Cryptic Genomic Aberrations Research Articles

Exon-Level Genomic Abnormalities Are Frequent in Cytogenetically Normal Patients with Philadelphia-Chromosome Negative Myeloproliferative Neoplasms (MPNs)

Polycythemia vera (PV) and essential thrombocythemia (ET) exhibit an abnormal karyotype in 10%-28% of patients (pts) while the frequency increases in up to 90% of pts with advanced forms of myelofibrosis (PMF) and MPN accelerated/blast phase. Similarly, Klampfl et al. (Blood,118(1) 167-176) demonstrated that an increase in the number of genomic abnormalities detected by chromosomal microarrays (CMA) is associated with disease progression. Although genomic aberrations in cytogenetically normal pts with MPN have been previously reported, this study focused on identifying cryptic genomic alterations with exon-level precision to genomically characterize pts with MPN that were karyotypically normal at the time of CMA analysis. We prospectively evaluated a cohort of 151 cytogenetically normal pts (23% PV, 35% ET, 21% PMF, and 21% post PV/ET MF) using a high-resolution chromosomal microarray (CMA) platform that detects both regions of copy-neutral loss of heterozygosity (cnLOH) and copy number alterations (CNAs) with exon-level resolution (Agilent's cancer-specific 2x400k aCGH+SNP). In order to uncover regions harboring genes that may have biological and/or clinical implications, we identified the minimal overlapping regions (MORs) containing CMA abnormalities occurring in at least 4 pts, using the "GRanges" package (Bioconductor). Genomic aberrations were documented in 69% (n=104) of pts. Of the 238 CMA abnormalities identified, 52 were gains, 38 were losses, and 148 were regions of cnLOH. Next-generation sequencing (NGS) was performed in 97% of these pts and 65% had JAK2, 15% had CALR, and 2% had MPL mutations. The remaining pts (n=25) were triple-negative, and 12 pts harbored other non-driver gene mutations. Only CMA lesions were identified in 6 pts (4%) and both NGS and CMA could not genomically detect abnormalities in 7 pts (5%). The most frequent MOR mapping to a single gene harbored JAK2 cnLOH which occurred in 25% of pts, consistent with previous reports. A statistical difference in the JAK2V617F variant allele frequency (VAF) in pts with 9p cnLOH (VAF=80%) and without 9p cnLOH (VAF=18%) (p<0.001) was observed. In pts with PV, a high JAK2 allele burden is associated with an aggressive phenotype (Vannucchi et al. Therapeutic Advances in Hematology 2010 2, 21-32). Alterations of 1q32.1 involving MDM4, a negative regulator of TP53, was the second most frequent MOR occurring in 14% of pts. We have recently demonstrated that gain of 1q results in overexpression of MDM4 and is associated with advanced forms of MPN (Marcellino et al, Blood Advances, 2018 2(24), 3581-3589). Four other recurrent MORs harboring a single gene, TAF15, RASA3, RHOC, and NCOR2 occurred in 8%, 5%, 4%, and 3% of pts, respectively. Each had intragenic gains. We previously reported alterations in these genes in the spleens of pts with MF (Zimran et al., Haematologica, 2018 (10)103). Our current results indicate that these same alterations are present in the bone marrow cells of cytogenetically normal MPN pts. Since these abnormalities are present in pts with advanced forms of MPN, alterations of these genes may represent markers for disease progression. Of the MORs harboring multiple genes, 3 regions involved 1p, 17q, and 18q in 4%, 3%, and 6% of pts, respectively. All pts had cnLOH of these regions and each harbored relevant genes that may be implicated in MPN; CDKN2C (1p), PPM1D (17q), and ASXL3(18q). Our results demonstrated that CMA abnormalities were present in 51% of pts with ET, 66% with PV, 87% with PMF, and 70%-100% with post ET/PV MF. CMA lesions were the sole alteration in 4% while 5% of chromosomally normal pts with an initial diagnosis of ET had neither somatic mutations nor CMA abnormalities. We provided evidence that recurrent genomic alterations can be identified in regions associated with disease progression, as well as novel regions harboring genes with potential pathobiological implications. We conclude that CMA is a vital tool in identifying cryptic genomic aberrations in cytogenetically normal pts with chronic phase MPN. Disclosures Hoffman: Merus: Research Funding. Ozbek:Viracor: Patents & Royalties: Biomarker Patent. Mascarenhas:Pharmaessentia: Consultancy, Membership on an entity's Board of Directors or advisory committees; Merus: Research Funding; Promedior: Research Funding; Janssen: Research Funding; CTI Biopharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Incyte: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Research Funding; Roche: Consultancy, Research Funding; Merck: Research Funding. Kremyanskaya:La Jolla: Consultancy; Incyte, Celgene, Constellation, Protagonist.: Research Funding.

