Recurrent Loss Of Heterozygosity Research Articles

Recurrent Loss of Heterozygosity in Pancreatic Neuroendocrine Tumors.

Chromosomal aneuploidies are prognostic markers across a wide variety of tumor types, and recent literature suggests that pancreatic neuroendocrine tumors are no different. In this study 214 patients with grade 1, 2, or 3 pancreatic neuroendocrine tumors had their tissue examined for chromosomal copy number alterations using next-generation sequencing. Univariate and multivariate statistical analyses were performed with all-cause mortality and disease-specific mortality as the end comparators. As such, the cohort stratified into 3 different clinically relevant chromosomal subgroups: an indolent subgroup characterized by loss of chromosome 11 in relative isolation, an aggressive subgroup characterized by losses of chromosomes 1, 2, 3, 6, 10, 11, 16, and 22 and with no loss of chromosomes 4, 5, 7, 12, 14, 17, 19, and 20, and finally a heterogeneous third group with a subset of cases that behave even more aggressively than the aforementioned.

Comprehensive Molecular Profiling of Desmoplastic Small Round Cell Tumor.

Desmoplastic small round cell tumor (DSRCT) is characterized by the EWSR1-WT1 t(11;22) (p13:q12) translocation. Few additional putative drivers have been identified, and research has suffered from a lack of model systems. Next-generation sequencing (NGS) data from 68 matched tumor-normal samples, whole-genome sequencing data from 10 samples, transcriptomic and affymetrix array data, and a bank of DSRCT patient-derived xenograft (PDX) are presented. EWSR1-WT1 fusions were noted to be simple, balanced events. Recurrent mutations were uncommon, but were noted in TERT (3%), ARID1A (6%), HRAS (5%), and TP53 (3%), and recurrent loss of heterozygosity (LOH) at 11p, 11q, and 16q was identified in 18%, 22%, and 34% of samples, respectively. Comparison of tumor-normal matched versus unmatched analysis suggests overcalling of somatic mutations in prior publications of DSRCT NGS data. Alterations in fibroblast growth factor receptor 4 (FGFR4) were identified in 5 of 68 (7%) of tumor samples, whereas differential overexpression of FGFR4 was confirmed orthogonally using 2 platforms. PDX models harbored the pathognomic EWSR1-WT1 fusion and were highly representative of corresponding tumors. Our analyses confirm DSRCT as a genomically quiet cancer defined by the balanced translocation, t(11;22)(p13:q12), characterized by a paucity of secondary mutations but a significant number of copy number alterations. Against this genomically quiet background, recurrent activating alterations of FGFR4 stood out, and suggest that this receptor tyrosine kinase, also noted to be highly expressed in DSRCT, should be further investigated. Future studies of DSRCT biology and preclinical therapeutic strategies should benefit from the PDX models characterized in this study. IMPLICATIONS: These data describe the general quiescence of the desmoplastic small round cell tumor (DSRCT) genome, present the first available bank of DSRCT model systems, and nominate FGFR4 as a key receptor tyrosine kinase in DSRCT, based on high expression, recurrent amplification, and recurrent activating mutations.

Recurrent loss of heterozygosity correlates with clinical outcome in pancreatic neuroendocrine cancer

Pancreatic neuroendocrine tumors (pNETs) are uncommon cancers arising from pancreatic islet cells. Here we report the analysis of gene mutation, copy number, and RNA expression of 57 sporadic well-differentiated pNETs. pNET genomes are dominated by aneuploidy, leading to concordant changes in RNA expression at the level of whole chromosomes and chromosome segments. We observed two distinct patterns of somatic pNET aneuploidy that are associated with tumor pathology and patient prognosis. Approximately 26% of the patients in this series had pNETs with genomes characterized by recurrent loss of heterozygosity (LoH) of 10 specific chromosomes, accompanied by bi-allelic MEN1 inactivation and generally poor clinical outcome. Another ~40% of patients had pNETs that lacked this recurrent LoH pattern but had chromosome 11 LoH, bi-allelic MEN1 inactivation, and universally good clinical outcome. The somatic aneuploidy allowed pathogenic germline variants (e.g., ATM) to be expressed unopposed, with RNA expression patterns showing inactivation of downstream tumor suppressor pathways. No prognostic associations were found with tumor morphology, single gene mutation, or expression of RNAs reflecting the activity of immune, differentiation, proliferative or tumor suppressor pathways. In pNETs, single gene mutations appear to be less important than aneuploidy, with MEN1 the only statistically significant recurrently mutated driver gene. In addition, only one pNET in the series had clearly actionable single nucleotide variants (SNVs) (in PTEN and FLCN) confirmed by corroborating RNA expression changes. The two clinically relevant patterns of LoH described here define a novel oncogenic mechanism and a plausible route to genomic precision oncology for this tumor type.

