Single Nucleotide Polymorphism Array Karyotyping Research Articles

P–558 SNP array versus karyotype for prenatal diagnosis in fetuses with abnormal ultrasound: a systematic review and meta-analysis

Abstract Study question Does Single nucleotide polymorphism (SNP) array provide a diagnostic advantage over conventional karyotype in prenatal diagnosis for fetuses with an abnormal ultrasound? Summary answer SNP array in the prenatal setting provides an incremental diagnostic yield over karyotype and the diagnostic accuracy is comparable with combined SNP array and karyotype What is known already Single nucleotide polymorphism and comparative genomic hybridization based arrays (aCGH) are the two chromosomal microarray (CMA) platforms available. Guidelines which recommend offering CMA instead of karyotyping for prenatal diagnosis are mainly based on studies that compared aCGH with karyotype. There is a paucity of reviews that critically appraise the role of SNP array as a prenatal diagnostic tool. We decided to estimate the incremental yield of SNP array over karyotype in detecting chromosomal abnormalities, and to determine the diagnostic accuracy of SNP alone compared with SNP array and karyotype in combination for prenatal diagnosis in fetuses with an abnormal ultrasound. Study design, size, duration We conducted a systematic review of studies comparing SNP array with karyotype for prenatal diagnosis in fetuses with an abnormal ultrasound. We performed a literature search in the electronic databases of EMBASE, PubMed, CENTRAL, CDSR, SCOPUS and Web of science for relevant studies published in the English language between January1996 and May 2020. We also hand searched the referenced list of included studies and performed a google search for grey literature to identify potential studies. Participants/materials, setting, methods The study population was women undergoing prenatal diagnosis for abnormal fetal ultrasound. Studies in which SNP array and karyotyping had been used in fetuses with abnormal ultrasound and which allowed for a 2 x 2 data extraction table were included. We estimated the incremental yields for SNP array over karyotype. For determining the diagnostic accuracy, we considered SNP array alone as the index test and combined karyotype & SNP array as the reference standard. Main results and the role of chance We included six studies for quantitative analysis. After pooling results, incremental yield of SNP array over normal karyotype was 10% (95% confidence interval, CI 4 to 16%) in fetuses with abnormal ultrasound while incremental yield of karyotype over SNP array was 1% (95% CI 0 to 2%). The agreement between SNP array and karyotype was 92%. Variant of uncertain significance (VUS) rates ranged from 4–8%. For SNP array alone versus combined SNP array and conventional karyotype, pooled sensitivity and specificity was 0.96 (95% CI 0.91 to 0.99) and 1.00 (95% CI 0.99 to 1.00) respectively. The Area under curve (AUC) was 0.99 indicating the discriminating ability of the SNP array was very high to identify the fetus with chromosomal abnormalities. Limitations, reasons for caution Only studies published in English were included. There was some degree of heterogeneity in inclusion criteria for the included studies. Wider implications of the findings: The current study suggests SNP array alone can replace conventional karyotype for prenatal diagnosis in fetus with an abnormal ultrasound. Limitations to adoption of SNP testing might include the requirement of requisite expertise to interpret the results and counsel patients appropriately, especially with the propensity of SNPs to identify VUS. Trial registration number Not applicable

Prognostic relevance of integrated genetic profiling in adult T-cell leukemia/lymphoma

AbstractAdult T-cell leukemia/lymphoma (ATL) is a heterogeneous group of peripheral T-cell malignancies characterized by human T-cell leukemia virus type-1 infection, whose genetic profile has recently been fully investigated. However, it is still poorly understood how these alterations affect clinical features and prognosis. We investigated the effects of genetic alterations commonly found in ATL on disease phenotypes and clinical outcomes, based on genotyping data obtained from 414 and 463 ATL patients using targeted-capture sequencing and single nucleotide polymorphism array karyotyping, respectively. Aggressive (acute/lymphoma) subtypes were associated with an increased burden of genetic and epigenetic alterations, higher frequencies of TP53 and IRF4 mutations, and many copy number alterations (CNAs), including PD-L1 amplifications and CDKN2A deletions, compared with indolent (chronic/smoldering) subtypes. By contrast, STAT3 mutations were more characteristic of indolent ATL. Higher numbers of somatic mutations and CNAs significantly correlated with worse survival. In a multivariate analysis incorporating both clinical factors and genetic alterations, the Japan Clinical Oncology Group prognostic index high-risk, older age, PRKCB mutations, and PD-L1 amplifications were independent poor prognostic factors in aggressive ATL. In indolent ATL, IRF4 mutations, PD-L1 amplifications, and CDKN2A deletions were significantly associated with shorter survival, although the chronic subtype with unfavorable clinical factors was only marginally significant. Thus, somatic alterations characterizing aggressive diseases predict worse prognosis in indolent ATL, among which PD-L1 amplifications are a strong genetic predictor in both aggressive and indolent ATL. ATL subtypes are further classified into molecularly distinct subsets with different prognosis. Genetic profiling might contribute to improved prognostication and management of ATL patients.

Clonal evolution in myelodysplastic syndromes.

During the past ten years, genome-wide analysis of genetic alterations in myelodysplastic syndromes (MDS) has improved our understanding of their pathogenesis. Especially, single nucleotide polymorphism array karyotyping and next-generation sequencing technologies (NGS) have unveiled frequent genetic changes in novel functional pathways, including DNA methylation, RNA splicing and cohesin complex formation, in MDS. Moreover, NGS shed light on the clonal evolution which occurs during the development and progression of MDS, pre-cancerous lesions of MDS, and the effects of germline mutations in MDS.