Identification of cancer gene fusions based on advanced analysis of the human genome or transcriptome

Many gene fusions have been recognized as important diagnostic and/or prognostic markers in human malignancies. In recent years, novel gene fusions have been identified in cases without prior knowledge of the genetic background. Accompanied by a powerful computational data analysis method, new genome-wide screening approaches were used to detect cryptic genomic aberrations. This review focused on advanced genomewide screening approaches in fusion gene identification, such as microarray-based approaches, next-generation sequencing, and NanoString nCounter gene expression system. The fundamental rationale and strategy for fusion gene identification using each biotech platform are also discussed.

Single Nucleotide Polymorphism Array (SNP-A) Karyotype Is the Best Predictor of Prognosis In Normal Cytogenetics Acute Myeloid Leukaemia (AML)

Abstract 411The herald of next generation sequencing has ushered an era for the discovery of frequent novel mutations such as DNMT3a, IDH1/2 and ASXL1 in acute myeloid leukaemia with a normal karyotype (AML-NK). The frequency of these mutations has been extensively documented; however, there is inconclusive evidence for their prognostic significance. Furthermore, whilst having a ‘normal’ cytogenetic karyotype, AML-NK has been characterized by SNP-A karyotyping revealing multiple cryptic genomic aberrations. To predict prognosis, mutations of FLT3, NPM1 and CEBPA as well as gene expression profiles have been used as biomarkers. Using 454 next generation sequencing (NGS), we sought to determine the prognostic significance of mutations in ASXL1, DNMT3a, FLT3, IDH1/2, NPM1 and TP53 as well as detect cryptic genomic aberrations using 250K SNP microarrays in 92 AML-NK patients (pts) uniformly treated from the UK MRC/NCRI AML clinical trials, excluding pts with induction deaths.We demonstrate the presence of 54 cryptic genomic aberrations in 37 pts consisting of 11 deletions (del) (8 pts), 10 gains (10 pts) and 33 regions of UPD (29 pts). Aberrations were excluded as CNV’s if there was >50% overlap with variants from the DGV and an internal cohort of 91 healthy subjects. The median size of del was 1.6Mb(0.18Mb-3.4Mb) with chromosome (chr) 4(q24) and 17(q11.2 and q22.1) deleted in two pts. One pt had micro del on chr12p13.2-p12.3 and 21q22.11-q22.12 affecting the ETV6 and AML1 genes, respectively, that was confirmed by FISH. Gains (median 0.37Mb(0.26–0.71Mb)) were identified on chr8 (4 pts). The median size of UPD’s was 83.3Mb(20.5Mb-158Mb) with interstitial UPD <20Mb excluded from the analysis. Chromosome 