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Dicentric chromosomes (DCs) are considered markers of cancer in various malignancies. However, they can be overlooked when conventional analysis or multicolor fluorescence in situ hybridization (mFISH) is used to detect complex karyotypes. We analyzed the karyotypes of 114 patients with acute myeloid leukemia (AML) and complex karyotypes and verified the presence of monosomies by FISH using multi-centromeric probes. Monosomy was detected in 63% of patients by G-banding/mFISH and confirmed in 55% of patients by centromeric FISH. FISH analysis indicated a high frequency of DCs that were previously considered monosomies. In some cases, it was apparent that the derivative monocentric chromosome was a primary DC. DCs were formed mostly by chromosomes 17 and 20. In conclusion, chromosome loss and unbalanced translocation suggest the presence of a hidden DC or its previous existence. DCs undergo several stabilizing changes and can induce other chromosomal aberrations and/or the formation of new DCs. This can result in the clonal evolution of abnormal cells, which is considered an independent prognostic marker of an unfavorable disease course and short survival.

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.

Dicentric chromosomes (DCs) have been described in many hematological diseases, including acute myeloid leukemia (AML). They are markers of cancer and induce chromosomal instability, leading to the formation of other chromosomal aberrations and the clonal evolution of pathological cells. Our knowledge of the roles and behavior of human DCs is often derived from studies of induced DCs and cell lines. It is difficult to identify all the DCs in the karyotypes of patients because of the limitations of metaphase cytogenetic methods. The aim of this study was to revise the karyotypes of 20 AML patients in whom DCs were found with conventional G-banding or multicolor fluorescence in situ hybridization (mFISH) with (multi)centromeric probes and to characterize the DCs at the molecular cytogenetic level. FISH analyses confirmed 23 of the 29 expected DCs in 18 of 20 patients and identified 13 others that had not been detected cytogenetically. Fourteen DCs were altered by other chromosomal changes. In conclusion, karyotypes with DCs are usually very complex, and we have shown that they often contain more than one DC, which can be missed with conventional or mFISH methods. Our study indicates an association between number of DCs in karyotype and very short survival of patients.

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).

Introduction: Chromothripsis is a recently identified genomic instability phenomenon that plays a role in the genesis and progression of cancer. It is a one-step catastrophic genomic event involving multiple chromosomal breakages and random DNA rejoining. This genetic abnormality can affect an entire chromosome, a chromosomal arm, or a single chromosomal region. Chromothripsis is associated with highly complex karyotypes and a very poor prognosis, and has been detected in a wide range of tumor entities, including hematological malignancies. However, this complex genomic abnormality has not been comprehensively studied in patients with myelodysplastic syndromes (MDS). The aim of the study was to assess the incidence, associated genetic features, and clinical significance of chromothripsis in a large homogeneous cohort of patients newly diagnosed with high-risk MDS and complex karyotypes. Methods: A detailed genome-wide analysis of fixed bone-marrow cells from adults with complex karyotypes (≥ 3 aberrations), identified with conventional G-banding at the diagnosis of MDS, was performed. The complex rearrangements were studied with integrative genetic methodologies: fluorescence in situ hybridization (FISH) with Vysis DNA probes (Abbott, Des Plaines, IL), multicolor FISH (mFISH) and/or multicolor banding (mBAND) methods with the 24XCyte and the XCyte color kits (MetaSystems, Altlussheim, Germany), and array-based comparative genomic hybridization with CytoChip Cancer SNP 180K (Illumina, San Diego, CA) or the SurePrint G3 Cancer CGH+SNP 4x180K Microarray (Agilent, Santa Clara, CA). A mutational analysis of the TP53 gene was also performed in selected cases using amplicon-based deep sequencing on a 454 GS Junior System (Roche, Basel, Switzerland) or the TruSight Myeloid Sequencing Panel on MiSeq sequencing instruments (Illumina). Results: In total, 265 patients with complex karyotypes and newly diagnosed high-risk MDS were included (131 females, 134 males; median age, 70 years). The hallmarks of chromothripsis were detected in 67.7% of cases, in both the main clones and one or more subclones. At the cytogenetic level, chromothripsis was apparent as multiple deletions, insertions, ring chromosomes, amplification of individual genes or chromosomal regions, and/or the formation of chaotically reassembled chromosomes. Chromothripsis affected almost all the chromosomes, except the Y chromosome. The most frequently involved were chromosomes 5 (33.3% of events), 7 (28.1% of events), 17 (18.9% of events), 11 and 12 (15.6% of events each). In samples with signs of chromothripsis, the higher frequency of aberrations on chromosome 17p was observed (loss of heterozygosity [LOH], copy-number neutral LOH [cnLOH] and/or homozygous mutation of TP53) (p = 0.05). Patients with chromothripsis had significantly worse overall survival (OS; median, 3 months). We also investigated the effect of chromothripsis on the survival of 183 patients treated with azacytidine. In this cohort, the median OS of chromothripsis-positive patients was 10.1 months (mean, 12.2 months), whereas the median OS of chromothripsis-negative patients was 17.3 months (mean, 31.0 months). Conclusions: Our results demonstrate that chromothripsis is a frequent genomic abnormality in patients with high-risk MDS, which influences the patient's prognosis and disease biology. Chromothripsis was associated with a higher frequency of LOH/cnLOH 17p, rapid disease progression, and short survival. The adverse outcomes may be attributable to the effects on the functions of many important genes. The OS of patients with high-risk MDS with chromotripsis may be slightly improved by azacytidine treatment, but the prognosis of these patients remains very poor. Therefore, a better understanding of the mechanistic basis of chromothripsis is extremely important and could lead to the development of new treatment strategies based on drugs that target the genes present in amplified or deleted regions and/or the DNA damage response pathways. This study was supported by research projects RVO-VFN64165, GACR P302/12/G157, AZV 16-27790A , Progres Q26 and Q28/LF1, GACR 18-01687S, MHCR 00023736 and UNCE/MED/016 . Disclosures No relevant conflicts of interest to declare.

