Conventional G-banding Cytogenetics Research Articles

Impact of SNP array karyotyping on the diagnosis and the outcome of chronic myelomonocytic leukemia with low risk cytogenetic features or no metaphases.

Chronic myelomonocytic leukemia (CMML) is a clonal hematopoietic disorder with heterogeneous clinical, morphological and genetic characteristics. Clonal cytogenetic abnormalities are found in 20-30% of patients with CMML. Patients with low risk cytogenetic features (normal karyotype and isolated loss of Y chromosome) account for ∼80% of CMML patients and often fall into the low risk categories of CMML prognostic scores. We hypothesized that single nucleotide polymorphism arrays (SNP-A) karyotyping could detect cryptic chromosomal alterations with prognostic impact in these subgroup of patients. SNP-A were performed at diagnosis in 128 CMML patients with low risk karyotypes or uninformative results for conventional G-banding cytogenetics (CC). Copy number alterations (CNAs) and regions of copy number neutral loss of heterozygosity (CNN-LOH) were detected in 67% of patients. Recurrent CNAs included gains in regions 8p12 and 21q22 as well as losses in 10q21.1 and 12p13.2. Interstitial CNN-LOHs were recurrently detected in the following regions: 4q24-4q35, 7q32.1-7q36.3, and 11q13.3-11q25. Statistical analysis showed that some of the alterations detected by SNP-A associated with the patients' outcome. A shortened overall survival (OS) and progression free survival (PFS) was observed in cases where the affected size of the genome (considering CNAs and CNN-LOHs) was >11 Mb. In addition, presence of interstitial CNN-LOH was predictive of poor OS. Presence of CNAs (≥1) associated with poorer OS and PFS in the patients with myeloproliferative CMML. Overall, SNP-A analysis increased the diagnostic yield in patients with low risk cytogenetic features or uninformative CC and added prognostic value to this subset of patients.

Fluorescence in situ hybridization of TP53 for the detection of chromosome 17 abnormalities in myelodysplastic syndromes

Conventional G-banding cytogenetics (CC) detects chromosome 17 (chr17) abnormalities in 2% of patients with de novo myelodysplastic syndromes (MDS). We used CC and fluorescence in situ hybridization (FISH) (LSI p53/17p13.1) to assess deletion of 17p in 531 patients with de novo MDS from the Spanish Group of Hematological Cytogenetics. FISH detected − 17 or 17p abnormalities in 13 cases (2.6%) in whom no 17p abnormalities were revealed by CC: 0.9% of patients with a normal karyotype, 0% in non-informative cytogenetics, 50% of patients with a chr17 abnormality without loss of 17p and 4.7% of cases with an abnormal karyotype not involving chr17. Our results suggest that applying FISH of 17p13 to identify the number of copies of the TP53 gene could be beneficial in patients with a complex karyotype. We recommend using FISH of 17p13 in young patients with a normal karyotype or non-informative cytogenetics, and always in isolated del(17p)

Utility of SNP Arrays in Chronic Myelomonocytic Leukemia with Low Risk Cytogenetic Features or No Metaphases

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 DisclosuresXicoy:Celgene: Honoraria. Sole:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Genomic Microarray Alterations Add Prognostic Power to the IPSS-R in MDS with Normal Karyotype