13 (q12.11-q34) and 6(p25.3-p22.1) were the most frequently affected with UPD observed in 15 and 5 pts, respectively. Of the 15 pts with UPD of chr13, 11 pts had this occurrence in small clones. Additional regions affected by UPD included chromosomes 1, 2, 4, 5, 7 and 11.NPM1, DNMT3a, FLT3-ITD, IDH2, FLT3-TKD, IDH1, TP53 and ASXL1 mutations occurred in 64%, 49%, 22%, 5%, 3%, 2% and 1% of patients, respectively with 13 cases having no detectable mutations. The R882 mutation hotspot of DNMT3a occurred in 33/45 cases with clone size ranging from 34–65% and occurred concurrently with mutations in NPM1 (31 pts), FLT3-ITD/TKD (30 pts) and IDH2 (9 pts). IDH1 (R130H) and IDH2 (R140Q and R172K) mutations were mutually exclusive and occurred in 23 pts, with 4 pts having concurrent FLT3, NPM1 and DNMT3a mutations. Only 1 patient had an ASXL1 mutation with a concurrent IDH2 mutation and 2 cases had mutation of TP53. FLT3-TKD/ITD mutations were exclusive of each other. Not all pts with FLT3-ITD mutation had UPD of chromosome 13. There was no correlation between genomic aberrations and mutations of any other assayed genes.The median duration of follow-up from time of diagnosis of the study group was 44 months (range 2–160). There were no significant correlations between the presence of genomic aberrations and age, sex, WBC, secondary disease or mutations in IDH1/2, FLT3-ITD or DNMT3a. Patients with FLT3-TKD were likely to have gains (2/5) compared to those with wild type (8/85, p=0.04). Patients with genomic aberrations had worse overall survival (OS) compared to pts without aberrations, (5 year OS 12% vs 40%, hazard ratio (HR) adjusted for other prognostic variables 2.56 (1.43–4.60), p=0.001); this was also seen when considering UPD on OS ((8% for those with UPD compared to 37% for those without UPD (adjusted HR 2.52(1.40–4.54) p=0.002) and cumulative incidence of relapse (CIR) (78% in pts with UPD compared to 54% for those without UPD (adjusted HR 4.04(1.84–8.90) p=0.0003). Adjusted analysis showed a dose effect for the number of UPD’s on OS (p=0.007) and relapse (p=0.001). In a model building analysis, adjusted for age, WBC, sex, secondary disease, performance status and mutations of IDH1, IDH2, FLT3-ITD/TKD, DNMT3a and NPM1, only UPD proved independently prognostic for relapse and survival. We conclude that cryptic genomic aberrations in uniformly treated AML-NK pts provide better prognostic value and outcome prediction than metaphase cytogenetics and mutation detection, thereby, helping to identify patient groups with poor prognosis and requiring specific therapeutic strategies. Disclosures:No relevant conflicts of interest to declare.