Gene amplification is a frequent genetic abnormality in solid tumors, and many oncogenes are activated in this way. In acute myeloid leukemia (AML), a frequent target of gene amplification is chromosome 11, particularly chromosome region 11q23, including the MLL (myeloid/lymphoid leukemia) gene. However, the number of other amplicons from the long arm of chromosome 11 has also been described. Duplication/amplification of chromosome 11 was found by cytogenetic methods in 10 of 119 newly diagnosed patients with AML. The amplification was presented as: amplification including only the 5' segment of the MLL gene (1 patient), trisomy 11 (3 patients), partial trisomy 11q (2 patients), isochromosome 11q (1 patient), and multiple amplification of specific regions (3 patients). In two cases, amplification involved parts of not only long arm but also of short arm of the chromosome 11: 11p15 and 11p11.1 to 11p13.

Unusual MLL gene rearrangements were found in bone marrow cells of four patients with acute myeloid leukemia. A combination of conventional and molecular cytogenetic methods were used to describe translocations t(9;12;11)(p22;p13;q23), t(11;19)(q23;p13.3), and t(10;11)(p12;23) and inverted insertion ins(10;11)(p12;q23.3q23.1). Partial nontandem duplication of the MLL gene was identified by reverse transcriptase-polymerase chain reaction in all cases. The duplication, which included MLL exons 2 through 8-9, was interrupted by a cryptic insertion of one or two exons from the respective MLL partner gene: MLLT10, MLLT3, or MLLT1.

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.