Genomic characterization of sarcomatoid transformation in clear cell renal cell carcinoma

The presence of sarcomatoid features in clear cell renal cell carcinoma (ccRCC) confers a poor prognosis and is of unknown pathogenesis. We performed exome sequencing of matched normal-carcinomatous-sarcomatoid specimens from 21 subjects. Two tumors had hypermutation consistent with mismatch repair deficiency. In the remainder, sarcomatoid and carcinomatous elements shared 42% of somatic single-nucleotide variants (SSNVs). Sarcomatoid elements had a higher overall SSNV burden (mean 90 vs. 63 SSNVs, P = 4.0 × 10(-4)), increased frequency of nonsynonymous SSNVs in Pan-Cancer genes (mean 1.4 vs. 0.26, P = 0.002), and increased frequency of loss of heterozygosity (LOH) across the genome (median 913 vs. 460 Mb in LOH, P < 0.05), with significant recurrent LOH on chromosomes 1p, 9, 10, 14, 17p, 18, and 22. The most frequent SSNVs shared by carcinomatous and sarcomatoid elements were in known ccRCC genes including von Hippel-Lindau tumor suppressor (VHL), polybromo 1 (PBRM1), SET domain containing 2 (SETD2), phosphatase and tensin homolog (PTEN). Most interestingly, sarcomatoid elements acquired biallelic tumor protein p53 (TP53) mutations in 32% of tumors (P = 5.47 × 10(-17)); TP53 mutations were absent in carcinomatous elements in nonhypermutated tumors and rare in previously studied ccRCCs. Mutations in known cancer drivers AT-rich interaction domain 1A (ARID1A) and BRCA1 associated protein 1 (BAP1) were significantly mutated in sarcomatoid elements and were mutually exclusive with TP53 and each other. These findings provide evidence that sarcomatoid elements arise from dedifferentiation of carcinomatous ccRCCs and implicate specific genes in this process. These findings have implications for the treatment of patients with these poor-prognosis cancers.

JAK2 V617F-Negative and MPL W515K/L-Negative Essential Thrombocythemia: A High Resolution SNP Array Study

BackgroundJAK2 V617F is the most common mutation in essential thrombocythemia (ET), occurring in approximately 50 % of cases. Second to JAK2 V617F is MPL W515K/L, accounting for about 10 % of cases. The molecular cause of the remaining ET cases is still largely unknown. AimsWe sought to investigate JAK2 V617F-negative and MPL W515K/L-negative ET for regions of copy number variations (CNV) and loss of heterozygosity (LOH). MethodsWe studied blood or bone marrow samples from a series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET cases. They were subjected to 2.7M SNP array by Affymetrix, which has 2,761,979 copy number markers including 400,103 SNP markers. The array data were analyzed for recurrent CNVs with Array Studio (OmicSoft), and for individual CNVs or recurrent LOHs (≥3 Mbs) with the Chromosome analysis suite (ChAS, Affymetrix). ResultsOnly 8 recurrent gains were identified, in 5/64 patients. Interestingly, the most common gain, occurring in 5 cases was a gain of chr7 q22.3, including the gene encoding Nicotinamide phosphoribosyltransferase (NAMPT). NAMPT is known to be overexpressed in several cancers such as multiple myeloma. It catalyzes the rate-limiting step of the nicotinamide adenine dinucleotide (NAD+) biosynthesis pathway. It is also required for cell growth and survival. We checked in the 5 patients for NAMPT amplification by quantitative PCR (qPCR) on genomic DNA in comparison to controls and by normalizing to ALB and RPPH1. We were able to validate the gain in 2/5 patients. The gain in these 2 patients was demonstrated to be acquired by qPCR of NAMPT in buccal swab DNA. Other recurrent gains involved regions of chromosomes 2, 5, 7, 12, 13, and 22. These gains included, amongst others, LCP1 on chr13 q14.3 and CYTIP on chr2 q24.1, occurring in 2/64 and in 3/64 respectively. We also checked for non-recurrent gains and losses in our cohort. This analysis generated a total of 8 CNVs in 6 different patients, comprising 5 regional gains in chromosomes 2, 8, 12, and 15 and 3 regional losses in chromosomes 5, 8 and 11. The array data were also analyzed for recurrent LOHs on ChAS, yielding 17 recurrent copy neutral LOHs (CN-LOH) in 35 patients (circos plot). The most common CN-LOH region was on chromosome 3 appearing in 8 patients. Other CN-LOH regions involved chromosomes 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, and 17 and they occurred in 2-5/64 patients. However, as small regions of CN-LOH can be constitutional, we suspect that most of these CN-LOH regions are not acquired. The largest region of CN-LOH observed was 12 Mbs in size. ConclusionsPrevious studies in unselected series of BCR-ABL1-negative myeloproliferative neoplasms have shown that copy number alterations are rare in ET as well as in polycythemia vera. In this series of 64 JAK2 V617F-negative and MPL W515K/L-negative ET patients we found recurrent gains not reported previously in the database of genomic variants in only 8% of patients, and small areas of CN-LOH in ∼55% of cases. However, most of the latter probably are constitutional. Our SNP array study provides further evidence that gains, losses or CN-LOH of small genomic regions do not play an essential role in the pathogenesis of the majority of JAK2 V617F-negative and MPL W515K/L-negative ET. However, the low frequency of megakaryocytes and unknown level of clonal involvement of the myeloid compartment in JAK2 V617F-negative and MPL W515K/L-negative ET bone marrow remain a caveat. Next generation sequencing technology is expected to bring new insights on the molecular pathogenesis of this elusive ET subset. [Display omitted] Circos plot showing the recurrent CN-LOHsLeft half represents a total of 35 patients carrying recurrent CN-LOHs and the right half represents the chromosomes and their associated properties. Right outermost layer depicts 1+log-gene density (min, 1; max 42) where cancer, OMIM and other genes are colored in red, blue and green respectively. Right middle and innermost layers designates SNP density (blue, values < 0,02; gray values, <0,06; red values >0,06; max scale 0,013) and absolute SNP numbers (min, 1; max, 12074) per windows of 50kb. Each 5Mb distance is marked with a tick underneath the innermost layer. Links from each chromosome are colored differently. Regions that are more confined on the same chromosome are least transparent and regions that are shared by more patients are drawn on top of lesser links. Disclosures:No relevant conflicts of interest to declare.