Two Novel Distinct Subtypes of Myeloid Neoplasms Molecularly Associated with Histone H3K36 Methylations

Myelodysplastic syndromes (MDS) and related disorders are a heterogeneous group of chronic myeloid neoplasms with a high propensity to acute myeloid leukemia. A cardinal feature of MDS, as revealed by the recent genetic studies, is a high frequency of mutations and copy number variations (CNVs) affecting epigenetic regulators, such as TET2, IDH1/2, DNMT3A, ASXL1, EZH2, and other genes, underscoring a major role of deregulated epigenetic regulation in MDS pathogenesis. Meanwhile, these mutations/deletions have different impacts on the phenotype and the clinical outcome of MDS, suggesting that it should be important to understand the underlying mechanism for abnormal epigenetic regulation for better classification and management of MDS. SETD2 and ASH1L are structurally related proteins that belong to the histone methyltransferase family of proteins commonly engaged in methylation of histone H3K36. Both genes have been reported to undergo frequent somatic mutations and copy number alterations, and also show abnormal gene expression in a variety of non-hematological cancers. Moreover, germline mutation of SETD2 has been implicated in overgrowth syndromes susceptible to various cancers. However, the role of alterations in these genes has not been examined in hematological malignancies including myelodysplasia.In this study, we interrogated somatic mutations and copy number variations, among a total of 1116 cases with MDS and myelodysplastic/myeloproliferative neoplasms (MDS/MPN), who had been analyzed by target deep sequencing (n=944), and single nucleotide polymorphism-array karyotyping (SNP-A) (n=222). Gene expression was analyzed in MDS cases and healthy controls, using publically available gene expression datasets.SETD2 mutations were found in 6 cases, including 2 with nonsense and 4 with missense mutations, and an additional 10 cases had gene deletions spanning 1.8-176 Mb regions commonly affecting the SETD2 locus in chromosome 3p21.31, where SETD2 represented the most frequently deleted gene within the commonly deleted region. SETD2 deletion significantly correlated with reduced SETD2 expression. Moreover, MDS cases showed a significantly higher SETD2 expression than healthy controls. In total, 16 cases had either mutations or deletions of the SETD2 gene, of which 70% (7 out of 10 cases with detailed diagnostic information) were RAEB-1/2 cases. SETD2 -mutated/deleted cases had frequent mutations in TP53 (n=4), SRSF2 (n=3), and ASXL1 (n=3) and showed a significantly poor prognosis compared to those without mutations/deletions (HR=3.82, 95%CI; 1.42-10.32, P=0.004).ASH1L, on the other hand, was mutated and amplified in 7 and 13 cases, respectively, of which a single case carried both mutation and amplification with the mutated allele being selectively amplified. All the mutations were missense variants, of which 3 were clustered between S1201 and S1209. MDS cases showed significantly higher expression of ASH1L compared to healthy controls, suggesting the role of ASH1L overexpression in MDS development. Frequent mutations in TET2 (n=8) and SF3B1 (n=6) were noted among the 19 cases with ASH1L lesions. RAEB-1/2 cases were less frequent (n=11) compared to SETD2-mutated/deleted cases. ASH1L mutations did not significantly affect overall survival compared to ASH1L-intact cases. Gene Set Expression Analysis (Broad Institute) on suppressed SETD2 and accelerated ASH1L demonstrated 2 distinct expression signatures most likely due to the differentially methylated H3K36.We described recurrent mutations and CNVs affecting two histone methyltransferase genes, which are thought to represent novel driver genes in MDS involved in epigenetic regulations. Given that SETD2 overexpression and reduced ASH1L expression are found in as many as 89% of MDS cases, deregulation of both genes might play a more role than expected from the incidence of mutations and CNVs alone. Although commonly involved in histone H3K36 methylation, both methyltransferases have distinct impacts on the pathogenesis and clinical outcome of MDS in terms of the mode of genetic alterations and their functional consequences: SETD2 was frequently affected by truncating mutations and gene deletions, whereas ASH1L underwent gene amplification without no truncating mutations, suggesting different gene targets for both methyltransferases, which should be further clarified through functional studies. DisclosuresAlpermann:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Shih:Novartis: Research Funding.

SNPs Array Karyotyping in Non-Hodgkin Lymphoma.

The traditional methods for detection of chromosomal aberrations, which included cytogenetic or gene candidate solutions, suffered from low sensitivity or the need for previous knowledge of the target regions of the genome. With the advent of single nucleotide polymorphism (SNP) arrays, genome screening at global level in order to find chromosomal aberrations like copy number variants, DNA amplifications, deletions, and also loss of heterozygosity became feasible. In this review, we present an update of the knowledge, gained by SNPs arrays, of the genomic complexity of the most important subtypes of non-Hodgkin lymphomas.

