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BACKGROUND Within the hematopoietic compartment, fibromodulin (FMOD) is almost exclusively expressed in chronic lymphocytic leukemia (CLL) lymphocytes. We set out to determine whether FMOD could be of help in diagnosing borderline lymphoproliferative disorders (LPD). METHODS We established 3 flow cytometry-defined groups (CLL [n = 65], borderline LPD [n = 28], broadly defined as those with CLLflow score between 35 and -20 or discordant CD43 and CLLflow, and non-CLL LPD [n = 40]). FMOD expression levels were determined by standard RT-PCR in whole-blood samples. Patients were included regardless of lymphocyte count but with tumor burden ≥40%. RESULTS FMOD expression levels distinguished between CLL (median 98.5, interquartile range [IQR] 37.8-195.1) and non-CLL LPD (median 0.012, IQR 0.003-0.033) with a sensitivity and specificity of 1. Most borderline LPDs were CD5/CD23/CD200-positive with no loss of B-cell antigens and negative or partial expression of CD43. 16/22 patients with available cytogenetic analysis showed trisomy 12. In 25/28 (89%) of these patients, FMOD expression levels fell between CLL and non-CLL (median 3.58, IQR 1.06-6.21). DISCUSSION This study could suggest that borderline LPDs may constitute a distinct group laying in the biological spectrum of chronic leukemic LPDs. Future studies will have to confirm these results with other biological data. Quantification of FMOD can potentially be of help in the diagnosis of phenotypically complex LPDs.

HemaSphere : open access journal of the European Hematology Association 5(12), e657 (2021). doi:10.1097/HS9.0000000000000657, Published by Wolters Kluwer Health, [Philadelphia, Pennsylvania]

OBJECTIVE Somatic mutations in ASXL1 seem to have a negative prognostic impact in patients with several myeloid neoplasms, including myelofibrosis (MF). The aim of this work was to determine the prevalence and profile of ASXL1 mutations in MF. METHODS We analyzed mutations in ASXL1 in 70 consecutive MF patients from 8 Spanish hospitals by means of Sanger sequencing, as well as JAK2, CALR, and MPL mutations. RESULTS ASXL1 mutations were found in 16/70 (23%) of cases, most commonly p.Gly646TrpfsX12 (5/16). Most mutations (13/16) were frameshift mutations. Of 54 ASXL1- wild-type patients, 32 (59%) had at least one single nucleotide polymorphism (SNP), 27 of them had g.78128C>T, g.79017A>C, and g.79085T>C [triple SNP (TSNP) patients]. The 5-yr overall survival probability of TSNP patients was 67% (95% CI, 43-91%) vs. 90% (95% CI, 77-100%) in ASXL1-WT patients (P = 0.152). CONCLUSION ASXL1 mutations were found in 23% of cases, p.Gly646TrpfsX12 being the most frequent. About 85% of mutations were found only in individual cases and 46% had not previously been reported, a pattern also seen in other series. Fifty percent of ASXL1-WT patients had a combination of three specific SNPs that might have a prognostic correlation that needs to be determined in larger series.

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.

// 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.

We read with interest the article by Flammiger et al. [1] on the study of a series of patients with aberrant T-cell phenotype of unclear significance by flow cytometry (FC). They divided their case...

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 ...

Fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). We have analyzed the role of plasma membrane transporters in nucleoside-derived drug bioavailability and action in CLL cells. Among the known plasma membrane transporters, we have previously observed a significant correlation between fludarabine uptake via ENT carriers and ex vivo sensitivity of CLL cells to fludarabine, although mRNA amounts of the equilibrative nucleoside transporters hENT1 and hENT2 do not show any predictive response to treatment. In this study, using polyclonal monospecific antibodies we have observed a significant correlation between the expression of hENT2 by Western blot and fludarabine uptake via hENT carriers and also with ex vivo sensitivity of CLL cells to fludarabine. These results suggest that the equilibrative nucleoside transporter hENT2 plays a role in fludarabine responsiveness in CLL patients.