Prognostic Significance of Cryptic Genomic Aberrations In AML with Normal Karyotype

AbstractAbstract 2729Acute myeloid leukaemia with a normal karyotype (AML-NK) is a heterogeneous clonal disorder of haematopoietic progenitor cells with an intermediate prognosis. Acquired mutations of FLT3, NPM1 and CEBPA as well as gene expression profiles have recently been used as biomarkers to further stratify prognostic subgroups, especially in AML-NK.We have previously shown that cryptic genomic aberrations in MDS cases are prognostically significant and regions of uniparental disomy (UPD), detectable by SNP array karyotyping, can harbour mutations in specific gene(s). We analysed 83 AML patients (pts), median age 52 (range 8–78) with normal cytogenetics, excluding pts who died within 30 days of entry, who had been treated on one of the UK MRC/NCRI AML clinical trial protocols, using high resolution 250K SNP microarrays to seek genomic aberrations. In addition, we sequenced the ‘mutation hotspot’ for IDH1/2 as well as screened for the presence of FLT3 ITD and NPM1 mutations. Genomic aberrations and mutational lesions were correlated with outcome data.To circumvent the lack of constitutional DNA, all aberrations overlapping by >50% with copy number variants from the Database of Genomic Variants as well variations observed in a cohort of 91 healthy subjects were excluded from further analysis. We demonstrate the presence of 69 cryptic genomic aberrations in 43 pts consisting of 12 deletions (del) (8 pts), 10 gains (10 pts) and 47 regions of UPD (35 pts). The median size of del was 1.6Mb (0.18Mb-3.4Mb) with chromosome 4 (q24) and 17 (q11.2 and q22.1) being the most affected. One pt had micro del on chromosome 12p13.2-p12.3 and 21q22.11-q22.12 affecting the ETV6 and AML1 genes respectively, suggestive of a t(12:21) aberration. Gains (median 0.37Mb (0.26–0.71Mb)) were identified predominantly on chromosome 8 (4 pts) with a common amplified region, 8q21.11. The median size of UPD's was 47Mb (2.3Mb-158Mb). Chromosome 13 was the most frequently affected with UPD of the entire chromosome observed in 17 pts. Interestingly, one pt had an interstitial region of UPD on q21.32-q21.33 (4.8Mb). Additional regions affected by UPD included chromosomes 6, 2, 5 in 5 pts and chromosome 11 in 4 pts.NPM1 mutations occurred in 55 pts (65%) and FLT3 ITD's occurred in 44 pts (53%). Of 39 pts with wild type FLT3, 22 had NPM1 mutations; of 28 pts with wild type NPM1, 11 had FLT3 ITD mutations. Conversely of the 44 pts with mutant FLT3, 33 had NPM1 mutations. Six pts had mutations of FLT3, NPM1 and IDH. Of note, only 2 of 17 pts with UPD of chromosome 13 did not have FLT3 mutations, suggesting the presence of additional pathological genes in this region. IDH1 (R130H) and IDH2 (R140Q and R172K) mutations occurred in 3 and 16 pts, respectively and were mutually exclusive. Interestingly, the R172K mutation occurred in only 3 pts. Of the 19 pts with IDH1/2 mutations, 9 and 12 pts respectively, had co-existing FLT3 ITD and NPM1 mutations.The median duration of follow-up from time of diagnosis of the study group was 29 months (range 2–160). There were no significant correlations between the presence of genomic aberrations and age, sex, WBC, secondary disease or IDH1/2 mutations; however, a significant difference was observed in the frequency of UPD in pts with mutant or wild type FLT3 (p=0.01) and NPM1 (p=0.008). A difference in overall survival (OS) was seen on comparison of pts with or without genomic aberrations, (5 year OS 10% vs 46%, hazard ratio (HR) 1.86 (1.01–3.44), p=0.05): significant differences were seen on univariate analyses when analysing gains (11% vs 27%, HR 3.07 (1.09–8.68) p=0.03), or del (0% vs 28%) HR 3.46 (1.02–11.8) p=0.05) separately, with some evidence that UPD also produced worse results (0% v 34% HR 1.82 (0.97–3.45) p=0.06). In multivariate analyses, adjusted for age, WBC, sex, secondary disease, performance status, IDH1, IDH2, FLT3 ITD and NPM1 all SNP array detected aberrations were independently prognostic for overall survival. Analysis combining all aberrations gave a HR of 2.99(1.33–0.75) p=0.006. When a model-building approach was used, after adjustment for baseline covariates, del p=0.005, gains p=0.002 and UPD p=0.02 all entered the model. We show, for the first time, that SNP microarray analysis in AML-NK pts reveals cryptic UPD and CN changes that may allow risk stratification and alter therapeutic strategies for this poor prognostic subgroup of patients. Disclosures:No relevant conflicts of interest to declare.

Identification of Novel Cryptic Chromosomal Abnormalities in Primary Myelofibrosis by Single-Nucleotide Polymorphism Oligonucleotide Microarray.