Abstract 4791: Prognostic model of lower grade gliomas

Abstract Background Lower grade gliomas (LGGs WHO grade II/III glioma) account for one third of all gliomas. Most LGGs generally show a slow progression, but some show a more aggressive clinical course, where several clinical/genetic factors, such as tumor size, presence of neurologic deficit before surgery and 1p19q LOH, have been reported to correlate patients’ survival. However, no large-scale studies prevent establishment of a reliable prognostication system. Methods Status of somatic mutations and copy number variations (CNVs) were investigated for 335 Japanese patients with LGG using whole exome/targeted sequencing and single nucleotide polymorphism-array karyotyping, respectively. Corresponding data were also publically available for 425 patients from the Cancer Genome Atlas (URL: http://cancergenome.nih.gov/). Correlation of genetic lesions and other parameters with overall survival (OS) was analyzed for combined 538 patients, which were divided into two sets, 269 training and 269 validation sets. First, the 269 patients of the training set were classified into 3 types according to the characteristic mutations and CNVs: Type 1 (mutated IDH with 1p/19q LOH), Type 2 (mutated IDH without 1p19q LOH), Type 3 (IDH wild type) patients. Using the LASSO Cox regression model, we built a classifier based on gene mutations, CNVs (over 5% of cases), sex, age, pathology, WHO grade and operation type (gross total resection or not). We validated the accuracy of this classifier in terms of prediction of OS in the independent group of 269 patients. Results Using the LASSO model, the patients in Type 1 and 3 were grouped into low- and high-risk groups, whereas Type 2 was not because no significant risk factors were extracted for Type 2 patients. Combining high-risk Type 1 and Type 2, between which OS was not significantly different, the entire training set was divided into 4 groups showing significantly different OS, low- and intermediate-risk groups and high- and very high-risk groups, respectively. In the training set, 5-year OS for low-, intermediate-, high-, and very high-risk groups was 100%, 82%, 49%, and 0%, which were 94%, 70%, 43%, and 14%, in the validation set, respectively. The performance of the new model was evaluated by receiver operator characteristic (ROC) analysis, which showed significantly higher accuracy compared to other models based solely on clinical/histological parameters, including pathology, WHO grade and Karnofsky performance status (KPS). Conclusion We established a new classifier based both on genetic and clinical parameters, which provides a reliable tool for predicting OS in LGGs patients and should be useful to guide therapy. Citation Format: Kosuke Aoki, Hiromichi Suzuki, Hideo Nakamura, Masahiro Mizoguchi, Tetsuya Abe, Satoru Miyano, Ichiro Takeuchi, Toshihiko Wakabayashi, Seishi Ogawa, Atsushi Natsume. Prognostic model of lower grade gliomas. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4791. doi:10.1158/1538-7445.AM2015-4791

High concordance of genomic and cytogenetic aberrations between peripheral blood and bone marrow in myelodysplastic syndrome (MDS).

Bone marrow (BM) genetic abnormalities in myelodysplastic syndrome (MDS) have provided important biological and prognostic information; however, frequent BM sampling in older patients has been associated with significant morbidity. Utilizing single-nucleotide polymorphism array (SNP-A) and targeted gene sequencing (TGS) of 24 frequently mutated genes in MDS, we assessed the concordance of genetic abnormalities in BM and peripheral blood (PB) samples concurrently from 201 MDS patients. SNP-A karyotype in BM was abnormal in 108 (54%) and normal in 93 (46%) patients, with 95% (190/201) having an identical PB karyotype. The median copy number (CN) for deletions was significantly lower in BM (CN:1.4 (1-1.9)) than in PB (CN:1.5 (1-1.95), P<0.001). Using TGS, 71% (130/183) patients had BM somatic mutations with 95% (124/130) having identical mutations in PB. The mutant allele burden was lower in PB (median 27% (1-96%)) compared with BM (median 29% (1-100%); P=0.14) with no significant difference in the number, types of mutations or World Health Organization subtype. In all patients with discordant SNP (n=11) and mutation (n=6) profiles between BM and PB, shared abnormalities were always present irrespective of treatment status. Overall, 86% of patients had a clonal aberration with 95% having identical SNP-A karyotype and mutations in PB, thus enabling frequent assessment of response to treatment and disease evolution especially in patients with fibrotic or hypocellular marrows.

Comparison of Peripheral Blood and Bone Marrow Molecular Profiling in Primary Myelodysplastic Syndromes (MDS)