Recurrent loss of heterozygosity in 1p36 associated with TNFRSF14 mutations in IRF4 translocation negative pediatric follicular lymphomas

Pediatric follicular lymphoma is a rare disease that differs genetically and clinically from its adult counterpart. With the exception of pediatric follicular lymphoma with IRF4-translocation, the genetic events associated with these lymphomas have not yet been defined. We applied array-comparative genomic hybridization and molecular inversion probe assay analyses to formalin-fixed paraffin-embedded tissues from 18 patients aged 18 years and under with IRF4 translocation negative follicular lymphoma. All evaluable cases lacked t(14;18). Only 6 of 16 evaluable cases displayed chromosomal imbalances with gains or amplifications of 6pter-p24.3 (including IRF4) and deletion and copy number neutral-loss of heterozygosity in 1p36 (including TNFRSF14) being most frequent. Sequencing of TNFRSF14 located in the minimal region of loss in 1p36.32 showed nine mutations in 7 cases from our series. Two subsets of pediatric follicular lymphoma were delineated according to the presence of molecular alterations, one with genomic aberrations associated with higher grade and/or diffuse large B-cell lymphoma component and more widespread disease, and another one lacking genetic alterations associated with more limited disease.

Investigation of tumor suppressor genes apart from VHL on 3p by deletion mapping in sporadic clear cell renal cell carcinoma (cRCC)

