%) as CLL versus reactive or normal. Sensitivity and specificity were 73% and 92% respectively and false positive and false negative cases were 26% and 8% respectively. Inclusion of a second discriminant function did not improve significantly the predictive power of the model. Conclusion, VCS data from flow-cytometry based automated blood cell counters can be used to differentiate CLL from normal or reactive lymphocytes by simple discriminant analysis classification.

Clinical-pathological sessions are a good method for solving diagnostic and/or therapeutic problems in patients with hemopathies. In these sessions, errors made during health care given to the patient can be detected. By analyzing how and why these errors are made, we can improve patient care and prevent further mistakes. The objective of this study is to describe 34 errors identified in 874 patients with hemopathies at clinical-pathological meetings in two centers, performed with the aim of solving a diagnostic problem and/or a therapeutic decision or due to a great interest of the case, in two institutions, over 22 years (1982–2004). An intererdisciplinary team of hematology specialists gathered every week at interactive sessions of about 45 minutes each, in both institutions. The methodology of sessions was: a description of the medical history of a patient in a one or two-page report and a revision of the different samples (peripheral blood, bone marrow and lymph node morphology, immunocytochemistry, flow-cytometry, cytogenetic and molecular studies) with the aid of a microscope and a TV monitor. A diagnostic and/or treatment were proposed at the end of the session. Eight-hundred and seventy-four reports were analyzed. All the diagnostics were classified: chronic lymphoproliferatives disorders (445), myeloproliferative and myelodysplastic syndromes (136), acute leukemias (136), other haematological diseases (74), non-haematological diseases (31), without a diagnosis after the meeting (52). We identified diagnostic (D) and therapeutic (T) mistakes and considered as the main causes of the medical error (mistake in the diagnosis and/or treatment): lack of expertise (LE), malpractice (MP), impetuosity (IM), bad logistic support (LS), inexplicable (IN). We divided the 22 years into two decades and each error was classified in one of these two groups. Our own mistakes (OM) and the errors made in other institutions (OI) were identified. A comparison between number of errors made in the first 11 years and the second 11 years was made using a Chi-square test. P