Introduction: Complex chromosomal aberrations (CCAs) are seen in approximately 20% of patients with newly-diagnosed MDS and are associated with poor prognosis. Bone marrow cells of MDS patients with CCAs are characterized by a high degree of genomic instability, which is connected with an increased risk of formation of different subclones with additional aberrations. The phenomenon of clonal evolution can be observed either at diagnosis or during the disease progression. At diagnosis it is manifested by the presence of two or more related subclones derived from one founder clone. These subclones can obtain a proliferative advantage leading to the clonal expansion. The aim of the study was to perform detailed genome-wide analyses of bone marrow cells of previously untreated MDS patients with CCAs, to investigate the clonal heterogeneity and to assess the frequency and clinical significance of related cytogenetic subclones with complex karyotypes. Methods: A comprehensive molecular cytogenetic analysis of fixed bone-marrow cells from 182 adults with CCAs (³3 aberrations) identified with conventional G-banding in the diagnosis of MDS was performed. The CCAs were studied through FISH with Vysis DNA probes (Abbott, Des Plaines, IL) and mFISH/mBAND methods (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). Results: Cytogenetic subclones were detected in 143 of 183 patients with complex karyotypes (78.6%). Among them, 98/183 cases (53.8%) displayed a defined number of subclones, whereas in 45/183 patients (24.7%) a precise definition of the individual subclones was not possible due to the high number of heterogeneous findings. Therefore these patients were subsumed as having composite karyotype. In 138/143 cases with clonal heterogeneity (94.9%) subclones showed related karyotypes. Twelve patients had one clone with an interstitial 5q deletion as the sole abnormality and other subclones with additional changes. One of these 12 cases displayed a subclone with complex karyotype in which deleted chromosome 5 was involved in an unbalanced translocation. Five patients (5.1%) had a combination of related clones with del(5q) and additional changes and unrelated subclones without this aberration. Complex aberrations were often associated with the loss of heterozygosity (LOH) of 17p (44%), however a difference in the frequency of LOH 17p in groups with and without clonal heterogeneity was not ascertained (p = 0.782). Moreover, the overall survival (OS) of patients with clonal heterogeneity did not differ from that of patients with one homogeneous complex aberrant clone (p = 0.391). Median OS in both groups was three months only. Conclusions: The results of this study confirmed clonal heterogeneity at the cytogenetic level as a frequent finding in patients with MDS and complex karyotypes. Although clonal diversity has been implicated as a driving mechanism of tumor development and progression and is usually associated with more aggressive disease, in this cohort of MDS cases with complex karyotypes, the presence of different subclones did not affect patients’ overall survival. It seems that the dismal prognosis of patients with CCAs is generally correlated with overall genomic instability and the complexity of aberrations. This may be manifested at different genetic levels by various phenomena such as increasing number of aberrations, accumulation of gene mutations and/or losses of heterozygosity of tumor-suppressor genes (including LOH TP53), involvement of deleted chromosome 5 in unbalanced rearrangements, extensive chromosome shattering, and clonal heterogeneity as well. Supported by RVO-VFN64165, GACR P302/12/G157/1, PRVOUK-P27/LF1/1, and MHCR 00023736. Disclosures No relevant conflicts of interest to declare.