BackgroundDisease karyotype is the parameter with the strongest prognostic impact in the IPSS-R. Around 50% of MDS patients have a normal karyotype. Conventional G-banding cytogenetics has a resolution of 5-10 Mb. SNP microarrays is a genomic array with a higher resolution than cytogenetics and allows the detection of small copy number alterations (CNA) and absence of heterozygosity (AOH).Our hypothesis is that in MDS patients with normal karyotype small cryptic alterations might exist, undetectable by conventional karyotyping, but with an impact on patient’s outcome. Our aim is to study a series of MDS patients with normal karyotype belonging to the IWG-PM consortium (MDS Foundation) by SNP microarrays. Herein, we describe the preliminary findings as recruitment is ongoing and complete results will be presented at the meeting.Patients and methodsWe have recruited microarray data of 125 MDS patients with normal karyotype from centers belonging to the IWG-PM of the MDS Foundation. All cases included were studied with SNP array from Affymetrix®.We collected clinical, biological and microarray data and we studied their impact on overall survival (OS) and acute myeloid leukemia (AML) evolution in 73 patients. The median age was 67.8 years (21.8-92.1), 51.2% were male and the most frequent subtype according 2008 WHO classification was refractory cytopenia with multilineage dysplasia (41.6%), followed by refractory anemia with excess of blasts-1 (RAEB-1) and -2, 18.4% and 16.8%, respectively. Median values at diagnosis were, for hemoglobin: 9.3g/dL, leukocytes count: 3.6x109/L, platelet count: 139x109/L, percentage of polymorphonuclear cells: 47%, and bone marrow blasts: 3.6%. Nineteen patients were analyzed with their matched germ-line control sample.The statistical analysis was performed with SPSS and R software. As level of significance, 5% (two-sided) was chosen. No adjustment for multiple testing was applied.ResultsFifty nine cases were found to be altered by microarrays (47.2%), with a median number of alterations of 1 (range: 1-8). CNA were detected in 36.8% of cases, in 8 cases CNA were larger than 5 Mb. AOH were detected in 18.4% of cases. The median size of the affected genome by CNA was 116 Kb, adding up CNA plus AOH was 24745.05Kb. There were significant differences, in distribution of FAB diagnosis and median percentage of bone marrow blasts, in patients altered by SNP arrays in comparison to patients with normal findings by this technique.The impact on the outcome has been studied in 73 patients. The median OS was 35.6 months. The results of SNP array analysis showed that alterations detected by microarrays had no significant impact on outcome (P=0.237 for OS and P=0.807 for AML evolution), but the median OS is higher in cases with a "real" (neither visible by cytogenetics nor submicroscopic alterations) normal karyotype (53.48 vs. 25.90 months). Of note, the presence of AOH has a strong impact on OS (53.48 vs. 16.13 months, P=0.004), less on AML (P=0.086); also its size, greater than 25Mb, has an impact on OS (P=0.012) and AML evolution (P=0.050).To compensate for differing risk distributions, we performed analyses adjusting for IPSS-R. Adjusted for risk, the microarray result adds prognostic power on OS (P=0.010) and AML evolution (P=0.059). As it was observed, the presence of AOH had prognostic impact on OS (P=0.008) and AML evolution (P=0.017). The affected genome size showed a significant impact on OS (P=0.006) and AML (P=0.024), four categories were defined: 0 Kb, >0-500 Kb, >500-20000 Kb and >20000 Kb.ConclusionsMDS patients with normal karyotypes and alterations detected by genomic microarrays have lower OS and shorter time to AML compared to those without CNA. While not significant in marginal analysis, it can be demonstrated if adjustment for IPSS-R is performed. Alterations and, particularly, AOH have an independent prognostic impact and add prognostic power to IPSS-R.AcknowledgmentsFinancial support: This work was supported, in part, by a grant from the Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras, Obra Social “La Caixa”, Celgene Spain and NHRI-EX103-10003NI,Taiwan.Special thanks to Dr. Al-Ali, Dr. Bäsecke, members of Grupo Español de SMD (GESMD) and MDS Foundation. DisclosuresNo relevant conflicts of interest to declare.

Immunophenotypic Features of Myeloid Stem Cell Neoplasms Associated with Monosomy 7