Abstract 1890Poster Board I-913 Background.Primary myelofibrosis (PMF) is a clonal myeloproliferative neoplasm (MPN) characterised by a proliferation of predominantly megakaryocytes and granulocytes in bone marrow that in fully developed disease is replaced by fibrous tissue.At molecular level, no specific defect has been identified yet. Cytogenetic abnormalities occur in up to 30% of patients, the commonest including del(13)(q12-22), der(6)t(1;6)(q21-23;p21.3), del (20q), and partial trisomy 1q. In addition, approximately 50% of patients with PMF exhibit a single, recurrent, somatic mutation in the gene encoding the cytoplasmic tyrosine kinase Janus kinase 2 (JAK2). However, such mutation is not specific, also occurring in other MPN.Recently a couple of reports dealt with single-nucleotide polymorphism (SNP) array karyotyping of MPD, including some PMF. Importantly, such studies could identify previously uncovered genetic lesions, highlighting the importance of novel high resolution technologies for the detection of formerly unknown, cryptic aberrations.In this study we performed high resolution karyotyping by SNP oligonucleotide microarray by using the most updated Affymetrix array (Genome-Wide Human SNP Array 6.0) in 20 cases of myelofibrosis (MF) in order to identify novel cryptic genomic aberrations. Methods.DNA (500 ng) was extracted from peripheral blood cells (PBMNC) of 14 primary and 6 secondary MF patients. PBMNC were depleted from lymphocytes by magnetic beads. Briefly, CD3+ cells were labeled with anti-CD3 MoAb directly coupled to magnetic microbeads (Miltenyi Biotech), washed and subsequently purified using Mini-MACS technology. After selection, cell present in the positive (CD3) and negative (PBMNC) fractions were counted and submitted to flow cytometry analysis. DNA was processed and hybridized to the Affymetrix SNP arrays 6.0 as for manufacturer instruction. A whole-genome copy number variation (CNV), genotyping, loss of heterozygosity (LOH) and uniparental disomy (UPD) analyses were performed using the Partek Suite 6.0. Ten lab-specific as well as 90 HapMap samples relative to Caucasian healthy donor were used as control reference. Genomic abnormalities were defined as recurrent when occurring in at least 25% of cases.JAK2 mutational status was assessed as reported, by alle-specific PCR. Clinical information and complete follow up were retrieved for all cases.Direct sequencing, FISH, qPCR and immunohistochemistry (IHC) has been chosen for validation. Results.In all patients we could detect several CNV. The median number of CNV was 60 (range, 34-72), including 46 amplifications (A) and 14 deletions (D). All commonest previously described abnormalities were detected. In addition, several formerly uncovered recurrent lesions were identified, mainly involving 1p, 1q, 2p, 4p, 4q, 5q, 6p, 6q, 7q, 8p, 9q 10q, 11p 11q, 12p, 14q, 15q, 16p, 16q, 17q, 18q, 19q, 20p, 22q. The median size of such CNV was 424,582 Kbp (1,379 Kbp-71,277 Mbp).We then compared JAK2+ vs. JAK2− cases. Of note, we found numerous definite aberrations (A or D) distinguishing the two groups and specifically affecting 16q23.1, 1p36.13, 3q26, 14q13.2, 5q33.2, 6q14.1, 7q33, 8p23.1, and 9p11.2.Grippingly, several genes of potential interest for PMF pathogenesis were identified within the involved loci, including RET, SCAPER, WWOX and SIRPB1. Among others, the product of such genes has been selected for validation by IHC. Similarly, many miRNA were recognized, which may deserve further investigation. Conclusions.By using a newly developed highly sensitive array we identified novel cryptic lesions in patients affected by MF. Future studies on larger series, as well as functional analyses will definitely assess their role in the pathogenesis of the disease. Of note, consistent differences were recorded in JAK2+ vs. JAK2−, supporting the hypothesis of different genetic mechanisms occurring in the two sub-groups. Acknowledgments:this work was supported by AIL Pesaro Onlus, Centro Interdipartimentale per la Ricerca sul Cancro “G. Prodi”, BolognAIL, AIRC, FIRB, RFO, Fondazione Cassa di Risparmio in Bologna, Fondazione della Banca del Monte e Ravenna, Progetto Strategico di Ateneo 2006.*GV and MRS equally contributed to this work. Disclosures:No relevant conflicts of interest to declare.

Multiple distinct clones may co-exist in different lineages in myelodysplastic syndromes

Using single nucleotide polymorphism (SNP) microarray with unfractionized bone marrow specimens, recent studies have demonstrated that multiple cytogenetically cryptic genomic aberrations, uniparental disomy (UPD) and/or copy number (CN) aberration, are present in patients with myelodysplastic syndromes (MDS). We hypothesize that various hematopoietic lineages in MDS may carry different cytogenetically cryptic genomic aberrations leading to lineage-specific manifestations of MDS. Flow cytometry sorting was performed to sort 12 MDS marrow samples into blastic, erythroid, immature myeloid and lymphoid fractions. The fractions with enough DNA underwent 250K SNP microarray analysis. Of importance, different chromosomal regions of UPD, deletions and/or gains were present in different fractions of same patients in all samples. Only small percentages (6.7%) of genomic aberrations were present in all fractions from same patients. These results suggest that multiple distinct clones may co-exist in different lineages in MDS and may contribute to cytopenias in specific lineages and the significant clinical heterogeneity observed in these patients. Further studies are warranted to confirm our findings and to investigate the lineage specific genomic lesions in MDS.