ObjectivesTo investigate the most recurrent deletion loci on 3p12-p26 by deletion mapping studies by PCR-LOH and BAC array-FISH in sporadic conventional renal cell carcinoma (cRCC) and further, to evaluate the their clinicopathologic significance in cRCC. Comparative allelotyping studies in cRCC and major epithelial carcinomas (MEC) such as lung, breast, and bladder tumors were also carried out to investigate the specificity of the targeted loci in cRCC. Subjects and methodsA total of 40 c-RCC patients were enrolled in this study, categorized in to 2 groups: group I comprises of patients of stages I and II and group II includes patients at stages III and IV. Loss of heterozygosity (LOH) studies were performed by PCR using 15 microsatellite markers of region 3p12-p26 on paired normal-tumor tissues. The recurrent LOH loci found in 27 cRCC tumors were further validated by BAC array-FISH using 23 serially mapped BAC clones. Simultaneously, the allelic deletion status of fragile histidine triad (FHIT) gene was studied by FISH in cRCC and major epithelial carcinoma (MEC) tumors. The numerical aberrations of chromosome 3 were also studied using the centromere enumeration probe (CEP) probe for chromosome 3 to validate the observed allelic losses by BAC array-FISH in cRCC as well as MECs. ResultsOur study revealed 3 affected regions of LOH on 3p in cRCC: 3p12.2-p14.1, 3p14.2-p21.1, and 3p24.2-p26.1 in both group I (stages I and II) and group II (stage III and IV). Comparative allelotyping studies revealed that except for LOH loci D3S2406 (20%), D3S1766 (14%), and D3S1560 (20%), remaining affected loci revealed retention of heterozygosity (ROH) in breast carcinomas. Lung and bladder tumors revealed ROH at all affected LOH loci. FISH with FHIT gene probe revealed deletions in cRCC (88%), breast (30%), and lung tumors (10%). FHIT gene deletions frequency was almost equal in both groups I and II (>70%), whereas a locus 3p13 (D3S2454) revealed the highest LOH in group II (83%) patients in comparison to group I (16%). BAC array-FISH studies in cRCC identified 15 recurrent deletion loci at crucial regions, 3p12.2, 3p14.2, 3p21.3, and 3p24.2-p26 with long continuous deletion of 3p14.1-p26.1 exclusively in patients of stages III and IV. Validation of LOH loci in breast carcinomas by BAC array-FISH with BAC clones mapped at these loci revealed comparatively lower deletion frequency for RP11-59E22 (3p12.2) (30%), RP11-759B7(3p21.1) (12%), and RP11-57D6 (3p25.2, proximal to VHL) (15%) than cRCC. ConclusionMolecular cytogenetic studies by BAC array-FISH was found to be more sensitive over LOH. Deletion patterns on 3p explored that deletion of FHIT and flanking loci may occur as an initiating event followed by deletions at 3p12.2, 3p21.31–3p21.32, and 3p24.2–3p26.1 in the initial stage of development of disease, while continuous large deletions of 3p21.3–3p26.1 and 3p14.1–3p26.1 occur as progressive deletion due to genetic instability. Lack of VHL along with flanking loci in 50% cRCC patients that included both groups I and II supported the hypothesis of both VHL dependent and VHL independent pathways in cRCC tumorigenesis. Comparative allelotyping studies in cRCC and MECs indicated association of specific targeted loci including VHL in cRCC. Further expansion of these studies with characterization of the genes at targeted loci and correlation with clinical outcome will explore the prognostic significance and also provide an insight into the mechanisms of tumor suppressive pathways in genitourinary cancers such as CRCC.

Losing balance: Hardy-Weinberg disequilibrium as a marker for recurrent loss-of-heterozygosity in cancer.

Identifying regions of loss-of-heterozygosity (LOH) in a tumor sample is a challenging problem. State-of-the-art computational approaches can infer LOH from single-nucleotide polymorphism (SNP) array data, but calling precise boundaries is complicated by normal-cell contamination and markers that are homozygous in the germline and therefore non-informative. More recently, the focus has shifted to pinpointing the loci recurrently affected by LOH events across multiple tumors. Recurrent LOH regions often harbor genes important for tumor suppression. Here, we propose a method that infers LOH rates across an entire sample set on an SNP-by-SNP basis. Our method achieves this by leveraging the straightforward principle that, by definition, LOH depletes heterozygotes, thereby disrupting Hardy-Weinberg equilibrium. We apply a statistical test for such LOH-influenced disruptions, and derive a maximum-likelihood estimator for the LOH rate based on the observed number of heterozygotes. This accounts for LOH in both its hemizygous deletion and copy-neutral forms, and does not make use of matched normal genotypes. Power simulations show high levels of sensitivity for the statistical test, and application to a control normal-tissue data set demonstrates a low false-discovery rate. We apply the method to three large publicly available tumor SNP array data sets, where it is able to localize tumor-suppressor gene targets of the LOH events. Inferred LOH rates are quite concordant across platforms/laboratories and between cell lines and tumors, but in a tumor type-dependent fashion. Finally, we produce rate estimates that are generally higher than previously published, and provide evidence that the latter are likely underestimates.

High Resolution Karyotyping of Large Granular Lymphocyte Leukemia by SNP Arrays Reveals Clonal Defects Leading to LOH at Loci Integral to Lymphocyte Function.