Introduction: Abnormalities involving chromosome 7 are common in myeloid stem cell neoplasms (MSN) including myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML) and are associated with poor prognosis. Immunophenotyping by flow cytometry is an important tool to identify abnormal myeloid blasts and assist diagnosis of MSN. However, immunophenotypic features of MSN with monosomy 7 or deletion 7q have not been described. Here we retrospectively analyzed the flow cytometry data of MSN cases with monosomy 7 or deletion 7q and summarized a characteristic immunophenotype that could help distinguish chromosome 7 anomalies from other cytogenetic abnormalities.Methods: Potential cases were identified by searching the UWMC pathology cytogenetic database from 2005 to 2013 for cases of MSN with monosomy 7 or deletion 7q detected by FISH or conventional G-banding cytogenetics. Only cases with concurrent flow cytometry analysis performed at UWMC Hematopathology Laboratory were included in this study. Flow cytometry was performed on a modified 4-laser, 10-color flow cytometer with the following antibodies used: CD4, CD5, CD7, CD13, CD14, CD15, CD16, CD19, CD33, CD34, CD38, CD45, CD56, CD64, CD71, CD117, CD123, and HLA-DR. Flow cytometry data were retrospectively analyzed to evaluate the antigen expression in myeloid blasts and maturing myelomonocytic cells.Results: 126 cases of MSN harboring monosomy 7 and 95 cases harboring deletion 7q, either as a single anomaly or in combination with other cytogenetic abnormalities, were identified that had concurrent flow cytometry data. 112 cases with monosomy 7 and 73 cases with deletion 7q had adequate myelomonocytic cells for evaluation and were included in the study.Increased CD14 on maturing myeloid cells was identified in 86 cases (77%) with monosomy 7. In addition to increased CD14 in the 86 cases, other immunophenotypic abnormalities were also identified, including presence of abnormal myeloid blasts (range 0-54%, median 7.3% of white cells); increased proportion of primitive blasts (CD34+/CD38-); aberrant expression of CD5 (22%), CD7 (72%), CD13 (48%), CD15 (19%), CD33 (50%), CD34 (37%), CD38 (57%), CD56 (41%), CD117 (54%), HLA-DR (59%) on the myeloid blasts; aberrant CD56 (43%) and CD64 (54%) expression on maturing myeloid cells; and aberrant CD56 expression on monocytes (41%). By contrast, only 6 cases with deletion 7q (8%) showed increased CD14 on maturing myeloid cells. Flow cytometry data from ten cases of regenerating marrow (day-28 marrow post therapy or post transplant) were also evaluated, and none of these cases showed increased CD14 on maturing myeloid cells. As previously reported, 10 of 15 cases receiving G-CSF therapy showed increased CD14 on myeloid cells, but without other immunophenotypic aberrancies.Conclusion: Our data demonstrate MSN cases with monosomy 7 were associated with a characteristic immunophenotypic profile, including increased CD14 on maturing myeloid components and aberrant expression of multiple antigens on the myeloid blasts. The aberrant immunophenotypic pattern may help identify patients with monosomy 7 at diagnosis even in the absence of abnormal myeloid blasts and may potentially help to monitor response following treatment. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Whole-Exome Sequencing in Myelodysplastic Syndromes with 5q Deletion

