Somatic Copy-neutral Loss Of Heterozygosity Research Articles

Germline GPR161 Mutations Predispose to Pediatric Medulloblastoma.

PURPOSEThe identification of a heritable tumor predisposition often leads to changes in management and increased surveillance of individuals who are at risk; however, for many rare entities, our knowledge of heritable predisposition is incomplete.METHODSFamilies with childhood medulloblastoma, one of the most prevalent childhood malignant brain tumors, were investigated to identify predisposing germline mutations. Initial findings were extended to genomes and epigenomes of 1,044 medulloblastoma cases from international multicenter cohorts, including retrospective and prospective clinical studies and patient series.RESULTSWe identified heterozygous germline mutations in the G protein-coupled receptor 161 (GPR161) gene in six patients with infant-onset medulloblastoma (median age, 1.5 years). GPR161 mutations were exclusively associated with the sonic hedgehog medulloblastoma (MBSHH) subgroup and accounted for 5% of infant MBSHH cases in our cohorts. Molecular tumor profiling revealed a loss of heterozygosity at GPR161 in all affected MBSHH tumors, atypical somatic copy number landscapes, and no additional somatic driver events. Analysis of 226 MBSHH tumors revealed somatic copy-neutral loss of heterozygosity of chromosome 1q as the hallmark characteristic of GPR161 deficiency and the primary mechanism for biallelic inactivation of GPR161 in affected MBSHH tumors.CONCLUSIONHere, we describe a novel brain tumor predisposition syndrome that is caused by germline GPR161 mutations and characterized by MBSHH in infants. Additional studies are needed to identify a potential broader tumor spectrum associated with germline GPR161 mutations.

Second-Hit, Postzygotic PMVK and MVD Mutations in Linear Porokeratosis

Linear porokeratosis features linear and whorled configurations of keratotic papules and plaques, with coronoid lamellae present on histologic examination. Because linear porokeratosis manifests in the lines of Blaschko representing the dorsoventral migration patterns of keratinocyte precursors, it has been suggested that postzygotic somatic mutation underlies the disease. However, no genetic evidence has supported this hypothesis to date. To identify genetic mutations associated with linear porokeratosis. Paired whole-exome sequencing of affected skin and blood/saliva samples from 3 participants from 3 academic medical centers with clinical and histologic diagnoses of linear porokeratosis. Whole-exome sequencing of paired blood/saliva and affected tissue samples isolated from linear porokeratosis lesions. Germline and somatic genomic characteristics of participants with linear porokeratosis. Of the 3 participants, 2 were male. Participant ages ranged from 5 to 20 years old. We found a combination of a novel germline mutation and a novel somatic mutation within affected tissue in all cases. One participant had a germline heterozygous PMVK c.329G>A mutation and a somatic copy-neutral loss of heterozygosity confined to the lesional skin, while a second had a germline heterozygous PMVK c.79G>T mutation and an additional PMVK c.379C>T mutation in the lesional skin. In a third participant, there was a germline splice-site mutation in MVD (c.70 + 5G>A) and a somatic deletion in MVD causing frameshift and premature codon termination within the lesional skin (c.811_815del, p.F271Afs*33 frameshift). Our findings suggest that linear porokeratosis is associated with the presence of second-hit postzygotic mutations in the genes that encode enzymes within the mevalonate biosynthesis pathway, and provide further evidence that the mevalonate pathway may be a potential target for therapeutic intervention in porokeratosis.

CDC73 Germline Mutation in a Family With Mixed Epithelial and Stromal Tumors

ObjectiveTo describe a family in which 3 members presented with mixed epithelial tumor of the kidney (MEST) and were found to possess a germline mutation in CDC73, a gene which is associated with hyperparathyroidism-jaw tumor syndrome (HPT-JT). Materials and MethodsBlood and tumor DNA from three family members who presented with a primary diagnosis of MEST was subjected to targeted gene sequencing to identify potential genetic components. ResultsA germline start codon mutation (p.M1I) in CDC73 was identified in all 3 family members who presented with MEST and 2 tumors from 1 patient demonstrated somatic copy-neutral loss of heterozygosity. Patients presented with no evidence of hyperparathyroidism or jaw tumors, but both female patients had hysterectomies at an early age due to excessive bleeding and numerous fibroids, which is common in HPT-JT. A germline p.M1I mutation has been previously reported in a family with clinical features of HPT-JT. ConclusionPatients with MEST may be at risk for HPT-JT and CDC73 germline mutation testing of MEST patients should be considered.