High definition cytogenetics and oligonucleotide aCGH analyses of cisplatin‐resistant ovarian cancer cells

Array comparative genomic hybridization (aCGH) is a key platform to assess cancer genomic profiles. Many structural genomic aberrations cannot be detected by aCGH alone. We have applied molecular cytogenetic analyses including spectral karyotyping, multicolor banding, and fluorescence in situ hybridization with aCGH to comprehensively investigate the genomic aberrations associated with cisplatin resistance in A2780 ovarian cancer cells. A2780 is a well-established model of chemotherapeutic resistance with distinct karyotypic abnormalities in the parental and cisplatin-resistant cells. Cytogenetic analysis revealed that two unbalanced translocations, der(8)t(1;8) and der(X)t(X;1), and loss of chromosome 13 were present only in the resistant line. Our aCGH analyses detected imbalances affecting an additional 10.59% of the genome in the cisplatin-resistant cells compared with the parental. DNA copy number changes included deletions at 1p10-p22.1, 8p23.3, and Xq13.1-pter, and a duplication of 8q11.22-q23. Cryptic genomic aberrations associated with concurrent localized changes of specific gene expression included a homozygous deletion of 0.38 Mb at 1p21.3 adjacent to SNX7, and an insertional transposition of 0.85 Mb from 13q12.12 into chromosome 22. This latter rearrangement led to an overexpression of four contiguous genes that flanked one of the breakpoint regions in chromosome 13. Furthermore, 17 genes showed differential expression correlating with genomic gain or loss between the resistant and parent lines, validated by a second expression array platform. These results highlight the integration of comprehensive profiling to determine relationships of genomic aberrations and genes associated with an in vitro drug resistance model in ovarian cancer. This article contains Supplementary Material available at http://www.interscience.wiley.com/jpages/1045-2257/suppmat.

Establishment and Characterization of a tel/abl Rearrangement Responsible for Imatinib Sensitivity in bcr/abl Negative Acute Lymphoblastic Leukemia.

Proliferation of Philadelphia (Ph) positive acute lymphoblastic leukemia (ALL) cells is driven by the bcr/abl oncoprotein which renders transformed cells independent of exogeneous growth factors. Bcr/abl can be targeted by fusion tyrosine kinase inhibitors such as imatinib. ALL is a heterogenous disease. Some cases of Ph negative ALL may be driven by oncogenic fusion kinases other than bcr/abl, with or without involvement of the abl-gene. Such kinases may also be susceptible to targeted inhibitors. At present however, few of such fusion kinases are known. This may be due to the lack of reliable in vitro culturing systems for primary ALL cells. Because of this, only a limited number of ALL cell lines are available to screen for alternative oncogenic fusion kinases. We have recently established an in vitro culturing system in which continuous proliferation (>1 year) was thus far induced in 11 out of 22 primary ALL samples, in the absence of exogenously added growth factors. Five of these 11 cultures were Ph negative by cytogenetics and bcr/abl negative by RT-PCR. Therefore, we first screened these lines for sensitivity to imatinib. One of the 6 Ph negative cell lines, LeidenALL-VG, derived from a 30-year old patient with Ph chromosome negative common ALL, displayed sensitivity to imatinib with an IC50 of 0.1 μM, comparable to the sensitivity of the bcr/abl positive cell lines. RT-PCR with primers specific for tel and abl revealed tel/abl fusion transcripts. Sequencing of the amplicons showed two splice variants of tel-abl: tel4/abl2 and tel5/abl2. Multicolor FISH based karyotyping using COBRA-FISH did not provide evidence for the suggested t(9;12) translocation, indicating cryptic tel/abl rearrangement. To map cryptic genomic aberrations, an array-comparative genomic hybridization array (array-CGH) was performed. A region at 9q34 spanning two array clones was gained (clones RP11-83J21 and RP11-143H20), corresponding with gain of a 300-1300 kb region containing the distal part of the abl gene. A region at 12p13 spanning one array clone was lost (clone RP11-59H1) corresponding with loss of a 1150 to 1750 kb region directly centromeric to the tel gene. To localize cryptic tel/abl rearrangement, fluorescent in situ hybridization (FISH) was performed using the distal tel and abl probes identified by array-CGH, as well as the clones proximal to these regions. FISH revealed an additional distal abl signal, co-localized with the proximal tel signal, on the short arm of an otherwise normal looking chromosome 12. With this, tel/abl rearrangement in LeidenALL-VG cells was characterized as a micro-insertion of one additional distal abl region into the tel region of a normal chromosome 12. Tel/abl rearrangement and imatinib sensitivity were confirmed in the primary cells. These results suggest that our serum-free approach to in vitro culture of primary ALL cells, in combination with molecular karyotyping tools such as array-CGH and COBRA-FISH, may allow identification and characterization of otherwise un-identified chromosomal aberrations, such as the cryptic tel/abl translocation illustrated here, that may be targeted by kinase inhibitors such as imatinib.