Large granular lymphocyte leukemia (LGL leukemia) is a semiautonomous clonal lymphoproliferation of cytotoxic T cells associated with various immune cytopenias. Within the spectrum of acquired immune-mediated bone marrow failure states, LGL leukemia can serve as monoclonal model of usually polyclonal T cell-mediated pathology. Mechanisms of unopposed clonal expansion of LGL cells in many aspects resemble true lymphoma and are not well understood. In addition to its reactive character, intrinsic clonal defects may be present in some patient with LGL, in particular those with more pronounced lymphoproliferative features. However, unlike in B-cell lymphomas, recurrent chromosomal abnormalities have not been frequently identified in LGL leukemia using traditional metaphase karyotyping techniques. We have applied Affymetrix 250K single nucleotide polymorphism-arrays (SNP-A) and 6.0 SNP-A in 28 patients with LGL leukemia, to elicit a far higher resolution of chromosomal content through genomic mapping of individual SNP and respective copy number analysis in LGL leukemia. SNP-A based cytogenetics have been applied successfully to MDS patients and has increased prognostic reliability. Blood mononuclear cells containing high proportion of clonal cells as determined by TCR Vβ flow cytometry were used as a source of DNA. For comparison, a large number of control blood and marrow specimens (N=119 for 6.0 and 124 for 250K SNP arrays) were analyzed. Data were processed using Genotyping Console v2.1 software (Affymetrix, Santa Clara, CA). After exclusion of known copy number variants (CNV) referenced in public databases and our own set of 178 normal controls, we found distinct chromosomal changes in 16/28 (57%) of LGL leukemia patients. Consensus regions of deletion/gain or uniparental disomy (UPD) were identified. The most common abnormalities included either UPD or copy number loss of chromosome 3q21.2–q21.31, which was identified in 6 (21%) patients. This region harbors CD86, the gene encoding the B7.2 protein responsible for T cell activation and regulation through costimulatory mechanisms. Copy number loss/UPD was also identified at chromosome 1p31.1–p32.3 in 4 (18%) patients. Interestingly, copy number gain in this same region was identified in 2 (9%) patients suggesting that this region may correspond to either a new, infrequent germ line encoded CNV or represents a somatic microdeletion. Recent studies indicated a role for SIL/SCL, which resides at this locus, in V(D)J recombination, lymphocyte development, and maturation. Additional conserved chromosomal abnormalities included copy number gains in 14q (11%) and copy number loss at 11p15 (14%), all possibly representing germ line or somatic CNV physiologically acquired during lymphocyte ontogenesis. We compared chromosomal lesions identified in our LGL cohort with clinical features including age, presence of neutropenia, anemia, thrombocytopenia, splenomegaly, immunophenotype, and degree of clonal expansion. No difference in clinical course was found between patients with and without cytogenetic abnormalities with regard to type and severity of cytopenias or size of the LGL clone. However, patients with 1p31.1–p32.3 deletions were found to have lesser degree of neutropenia compared to patients without 1p31.1–p32.3 deletions (p<0.001). While these data may reflect chromosomal variants and random somatic abnormalities, the recurrent loss of heterozygosity and gains within several loci with known regulatory function for lymphocyte biology suggests the involvement of these defects in the mechanism of clonal evolution.

Chromosome copy number analysis in screening for prognosis‐related genomic regions in colorectal carcinoma

Colorectal carcinoma (CRC) remains the major cause of cancer death in humans. Although chromosomal structural anomaly is presumed to play an important role in the carcinogenesis of CRC, chromosomal copy number alterations (CNA) and loss of heterozygosity (LOH) have not yet been analyzed extensively at high resolution in CRC. Here we aim to identify recurrent CNA and LOH in human CRC with the use of single nucleotide polymorphism-typing microarrays, and to reveal their relevance to clinical outcome. Surgically resected CRC specimens and paired normal mucosa were obtained from a consecutive series of 94 patients with CRC, and both of them were subjected to genotyping with Affymetrix Mapping 50K arrays. CNA and LOH were inferred computationally on every single nucleotide polymorphism site by integrating the array data for paired specimens. Our large dataset reveals recurrent CNA in CRC at chromosomes 7, 8, 13, 18, and 20, and recurrent LOH at chromosomes 1p, 4q, 5q, 8p, 11q, 14q, 15q, 17p, 18, and 22. Frequent uniparental disomy was also identified in chromosomes 8p, 17p, and 18q. Very common CNA and LOH were present at narrow loci of <1 Mbp containing only a few genes. In addition, we revealed a number of novel CNA and LOH that were linked statistically to the prognosis of the patients. The precise and large-scale measurement of CNA and LOH in the CRC genome is efficient for pinpointing prognosis-related genome regions as well as providing a list of unknown genes that are likely to be involved in CRC development.

Acquisition of New In Vivo Genomic Change over Time in Multiple Myeloma.