Survivin is a member of the inhibitor of apoptosis protein family that is expressed in G2/M phase. Survivin is overexpressed and associated with parameters of poor prognosis in different human tumors. The role of survivin in the pathogenesis of mantle cell lymphoma (MCL) was examined in a series of typical and blastoid tumors. Survivin was detected as a nuclear pattern in a variable number of tumor cells. Mitotic figures were always positive with a strong delineation of the chromosomes. Western blot analysis confirmed the presence of survivin only in nuclear fractions. Protein expression detected by immunohistochemistry correlated with mRNA levels analyzed by quantitative real-time reverse transcription-polymerase chain reaction (P < 0.0001). Survivin expression levels were higher in blastoid MCL variants (P < 0.0001) and were associated with the proliferative activity (P = 0.001), but not with the ploidy status of the tumors. The number of apoptotic cells was independent of survivin or Ki-67 expression. Overall survival was significantly shorter in patients with high survivin expression. However, in a multivariate analysis, proliferative index was a better predictor of survival than survivin score. These findings indicate that survivin is commonly expressed in MCL with a nuclear and mitotic pattern. The expression levels are strongly associated with the proliferative activity of the tumors and the survival of the patients, suggesting a potential role in cell cycle regulation and tumor progression.

The mutational status of immunoglobulin heavy-chain variable-region (IgVH) genes in the leukemic cells of chronic lymphocytic leukemia (CLL) is an important prognostic factor in the disease. We investigated whether the expression of ZAP-70 by CLL cells correlated with the IgVH mutational status, disease progression, and survival.The expression of ZAP-70 was analyzed in T-cell and B-cell lines and in peripheral-blood samples from 56 patients with CLL with the use of flow cytometry, Western blotting, and immunohistochemistry. The results were correlated with the IgVH mutational status and clinical outcome.ZAP-70 was detected by flow-cytometric analysis in cells of T-cell lineage and in leukemic cells from 32 of 56 patients with CLL. In all patients in whom at least 20 percent of the leukemic cells were positive for ZAP-70, IgVH was unmutated, whereas IgVH mutations were found in 21 of 24 patients in whom less than 20 percent of the leukemic cells were positive for ZAP-70 (P0.001). Concordant results were obtained when ZAP-70 expression was assessed by immunohistochemistry or Western blotting. The level of ZAP-70 expression did not change over time (median, 37 months) in sequential samples from 30 patients with CLL. Patients with Binet stage A CLL who had at least 20 percent ZAP-70-positive leukemic cells had more rapid progression and poorer survival than those with less than 20 percent ZAP-70-positive cells.Among patients with CLL, expression of ZAP-70, as detected by flow-cytometric analysis, correlated with IgVH mutational status, disease progression, and survival.

The role of Bax and Bak, 2 proapoptotic proteins of the Bcl-2 family, was analyzed in primary B-cell chronic lymphocytic leukemia (CLL) cells following in vitro treatment with fludarabine, dexamethasone, or the combination of fludarabine with cyclophosphamide and mitoxantrone (FCM). A strong correlation was found between the number of apoptotic cells and the percentage of cells stained with antibodies recognizing conformational changes of Bax (n = 33;r = 0.836; P < .001) or Bak (n = 10;r = 0.948; P