Whole-exome sequencing identifies germline mutation in TP53 and ATRX in a child with genomically aberrant AT/RT and her mother with anaplastic astrocytoma.

Brain tumors typically arise sporadically and do not affect several family members simultaneously. In the present study, we describe clinical and genetic data from two patients, a mother and her daughter, with familial brain tumors. Exome sequencing revealed a germline missense mutation in the TP53 and ATRX genes in both cases, and a somatic copy-neutral loss of heterozygosity (LOH) in TP53 in both atypical teratoid/rhabdoid tumor (AT/RT) and astrocytoma tumors. ATRX mutation was associated with the loss of ATRX protein expression. In the astrocytoma case, R132C missense mutation was found in the known hotspot site in isocitrate dehydrogenase 1 (IDH1) and LOH was detected in TP53. The mother carried few other somatic alterations, suggesting that the IDH1 mutation and LOH in TP53 were sufficient to drive tumor development. The genome in the AT/RT tumor was atypically aneuploid: Most chromosomes had experienced copy-neutral LOH or whole-chromosome gains. Only Chromosome 18 had normal diploid status. INI1/hSNF5/SMARCB1 was homozygously deleted in the AT/RT tumor. This report provides further information about tumor development in a predisposed genetic background and describes two special Li–Fraumeni cases with a familial brain tumor.

Somatic copy-neutral loss of heterozygosity and copy number abnormalities in Malaysian sporadic colorectal carcinoma patients

Colorectal cancer is one of the most common cancers in many countries, including Malaysia. The accumulation of genomic alterations is an important feature of colorectal carcinogenesis. A better understanding of the molecular events underlying the stages of colorectal carcinogenesis might be helpful in the detection and management of the disease. We used a commercially available single-nucleotide polymorphism genotyping array to detect both copy number abnormalities (CNAs) and copy-neutral loss of heterozygosity (LOH) in sporadic colorectal carcinomas. Matched tumor and normal tissues of 13 colorectal carcinomas (Dukes' stages A-D) were analyzed using a 250K single nucleotide polymorphism array. An additional assay was performed to determine the microsatellite instability status by using the National Cancer Institute-recommended BAT-26 panel. In general, copy number gain (92.3%) was most common, followed by copy number loss (53.8%) and copy-neutral LOH (46.2%). Frequent CNAs of gains and losses were observed on chromosomes 7p, 8, 13q, 17p, 18q, and 20q, and copy-neutral LOH was observed on chromosomes 2, 6, 12, 13q, 14q, 17, 20p, 19q, and 22q. Even though genomic alterations are associated with colorectal cancer progression, our results showed that DNA CNAs and copy-neutral LOH do not reflect disease progression in at least 50% tumors. Copy-neutral LOH was observed in both early and advanced tumors, which favors the involvement of these genomic alterations in the early stages of tumor development.

Myelodysplasia and leukemia of Fanconi anemia are associated with a specific pattern of genomic abnormalities that includes cryptic RUNX1/AML1 lesions

AbstractFanconi anemia (FA) is a genetic condition associated with bone marrow (BM) failure, myelodysplasia (MDS), and acute myeloid leukemia (AML). We studied 57 FA patients with hypoplastic or aplastic anemia (n = 20), MDS (n = 18), AML (n = 11), or no BM abnormality (n = 8). BM samples were analyzed by karyotype, high-density DNA arrays with respect to paired fibroblasts, and by selected oncogene sequencing. A specific pattern of chromosomal abnormalities was found in MDS/AML, which included 1q+ (44.8%), 3q+ (41.4%), −7/7q (17.2%), and 11q− (13.8%). Moreover, cryptic RUNX1/AML1 lesions (translocations, deletions, or mutations) were observed for the first time in FA (20.7%). Rare mutations of NRAS, FLT3-ITD, MLL-PTD, ERG amplification, and ZFP36L2-PRDM16 translocation, but no TP53, TET2, CBL, NPM1, and CEBPα mutations were found. Frequent homozygosity regions were related not to somatic copy-neutral loss of heterozygosity but to consanguinity, suggesting that homologous recombination is not a common progression mechanism in FA. Importantly, the RUNX1 and other chromosomal/genomic lesions were found at the MDS/AML stages, except for 1q+, which was found at all stages. These data have implications for staging and therapeutic managing in FA patients, and also to analyze the mechanisms of clonal evolution and oncogenesis in a background of genomic instability and BM failure.