AMM and MA – Joint first authorsDiagnosis of myelodysplastic syndromes (MDS) relies on demonstrating peripheral blood (PB) / bone marrow (BM) dysplasia and cytogenetic abnormalities, forming the backbone of the revised International Prognostic Scoring System (IPSS-R). However, assessment of dysplasia is operator dependent and metaphase cytogenetic analysis (MC) is often normal or uninformative creating diagnostic challenges, particularly in patients with early or low risk MDS. Impressive advances have taken place in the last decade in the identification and chronicling of the genetic lesions leading to phenotypic diversity of MDS. We sought to evaluate the value of single nucleotide polymorphism array (SNP-A) based cytogenetic assessment and high throughput sequencing of the 24 genes most frequently mutated in MDS to determine if genetic abnormalities in the BM are reflected in the PB thus enabling easy assessment of response to treatment and/or disease progression.A MiSeq based gene panel comprising of 24 frequently mutated MDS genes: ASXL1, CBL, CEBPA, DNMT3A, ETV6, EZH2, FLT3, GATA2, IDH1, IDH2, JAK2, KDM6, KIT, KRAS, NPM1, NRAS, RUNX1, SF3B1, SRSF2, STAG2, TET2, TP53, U2AF1, ZRSR2 and CytoHD/750K SNP-A karyotyping was applied to both PB and BM concurrent samples. Genomic aberrations and non-synonymous variant calls were filtered using public databases to exclude polymorphisms.PB and BM from 201 MDS patients [median 62 years (17-88)] followed up for median 21 months (0.3-171) was analysed. Sixty (30%) patients received supportive care only whilst others were treated with MDS/AML directed therapies. The WHO subtypes were: 5q- syndrome (n=26), refractory cytopenia/s (RA/RCMD, n=53), refractory anaemia with excess blasts/acute myeloid leukaemia (RAEB/AML, n=51), refractory anaemia with ringed sideroblasts (RARS/RCMD-RS, n=20) and other subtypes (including myeloproliferative and hypoplastic MDS, n=51). Based on the IPSS-R risk groups 62% had low risk disease: classified as very low (n=35), low (n=89), intermediate (n=42), high (n=16) and very high (n=19) risk groups. Metaphase cytogenetic analysis was normal (NK-MC) in 113(56%), abnormal (AK-MC) in 65(32%) patients and in 23(12%) MC failed. SNP-A was informative in all patients identifying a normal (NK-SNP) in 93(46%) and abnormal (AK-SNP) in 108(54%) patients, respectively. A comparison of BM and PB by SNP-A, showed 190 patients having an identical karyotype (95% concordance). BM SNP-A identified 36 (32%) patients with SNP-A abnormalities not detected by MC, changing the IPSS-R in 49 (24%) patients overall. Inclusion of SNP-A abnormalities changed the IPSS-R risk group in 28 (25%) of NK-MC patients; from good to intermediate (n=21), poor (n=5) and very poor (n=2) groups, respectively. In 9/11 patients with discordant SNP-A karyotypes between BM and PB, the IPSS-R remained unchanged. We found no difference in the clonal burden between PB and BM for gains and CN-LOH. However, for deletions, the clonal size was significantly lower in BM [median 1.4 (1 – 1.9)], than PB [median 1.5 (1 – 1.95), p<0.001].Mutational profiling from 183 patients revealed 248 and 240 mutations in BM and PB, respectively. BM and PB mutation profiles were identical in 171 patients (93% concordance) (Fig 1). In 11/12 patients with discordant mutation profiles, additional identical mutations and SNP-A abnormalities were noted in both BM and PB. Similarly, clone size was lower (p= 0.53) in the PB [median 27%, (1%-96%)] compared to BM [median 30%, (1%-100%)] and, in addition, there was no difference in the overall number or the type of mutation between the BM and PB. Interestingly, analysis of known poor risk MDS gene mutations (ASXL1, EZH2, RUNX1, P53 and U2AF1) identified 85 mutations in BM compared to 80 in the PB. In 5 patients where these mutations were discordant, additional identical mutations were detected in both BM and PB. Patients with ASXL1 and TP53 mutations had significantly lower clonal burden in PB (p<0.001) compared to BM in contrast to RUNX1 and EZH2 where there was no difference in the clone size. Using PB we were able to show that both SNP-A defects and gene mutations were efficiently detected when compared with BM.This study confirms that PB can reliably substitute for BM for all molecular assays in MDS; which is highly relevant for the clinical assessment of this relatively elderly group of patients. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Genomic Landscape of Pediatric CBF-AML By SNP-Array Karyotyping and Extensive Mutational Analysis