Dear Editor, Chronic myelomonocytic leukemia (CMML) has been classified into a new category of myelodysplastic/myeloproliferative diseases (MDS/MPN) according to the last WHO classification of myeloid malignancies [1]. However, some patients with CMML show proliferative features (MP-CMML) such as leukocytosis and organomegaly, making them clinically and biologically similar to myeloproliferative neoplasms (MPN). At biological level, JAK2V617F mutations in MP-CMML have been described at a lower frequency than in MPN [2]. Recently, CALR gene mutations have been described in 67–88 % JAK2V617F and MPL-negative MPN; these cases also have a good prognosis [3–7]. The role of CALR mutations in CMML is not well known, and the few studies carried out so far have not focused on MP subtype [3, 4, 8]. The objective of this study was to characterize the frequency, type, and prognostic implications of CALR mutations in MP-CMML. CALR exon 9 mutations were studied in 30 patients (22 males, median age 66 years) diagnosed of CMML according toWHO classification [1], and all of them belonged to MP-CMML according to the French-American-British Co-operative Leukaemia Group (Table 1). All patients provided informed consent for sample collection. Biological analyses were carried out in accordance with the Declaration of Helsinki. Mutation analysis was performed as previously described [3]. An informative result was obtained in 29/30 patients, and no CALR exon 9 mutation was found. Although a small percentage of mutated MP-CMML patients was expected [4], no mutations were detected in the samples studied. These results are in concordance with those of Klampfl T et al. [3] and Hou et al. [8], who found no CALR mutations in 64 and 48 CMML patients, respectively (subtype not specified), but they do differ from the results of Nangalia et al., who found 1/33 CMML mutated patient (subtype not specified) [4]. There is also another study [9] of other MDS/ MPN groups (atypical CML and unclassifiable MDS/MPN) where CALR mutations were not seen in either entity (n=30). Therefore, outside essential thrombocythemia and primary myelofibrosis, CALR mutations are only found in (1) a few patients with MDS/MPN, most of them defined as having refractory anemia with ring sideroblasts associated with marked thrombocytosis, even though the percentage described is very variable depending on the series (0 to 12.5 %) [3, 4, 10] and (2) a few MDS patients, mainly with refractory anemia with ring sideroblasts, refractory anemia, and refractory anemia with excess blasts [3, 4, 8, 10]. This observation could indicate a relationship between CALR mutations and the thrombocytosis phenotype within myeloid neoplasms, suggesting that calreticulin mutations primarily affect the biology of megakaryocytes [11]. L. Zamora (*) :M. Cabezon : S. Marce : L. Palomo : F. Milla : E. Feliu : B. Xicoy ICO Badalona, Hospital Germans Trias i Pujol, Institut de Recerca contra la Leucemia Josep Carreras, Badalona, Spain e-mail: lzamora@iconcologia.net

Mechanisms involving the in vitro effect of rituximab in cells from 55 patients with B-cell lymphoproliferative disorders were investigated. No cytotoxic effect was observed when cells were incubated with rituximab alone, but in the presence of human AB serum rituximab induced complement-dependent cell death (R-CDC). A cytotoxic effect was observed in cells from 9 of 33 patients with B-cell chronic lymphocytic leukemia, 16 of 16 patients with mantle-cell lymphoma, 4 of 4 patients with follicular lymphoma, and 2 of 2 patients with hairy-cell leukemia. R-CDC was observed in cells from patients expressing more than 50 × 103 CD20 molecules per cell, and directly correlated with the number of CD20 molecules per cell. Preincubation with anti-CD59 increased the cytotoxic effect of rituximab and sensitized cells from nonsensitive cases. Neither cleavage of poly-ADP ribose polymerase (PARP) nor activation of caspase-3 was observed in R-CDC. In addition, no cells with a hypodiploid DNA content were detected and R-CDC was not prevented by a broad-spectrum caspase inhibitor, suggesting a caspase-independent mechanism. Incubation with rituximab in the presence of AB serum induced a rapid and intense production of reactive oxygen species (ROS). R-CDC was blocked by the incubation of cells with N-acetyl-L-cysteine (NAC) or Tiron, 2 ROS scavengers, indicating that the cytotoxic effect was due to the generation of superoxide (O2−) radicals. In conclusion, the results of the present study suggest that CD20, CD59, and complement have a role in the in vitro cytotoxic effect of rituximab, which is mediated by a caspase-independent process that involves ROS generation.

