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The karyotype of bone-marrow cells at the time of diagnosis is one of the most important prognostic factors in patients with myelodysplastic syndromes (MDS). In some cases, the acquisition of additional genetic aberrations (clonal evolution [CE]) associated with clinical progression may occur during the disease. We analyzed a cohort of 469 MDS patients using a combination of molecular cytogenomic methods to identify cryptic aberrations and to assess their potential role in CE. We confirmed CE in 36 (8%) patients. The analysis of bone-marrow samples with a combination of cytogenomic methods at diagnosis and after CE identified 214 chromosomal aberrations. The early genetic changes in the diagnostic samples were frequently MDS specific (17 MDS-specific/57 early changes). Most progression-related aberrations identified after CE were not MDS specific (131 non-MDS-specific/155 progression-related changes). Copy number neutral loss of heterozygosity (CN-LOH) was detected in 19% of patients. MDS-specific CN-LOH (4q, 17p) was identified in three patients, and probably pathogenic homozygous mutations were found in TET2 (4q24) and TP53 (17p13.1) genes. We observed a statistically significant difference in overall survival (OS) between the groups of patients divided according to their diagnostic cytogenomic findings, with worse OS in the group with complex karyotypes (P = .021). A combination of cytogenomic methods allowed us to detect many cryptic genomic changes and identify genes and genomic regions that may represent therapeutic targets in patients with progressive MDS.

Deletion 20q is a recurrent abnormality in myeloid malignancies. In our previous study, we identified fusion of the additional sex combs-like 1 (ASXL1) and teashirt zinc finger homeobox 2 genes in a patient with myelodysplastic syndrome. The objective of this study was to determine the frequency of ASXL1 breakpoints in a cohort of 36 patients with deletion 20q as the sole cytogenetic aberration. A combination of molecular cytogenetic methods was used to confirm ASXL1 gene alterations in 19 of the 36 patients, and the determination of ASXL1 gene changes in patients with deletion 20q revealed clinical and prognostic impacts.

MDS with complex chromosomal aberrations (CCA) are characterized by short survival and a high rate of transformation to AML. A comprehensive genome-wide analysis of bone-marrow cells of 157 adults with newly diagnosed MDS and CCA revealed a large spectrum of nonrandom genomic changes related to the advanced stages of MDS. Chromosome shattering, probably resulting from chromothripsis, was found in 47% of patients. Deleted chromosome 5 was unstable and often involved in different types of cryptic unbalanced rearrangements. No true monosomy 5 was observed. Patients with CCA involving deleted chromosome 5 had an extremely poor prognosis (median overall survival, 2 months).

Complex karyotypes are seen in approximately 20% of patients with myelodysplastic syndromes (MDS) and are associated with a high risk of transformation to acute myeloid leukemia and poor outcomes in patients. Copy number neutral loss of heterozygosity (CN-LOH, i.e., both copies of a chromosomal pair or their parts originate from one parent) might contribute to increased genomic instability in the bone-marrow cells of patients with MDS. The pathological potential of CN-LOH, which arises as a clonal aberration in a proportion of somatic cells, consists of tumor suppressor gene and oncogene homozygous mutations. The aim of our study was to evaluate the frequency of CN-LOH at 17p in bone-marrow cells of newly diagnosed MDS patients with complex chromosomal aberrations and to assess its correlation with mutations in the TP53 gene (17p13.1). CN-LOH was detected in 40 chromosomal regions in 21 (29%) of 72 patients analyzed. The changes in 27 of the 40 regions identified were sporadic. The most common finding was CN-LOH of the short arm of chromosome 17, which was detected in 13 (18%) of 72 patients. A mutational analysis confirmed the homozygous mutation of TP53 in all CN-LOH 17p patients, among which two frameshift mutations are not registered in the International Agency for Research on Cancer TP53 Database. CN-LOH 17p correlated with aggressive disease (median overall survival 4 months) and was strongly associated with a complex karyotype in the cohort studied, which might cause rapid disease progression in high-risk MDS. No other CN-LOH region previously recorded in MDS or AML patients (1p, 4q, 7q, 11q, 13q, 19q, 21q) was detected in our cohort of patients with complex karyotype examined at the diagnosis of MDS. The LOH region appeared to be balanced (i.e., with no DNA copy number change) when examined with conventional and molecular cytogenetic methods. Therefore, a microarray that detects single-nucleotide polymorphisms is an ideal method with which to identify and further characterize CN-LOH. Our data should specify the prognosis and should lead to the identification of potential targets for therapeutic interventions.