Multiple myeloma (MM) cells present with significant genetic abnormality. This genetic instability is considered responsible for not only development of malignant phenotype but also progression of myeloma as well as development of drug resistance. We have previously demonstrated that MM cell lines have elevated homologous recombination activity that leads to acquisition of new genomic changes over time and is associated with development of drug resistance (Blood 2004; 104: 3409). However, such genomic evolution in patient samples has not been documented. Here, we have performed a genome wide loss of heterozygosity (LOH) assay, using high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci, and identify areas of amplification and losses to determine ongoing development of new changes that may reflect instability. We have evaluated 17 MM patients with purified myeloma cells obtained at two time points, at least six months apart. CD138–expressing myeloma cells from these patients were purified and their peripheral blood mononuclear cells were also obtained. Genomic DNA from these cells was digested with StyI, PCR amplified and hybridized to 250K SNP array. Results from CD138+ myeloma cells from two time points or more were compared with each other using the dChip software for LOH and copy number analysis. During the LOH analysis, we have adapted a tumor vs. tumor comparison to track all the new SNPs affected. We have observed that myeloma cells acquired new LOH loci in the subsequent samples. These new areas of genetic changes were recurrent; for example on chromosome 13, an average area of recurrent LOH of 87 Mb was found to be present among three patients. We were able to observe recurrence of significant copy changes on several chromosomes with/without LOH. The reproducible area of new acquisition of LOH and/or significant copy changes interestingly involved areas on chromosomes 1p, 1q, 8p, 9q, 13q, 15, 16q, 20p, 21q, and X that may have significant role in the pathogenesis and progression of the disease. We are currently analyzing the gene expression profile from these time points to identify expression changes correlating with the observed genomic changes on follow up samples. In summary we demonstrate continued acquisition of new genomic changes in vivo in MM over time and provide a molecular basis for evolution of more aggressive disease able to escape therapeutic interventions. The mechanism governing such evolution is an important target to not only inhibit progression of the disease but also development of drug resistance.

Genomic Profiling of Chronic Lymphocytic Leukemia Identifies Novel Genetic Subtypes.

CLL is the most common leukemia in the Western world and displays considerable clinical heterogeneity. Genetic approaches to elucidate defects in CLL genomes have resulted in the identification of recurrent chromosomal abnormalities with prognostic importance. A genome-wide high-density unbiased view of chromosomal copy number changes or loss of heterozygosity (LOH) may allow for further refinements of genomics-based risk prognostication. We have conducted a genomic profiling study using 50KSNP-oligonucleotide arrays (Affymetrix) on FACS-sorted CD19+ cells and paired buccal DNA from 120 patients with typical CLL (CD5+/CD23+) at various stages of presentation enrolled in a clinical translational protocol. We have found novel genetic subtypes, including novel regions of recurrent LOH and chromosomal copy loss on chromosomes 3p, 14q and 18p at frequencies of 3–6%. Fine mapping of the clinically important regions del(6q), del(11q), del(13q14) and del(17p) suggests molecular heterogeneity of these lesions. Anatomic criteria suggest existence of multiple distinct subtypes of del13q14. Furthermore, the clinically important del(17p) is associated with almost uniform deletion of most of 17p, occasionally displays copy-neutral LOH (undetectable by FISH) with p53 mutations and occurs without p53 mutations in a subset of patients as previously reported. Finally, using a genome-wide composite genomic instability score of copy gains and losses we found a subset of CLL cases (18%) with high genomic instability values (greater than 2 lesions per genome). These findings may have implications for CLL risk-adapted therapies.

Chromosomal Deletions and Amplifications in Multiple Myeloma Detected by 500K Single Nucleotide Polymorphism Array Analysis.

Genetic aberrations, such as deletions and amplifications are among the major pathogenetic mechanisms underlying malignant transformation and progression. Analysis of chromosomal aberrations is particularly important as amplifications of oncogenes and deletions of tumor suppressor genes are major steps in the “multi-hit” process of tumorigenesis. Genome-wide molecular analyses, such as loss of heterozygosity (LOH) profiling and comparative genomic hybridization (CGH) have significantly enhanced our ability to detect chromosomal aberrations in cancer cells and assess their role in tumorigenesis. The recent introduction of high-density oligonucleotide arrays capable of measuring up to 500K single nucleotide polymorphisms (SNP) loci is able to analyze small areas of gains or losses. Here we describe the use of high-density array representing upto 500K SNPs to not only characterize SNPs but also evaluate genome-wide areas of gains and losses in myeloma. We purified CD138+ myeloma cells from 5 patients and also obtained their peripheral blood mononuclear cells. Purified genomic DNA from these cells was digested with Sty and Nsp restriction enzymes, PCR amplified and hybridized to 500K SNP array. Results from DNA obtained from CD138+ myeloma cells were compared with DNA from normal peripheral blood mononuclear cells from the same patient using the dChip software for LOH and copy number analysis. Areas of deletions and amplifications were identified and correlated with gene expression profile obtained from CD138+ myeloma cells of the same patient. The preliminary analysis identified various areas of amplifications and deletions. The significant areas of structural gains or losses included Chromosome 1, 6, 8, 11, 12, 13, 14, 15, 22, X among others and ranged in size from 1 Mb to the loss of entire chromosome (13q−). In this small sample we have identified recurrent LOH shared by 2 or more samples including 1p21, 6q, 12p13 and 13q. The derived CGH results correlated with known cytogenetic abnormalities in the patients, with identification of additional areas of abnormality not detected by conventional cytogenetics or FISH. These structural changes also corresponded with the results from gene expression profiling of MM cells from same patients, thus validating the technique and its application. A large series of patient samples are now under investigation using 500K SNP chip to define regions of homozygous deletions as well as amplification. In conclusion, these results demonstrate that SNP array analysis provides a powerful new tool for the analysis of allelic imbalance in myeloma. Furthermore, this technique can identify novel candidate loci to study in pathogenesis of myeloma and advance identification of potential therapeutic targets for this fetal disease.