Introduction In the last years genome wide profilings have identified recurrent Copy Number Alterations (CNA) in genes potentially involved in the pathogenesis of Acute Lymphoblastic Leukemia (ALL). These studies have identified deletions in B-cell development genes (IKZF1, EBF1, PAX5, TCF3, etc.), cell cycle regulation genes (CDKN2A/B, RB1, TP53, etc.), glucocorticoid resistance genes (BTG1, CREBBP) and growth factor receptors genes (CRLF2, CSF2RA, IL3RA) among others. Some of these CNA (i.e. IKZF1, CDKN2A, CRLF2) have been reported to have prognostic significance in several pediatric series but there are very few data regarding their impact in B-lineage adult ALL. Our aim was to analyze the frequency and prognostic significance of CNA in a series of 125 B-lineage adult ALL patients treated according to risk-adapted protocols from the Spanish PETHEMA Group. Methods Bone marrow or peripheral blood (with significant blast burden) samples from 125 B-lineage adult ALL patients enrolled in risk-adapted protocols from the PETHEMA Group were analyzed at diagnosis. 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. Results The median age [range] was 40 [15-74] years, 71 (57%) males, median WBC count 12.11 x109/L [0.4-388]. Immunophenotype: pro-B 14 (11%), common 71 (58%), pre-B 26 (21%), mature-B 10 (8%), unavailable 2 (2%). Cytogenetics: normal 16 (13%), hyperdiploid 6 (5%), hypodiploid 2 (2%), t(9:22) 20 (16%), t(1;19) 8 (6%), 11q23/MLL 11 (9%), 8q24/C-MYC 7 (5%), complex 1 (1%), iAMP21 2 (2%), other translocations or deletions 31 (25%), no growth 20 (16%). CNA frequencies of the 125 patients are shown in the table. IKZF1 deletions were significantly associated with EBF1 deletions, high WBC count and Philadelphia (Ph) chromosome. In the IKZF1 deleted cohort whole gene deletions were as frequent as Ik6 isoforms (28% each). A high codeletion rate was detected in genes located in 9p (CDKN2A/B with PAX5, CDKN2A/B with hsa-miR-31 and PAX5 with hsa-miR-31). CDKN2A/B also showed concomitant deletions with ETV6 while PAX5 showed codeletions with BTG1. CDKN2A/B and PAX5 deleted patients had higher WBC counts than non-deleted individuals. Clinical follow-up data was available for 123 patients of the whole series and for the 105 patients of the Ph-negative cohort. Multivariate analysis showed that advanced age, BTG1 deletions and EBF1 deletions were negative prognostic factors for achieving Complete Remission (CR) and WBC count and IKZF1 deletions significantly reduced CR duration in both cohorts. Interestingly, there were significant differences in relapse rates between whole and partial gene IKZF1 deletions. IKZF1 haploinsufficient patients had a probability of CR duration at 3 years of 83% ± 30% vs. 6% ± 12% of partial gene deletion carriers. Advanced age and IKZF1 deletions were predictors for overall survival in the Ph-negative cohort and age>30 years, IKZF1 deletions and hsa-miR-31 deletions were associated with poor prognosis in the whole series. Conclusions In B-lineage adult ALL, deletions of IKZF1, EBF1, BTG1 or hsa-miR-31 are markers with prognostic significance in addition to age and WBC count. Patients with partial IKZF1 gene deletions have a significantly higher probability of relapse than those with whole gene loss. These genetic abnormalities could help to better define prognostic subgroups in adult patients with B-lineage ALL. Supported by the grants PI10/01417 and RD12-0036-0029 from Instituto Carlos III and a grant from the Spanish Society of Hematology and Hemotherapy (2012). Disclosures: No relevant conflicts of interest to declare.

In up to 5–15% of studies of lymphoproliferative disorders (LPD), flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, two clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and nine TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (four IGH, 10 TCRγ), albeit nonconclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In eight IGH samples, the results of PCR techniques were noninformative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and nonconclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 International Clinical Cytometry Society

