Resolution Single Nucleotide Polymorphism Array Research Articles

Diagnostic application of high resolution single nucleotide polymorphism array analysis for children with brain tumors

Single nucleotide polymorphism (SNP) array analysis is currently used as a first tier test for pediatric brain tumors at The Children's Hospital of Philadelphia. The results from 100 consecutive patients are summarized in the present report. Eighty-seven percent of the tumors had at least one pathogenic copy number alteration. Nineteen of 56 low grade gliomas (LGGs) demonstrated a duplication in 7q34, which resulted in a KIAA1549-BRAF fusion. Chromosome band 7q34 deletions, which resulted in a FAM131B-BRAF fusion, were identified in one pilocytic astrocytoma (PA) and one dysembryoplastic neuroepithelial tumor (DNT). One ganglioglioma (GG) demonstrated a 6q23.3q26 deletion that was predicted to result in a MYB-QKI fusion. Gains of chromosomes 5, 6, 7, 11, and 20 were seen in a subset of LGGs. Monosomy 6, deletion of 9q and 10q, and an i(17)(q10) were each detected in the medulloblastomas (MBs). Deletions and regions of loss of heterozygosity that encompassed TP53, RB1, CDKN2A/B, CHEK2, NF1, and NF2 were identified in a variety of tumors, which led to a recommendation for germline testing. A BRAF p.Thr599dup or p.V600E mutation was identified by Sanger sequencing in one and five gliomas, respectively, and a somatic TP53 mutation was identified in a fibrillary astrocytoma. No TP53 hot-spot mutations were detected in the MBs. SNP array analysis of pediatric brain tumors can be combined with pathologic examination and molecular analyses to further refine diagnoses, offer more accurate prognostic assessments, and identify patients who should be referred for cancer risk assessment.

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.

Implementation of high resolution single nucleotide polymorphism array analysis as a clinical test for patients with hematologic malignancies

Single nucleotide polymorphism-based oligonucleotide arrays have been used as a research tool to detect genomic copy number changes and allelic imbalance in a variety of hematologic malignancies and solid tumors. The high resolution, genome-wide coverage, minimal DNA requirements, and relatively short turnaround time are advantageous for use in a clinical setting. We validated the Illumina HumanHap550 BeadChip array for clinical use by analyzing 127 pediatric leukemia and lymphoma samples that had previously been characterized by means of standard cytogenetic analysis and fluorescence in situ hybridization. A higher resolution Illumina HumanHap610 BeadChip array was ultimately used for clinical testing. To date, 180 samples from children with a suspected or confirmed hematologic malignancy have been analyzed. Of the 180 clinical samples, 130 (72%) bone marrow or lymphoma specimens had aberrations revealed by the array that were not seen in the karyotypes. These typically included deletions in genes associated with B- or T-cell malignancies, such as CDKN2A/B, PAX5, and IKZF1. There were also 75 regions of copy number neutral loss of heterozygosity (>5 Mb threshold) detected in 49 samples in this cohort, which could be categorized as constitutional or acquired abnormalities. On the basis of our experience in the last 2 years, we suggest that single nucleotide polymorphism arrays are a valuable addition to, but not a replacement for, standard cytogenetic approaches for hematologic malignancies.

A High Resolution Analysis of Chromosome 21 Amplification In Myeloid Malignancies Reveals An Association with a Specific Cytogenetic Subgroup and Enhanced ERG Gene Expression.