Karyotype represents one of the most valuable prognostic markers for MDS patients with profound impact on differential diagnosis and therapeutic decisions despite of the fact that it is a heterogenous patient population with diverse pathogenesis and clinical course. In approximately 10–20% of MDS patients complex chromosomal aberrations (CCA; three or more numerical/structural cytogenetic aberrations) are present at diagnosis and this finding is associated with the treatment resistance and poor outcome. Precise identifications of chromosomal regions involved in CCA could help in detection of cryptic, recurrent, prognostically relevant aberrations and in identification of candidate genes involved in leukemogenesis and progression of the disease. In this study we used various modifications of molecular cytogenetic techniques to specify CCA more precisely and to determine exact localization and frequency of chromosomal breakpoints. During the last seven years we examined bone marrow samples of 630 adults with MDS and in 81 of them (12,9%) complex karyotypes were found (in 73 patients at diagnosis and in 8 cases during the disease progression). Out of 73 patients with CCA at diagnosis were 43 males and 30 females, age range 22 to 82 years (median 65,5 years). Primary MDS was diagnosed in 63 (86.3%) of them, in 10 cases therapy related MDS was ascertained. Diagnoses according to the WHO classifications: MDS NS 3x, RA 2x, RA/RCMD 6x, RCMD 5x, RAEB I 20x, RAEB II 20x, MDS-AML 16x and RARS 1x. 63 patients died, 10 patients are still alive (four of them after stem cell transplatation). Chromosomal aberrations found by G-banding were verified by FISH with appropriate locus specific probes (Abbott-Vysis, Des Plaines, Illinois, USA), and by multiplex FISH (mFISH/mBAND) with the “XCyte” probe kits (MetaSystems, Altlussheim, Germany). Deletion of 5q31 region was proved in 63 patients (86.3%). In 29 cases mFISH analyses showed that parts of deleted No. 5 were translocated into other chromosomes. The most recurrent chromosomal partners of No. 5 in unbalanced translocations were Nos. 17 (9x), 12 (5x), 7 (5x), 2 (4x) and 3 (4x). Monosomy of No. 5 was confirmed in one case only, thus it was proved that monosomy 5 is not separated diagnostic unit in CCA. Except No. 5 the most frequently involved in CCA were chromosomes 7 (36x), 3 (33x), 12 (31x), 17 (31x) and 11 (23x). Presence of CCA at diagnosis was connected with poor response to the therapy and with short overall survival. For survival analysis patients were classified into three groups according to the molecular cytogenetic findings: del(5)(q31) and additional CCA – 33 cases, deleted chromosome 5 involved in CCA – 29 cases, CCA without deletion of 5q – 10 cases. The worst overall survival was found in group 2 (median 3 months), followed by group 1 (median 5 months) and 3 (median 6 months). Patients with MDS and CCA should be considered as a unique entity with extremely poor prognosis. Exact molecular cytogenetic analysis by multiplex FISH of all chromosomal aberrations which are involved in CCA will lead to selection of the patients into new prognostic groups. Exploring the underlying mechanism of leukemogenesis could improve the therapeutic outcome for these patients and could contribute to the development of a new targeted treatment strategy.

Introduction: Inmyelodysplastic syndromes (MDS) the karyotype is one of the most important predictor of disease advancement, response to the treatment and patients' outcome. Clonal cytogenetic abnormalities are detected in the bone marrow cells in approximately 50% MDS patients. The interstitial deletion of the long arm of chromosome 5-del(5q)-is the most common finding, accounting for roughly 30% of abnormal karyotypes. According to IPSS-R, MDS with isolated del(5q) are associated with a favorable clinical course and are recognized as a specific subtype of MDS. However in some cases, acquisition of additional genetic aberrations may occur during the course of the disease and it has been proved it negatively influences outcome of MDS patients. The aim of the study was: (1) to evaluate the frequency of cytogenetic clonal evolution during the course of the disease in MDS patients with isolated del(5q), (2) to analyze the pattern of acquired cytogenetic abnormalities, and (3) to assess the impact of clonal evolution on transformation to acute myeloid leukemia (AML) and/or overall survival (OS). Patients and Methods: A detailed retrospective and/or prospective genome-wide analysis of fixed bone-marrow cells of 184 adults with del(5q), identified with conventional G-banding at the diagnosis of MDS, was performed during the course of their disease. The chromosomal aberrations were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods using the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with CytoChip Cancer SNP 180K (BlueGnome, Cambridge, UK) or with Illumina Human CytoSNP-12 arrays (Illumina, San Diego, CA). Amplicon deep sequencing of TP53 mutations (exons 4-11) was performed on a Roche 454 GS Junior system (Roche, Indianapolis, IN) using oligonucleotide primer plate assays validated according to the IRON-II (Interlaboratory Robustness Of Next generation sequencing). Results: Cytogenetic clonal evolution was observed in 21/184 MDS patients with isolated del(5q) (11.4%). Abnormalities most frequently acquired during the course of the disease were non-balanced rearrangements involving chromosomes 5, 7, 3, 17, 12 and 11. Deleted chromosome 5 was highly unstable and was often involved in different types of cryptic unbalanced rearrangements (translocations, insertions). Fragmentation of 5q was present as well.Clonal evolution was frequently associated with TP53 mutations and/or unbalanced aberrations of 17p.The median time between the first and the last evaluation was 23 months (range 2-83 months). Clonal evolution was detected between 2 and 83 months (median 14 months) after first cytogenetic evaluation. Median OS was 35 months (range 6-89 months), median survival from the first emergence of cytogenetic clonal evolution was 7.5 months (range 1-31 months). Progression to RAEB 1 or RAEB 2 was detected in 10 patients, 11 patients transformed into AML. Conclusions: Clonal evolution was detected in 11.4% of patients with MDS and isolated del(5q). The pattern of acquired abnormalities was similar to the changes previously described in high risk MDS at primary evaluation. The emergence of specific clones with additional genetic aberrations frequently correlated with altered clinical parameters. Cytogenetic clonal evolution was strongly associated with higher frequency of loss of heterozygosity (LOH) of 17p and/or TP53 mutations, shorter OS, rapid disease progression and transformation to AML. This data substantiate a need for regular molecular cytogenetic monitoring to guide clinical treatment decision in MDS with isolated del(5q). This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, PRVOUK-P27/LF1/1, and MHCR 00023736. Disclosures No relevant conflicts of interest to declare.