In up to 5-15% of studies of lymphoproliferative disorders (LPD) flow cytometry (FCM) or immunomorphologic methods cannot discriminate malignant from reactive processes. The aim of this work was to determine the usefulness of PCR for solving these diagnostic uncertainties. We analyzed IGH and TCRγ genes by PCR in 106 samples with inconclusive FCM results. A clonal result was registered in 36/106 studies, with a LPD being confirmed in 27 (75%) of these cases. Specifically, 9/9 IGH clonal and 16/25 TCRγ clonal results were finally diagnosed with LPD. Additionally, 2 clonal TCRγ samples with suspicion of undefined LPD were finally diagnosed with T LPD. Although polyclonal results were obtained in 47 of the cases studied (38 IGH and 9 TCRγ), hematologic neoplasms were diagnosed in 4/38 IGH polyclonal and in 1/9 TCRγ polyclonal studies. There were also 14 PCR polyclonal results (4 IGH, 10 TCRγ), albeit non-conclusive. Of these, 2/4 were eventually diagnosed with B-cell lymphoma and 3/10 with T-cell LPD. In 8 IGH samples the results of PCR techniques were non-informative but in 3/8 cases a B lymphoma was finally confirmed. We concluded that PCR is a useful technique to identify LPD when FCM is inconclusive. A PCR clonal B result is indicative of malignancy but IGH polyclonal and non-conclusive results do not exclude lymphoid neoplasms. Interpretation of T-cell clonality should be based on all the available clinical and analytical data. © 2013 Clinical Cytometry Society.

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.

Background and objectives Tyrosine kinase inhibitors (TKI) have improved the management of patients with chronic myeloid leukemia (CML). However, a significant proportion of patients do not achieve the optimal response or are resistant to TKI. ABL kinase domain mutations have been extensively implicated in the pathogenesis of TKI resistance. Treatment with second-generation TKI has produced high rates of hematologic and cytogenetic responses in mutated ABL patients. The aim of this study was to determine the type and frequency of ABL mutations in patients who were resistant to imatinib or had lost the response, and to analyze the effect of second-generation TKI on their outcome. Patients and methods The presence of ABL mutations in 45 CML patients resistant to imatinib was evaluated by direct sequencing and was correlated with the results of the cytogenetic study (performed in 39 cases). The outcome of these patients after therapy with nilotinib or dasatinib was analyzed. Results ABL mutations were detected in 14 out of 45 resistant patients. Patients with clonal cytogenetic evolution tended to develop mutations more frequently than those without clonal evolution. Nine out of the 15 patients with ABL mutation responded to a treatment switch to nilotinib (n = 4), dasatinib (n = 2), interferon (n = 1) or hematopoietic stem cell transplantation (n = 2). Conclusion The frequency of ABL mutations in CML patients resistant to imatinib is high and is more frequent among those with clonal cytogenetic evolution. The change to second-generation TKI can overcome imatinib resistance in most of the mutated patients.

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.

Nucleoside transporters might play a relevant role in the intracellular targeting of many nucleoside analogs used in anticancer therapy. Two gene families (SLC28 and SLC29) encode the two types of human nucleoside transporters, concentrative nucleoside transporter (CNT) and equilibrative nucleoside transporter (ENT) proteins. Chronic lymphocytic leukemia (CLL) cells express both SLC28- and SLC29-related mRNA, although transport function seems to be mostly related to ENT-type transporters. Here we have analyzed the role of nucleoside transporters in nucleoside-derived drug bioavailability and action in mantle cell lymphoma (MCL) cells.The relative amounts of hENT1 and hENT2-related mRNA and protein were analyzed in five MCL cell lines and 20 primary MCL tumors by real-time quantitative reverse transcriptase polymerase chain reaction and western blots. Cell viability, measured by annexin V-FITC staining, and nucleoside-derived drug transport were also studied.MCL cells express higher levels of hENT1 protein than do CLL cells, and a good correlation was found between protein and mRNA levels of hENT1, thus indirectly suggesting that hENT1 might be transcriptionally regulated in MCL cells. More importantly, a significant correlation between these two parameters, drug uptake and sensitivity to gemcitabine, was also observed.These results further support the concept that nucleoside transporters are implicated in the therapeutic response to nucleoside analogs, and suggest a particular and novel role for hENT1 in the genotoxic response to selected nucleoside analogs, such as gemcitabine, in MCL cells.