High-Resolution Genomic Profiling of Adult and Pediatric Core Binding Factor Acute Myeloid Leukemia Reveals New Recurrent Genomic Aberrations

AbstractAbstract 849Core-binding-factor (CBF) acute myeloid leukemia (AML) defined by the presence of t(8;21)(q22;q22) or inv(16)(p13.1q22)/t(16;16)(p13.1;q22) is associated with favorable outcome. However, about 30–40% of patients are not cured by current treatment approaches. Secondary genetic changes are believed to cause clinical heterogeneity. To identify new secondary genetic lesions, we performed high-resolution, genome-wide analysis of copy number aberrations (CNA) and copy neutral loss of heterozygosity (CN-LOH) using Affymetrix 6.0 single nucleotide polymorphism (SNP) microarrays in 300 adult and pediatric CBF AMLs; t(8;21), n=157 (adult, n=114; pediatric, n=43); and inv(16), n=143 (adult, n=104; pediatric, n=39). Germline control DNA from remission bone marrow or peripheral blood was available for paired analysis in 175 patients. In addition, for 42 patients matched relapse samples were analyzed. Data were processed using reference alignment, dChipSNP and circular binary segmentation. Paired analysis revealed a median of 1.28 somatic CNAs per case [t(8;21): 1.14, range: 0–5; inv(16): 1.45, range: 0–9], with deletions more common than gains [t(8;21): 0.94 losses/case vs. 0.2 gains/case; inv(16): 0.95 vs. 0.5]. Recurrent deletions were detected at chromosomal bands 7q36.1 (n=23), 9q21.13 (n=15), 11p13 (n=7), 17q11.2 (n=6), 10q24.32 (n=2), and at the chromosomal breakpoints of t(8;21) and inv(16) on 8q21.3 (n=9), 21q22 (n=16), 16p13.11 (n=28), and 16q22.11 (n=21). Deletions at 7q, 11p and 17q were validated using FISH analysis. Minimally deleted regions (MDR) less than 1.5 Mb were identified at 7q36.1 (647 Kb, 4 genes), 9q21.13 (1125 Kb, 9 genes), 11p13 (130 Kb, 1 gene), and 17q11.2 (902 Kb, 11 genes), with each region containing a putative tumor suppressor (e.g., MLL3 on 7q, FRMD3 on 9q, WT1 on 11p, and NF1 on 17q). Sequence analysis of MLL3 in 23 cases with del(7q), 1 case with 7q CN-LOH, and 23 randomly selected cases identified a MLL3 truncating mutation leading to a premature stop codon in a case that lacked a 7q alteration. The del(11p13) contained only WT1 and primarily affected inv(16)-cases (5 of 7). Sequence analysis of WT1 in four cases with del(11p) revealed an additional frame shift mutation in the remaining allele in one case. Sequence analysis of WT1 in an additional 103 inv(16)-containing cases revealed mutations in 10 (9%) of the cases. The MDR at 17q11.2 was exclusively identified in inv(16)-containing cases (n=6) and included the tumor supressor NF1. Sequence analysis of all coding exons in NF1 in 4 additional inv(16)-containing AMLs revealed no additional mutation. Recurrent gains were identified at 22q11.21-q13.33 (n=20; 32 Mb), 8q24.21 (n=14; 138 Kb), 13q21.1-q34 (n=6; 14 Mb), and 11q25 (n=5; 368 Kb). The smallest gains were identified at 8q24.21 and 11q25, both containing only a single non-coding RNA gene (CCDC26 and LOC283177, respectively). Somatic CN-LOH were uncommon and only found at 1p36.33-p12 (n=2), 4q (n=2), and 19p (n=2). In a comparative analysis of paired diagnostic and relapse samples, novel CNAs at the time of relapse were identified at 3q13.31 (n=5), 5q (n=2), 17p (n=2), and 17q (n=2). The MDR at 3q was only 46 kb in size and contains a single transcript that has been connected to LSAMP, a putative tumor suppressor located 404 Kb upstream of the deletion. In summary, our data provide a comprehensive profiling of copy number alterations in pediatric and adult CBF AML. These data demonstrate a very low number of CNAs, with no significant differences noted between pediatric and adult cases. Interestingly, a number of novel recurrent secondary genetic alterations are identified. Exploring the biological role of these lesions in leukemogenesis and drug resistance should provide important insights into the CBF leukemias. Disclosures:No relevant conflicts of interest to declare.