Background. Core binding factor (CBF) acute myeloid leukemia (AML) includes AML with t(8;21) and inv(16) leading to RUNX1-RUNX1T1 or CBFB-MYH11 fusion genes. These recurrent genetic abnormalities are both associated with disruption of genes encoding subunits of the CBF, a heterodimeric transcription factor involved in hematopoiesis. Although the fusion proteins appear to be crucial for the leukemogenic process, considerable experimental evidence indicates that they are not sufficient to induce AML on their own. Due to their high sensitivity to chemotherapy with high complete remission rates and their relatively favorable outcome, CBF-AML is considered to have a good prognosis. Nonetheless, about 30-40% of these patients relapse after standard intensive chemotherapy. In this context, identification of additional genetic or molecular abnormalities could allow better understanding of CBF-AML leukemogenesis, prediction of clinical outcome and identification of novel therapeutic targets.Methods. This study focuses on 73 patients with CBF-AML [43 t(8;21) and 30 inv(16)-AML] enrolled in the pediatric trial ELAM02. Single nucleotide polymorphism array (SNP-A) was performed for all patients using Cytoscan® HD arrays according to the manufacturer instructions. In order to distinguish somatic from constitutional SNP-A lesions, we excluded known copy number abnormalities (CNA) if there was >50% overlap with variants from public database, except for breakpoints-related alterations. Interstitial uniparental disomies (UPD) <10 Mb and telomeric UPD <5 Mb were considered as constitutional and excluded from the analysis. Additionally, extensive mutational analysis (including 45 genes frequently reported to be mutated in myeloid malignancies) were performed for 37 and 25 patients with t(8;21) and inv(16)-AML respectively. Two different technologies of next generation sequencing (NGS) were used, allowing direct validation.Results. Among the 73 cases, 145 SNP-A lesions were found in 58 patients (81%) with a median of 2 lesions per case (range, 0-8). CNA was more frequent (84 losses, 47 gains) than UPD (n=14). No significant difference was noted between the number of CNA and UPD in inv(16) and t(8;21)-AML. Small lesions were common at breakpoints involved in the t(8;21) and inv(16) (respectively 4/43 and 6/30). Additional recurrent CNA mostly involved entire chromosomes, chromosomal arms or large chromosomal regions. Del(9q) and loss of sex chromosome were restricted to t(8;21)-AML (respectively 6/43 and 20/43). Trisomy 22 was restricted to inv(16)-AML (2/30). Other recurrent CNA included trisomy 8 (3/43 vs 1/30) and gains of 13q (2/43 vs 1/30) in both subtypes, gains of 1q and del(2q) in t(8;21)-AML (each 2/43). Del(7q) was among the most common aberrations regardless of subtype (7/43 and 7/30). The minimally deleted region of 7q contained 57 genes including MLL3 and EZH2. Additionally, we found focal deletions of IKZF1 in one patient, NF1 in another and 3 deletions of CCDC26. Except for known mutations (KIT, RAS, FLT3), NGS did not reveal any other alterations in inv(16)-AML. By contrast, t(8;21)-AML was marked by the frequency of mutations in ASXL1/2 (8%/24%) and cohesin genes SMC1A, SMC3, RAD21, STAG2, NIPBL (27% combined). Mutations were also detected in epigenetic-related genes EZH2 (5%), TET2 (8%), IDH1/2 (5%) and WT1(11%).Conclusions. SNP-A karyotyping of 73 pediatric CBF-AML revealed several recurrent alterations, with differing distribution between the 2 subgroups. Moreover, t(8;21) and inv(16)-AML appeared to have distinct mutational profiles, leading us to consider them separately for future studies. We recently reported high frequency of ASXL mutations in t(8;21)-AML and their absence in inv(16)-AML (Micol, Duployez and Boissel et al, Blood 2014). We now report high frequency of mutations in cohesin genes with the same distribution. Recent description of functional relations between cohesin and polycomb proteins, together with our results, suggest an important pathway in t(8;21) leukemogenesis. Concurrent ASXL and cohesin mutations were found in several patients, suggesting they could cooperate in some cases. Interestingly, ASXL mutations were exclusive of del(7q), suggesting that disruption of the ASXL-associated proteins MLL3 and EZH2 could be of great interest in the physiopathology of t(8;21)-AML. Finally, correlations with clinical outcome are in progress. DisclosuresNo relevant conflicts of interest to declare.

High‐resolution genomic copy number profiling of primary intraocular lymphoma by single nucleotide polymorphism microarrays

Primary intraocular lymphoma (PIOL) is a rare lymphoma. Because of difficulties in obtaining tissue samples, little is known about the disease's genetic features. In order to clarify these features, we carried out single nucleotide polymorphism array karyotyping of IOL using genomic DNA extracted from vitreous fluid. We analyzed 33 samples of IOLs consisting of 16 PIOLs, 12 IOLs with a central nervous system (CNS) lesion at diagnosis (IOCNSL), and five secondary IOLs following systemic lymphoma. All were B-cell type. We identified recurrent copy number (CN) gain regions in PIOLs, most frequently on chromosome 1q followed by 18q and 19q. Chromosome 6q was the most frequent loss region. Although these CN gain regions of PIOL were in common with those of IOCNSL, loss of 6q22.33 containing PTPRK and 9p21.3 containing CDKN2A were more frequently deleted in IOCNSL. Large CN loss in 6q was detected in three of four PIOL patients who had early CNS development and short survival periods, whereas long-term survivors did not have such deletions. There was a correlation between gain of the IL-10 gene located on 1q and intravitreal interleukin-10 concentration, which was higher in IOL than in benign uveitis. The results suggest that IOCNSL is a highly malignant form of PIOL that infiltrates into the CNS at an early stage. They also indicate that genetic differences between PIOL and primary CNS lymphoma need to be clarified.

Single nucleotide polymorphism array karyotyping: A diagnostic and prognostic tool in myelodysplastic syndromes with unsuccessful conventional cytogenetic testing

Cytogenetic aberrations identified by metaphase cytogenetics (MC) have diagnostic, prognostic, and therapeutic implications in myelodysplastic syndromes (MDS). However, in some MDS patients MC study is unsuccesful. Single nucleotide polymorphism array (SNP-A) based karyotyping could be helpful in these cases. We performed SNP-A in 62 samples from bone marrow or peripheral blood of primary MDS with an unsuccessful MC study. SNP-A analysis enabled the detection of aberrations in 31 (50%) patients. We used the copy number alteration information to apply the International Prognostic Scoring System (IPSS) and we observed differences in survival between the low/intermediate-1 and intermediate-2/high risk patients. We also saw differences in survival between very low/low/intermediate and the high/very high patients when we applied the revised IPSS (IPSS-R). In conclusion, SNP-A can be used successfully in PB samples and the identification of CNA by SNP-A improve the diagnostic and prognostic evaluation of this group of MDS patients.

Utility of peripheral blood for cytogenetic and mutation analysis in myelodysplastic syndrome

AbstractRecent studies have shown that more than 80% of bone marrow (BM) samples from patients with myelodysplastic syndrome (MDS) harbor somatic mutations and/or genomic aberrations, which are of diagnostic and prognostic importance. We investigated the potential use of peripheral blood (PB) and serum to identify and monitor BM-derived genetic markers using high-resolution single nucleotide polymorphism array (SNP-A) karyotyping and parallel sequencing of 22 genes frequently mutated in MDS. This pilot study showed a 100% SNP-A karyotype concordance and a 97% mutation concordance between the BM and PB. In contrast, mutation analysis using Sanger sequencing of PB and serum-derived DNA showed only 65% and 42% concordance to BM, respectively. Our results show the potential utility of PB as a surrogate for BM for MDS patients, thus avoiding the need for repeated BM aspirates particularly in elderly patients and those with fibrotic or hypocellular marrows.