Purpose: To determine the methylthioadenosine phosphorylase (MTAP) gene alterations in mantle cell lymphoma (MCL) and to investigate whether the targeted inactivation of the alternative de novo AMP synthesis pathway may be a useful therapeutic strategy in tumors with inactivation of this enzyme. Experimental Design: MTAP gene deletion and protein expression were studied in 64 and 52 primary MCL, respectively, and the results were correlated with clinical behavior. Five MCL cell lines were analyzed for MTAP expression and for the in vitro sensitivity to l-alanosine, an inhibitor of adenylosuccinate synthetase, and hence de novo AMP synthesis. Results: No protein expression was detected in 8 of 52 (15%) tumors and one cell line (Granta 519). Six of these MTAP negative tumors and Granta 519 cell line had a codeletion of MTAP and p16 genes; one case showed a deletion of MTAP, but not p16, and one tumor had no deletions in neither of these genes. Patients with MTAP deletions had a significant shorter overall survival (mean, 16.1 months) than patients with wild-type MTAP (mean, 63.6 months; P < 0.0001). l-Alanosine induced cytotoxicity and activation of the intrinsic mitochondrial-dependent apoptotic pathway in MCL cells. 9-β-d-Erythrofuranosyladenine, an analogue of 5′-methylthioadenosine, selectively rescued MTAP-positive cells from l-alanosine toxicity. Conclusions: MTAP gene deletion and lack of protein expression are associated with poor prognosis in MCL and might identify patients who might benefit from treatment with de novo AMP synthesis pathway–targeted therapies.

Mantle cell lymphoma (MCL) is a mature B-cell proliferation characterized by the presence of translocation t(11;14)(q13;q32), an aggressive clinical course, and poor response to chemotherapy. The majority of drugs currently used in the treatment of lymphoproliferative disorders induce cell death by triggering apoptosis, but few data concerning drug-induced apoptosis in MCL have been reported. We have analysed the mechanisms of drug-induced cell death in four cell lines with the t(11;14) and in primary cells from 10 patients with MCL. Mitoxantrone, a topoisomerase II inhibitor, induced a strong cytotoxic effect in three cell lines (JVM-2, REC-1, and Granta 519), and in primary MCL cells. This cytotoxic effect due to apoptosis induction was observed despite the presence of either p53 or ATM abnormalities. However, no cytotoxic effect was detected after incubation with DNA-damaging agents in the NCEB-1 cell line, carrying p53 and ATM alterations, despite the presence of functional mitochondrial machinery. These results support that mitoxantrone can be effective in the treatment of MCL but that this activity requires the integrity of functional DNA-damage response genes.

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.

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.

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 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.

High levels of Mcl-1 have been related to a poor response to therapy in patients with CLL. Genetic polymorphisms in the promoter region of the Mcl-1 gene consisting in insertions of 6 (+6) or 18 (+18) bp have been described in 30% to 60% of patients with CLL. The biological and clinical translation of such polymorphisms is controversial. We have analysed this issue in a cohort of newly diagnosed patients with CLL (14 F/ 46 M; 80% in B or C Binet’s stage), treated with a combination of fludarabine (F), cyclophospamide (C) and mitoxantrone (M). Polymorphisms were correlated with: Mcl-1 RNA expression as assessed by quantitative PCR, ex vivo cytotoxicity against F, C, and M, alone or in combination, using the MTT assay, and clinical response to treatment. The frequency of the wild type (wt), +6, and +18 allel was 56%, 12%, and 32 %, respectively (table). Twenty-one of 60 (35%) patients were homozygous for wt allel. Quantification of Mcl-1 RNA expression was performed in 18 patients. Wt/wt patients (n=6) tended to have lower Mcl-1 gene expression than the remaining patients (1.0 AU ±0.4 vs 2.2 AU ±2.8). Ex vivo cytotoxicity was different according to the Mcl-1 genotypes for F, M, F+M, and C+F, but not for FCM. The highest cytotoxicity was observed in patients with +18/+18 genotype (n=7) while those with wt/wt (n=11) presented intermediate cytotoxicity and those with other genotypes (n=17) had the lowest cytotoxicity. Twenty-eight patients achieved CR, 22 PR, and 5 failed to respond. No relationship was found between Mcl-1 polymorphisms and response to therapy (table). Interestingly, however, the ex vivo response to F (58.2% ±16 vs 45.7% ±16, p=0.05) and F+M (87% ±9 vs 76% ±15.5, p=0.04) was greater in the 13 patients who achieved MRD-negative CR. After a median follow up of 28 months (range: 6–53), 16 of 38 patients have progressed. No relationship was found between Mcl-1 polymorphism and progression after treatment. In summary, in this series of patients with advanced CLL homogeneously treated the frequency of insertions in the promoter region of Mcl-1 gene was high. Although Mcl-1 polymorphisms were associated with the ex vivo response to antileukemic drugs, they did not correlate with response to treatment and disease progression. Distribution of Mcl-1 Polymorphisms and Response to Treatment Allel Allelic Frequency Genotype Patients CR (MRD- CR) wt/wt 21 (35%) 9 ( 4 ) wt 68 (56%) +6/+6 2 ( 3%) 0 ( 0 ) +18/+18 7 (12%) 4 ( 3 ) +6 14 (12%) wt/+6 6 (10%) 4 ( 2 ) wt/+18 20 (33%) 9 ( 3 ) +18 38 (32%) +6/+18 4 ( 7%) 2 ( 1 )