Abstract 1623 Poster Board I-649 Clonal cytogenetic abnormalities are detected in approximately 50% of patients with primary MDS and have a strong impact on disease outcome. More prevalent are unbalanced chromosomal aberrations reflecting a gain or loss of genetic material, whereas balanced rearrangements are rather rare. The most recurrent structural karyotypic abnormality is an interstitial deletion of the long arm of chromosome 5 occurring in 30% of primary MDS cases, either as isolated aberration (10%) or combined with additional cytogenetic changes (20%). An isolated del(5q) is generally associated with a better prognosis, whereas patients with one or more additional chromosomal abnormalities have a significantly shorter overall survival. The worst prognosis is observed in patients with del(5q) occurring in the context of a complex chromosomal aberrations (CCA; 33 abnormalities). Identifications of chromosomal regions involved in CCA in patients with MDS is very important and could yield detection of cryptic, recurrent, prognostically relevant aberrations and identification of candidate genes involved especially in progression of the disease. Detailed molecular cytogenetic analyses of CCA might lead to better understanding of molecular etiology of MDS with its therapeutic consequences. The aim of this study was to assess prognostic relevance of CCA in a cohort of patients with primary MDS and del(5q), to evaluate involvement of specific chromosomes and/or chromosomal regions in CCA and to establish the exact localization of chromosomal breakpoints. During 2002 and 2009, we examined bone marrow samples of 720 adults with MDS. Out of these, a total of 70 patients with primary disease and del(5q) associated with the CCA were identified at the time of initial diagnosis - 32 women (46%) and 38 men (54%), with a median age of 66 years (range 22 - 83). Diagnoses according to the WHO classification were as follows: RA (3%), RCMD (14%), RAEB I (27%), RAEB II (35%), MDS-AML (17%), CMML (3%) and MDS-U (1%). For precise analyses of chromosomal regions and breakpoints involved in CCA, various modifications of molecular cytogenetic techniques were used: FISH with locus specific probes (Abbott Vysis), CGH, mFISH/mBAND (MetaSystems) and/or array CGH (BlueGnome, NimbleGene). In 40 cases, mFISH analyses showed that parts of deleted chromosome 5 were translocated to other chromosomes. Fragmentation of 5q was also frequently found. Interestingly, no patient with monosomy 5 was identified in this study. Even in patients with suspect monosomy 5 detected by conventional cytogenetics, further detailed molecular cytogenetic analyses revealed parts of chromosome 5 material were retained in means of insertions within CCA. The most recurrent partners of del(5q) in unbalanced translocations were chromosomes 12 (23%), 3 (21%) and 17 (21%). The most common chromosome 5 breakpoints were 5q33 (47%), 5q13 (39%) and 5q11 (13%), the most frequently encountered deletion was q13q33 (30%). Except the chromosome 5, most often in CCA involved chromosomes were 17 (53%), 12 (49%), 7 (47%), 3 (43%) and 11 (31%), and the most recurrent breakpoints were at regions 12p11 (16%), 7q11 (10%), 7q21 (9%) and 7p11 (9%). Finding of CCA at diagnosis were associated with poor response to the therapy and short overall survival (median 4 months). Patients were classified into two groups according to the results of molecular cytogenetic analyses: group A (n=30) – del(5q) and additional CCA, and group B (n=40) – deleted chromosome 5 involved in CCA. The shortest overall survival was found in group B (median 3 months versus 5 months in group A). In conclusion, the involvement of specific chromosomes and/or chromosomal regions in CCA associated with del(5q) is nonrandom. Deleted chromosome 5 is very unstable and is often involved in different types of cryptic unbalanced rearrangements (translocation, insertions). Monosomy of chromosome 5, quoted in the literature, does not actually exist in MDS. Patients with del(5q) involved in CCA should be considered as a unique entity with extremely poor prognosis. Supported by grants NR/9227-3, NR/9481-3, MZO VFN2005, MSM 0021620808 and MSM LC535 Disclosures No relevant conflicts of interest to declare.