A Recurrent Pattern of Acquired Genomic Abnormalities In Myelodysplasia and Leukemia of Fanconi Anemia Includes Cryptic RUNX1/AML1 abnormalities

AbstractAbstract 975Fanconi anemia (FA) is a rare genetic condition characterized by congenital abnormalities, chromosome fragility, progressive bone marrow failure during childhood, and cancer susceptibility. FA patients experience a high risk to develop myelodysplasia (MDS) and secondary-type acute myeloid leukemia (AML) during their teens or in young adulthood. Severity of the cytopenia, excess of blast cells and presence of a cytogenetic clone in the bone marrow are usual criteria to undertake hematopoietic stem cell transplantation. In order to investigate the pattern of chromosomal and genomic abnormalities during bone marrow progression in FA and their association to MDS/AML, we analyzed bone marrow samples from FA patients using a wide panel of chromosomal and molecular techniques including DNA microarrays and oncogene sequencing. This series of FA patients was enriched in patients older than 18 year-old and/or with morphological or karyotypic abnormalities on the follow up BM aspirate. 57 FA patients were included, aged 4 to 57 yo (median 18); FA groups were FA-A (n=49), FA-G (n=1), FA-D2 (n=1), FA-D1 (n=1) and undertermined (n=5). Bone marrow morphology was hypoplastic/aplastic anemia (n=20), MDS (n=18, mainly RCMD and RAEB according to the WHO 2008 classification), AML (n=11), or no abnormality except the usual mild dyserythropoiesis of FA (n=8). Bone marrow samples were analyzed by karyotype, FISH, high density array-CGH and/or SNP-arrays with respect to the paired fibroblast DNAs, and by sequencing of selected oncogenes and tumor suppressor genes. A specific pattern of genomic abnormalities due to unbalanced translocations was found in the 29 MDS/AML, which included 1q+ (44.8%), 3q+ (41.3%), -7/7q (17.2%), and 11q- (13.8%). Moreover, cryptic abnormalities (translocations, deletions or mutations) of the RUNX1/AML1 gene were evidenced for the first time in FA, in 6 out of the 29 patients with MDS or AML (20.7%). By contrast, mutations of FLT3-ITD, MLL-ITD, and N-RAS, but not TP53, CBL, TET2, CEBPa, NPM1, and FLT3-TKD, were rarely found. Frequent homozygosity regions were evidenced by SNP-array in 11 patients, but the analysis of the paired fibroblast DNA and the constitutional FANC mutations demonstrated that they were not related to somatic copy-neutral loss of heterozygosity but to consanguinity. Importantly, the RUNX1/AML1 and other chromosomal/genomic abnormalities were found at the MDS and AML stages only, except for 1q+ which could be found at any stages including normal bone marrow morphology. In our experience 1q+ does not predict systematically a transformation into MDS/AML in the following years. These data have important implications, not only for the cytogenetic staging of the bone marrow cells in FA patients with an impact for therapeutic managing, but also as a basis to investigate the multistep clonal selection and related oncogenesis in patients with hypoplastic bone marrow and genomic instability, with potential relevance for non-FA patients. Disclosures:Gluckman:Cord-use: Membership on an entity's Board of Directors or advisory committees.