INTRODUCTIONMyelodysplastic syndromes (MDS) are a heterogeneous group of clonal myeloid stem cell disorders that are highly prevalent in elderly populations. MDS are characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and increased risk of transformation to acute myeloid leukemia (AML; 20–30% of patients with MDS). Around 50% of MDS patients carry at least one karyotypic aberration. The interstitial deletion of the long arm of chromosome 5 ([del(5q)] is the most common aberration, accounting for almost 30% of abnormal MDS karyotype. Various studies supports a favorable prognosis of MDS with isolated del(5q) with an excellent response to lenalidomide treatment. In order to describe the molecular events associated with MDS and del(5q) we performed whole-exome sequencing (WES)(assessing 334,378 exons) of tumor-normal paired samples from 20 MDS patients to unravel the genetic basis of MDS with del(5q). The analysis is ongoing and the complete results will be presented in the meeting.METHODSA total of 50 samples from 20 patients with MDS, with del(5q) were collected. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss del(5q)) and SNP arrays with Cytoscan HD (Affymetrix). These samples included: 20 tumor samples at diagnosis, 20 control samples and 10 samples after diagnosis, during lenalidomide treatment (5) or at the moment of relapse (5) in order to compare the genetic status before and during the treatment. Genomic DNA from tumor cells was obtained from bone marrow (BM) samples or from PB granulocytes. As a source of constitutional DNA we used CD3+T cells from each patient by isolating by magnetic-activated cell sorting.WES targeted capture was carried out on 7μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4.Libraries were sequenced on an Illumina HiSeq2000. Sequencing data will be analyzed using an in-house bioinformatics pipeline as previously reported.RESULTSOur preliminary analysis of these 20 new patients by WES confirmed our previous analyses with mutations in well described genes as ASXL1, JAK2 and TET2, but not in genes RUNX1, SF3B1 and SRSF2. In those patients we found two patients with missense mutation in TP53, one of the patients had an isolated del(5q) and is receiving lenalidomide treatment, and the other one had a complex karyotype. According to our prior analyses, in which 249 non-silent somatic variants were detected, we look forward to validate these mutations in this new series of patients.CONCLUSIONSWe envision to validate these previous results with the new sequencing data of more patients with MDS and del(5q). We expect to measure somatic mutations that vary in abundance after lenalidomide treatment, potentially identifying mutations associated with resistance or relapse.ACKNOWLEDGEMENTS: This work has been supported (in part) by a grants from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); 2014 SGR225 (GRE) Generalitat de Catalunya; Fundació Internacional Josep Carreras; Obra Social “la Caixa”; Sociedad Española de Hematología y Hemoterapia (SEHH)and Celgene Spain.FOOTNOTESRafael Bejar and Francesc Sole contributed equally. DisclosuresDíez-Campelo:Novartis, Celgene: Honoraria, Research Funding. Xicoy:Celgene: Honoraria. Cañizo:Celgene, Jansen-Cilag, Arry, Novartis: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding. sanchez-Garcia:Celgene: Honoraria, Research Funding. Bejar:Celgene: Membership on an entity’s Board of Directors or advisory committees; Genoptix Medical Laboratory: Consultancy, Honoraria, Licensed IP, no royalties Patents & Royalties, Membership on an entity’s Board of Directors or advisory committees. Sole:Celgene: Consultancy, Membership on an entity’s Board of Directors or advisory committees, Research Funding.

Interstitial 13q14 deletions detected in the karyotype and translocations with concomitant deletion at 13q14 in chronic lymphocytic leukemia: different genetic mechanisms but equivalent poorer clinical outcome.

Deletion of 13q14 as the sole abnormality is a good prognostic marker in chronic lymphocytic leukemia (CLL). Nonetheless, the prognostic value of reciprocal 13q14 translocations [t(13q)] with related 13q losses has not been fully elucidated. We described clinical and biological characteristics of 25 CLL patients with t(13q), and compared with 62 patients carrying interstitial del(13q) by conventional G-banding cytogenetics (CGC) [i-del(13q)] and 295 patients with del(13q) only detected by fluorescence in situ hybridization (FISH) [F-del(13q)]. Besides from the CLL FISH panel (D13S319, CEP12, ATM, TP53), we studied RB1 deletions in all t(13q) cases and a representative group of i-del(13q) and F-del(13q). We analyzed NOTCH1, SF3B1, and MYD88 mutations in t(13q) cases by Sanger sequencing. In all, 25 distinct t(13q) were described. All these cases showed D13S319 deletion while 32% also lost RB1. The median percentage of 13q-deleted nuclei did not differ from i-del(13q) patients (73% vs. 64%), but both were significantly higher than F-del(13q) (52%, P < 0.001). Moreover, t(13q) patients showed an increased incidence of biallelic del(13q) (52% vs. 11.3% and 14.9%, P < 0.001) and higher rates of concomitant 17p deletion (37.5% vs. 8.6% and 7.2%, P < 0.001). RB1 involvement was significantly higher in the i-del(13q) group (79%, P < 0.001). Two t(13q) patients (11.8%) carried NOTCH1 mutations. Time to first treatment in t(13q) and i-del(13q) was shorter than F-del(13q) (67, 44, and 137 months, P = 0.029), and preserved significance in the multivariate analysis. In conclusion, t(13q) and del(13q) patients detected by CGC constitute a subgroup within the 13q-deleted CLL patients associated with a worse clinical outcome.

Genetic abnormalities in chronic lymphocytic leukemia: where we are and where we go.