Background Aberrations of chromosome 5 are common cytogenetic findings in bone marrow cells of patients with myelodysplastic syndromes (MDS). The most frequent is the interstitial deletion of the long arm of chromosome 5 [del(5q)] - occurring as sole abnormality or in combination with additional aberrations in ∼30% of primary and up to 50% of secondary MDS. Extent and breakpoints of deleted segment differs among patients. A critical area deleted in all cases (the common deleted region, CDR) has been localized between bands 5q31.1 and 5q32. Loss of the whole chromosome 5 was described in about 3-8% MDS cases. However, in recent studies it has been shown that many suspected monosomies 5 are in fact cryptic translocations or insertions, undetectable by conventional G-banding. The aim of the study was to perform detailed genome wide analyses of bone marrow cells of MDS patients with suspected monosomy 5 detected by conventional G-banding at diagnosis, to verify the loss of genetic material of chromosome 5, to compare the breakpoints and extent of chromosome 5 deletions in individual patients, and to assess real existence and clinical significance of monosomy 5 in MDS. Patients and methods In the period 2002–2012, bone-marrow samples from 870 adults with newly diagnosed MDS were examined and 80 patients (9.2%) with suspected monosomy 5 were identified (50 males, 30 females; median age 67 years). Karyotypes were analyzed by conventional G-banding, FISH with Vysis DNA probes (Abbott, Des Plaines, IL), and mFISH/mBAND (MetaSystems, Altlussheim, Germany). Genomic imbalances were identified with oligonucleotide CGH-SNP arrays (BlueGnome, Cambridge, UK) or with SNP arrays (Illumina, San Diego, CA). Results All 80 patients presented a complex karyotype with confirmed del(5)(q31). The most frequent breakpoints on chromosome 5 involved bands 5q14.3, 5q34, 5q33.3, 5q11.2, and 5q13.2. On 5p, breaks occurred sporadically (recurrent breakpoints at 5p12, 5p14.1, and 5p15.3). The extent of del(5q) ranged from 23.55 Mb to 131.4 Mb (whole arm). The most proximal and most distal breakpoints were located at 5q11.1 and 5q35.3, respectively. The CDR occurred between bands 5q31.1 and 5q32 (15.1 Mb). No patient with pure monosomy 5 was found. In all cases, parts of the deleted chromosome 5 were fragmented and inserted elsewhere in the genome. A commonly retained region conserved in all patients was established at 5p11.1–p14.2 (22.31 Mb). The most recurrent partners of deleted chromosome 5 in cryptic unbalanced rearrangements were chromosomes 17, 3, 7, and 18. Patients with deleted chromosome 5 involved in complex aberrations have an extremely poor prognosis (in the present cohort, a median OS of only two months). Discussion and conclusions Based on these results, we believe that pure monosomy 5, quoted in the literature, in MDS does not actually exist. It seems that the primary interstitial deletion of 5q is likely the cause of increased genomic instability, which may, under certain circumstances, lead to fragmentation and disintegration of the whole chromosome 5 and to the emergence of additional complex rearrangements. Mechanism responsible for fragmentation of deleted chromosome 5 remains unclear. One of possible explanations might be the phenomenon called chromothripsis, whereby one or more chromosomes or chromosomal regions shatter into pieces in a single catastrophic event. Some of these pieces are then stitched haphazardly together by DNA repair machinery, whereas some of them are lost. This process is resulting in severe genomic damage and fast disease progression. MDS patients with deleted chromosome 5 involved in complex rearrangements should be considered as a unique entity with extremely poor prognosis. Supported by grants RVO-VFN64165/2012, GACR P302/12G157/1, PRVOUK-P27/LF1/1 and MHCR 00023736. Disclosures: No relevant conflicts of interest to declare.

Background Constitutional translocations between sex chromosomes are rather rare in humans with breakpoints at Xp11 and Yq11 as the most frequent. Breakpoints on the short arm of the Y chromosome form one subgroup of t(X;Y), giving rise to a derived chromosome with the centromeres of both the X and Y chromosomes, dic(X;Y). Here, we report a rare congenital chromosomal aberration, 46,X,dic(X;Y)(p22.33;p11.32)[20]/45,X[10], in an adult male. Case presentation Primary myelofibrosis, a malignant haematological disease, was diagnosed in a 63-year-old man following liver transplantation after hepatocellular carcinoma. By the analysis of the bone marrow sample, the karyotype 46,X,dic(X;Y)(p22.33;p11.32) was detected in all the mitoses analysed and verified with multicolour fluorescence in situ hybridization (mFISH). A cytogenetic examination of stimulated peripheral blood cells revealed the constitutional karyotype 46,X,dic(X;Y)(p22.33;p11.32)[20]/45,X[10]. The cell line 45,X was confirmed with FISH in 35 % of interphase nuclei. The SRY locus was present on the dicentric chromosome. A CGH/SNP array (Illumina) revealed a gain of 153,7 Mbp of the X chromosome and a 803-kbp microdeletion (including the SHOX gene), which were also confirmed with FISH. SHOX encodes a transcriptional factor that regulates the growth of the long bones. The deletion of the SHOX gene together with the Madelung deformity of the forearm and the short stature of the proband led to a diagnosis of Léri-Weill dyschondrosteosis (LWD). The gain of almost the whole X chromosome (153,7 Mbp) was considered a variant of Klinefelter syndrome (KS). The levels of gonadotropins and testosterone were consistent with gonadal dysfunction. A malformation of the right external ear was detected. Conclusions We have reported a structural aberration of the sex chromosomes, dic(X;Y)(p22.33;p11.32). The related genomic imbalance is associated with two known hereditary syndromes, LWD and a KS variant, identified in our proband at an advanced age. Because the breakpoints did not involve cancer genes, we inferred that the two malignancies in the proband were not caused by this abnormality. The possible influence of SHOX haploinsufficiency on the growth regulation of auricular chondrocytes is discussed.

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.