TP53 mutations in myelodysplastic syndrome are strongly correlated with aberrations of chromosome 5, and correlate with adverse prognosis

This study aimed to determine the incidence/prognostic impact of TP53 mutation in 318 myelodysplastic syndrome (MDS) patients, and to correlate the changes to cytogenetics, single nucleotide polymorphism array karyotyping and clinical outcome. The median age was 65 years (17-89 years) and median follow-up was 45 months [95% confidence interval (CI) 27-62 months]. TP53 mutations occurred in 30 (9.4%) patients, exclusively in isolated del5q (19%) and complex karyotype (CK) with -5/5q-(72%), correlated with International Prognostic Scoring System intermediate-2/high, TP53 protein expression, higher blast count and leukaemic progression. Patients with mutant TP53 had a paucity of mutations in other genes implicated in myeloid malignancies. Median overall survival of patients with TP53 mutation was shorter than wild-type (9 versus 66 months, P < 0.001) and it retained significance in multivariable model (Hazard Ratio 3.8, 95%CI 2.3-6.3,P < 0.001). None of the sequentially analysed samples showed a disappearance of the mutant clone or emergence of new clones, suggesting an early occurrence of TP53 mutations. A reduction in mutant clone correlated with response to 5-azacitidine, however clones increased in non-responders and persisted at relapse. The adverse impact of TP53 persists after adjustment for cytogenetic risk and is of practical importance in evaluating prognosis. The relatively common occurrence of these mutations in two different prognostic spectrums of MDS, i.e. isolated 5q- and CK with -5/5q-, possibly implies two different mechanistic roles for TP53 protein.

Genome-wide single-nucleotide polymorphism array-based karyotyping in myelodysplastic syndrome and chronic myelomonocytic leukemia and its impact on treatment outcomes following decitabine treatment

Decitabine is a hypomethylating agent with proven clinical efficacy in myelodysplastic syndrome (MDS). The current study analyzed the role of single nucleotide polymorphism array (SNP-A)-based karyotyping in prediction of clinical outcome in MDS or chronic myelomonocytic leukemia (CMML) patients following decitabine therapy. A total of 61 MDS/CMML patients treated with decitabine were evaluated with Genome-Wide Human SNP 6.0 Array using DNAs derived from marrow samples. The primary endpoint was the best response rate including complete (CR) and partial response (PR) with overall (OS) and event-free survival (EFS) as secondary endpoints. Best response was noted in 14 patients (26.4 %) out of 53 evaluated patients including 12 CR and two PR with median follow-up of 21.6 months. A total of 81 abnormal SNP lesions were found in 25 out of 61 patients (41.0 %). The patients carrying abnormal SNP lesions showed an inferior CR/PR rate (p = 0.002) and showed a trend of worse OS (p = 0.02 in univariate, p = 0.09 in multivariate) compared to those without SNP lesions, but not were associated with inferior EFS. The presence of abnormal SNP lesions in MDS was associated with adverse outcomes following decitabine therapy. Further study is strongly warranted to establish the role of SNP-A karyotyping in MDS.

Utility of Peripheral Blood for Cytogenetic and Mutation Analysis in Myelodysplastic Syndrome