Using Combined High Density SNP/CNV Arrays as a Clinical Karyotyping Tool in Myeloid Malignancies.

We have evaluated various types of single nucleotide polymorphism arrays (SNP-A) as a karyotyping platform in over 600 cases of bone marrow failure and various myeloid disorders including MDS, MDS/MPD, and AML and in over 360 controls. We have shown that SNP-A not only reliably confirms chromosome gains and losses identified by metaphase cytogenetics (MC) but also allows for detection of previously cryptic chromosomal lesions. Moreover, through the ability of combining copy number measurement with genotyping, SNP-A also enables detection of copy-neutral loss of heterozygosity (LOH), a lesion that cannot be recognized using traditional MC. We have previously shown that this type of lesion occurs frequently in myeloid disorders. Recently, ultra-high density SNP-A were introduced (Affymetrix 6.0 array platform) with modified chemistry and a combination of SNP (>906,600) and copy number variant probes (CNV >946,000) to allow for precise delineation of the location and size of submicroscopic chromosomal defects, copy-neutral LOH and germline CNVs. This technology will likely supplant previous SNP-A platforms in performance and opens a new era in clinical cytogenetic diagnostics. In this pilot trial, we applied the Affymetrix SNP 6.0 array to samples obtained at diagnosis from 193 patients (59 AML, 115 MDS, 19 MDS/MPD). For 95 samples, both Affymetrix 250K and 6.0 arrays were applied; 6.0 SNP-A allowed for more identification of 33 regions of copy number aberrations and 11 copy-neutral LOH, suggesting a higher resolution of this technology over 250K arrays. By MC, 43% (71/167) of patients had chromosomal aberrations including a complex karyotype; 48% (81/167) had a normal karyotype and 9% (15/167) showed no growth. By 6.0 SNP-A, aberrations were detected in 78% (150/193) of patients. Significantly, among the samples with either a normal karyotype or non-informative results, aberrations were found by SNP-A in 57% and 73%, respectively. In an illustrative case with a balanced translocation by MC, copy number changes were found at the junctions, indicating that the translocation breakpoints contained small deletions. Comparison of 6.0 SNP-A results with MC showed concordance for detection of MC-defined lesions. However, 9% (15/167) of defects were only detected by MC and could not be detected by SNP 6.0 array in patients with a low proportion of abnormal metaphases. Gains and losses detected by SNP-A 6.0 required a sensitivity threshold of 20% clonal cells. We identified recurrent lesions not previously reported as being CNV. These comprised 238 regions of loss, 257 regions of gain, and 97 regions of copy-neutral LOH. When feasible (or if indicated to resolve a discordance with MC or to exclude CNV), DNA from sorted CD3+ cells was used as a reference. Recurrent losses were located in 37%(71/193) patients at 5q, 7p, 7q, 13q, 17p, 20q, 1p, 12p, 3q and 4q. Recurrent gains in 28%(55/193) patients included 13q, 11p, 11q, 21q, 3p, 8p, 8q, 9q, 19p and 5p. Somatic copy-neutral LOH was identified in 20%(39/193) patients at 13q, 1p, 5q, 6p, 7q, 11q, 17q, 3p, 4q, and 12q. Homozygous deletion was observed in one patient with a 6 Mb loss at 5q34 (not visible on MC) within a heterozygous 5q11.2–5q35.3 deletion Copy neutral LOH was found in 29% (56/193) patients overall. AML patients appeared to have more frequent copy neutral LOH(34%) than MDS (24%), but not significant (P=0.2). The 5%(9/193) of patients had more than 2 chromosomes with copy-neutral LOH. Among the samples showing a normal karyotype, 20% (16/81) of patients showed copy -neutral LOH as the sole aberration without additional copy number aberrations. To our knowledge, this is the first systematic application of SNP-A 6.0 in a large cohort of patients with myeloid malignancies. Our results demonstrate that this technology complements, but does not replace conventional MC for identification of abnormalities in myeloid neoplasia.