Chromosomal abnormalities in chronic lymphocytic leukemia (CLL) are detected in up to 80% of patients. Among them, deletions of 11q, 13q, 17p, and trisomy 12 have a known prognostic value and play an important role in CLL pathogenesis and evolution, determining patients outcome and therapeutic strategies. Standard methods used to identify these genomic aberrations include both conventional G-banding cytogenetics (CGC) and fluorescence in situ hybridization (FISH). Although FISH analyses have been implemented as the gold standard, CGC allows the identification of chromosomal translocations and complex karyotypes, the latest associated with poor outcome. Genomic arrays have a higher resolution that allows the detection of cryptic abnormalities, although these have not been fully implemented in routine laboratories. In the last years, next generation sequencing (NGS) methods have identified a wide range of gene mutations (e.g., TP53, NOTCH1, SF3B1, and BIRC3) which have improved our knowledge about CLL development, allowing us to refine both the prognostic subgroups and better therapeutic strategies. Clonal evolution has also recently arisen as a key point in CLL, integrating cytogenetic alterations and mutations in a dynamic model that improve our understanding about its clinical course and relapse.

Whole-Exome Sequencing In Myelodysplastic Syndromes With 5q- and Normal Karyotype

IntroductionMyelodysplastic Syndromes (MDS) are a heterogeneous group of clonal myeloid stem cells disorders with high prevalence in the elderly characterized by inefficient hematopoiesis, peripheral blood (PB) cytopenias, and an increased risk of transformation to acute myeloid leukemia (AML). The karyotype is the clinical parameter with the strongest prognostic impact according the IPSS-R (Greenberg et al., 2012). The most frequent cytogenetic alteration is the chromosome 5q deletion (del[5q]) which as a single anomaly, confers a good prognosis and predicts an excellent response to lenalidomide. Whether other genetic abnormalities routinely cooperate with del(5q) is not known. Whole-exome sequencing (WES) is a powerful tool to identify somatic mutations in protein coding genes that might cooperate with del(5q).In order to better understand the genetic basis of MDS with del(5q), we performed whole-exome sequencing (assessing 334,378 exons) of tumor-normal paired samples from 21 MDS patients. Herein we describe the preliminary findings. The analysis is ongoing and the complete results will be presented in the meeting. MethodsA total of 21 patients with MDS (16 with del(5q) as a sole abnormality, 3 with del(5q) and additional alterations and 2 with normal karyotype) were included in our study. We examined a total of 25 tumor samples (21 diagnostic bone marrow (BM) samples with matched CD3+ cells as a controls, additional BM samples from 3 patients during lenalidomide treatment and 1 bone marrow sample from a del(5q) patient after AML progression). DNA was extracted from BM samples and from isolated peripheral blood CD3+ cells (magnetic-activated cell sorting (MACS), MiltenyiBiotec GmbH, Germany). The purity of CD3+ cells was assessed by FC 500 flow cytometer (Beckman Coulter, Hialeah, Fl, USA). Only DNA that fulfilled quality controls required by WES were submitted. For each diagnostic sample, we performed Conventional G-banding cytogenetics and fluorescence in situ hybridization (FISH, to confirm or dismiss 5q deletions).Whole-exome targeted capture was carried out on 3 μg of genomic DNA, using the SureSelect Human Exome Kit 51Mb version 4 (Agilent Technologies, Inc., Santa Clara, CA, USA). The captured and amplified exome library was sequenced with 100 bp paired-end reads on an Illumina HiSeq2000. Whole-exome sequencing data were analyzed using an in-house bioinformatics pipeline as previously reported. Somatic mutations identified as alterations present in tumor but not in the matched CD3+ sample were validated by Sanger sequencing. ResultsIn our preliminary analysis of WES from 12 patients (10 patients with 5q- and 2 patients with normal karyotype), a total of 249 non-silent somatic variant candidates were identified, of which 146 were confirmed as somatic mutations. Recurrent mutations were observed in three genes (ASXL1, NBPF10 and SF3B1) in 3 different patients. Seven genes (HRNR, JAK2, POTEG, MUC5B, PHLDA, TTN, ZNF717) were mutated in two patients. Mutations in several genes known to be mutated in MDS (ASXL1, JAK2, RUNX1, SF3B1, SRSF2 and TET2) were also identified. Patients with the 5q deletion had an average of 11 mutations whereas patients with normal karyotype had a higher mean (14.5). Mutated genes identified in both groups were HRNR, JAK2, MUC5B, NBPF10 and SF3B1. No mutations in TP53 were detected in this subset. Pathway analysis of the complete list of somatically mutated genes will be carried out once all 21 patients are analyzed. The four in-treatment samples will be examined from their matched diagnostic samples. ConclusionsWhole-exome sequencing of largely del(5q) MDS patient samples identified both known and previously unreported somatic mutations. Analysis of additional samples will allow a more complete description of the genes and pathways that may cooperate with del(5q) in the development and progression of MDS. AcknowledgmentsFinancial support: This work has been supported (in part) by a grant from Instituto de Salud Carlos III, Ministerio de Sanidad y Consumo, Spain (PI 11/02010); by Red Temática de Investigación Cooperativa en Cáncer (RTICC, FEDER) (RD07/0020/2004; RD12/0036/0044); Acción COST BM0801: European Genomics and Epigenomics Study on MDS and AML; Sociedad Española de Hematología y Hemoterapia (SEHH) and MDS Celgene. FootnotesRafael Bejar and Francesc Sole contributed equally. Disclosures:Díez-Campelo:Novartis and Celgene: Honoraria, Research Funding. Cañizo:Celgene Jansen-Cilag Arry Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sanchez:Celgene: Honoraria, Research Funding. Bejar:Genoptix: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees. Solé:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding.