Nucleoside derivatives are widely employed in the treatment of hematological malignancies. In particular, fludarabine is considered the treatment of choice for most patients with chronic lymphocytic leukemia (CLL). Anticancer therapy using nucleoside-derived analogs is dependent on drug uptake and metabolic activation. Although some enzymes have been shown to be suitable biomarkers of drug metabolism and may determine response to therapy, the role of transporters in determining nucleoside-derived drug bioavailability is less known. The uptake of these drugs into cells is mediated by one or more of the different nucleoside transporters that have recently been cloned. These proteins belong to two non-related gene families, SLC28 and SLC29, encoding CNT (Concentrative Nucleoside Transporters) and ENT (Equilibrative Nucleoside Transporters) proteins, respectively. In primary CLL cells we have described a high variability in ENT1, ENT2, CNT2 and CNT3 mRNA expression, whereas no CNT1 mRNA is detected. We have also shown that, in CLL cells, the uptake of fludarabine relies mainly ENT-type carriers and that ex vivo sensitivity significantly correlates with this ENT mediated uptake. In order to elucidate the role of ENT1 and ENT2 in the uptake and cytotoxicity of fludarabine, we have analyzed ENT1 and ENT2 expression by real time RT-PCR and Western Blot in cells from 19 CLL patients. ENT1 and ENT2 mRNA expression showed a high variability. ENT2 mRNA levels showed a range of variability of nearly 4-fold, whereas ENT1 variability was of 19-fold. Polyclonal antibodies against ENT1 and ENT2 consistently identified a single band of 50–55 kDa in all samples. Semiquantitative analysis of ENT1 and ENT2 expression was achieved by calculating the densitometry ratios versus α-tubulin, in a range of protein concentrations in which densitometric signal has been previously shown to be linear. Contrary to the mRNA expression, Western Blot analysis of ENT1 showed much less variability among patients (three-fold) than ENT2 (11-fold). The amounts of ENT1 protein did not significantly correlate with ENT1 mRNA expression, transporter function, or ex vivo fludarabine cytotoxicity. In contrast, although ENT2 protein does not bear any significant relationship with ENT2 mRNA, its expression correlates with transport activity. Moreover, ex-vivo sensitivity to fludarabine, at a pharmacological concentration (7.5 μM), directly correlated with the amount of ENT2 protein. Similar correlations were observed when the drug was tested at 3 and 15 μM. In summary, this study strongly suggests that fludarabine action on CLL cells is determined by the equilibrative nucleoside transporter ENT2. This is the first time ENT2 has been implicated in the therapeutic response to a nucleoside analog and might be useful to predict fluradabine response in CLL patients.