Prevalence and prognostic significance of allelic imbalance by single-nucleotide polymorphism analysis in low-risk myelodysplastic syndromes

AbstractLow-risk myelodysplastic syndrome (MDS) with normal cytogenetics accounts for approximately 50% of MDS patients. There are no pathognomonic markers in these cases and the diagnosis rests on cytomorphologic abnormalities in bone marrow and/or peripheral blood. Affymetrix high-resolution single-nucleotide polymorphism (SNP) genotyping microarrays allow detection of cytogenetically cryptic genomic aberrations. We have studied 119 low-risk MDS patients (refractory anemia [RA] = 22; refractory cytopenia with multilineage dysplasia [RCMD] = 51; refractory anemia with ringed sideroblasts [RARS] = 12; refractory cytopenia with multilineage dysplasia with ringed sideroblasts [RCMD-RS] = 12; 5q− syndrome = 16; refractory anemia with excess blasts [RAEB] = 6) using SNP microarrays to seek chromosomal markers undetected by conventional cytogenetics. Loss of heterozygosity (LOH) detected by 50K arrays was verified using 250K and 500K arrays. We demonstrate the presence of uniparental disomy (UPD) in 46%, deletions in 10%, and amplifications in 8% of cases. Copy number (CN) changes were acquired, whereas UPDs were also detected in constitutional DNA. UPD on 4q was identified in 25% of RARS, 12% of RCMD with normal cytogenetics, 17% of RAEB, and 6% of 5q− syndrome cases. Univariate analysis showed deletions (P = .04) and International Prognostic Scoring System (IPSS; P < .001) scores correlated with overall survival; however, on multivariate analysis only IPSS scores retained prognostic significance (P < .001). We show, for the first time, that SNP microarray analysis in low-risk MDS patients reveals hitherto unrecognized UPD and CN changes that may allow stratification of these patients for early therapeutic interventions.

Fortuitous detection of a submicroscopic deletion at 1q25 in a girl with Cornelia‐de Lange syndrome carrying t(5;13)(p13.1;q12.1) by array‐based comparative genomic hybridization

We report on a 2-year-old Japanese girl with Cornelia-de Lange syndrome (CdLS) who had mental and growth retardation, together with characteristic facial anomalies and mild extremity malformations. She had a balanced chromosomal translocation, 46,XX,t(5;13)(p13.1;q12.1) de novo. Surprisingly, this was the same translocation that had provided a clue to the identification of a major causative gene for CdLS, NIPBL [Krantz et al., 2004; Tonkin et al., 2004]. Using fluorescence in situ hybridization (FISH), the breakpoint was confirmed to lie within NIPBL at 5p13.1. Furthermore, array-based comparative genomic hybridization (array-CGH) demonstrated a cryptic 1-Mb deletion harboring six known genes at 1q25-q31.1. A FISH analysis of her parents confirmed that the deletion was de novo. Although patients with interstitial deletions at 1q are rare, some of their features were similar to those observed in our patient, indicating that her clinical manifestations are likely to be affected by not only the disruption of NIPBL but also the concomitant microdeletion at 1q25-q31.1. The present case suggests that array-CGH can uncover cryptic genomic aberrations affecting atypical phenotypes even in well-known congenital disorders.