Response to lenalidomide in myelodysplastic syndromes with del(5q): influence of cytogenetics and mutations

Lenalidomide is an effective drug in low-risk myelodysplastic syndromes (MDS) with isolated del(5q), although not all patients respond. Studies have suggested a role for TP53 mutations and karyotype complexity in disease progression and outcome. In order to assess the impact of complex karyotypes on treatment response and disease progression in 52 lenalidomide-treated patients with del(5q) MDS, conventional G-banding cytogenetics (CC), single nucleotide polymorphism array (SNP-A), and genomic sequencing methods were used. SNP-A analysis (with control sample, lymphocytes CD3+, in 30 cases) revealed 5q losses in all cases. Other recurrent abnormalities were infrequent and were not associated with lenalidomide responsiveness. Low karyotype complexity (by CC) and a high baseline platelet count (>280×10(9) /l) were associated with the achievement of haematological response (P=0·020, P=0·013 respectively). Unmutated TP53 status showed a tendency for haematological response (P=0·061). Complete cytogenetic response was not observed in any of the mutated TP53 cases. By multivariate analysis, the most important predictor for lenalidomide treatment failure was a platelet count <280×10(9) /l (Odds Ratio=6·17, P=0·040). This study reveals the importance of a low baseline platelet count, karyotypic complexity and TP53 mutational status for response to lenalidomide treatment. It supports the molecular study of TP53 in MDS patients treated with lenalidomide.

Application of FISH 7q in MDS patients without monosomy 7 or 7q deletion by conventional G-banding cytogenetics: Does −7/7q− detection by FISH have prognostic value?

Chromosomal abnormalities are detected in 40–60% of patients with de novo myelodysplastic syndromes (MDS). This study used the FISH technique in 773 patients with de novo MDS without evidence of monosomy 7 (−7) or 7q deletion (7q−) by conventional G-banding cytogenetics (CC) to analyze their prognostic impact by FISH alone. FISH detected −7/7q− in 5.2% of patients. Presence of −7/7q− was associated with shorter overall survival than absence of such aberrations. Our results suggest that FISH 7q could be beneficial in patients with intermediate WHO morphologic risk stratification and no evidence of −7/7q− by CC.

Genomic arrays in chronic lymphocytic leukemia routine clinical practice: are we ready to substitute conventional cytogenetics and fluorescence in situ hybridization techniques?