Abstract 1707With the advent of high throughput and high resolution techniques, >80% of myelodysplastic syndrome (MDS) patients harbour somatic mutations and/or genomic aberrations, which provide diagnostic and prognostic utility; however, frequent bone marrow (BM) aspirates are required. In a significant minority, the BM is hypocellular and fibrotic with suboptimal in vitro growth and, additionally, the procedure causes discomfort particularly in the elderly. This led us to investigate the use of peripheral blood (PB) and serum to identify and monitor BM derived genetic markers using high resolution single nucleotide polymorphism array (SNP-A) karyotyping and 454 parallel sequencing (454-PS) of a 22 gene myeloid panel comprising of all the exons of DNMT3a, RUNX1, CEBPα, TP53, EZH2, TET2 and ZRSR2 and mutations ‘hotposts' for NPM1, FLT3, ASXL1, IDH1, IDH2, MPL, JAK2, BRAF, cCBL, NRAS, KRAS, C-KIT, SF3B1, SRSF2, and U2AF1.We selected 23 MDS patients with concurrent BM and PB samples and detected 45 mutations in TET2, SF3B1, ASXL1, TP53, DNMT3a, FLT3, U2AF1, NRAS, cCBL, JAK2 and IDH2 in BM and subsequently analysed their PB using 454-PS with independent validation performed by Sanger sequencing (SS). All the mutations identified in BM were detected in PB with the exception of a single NRAS (BM-11%). Nine patients had a single mutation in SF3B1, ASXL1, TP53, TET2, DNMT3a and U2AF1 with the remaining patients having multiple coexisting mutations. Overall there was no significant difference in the mutation burden between the PB (median 25%(1.5%-50%)) and the BM (median 33%(5%-68%)). Concurrent analysis of unamplified and whole genome amplified PB DNA from 3 patients with mutations in TET2, U2AF1, ASXL1 and NRAS showed no difference in their mutation profile. As expected, in three patients post therapy the mutation burden in the PB was lower than in the pre-treatment BM sample.The lower mutation burden and sequence context in the PB contributed significantly to the quality of SS analysis. Prior knowledge of the mutation site resulted in 98% concordance (smallest clone size - 1.5%) between BM and PB, however, a blind approach decreased this to 84% (smallest clone size - 15%). In addition, serum was available from 14 patients (22 known mutations in BM) and SS of serum DNA identified 12 mutations correctly (U2AF1, FLT3, SF3B1, TET2, TP53, ASXL1, DNMT3a and IDH2, 4 as wildtype (TET2, cCBL, ASXL1 and SF3B1) and 6 failed to amplify (ASXL1, TP53 and TET2) without any preference for specific genes and the failure attributed to the quantity of serum DNA.Karyotype aberrations in PB were assessed using Affymetrix SNP 6.0 arrays on 31 MDS patients; normal karyotype (n=11), del5q (n=9), del7q/-7 (n=5), trisomy 8 (n=2), complex (n=2), isodiXq13 (n=1) and t(2:4)(q33;q27). An overall karyotype concordance of 94% was observed in PB with the 2 discordant cases showing normal karyotype in PB and BM by SNP-A but having monosomy 7 (partial cytogenetic remission after 5-azacitidine) and t(2:4)(q33;q27) respectively in their BM by MC. The mean copy number (CN) in the PB was lower than BM (PB vs BM); deletions (CN of 1.8 vs 1.6) and gains(CN of 2.2 vs 2.4), implying a smaller abnormal clone in the PB. Concurrent SNP-A karyotype from BM from 9 patients was concordant with SNP-A karyotype from PB, although a patient with complex karyotype determined by SNP-A in the BM and having 30 aberrations had only 15 aberrations detected in the PB.To determine if the 5q deletion was lineage restricted, we enriched PB for CD3+, CD19+ and CD3-CD19- populations from 4 patients. FISH and SNP-A karyotyping showed the presence of the 5q deletion using both techniques in all three fractions, however at a lower level in PB lymphocytes indicating the presence of a smaller clone.In conclusion, our study showed an excellent concordance between BM and PB, both for karyotype and mutational analyses using high resolution SNP-A karyotyping and 454-PS suggesting its clinical utility as a surrogate for BM, thereby, avoiding the discomfort of repeated BM aspirates and help in monitoring response to therapy more frequently. Disclosures:No relevant conflicts of interest to declare.

Single Nucleotide Polymorphism Array Lesions, TET2, DNMT3A, ASXL1 and CBL Mutations Are Present in Systemic Mastocytosis

We hypothesized that analysis of single nucleotide polymorphism arrays (SNP-A) and new molecular defects may provide new insight in the pathogenesis of systemic mastocytosis (SM). SNP-A karyotyping was applied to identify recurrent areas of loss of heterozygosity and bidirectional sequencing was performed to evaluate the mutational status of TET2, DNMT3A, ASXL1, EZH2, IDH1/IDH2 and the CBL gene family. Overall survival (OS) was analyzed using the Kaplan-Meier method. We studied a total of 26 patients with SM. In 67% of SM patients, SNP-A karyotyping showed new chromosomal abnormalities including uniparental disomy of 4q and 2p spanning TET2/KIT and DNMT3A. Mutations in TET2, DNMT3A, ASXL1 and CBL were found in 23%, 12%, 12%, and 4% of SM patients, respectively. No mutations were observed in EZH2 and IDH1/IDH2. Significant differences in OS were observed for SM mutated patients grouped based on the presence of combined TET2/DNMT3A/ASXL1 mutations independent of KIT (P = 0.04) and sole TET2 mutations (P<0.001). In conclusion, TET2, DNMT3A and ASXL1 mutations are also present in mastocytosis and these mutations may affect prognosis, as demonstrated by worse OS in mutated patients.

A common 8q (MYC) amplification detected in a multifocal anaplastic astrocytoma by SNP array karyotyping

The distinction of multifocal versus multicentric gliomas can conceivably have important therapeutic implications. We present a 27-year-old man with two radiologically distinct non-enhancing infiltrative masses in the anterior frontal lobe and the posterior temporoparietal region. No intervening disease was evident on MRI modalities; the lesions were stable over a period of many months. He underwent two separate resections a few months apart. Given the question of whether his tumors represented two de novo primary multicentric tumors or one multifocal tumor, single nucleotide polymorphism (SNP) array karyotyping and in situ hybridization studies were performed on both tumors. The two tumor profiles looked remarkably similar, histologically and genetically: both were anaplastic astrocytomas with a common 33Mb gain/ amplification of 8q23.3-q24.3, including MYC amplification, suggesting a monoclonal origin. The temporoparietal neoplasm showed several additional genetic alterations. This case illustrates that even with today's advanced neuroimaging modalities, extensive radiologically invisible tumor may be present between seemingly separate sites of glioma involvement. Thus modern global genomic studies of such tumors may help distinguish whether multiple tumors represent one extensive neoplasm with microscopically invasive disease or multiple genetically distinct tumors.