Genomic Profiling Maps Loss of Heterozygosity and Defines the Timing and Stage Dependence of Epigenetic and Genetic Events in Wilms' Tumors

To understand genetic and epigenetic pathways in Wilms' tumors, we carried out a genome scan for loss of heterozygosity (LOH) using Affymetrix 10K single nucleotide polymorphism (SNP) chips and supplemented the data with karyotype information. To score loss of imprinting (LOI) of the IGF2 gene, we assessed DNA methylation of the H19 5' differentially methylated region (DMR). Few chromosomal regions other than band 11p13 (WT1) were lost in Wilms' tumors from Denys-Drash and Wilms' tumor-aniridia syndromes, whereas sporadic Wilms' tumors showed LOH of several regions, most frequently 11p15 but also 1p, 4q, 7p, 11q, 14q, 16q, and 17p. LOI was common in the sporadic Wilms' tumors but absent in the syndromic cases. The SNP chips identified novel centers of LOH in the sporadic tumors, including a 2.4-Mb minimal region on chromosome 4q24-q25. Losses of chromosomes 1p, 14q, 16q, and 17p were more common in tumors presenting at an advanced stage; 11p15 LOH was seen at all stages, whereas LOI was associated with early-stage presentation. Wilms' tumors with LOI often completely lacked LOH in the genome-wide analysis, and in some tumors with concomitant 16q LOH and LOI, the loss of chromosome 16q was mosaic, whereas the H19 DMR methylation was complete. These findings confirm molecular differences between sporadic and syndromic Wilms' tumors, define regions of recurrent LOH, and indicate that gain of methylation at the H19 DMR is an early event in Wilms' tumorigenesis that is independent of chromosomal losses. The data further suggest a biological difference between sporadic Wilms' tumors with and without LOI.

Region-specific detection of neuroblastoma loss of heterozygosity at multiple loci simultaneously using a SNP-based tag-array platform.

Many cancers are characterized by chromosomal aberrations that may be predictive of disease outcome. Human neuroblastomas are characterized by somatically acquired copy number changes, including loss of heterozygosity (LOH) at multiple chromosomal loci, and these aberrations are strongly associated with clinical phenotype including patient outcome. We developed a method to assess region-specific LOH by genotyping multiple SNPs simultaneously in DNA from tumor tissues. We identified informative SNPs at an average 293-kb density across nine regions of recurrent LOH in human neuroblastomas. We also identified SNPs in two copy number neutral regions, as well as two regions of copy number gain. SNPs were PCR-amplified in 12-plex reactions and used in solution-phase single-nucleotide extension incorporating tagged dideoxynucleotides. Each extension primer had 5' complementarity to one of 2000 oligonucleotides on a commercially available tag-array platform allowing for solid-phase sorting and identification of individual SNPs. This approach allowed for simultaneous detection of multiple regions of LOH in six human neuroblastoma-derived cell lines, and, more importantly, 14 human neuroblastoma primary tumors. Concordance with conventional genotyping was nearly absolute. Detection of LOH in this assay may not require comparison to matched normal DNAs because of the redundancy of informative SNPs in each region. The customized tag-array system for LOH detection described here is rapid, results in parallel assessment of multiple genomic alterations, and may speed identification of and/or assaying prognostically relevant DNA copy number alterations in many human cancers.

Microsatellite allelotyping differentiates chromophobe renal cell carcinomas from renal oncocytomas and identifies new genetic changes.

The diagnosis of renal oncocytomas (ROs) and chromophobe renal cell carcinomas (RCCs) based on histological features is often uncertain. To assess the value of genetic analysis in their differential diagnosis we analysed 27 ROs and 21 chromophobe RCCs by microsatellite allelotyping. Markers at the short and long arms of chromosomes specifically involved in the genetic changes of the four main types of renal cancers were selected. Allelic changes were identified by automated sequencing. Allelic changes at chromosome 1p occurred in 8/26 (31%) and at chromosome 14q in 4/27 (15%) ROs. Loss of heterozygosity (LOH) at chromosomes 1, 2, 6, 10, 13, 17 and 21 were seen in 90%, 90%, 96%, 86%, 85%, 90% and 72% of the chromophobe RCCs, respectively. Alterations of at least three of these chromosomal sites were detected in each chromophobe RCC. In addition, we found recurrent LOH at chromosomes 9p23 (43%), 18q22 (30%), 5q22 (28%) and 8p (28%) in chromophobe RCCs. Chromophobe RCCs can be differentiated from ROs by analysing specific chromosomal regions with microsatellites.