Chronic lymphocytic leukemia (CLL) is characterized by a highly variable clinical course. Del(11q) and del(17p), routinely studied by conventional G-banding cytogenetics (CGC) and fluorescence in situ hybridization (FISH), have been related to progression and shorter overall survival. Recently, array-based karyotyping has gained acceptance as a high-resolution new tool for detecting genomic imbalances. The aim of the present study was to compare genomic arrays with CGC and FISH to ascertain whether the current techniques could be substituted in routine procedures. We analyzed 70 patients with CLL using the Cytogenetics Whole-Genome 2.7M Array and CytoScan HD Array (Affymetrix), CGC and FISH with the classical CLL panel. Whereas 31.4% and 68.6% of patients presented abnormalities when studied by CGC and FISH, respectively, these rates increased when arrays were also analyzed (78.6% and 80%). Although abnormality detection is higher when arrays are applied, one case with del(11q) and three with del(17p) were missed by genomic arrays due to their limited sensitivity. We consider that the complete substitution of CGC and FISH by genomic arrays in routine laboratories could negatively affect the management of some patients harboring 11q or 17p deletions. In conclusion, genomic arrays are valid to detect known and novel genomic imbalances in CLL, but should be maintained as a complementary tool to the current techniques.

Do the SLC25A38 or ALAS2 Genes Play a Role In the Phenotypic Expression of Ringed Sideroblasts In Acquired Myelodysplastic Syndrome?

AbstractAbstract 4954 Background.The SLC25A38 gene has recently been identified to play a role in the pathogenesis of congenital sideroblastic anemia (CSA). The erythroid specific mitochondrial carrier family protein SLC25A38 is important for the biosynthesis of heme. The ALAS2 gene is also frequently mutated in CSA. Refractory anemia with ringed sideroblasts (RARS) is an acquired myelodysplastic condition characterized by lineage dysplasia with an excess number of ringed sideroblasts in the marrow. A genetic cause for the expression of ringed sideroblasts in RARS or other myleodysplasias has not been clearly determined. We examine whether loss of function mutations in SLC25A38 or ALAS2 genes are associated with acquired myelodysplastic syndromes (MDS) with ringed sideroblasts. Methods.Participants had to have adequate banked DNA or bone marrow from diagnosis and meet WHO 2001 criteria of MDS and a high percentage of ringed sideroblasts. Medical records were retrospectively examined for patient demographics and outcomes. All diagnostic bone marrows were reviewed to confirm the diagnosis and the percentage of ringed sideroblasts and blasts was recorded. Cytogenetic findings were also recorded. DNA was extracted as needed by standard techniques. Coding exons and exon/intron boundaries of SLC25A38/ALAS2 were PCR-amplified using primers designed with Primer3 (http://frodo.wi.mit.edu/) from each affected individual. These products were then sequenced using the ABI 3130 xl electrophoresis instrument (Applied Biosystems). Sequence chromatograms were interpreted using the MutationSurveyor program from SoftGenetics, Inc., with gene annotations from GenBank looking for mutations predicted to result in loss of function. Results.12 samples were identified from patients diagnosed between 2003 and 2008. The diagnosis by WHO 2001 classification of myelodysplastic syndrome was refractory anemia with ringed sideroblasts [RARS] (n=7), refractory cytopenia with multilineage dysplasia (RCMD-RS] (n=4) and refractory anemia with ringed sideroblasts with thrombocytosis [RARS-T] (n=1). The median age at diagnosis was 82 years (range 58–94 years). Participants were males (n=9) and females (n=3). The clinical status was as follows: Remission (n = 1), Active Disease (n = 7), and Unknown (n = 4). Treatment provided to patients included transfusion supportive care only (n =2), erythropoietin (n= 3) and MDS directed drug therapy (n=1) and unknown (n=5). For those with blood counts available at diagnosis, the median white blood cell count was 8 × 109/L (range 4.7–10.9), the hemoglobin was 94 g/L (range 85–112) and the platelets were 327 × 109/L (range 3–951). The absolute neutrophil count was 5.3 × 109/L (range 3.1–7) and the absolute lymphocyte count was 1.9 × 109/L (range 0.7 – 2.2). The median percentage ringed sideroblast count in the diagnostic bone marrow was 51% (range 15–81%). Conventional G-banding cytogenetics showed a definitive abnormality in only one of the 12 patients. Mutations predicted to result in complete loss of function occurred in 0/12 in the SLC25A38 gene and 0/12 in the ALAS2 gene. One previously unreported variant of unknown significance at SLC25A38 E03 (cDNA position #239C>G; 80T>R) was identified in homozygous form in a patient with RARS and normal cytogenetics. Conclusions.Variations in the coding regions of SLC25A38 or ALAS2 genes were not obviously associated with mutations predicted to result in complete loss of function in this cohort of patients with acquired myelodysplastic syndromes with ringed sideroblasts. We did identify one unique variant in homozygous form but its significance is uncertain. We plan to expand this study to confirm these findings. Whilst loss of complete function of these genes does not appear to be associated with acquired ringed sideroblasts, we have not ruled out a contribution of functional mutations resulting in reduced expression of these genes. Further examination of this question may clarify the physiological understanding of ringed sideroblasts in acquired MDS, which in turn may represent a target for therapy. Disclosures:No relevant conflicts of interest to declare.