Topography, Clinical, and Genomic Correlates of 5q Myeloid Malignancies Revisited

Interstitial deletions of chromosome 5q are common in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), pointing toward the pathogenic role of this region in disease phenotype and clonal evolution. The higher level of resolution of single-nucleotide polymorphism array (SNP-A) karyotyping may be used to find cryptic abnormalities and to precisely define the topographic features of the genomic lesions, allowing for more accurate clinical correlations. We analyzed high-density SNP-A karyotyping at diagnosis for a cohort of 1,155 clinically well-annotated patients with malignant myeloid disorders. results: We identified chromosome 5q deletions in 142 (12%) of 1,155 patients and uniparental disomy segments (UPD) in four (0.35%) of 1,155 patients. Patients with deletions involving the centromeric and telomeric extremes of 5q have a more aggressive disease phenotype and additional chromosomal lesions. Lesions not involving the centromeric or telomeric extremes of 5q are not exclusive to 5q- syndrome but can be associated with other less aggressive forms of MDS. In addition, larger 5q deletions are associated with either del(17p) or UPD17p. In 31 of 33 patients with del(5q) AML, either a deletion involving the centromeric and/or telomeric regions or heterozygous mutations in NPM1 or MAML1 located in 5q35 were present. Our results suggest that the extent of the affected region on 5q determines clinical characteristics that can be further modified by heterozygous mutations present in the telomeric extreme.

Single-Nucleotide Polymorphism Array Karyotyping in Clinical Practice: Where, When, and How?

Single-nucleotide polymorphism array (SNP-A) karyotyping is a new technology that has enabled genome-wide detection of genetic lesions in human cancers, including hematopoietic neoplasms. Taking advantage of very large numbers of allele-specific probes synthesized on microarrays at high density, copy number alterations as well as allelic imbalances can be sensitively detected in a genome-wide manner at unprecedented resolutions. Most importantly, SNP-A karyotyping represents the only platform currently available for genome-scale detection of copy neutral loss of heterozygosity (CN-LOH) or uniparental disomy (UPD), which is widely observed in cancer genomes. Although not applicable to detection of balanced translocations, which are commonly found in hematopoietic malignancies, SNP-A karyotyping technology complements and even outperforms conventional metaphase karyotyping, potentially allowing for more accurate genetic diagnosis of hematopoietic neoplasms in clinical practice. Here, we review the current status of SNP-A karyotyping and its application to hematopoietic neoplasms.

Genome-Wide Single-Nucleotide Polymorphism-Array Can Improve Prognostic Stratification of Core Binding Factor Acute Myeloid Leukemia, Especially in the Subgroup with Inv(16)/t(16;16) or without D816 C-KIT Mutation,

Abstract 3515 Background:The core binding factor (CBF) AML can be achieved long-term remission with high dose cytarabine-based chemotherapy alone. However, those with C-KIT gene mutation (esp. D816 C-KIT mutation) showed worse treatment outcomes compared to those with wild type C-KIT gene. The remaining cases without D816 C-KIT mutation is around 75% of CBF AML, which implies requirement of more sophisticated dissection of the patients according to their prognosis. Single nucleotide polymorphism (SNP) array (SNP-A) could detect cryptic abnormal genomic lesions, not identified by metaphase cytogenetics(MC). In this study, we analyzed the prognostic value of SNP-A based karyotyping combined with MC and its association with C-KIT mutation to facilitate further stratification of CBF AML patients. Methods and Materials:A total of 98 CBF AML patients were included and of whom, 63 (64%) and 35 patients (36%) were t(8;21) and inv(16)/t(16;16), respectively. Genome-Wide Human SNP 6.0 Array (Affymetrix, CA, USA) was performed using DNAs from marrow samples taken at diagnosis. Results:A total of 40 abnormal genomic lesions in 25 patients (26%) were detected by SNP-A karyotyping analysis, with a mean of 1.6 lesions per affected case (median size 33.6 Mb; range 0.4–145.9 Mb), including 3 CN-LOH lesions, 17 gain lesions, and 20 loss lesions. Survival of the patients with abnormal lesion(s) detected by SNP-A or/and MC was worse than those without any lesions in terms of 2 years’ overall survival (OS; 57.5% vs 76.4%, p=0.028), event-free (EFS; 45.7% vs 66.2%, p=0.072) and leukemia free survival (LFS; 49.0% vs 77.4%, p=0.015). In contrast, MC alone could not stratify patients according to their long-term prognosis. Especially, in the subgroup with inv(16)/t(16;16), survival of patients with abnormal SNP-A/MC lesion showed worse than that of those without lesion (40.9±12.7% vs 80.2±10.4% at 2 yrs, p=0.040), but not in the subgroup with t(8;21) (66.85±9.1% vs 74.4±7.8% at 2 yrs, p=0.240). As for the subgroup with D816 C-KIT mutation, there were no differences of OS (p=0.417), EFS (p=0.380) and LFS (p=0.218) according to the presence of abnormal lesions detected by either SNP-A or MC. However, in the subgroup without D816 C-KIT mutation, those with abnormal lesions detected by either SNP-A or MC showed worse survival compared to those without abnormal lesions with respect to OS (61.6±8.7% vs 82.7±5.6% at 2 yrs, p=0.038). Multivariate analysis confirmed prognostic impact of abnormal SNP/MC lesions on OS (HR 2.743, p=0.020), EFS (HR 2.434, p=0.025), and LFS (HR 3.350, p=0.012). Conclusion:This study suggests that combined use of SNP-A with MC in the initial evaluation of CBF AML can provide an important prognostic value, especially in the inv(16)/t(16;16) subgroup or in the patients without having D816 C-KIT mutation. Disclosures:No relevant conflicts of interest to declare.