AbstractAbstract 1687Genetic alterations including chromosomal translocation, somatic mutation, and gene amplification are thought to play a key role in oncogenesis. Gains of whole or segmental chromosome 21 (Ch21) are observed in many types of myeloid malignancies and are often associated with acute megakaryoblastic leukemia (AMKL). In Down syndrome, transient abnormal myelopoiesis and acute lymphoblastic leukemia can be observed, but the prevalence of AMKL is striking. In rare Down syndrome patients, a subcytogenetic Ch21 minimal amplified region is observed and always found to include ERG as well as the RUNX1 gene locus. Recently, gain of ERG gene copy number has been demonstrated to induce leukemia in mouse models and mutations in RUNX1 have been reported in patients with myeloid malignancies with somatic trisomy 21. The pathogenic gene(s) driving malignant disease in congenital and/or somatic gain of Ch21 are poorly understood. We applied high resolution single nucleotide polymorphism array (SNP-A) to study whether small copy number gains are present on Ch21, which cannot be seen by metaphase cytogenetics. We also tested for potential synergistic karyotypic abnormalities in the patients with gain of Ch21 gene segments.We screened a large cohort of 522 patients with myeloid malignancies by SNP-A platform, and detected 36 events that included whole or partial amplification of Ch21 in 32 cases (6%). The affected length was between 215,063 and 46,944,323 bp and the average was 30,732,002. These include 13 congenital lesions (AMKL evolving in Down syndrome), and 23 somatic alterations. Among the AMKL cohort of 34 cases, gains of Ch21 were observed in 15/25 (60%) juvenile and 2/9 (22%) adult cases. A minimal consensus amplification region was defined from nt38637816 to nt38852879 on Ch21 and this region included ERG. Amplification of ERG was identified in 30/36 of the Ch21 gain lesions studied. Although we sequenced all exons of the ERG gene in all cases with Ch21 gain, no mutation was detected. Based on the possibility that gene amplification leads to increased gene expression, ERG mRNA levels were investigated. CD34+ cells showed the highest ERG expression among hematopoietic cell types. When CD34+ cells from acute myeloid leukemia (AML) patients with somatic trisomy 21, with normal copy of Ch21 and healthy donors were investigated by real time PCR, relative expression of ERG was the highest in trisomy 21 patients among three groups. Based on our previous work and that of others, we tested the mutational status of RUNX1 in the 23 cases with Ch21 amplification that included RUNX1. Mutations were found in 2/23 (9%) accompanied by trisomy 21. No mutation was found in patients with Down syndrome. In one mutant case, a homozygous missense mutation, (L56S) was identified and associated with uniparental trisomy that included RUNX1. The second mutant case (W106L) was in a patient with a 45,XY,-7,i(21)(q10) karyoptype. The mutation was duplicated but was not associated with loss of heterozygosity (LOH). When RUNX1 gene expression in the cases with and without trisomy 21 using CD34 positive bone marrow cells was investigated, no significant difference in relative RUNX1 mRNA levels between trisomy 21 and cases with diploid Ch21 was found. Finally, we evaluated whether additional chromosomal lesions were associated with a gain of Ch21 gene segments. Recurrent losses were detected on chromosome 1, 2, 3, 5, 7, 9, and 17. Deletions of 5q were frequent in the cases with somatic gain of Ch21 (47%; 8/17), while no del5q was detected in the cases with Down syndrome. Conversely, LOH17p (3 uniparental disomies (UPDs) and 2 deletions) was found in both somatic and congenital cases (5/32), with one case of deletion17p associated with a hemizygous p53 mutation. In addition, UPD11q was accompanied by a CBL homozygous mutation in a RAEB case with somatic trisomy 21. Del7q was also observed in both groups (4 in somatic and 3 in congenital cases), including a 7q36.1 microdeletion associated with EZH2 in AMKL with Down syndrome.In sum, our study demonstrates that high resolution SNP-A analysis focused on Ch21 gene segments revealed frequent cryptic somatic gain lesions and a uniparental trisomy. ERG was the sole gene located in the minimally shared gain lesions and is overexpressed in a wild type form in AML cases with somatic trisomy 21. RUNX1 mutations were found in 3 or 2 identical alleles of somatic trisomy 21 cases but are absent in most cases of trisomy 21. Disclosures:No relevant conflicts of interest to declare.

Characterization of chromosome arm 20q abnormalities in myeloid malignancies using genome‐wide single nucleotide polymorphism array analysis

Deletion of the long arm of chromosome 20 is a common abnormality associated with myeloid malignancies. We characterized abnormalities of chromosome 20 as defined by metaphase cytogenetics (MC) in patients with myeloid neoplasms to define commonly deleted regions (CDR) and commonly retained regions (CRR) using genome-wide, high resolution single nucleotide polymorphism array (SNP-A) analysis. We reviewed the MC results of a cohort of 1,162 patients with myeloid malignancies, including myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasia (MDS/MPN), and acute myeloid leukemia (AML). We further analyzed a subcohort of 532 patients by SNP-A using the Affymetrix Genome-Wide Human SNP Array 6.0 and GeneChip Human Mapping 250K Nsp arrays. By MC, 5% (54/1,162) harbored a deletion of 20q; in 30% (16/54), del(20q) was the sole cytogenetic abnormality. By SNP-A analysis, we identified del(20q) in 23 patients, 3 not detected by MC. In four cases, monosomy 20 with a marker chromosome by MC was proven to be an interstitial deletion of 20q by SNP-A. We defined 2 CDR and 2 CRR on chromosome arm 20q: CDR1 spanned 2.5 Mb between bands 20q11.23 and 20q12, while CDR2 encompassed 1.8 Mb within 20q13.12. CRR1 spanned 1.9 Mb within 20q11.21 and CRR2 encompassed 2.5 Mb within 20q13.33. In contrast to other chromosomes frequently affected by deletions, no somatic copy neutral loss of heterozygosity (CN-LOH) was detected. Our data suggest that SNP-A is useful for the detection of cryptic aberrations of chromosome 20q and allows for a more precise characterization of complex karyotypes. Furthermore, SNP-A allowed definition of a CDR on 20q.