Early onset of acute lymphoblastic leukemia with MLL rearrangement after autologous stem cell transplantation for multiple myeloma

Dear Editor, We would like to present our experience with a patient with multiple myeloma (MM) who developed acute lymphoblastic leukemia (ALL) with mixed lineage leukemia gene (MLL) rearrangement only 1 year after administration of alkylating agents. A 61-year-old woman presented with fatigue and was introduced to our hospital in January 2007. Serum immunoelectrophoresis showed increased monoclonal IgD (115.0 mg/dL) and λ light chain, and urine immunoelectrophoresis showed increased monoclonal Bence–Jones protein (4,360 mg/g creatinine). The bone marrow (BM) was infiltrated by malignant plasma cells (50% of nucleated cells) and conventional G-banding cytogenetics revealed normal karyotype (46,XX [20/20]). A diagnosis of MM was made. After four cycles of high-dose dexamethasone, we mobilized and collected her peripheral blood stem cells using cyclophosphamide (4 g/m) and granulocyte-colonystimulating factor in July 2007. She underwent autologous stem cell transplantation (ASCT) in August 2007 with highdose melphalan (200 mg/m) as a conditioning regimen. She made recovery without severe complication. We evaluated the response of treatment as very good partial remission. In July 2008, although she had no symptom, investigation revealed pancytopenia. The BM was heavily infiltrated by blasts (94%) with the immunophenotype as B cell lymphoblasts: CD19+, CD79a+, κ−, l−. G-banding chromosomal cytogenetics revealed chromosomal abnormalities: 46,XX,t(1;11)(p32–34;q23) [4/6]. Fluorescence in situ hybridization (FISH) examination showed split signals of MLL gene. A diagnosis of B-ALL with MLL rearrangement was made. She was commenced on the JALSG 202-O protocol (Clinicaltrials.gov; study ID:NCT00131027) and achieved hematological complete remission. We performed IgH gene rearrangement studies on genomic DNA extracted from the BM aspirates at the time of diagnosis of MM and that of ALL. Southern blotting (SRL inc., Nakagyo-ku, Kyoto, Japan) test showed different monoclonal bands on DNA extracted from both BM aspirates (Fig. 1). From these findings, we concluded that her MM and ALL were derived from different clones. Lau et al. [1] reported a patient with MM that terminated in ALL. Lau indicated that two diseases arose from different clones by IgH gene rearrangement analysis. Cases of secondary ALL supposed to be Topoisomerase II inhibitor-related were previously reported [2], and Lau’s case was administered doxorubicin. Although we did not administer Topoisomerase II inhibitor, we detected chromosomal abnormality of 11q23 that is typically determined from cases of Topoisomerase II inhibitor-related leukemia [2]. To our knowledge, this is the first report describing alkylating-agent-related ALL with MLL rearrangement. Our hypothesis is that her lymphoid progenitor cells had initial instability or a mutation that cannot be detected by conventional G-banding test, and different types of DNA modification separately led to MM and ALL. We suppose modification leading to ALL was MLL rearrangement. This is consistent with the reports that MLL rearrangement is Ann Hematol (2009) 88:813–814 DOI 10.1007/s00277-008-0680-8