Disruption of β-catenin pathway or genomic instability define two distinct categories of liver cancer in transgenic mice

Background & Aims : Human liver cancer can be divided into 2 categories that are characterized by activation of β-catenin and genomic instability. Here we investigate whether similar categories exist among 5 transgenic models of liver cancer, including c-myc, transforming growth factor-α, E2F-1, c-myc/transforming growth factor-α, and c-myc/E2F-1 mice. Methods : The random amplified polymorphic DNA method was used to assess the overall genomic instability, and chromosomal loci affected by genomic alterations were determined by microsatellite analysis. β-Catenin mutations and deletions were analyzed by polymerase chain reaction and sequencing screening. Cellular localization of β-catenin and expression of α-fetoprotein, a prognostic marker of hepatocellular carcinoma, were investigated by immunohistochemistry. Results : Liver tumors from the transgenic mice could be divided into 2 broad categories characterized by extensive genomic instability (exemplified by the c-myc/transforming growth factor-α mouse) and activation of β-catenin (exemplified by the c-myc/E2F-1 mouse). The c-myc/transforming growth factor-α tumors displayed extensive genomic instability with recurrent loss of heterozygosity at chromosomes 1, 2, 4, 6, 7, 9, 12, 14, and X and a low rate of β-catenin activation. The genomic instability was evident from the early dysplastic stage and occurred concomitantly with increased expression of α-fetoprotein. The c-myc/E2F-1 tumors were characterized by a high frequency of β-catenin activation in the presence of a relatively stable genome and low α-fetoprotein levels. Conclusions : We have identified 2 prototype experimental models, i.e., c-myc/transforming growth factor-α and c-myc/E2F-1 mice, for the 2 categories of human hepatocellular carcinoma characterized by genomic instability and β-catenin activation, respectively. These mouse models will assist in the elucidation of the molecular basis of human hepatocellular carcinoma.

Genome scanning with array CGH delineates regional alterations in mouse islet carcinomas

Carcinomas that develop in the pancreatic islets of transgenic mice expressing the SV40 T-antigens (Tag) under transcriptional control of the rat insulin II promoter (RIP) progress through well-characterized stages that are similar to aspects of human tumor progression, including hyperplastic growth, increased angiogenesis and reduced apoptosis. The latter two stages have been associated with recurrent loss of heterozygosity (LOH) and reduced genome copy number on chromosomes 9 (LOH9) and 16 (LOH16), aberrations which we believe contribute to these phenotypes. Earlier analyses localized LOH9 to approximately 3 Mb and LOH16 to approximately 30 Mb (both syntenic with human 3q21-q25) but were limited by low throughput and a lack of informative polymorphic markers. Here we show that comparative genomic hybridization to DNA microarrays (array CGH) overcomes these limitations by allowing efficient, genome-wide analyses of relative genome copy number. The CGH arrays used in these experiments carried BACs distributed at 2-20-MB intervals across the mouse genome and at higher density in regions of interest. Using array CGH, we further narrowed the loci for LOH9 and LOH16 and defined new or previously unappreciated recurrent regions of copy-number decrease on chromosomes 6, 8 and 14 (syntenic with human chromosomes 12p11-p13, 16q24.3 and 13q11-q32, respectively) and regions of copy-number increase on chromosomes 2 and 4 (syntenic to human chromosomes 20q13.2 and 1p32-p36, respectively). Our analyses of human genome sequences syntenic to these regions suggest that CYP24, PFDN4, STMN1, CDKN1B, PPP2R3 and FSTL1 are candidate oncogenes or tumor-suppressor genes. We also show that irradiation and genetic background influence the spectrum of aberrations present in these tumors.

Loss of heterozygosity and HIV infection in patients with anal squamous-cell carcinoma.

This study was designed to determine whether loss of heterozygosity and/or microsatellite instability correlate with HIV infection and tumor recurrence after chemoradiation therapy in patients with squamous-cell carcinoma of the anus. The molecular mechanisms leading to the progression of HIV-related squamous-cell carcinoma of the anus are poorly understood. In particular, genetic alterations responsible for resistance to chemoradiation have important clinical and functional implications. In a case-control study, we analyzed normal and tumor DNA samples of four patients with squamous-cell carcinoma of the anus who were successfully treated with chemoradiotherapy and four patients with radio-resistant squamous-cell carcinoma of the anus who required abdominoperineal resection for local recurrence. To determine the presence of microsatellite instability, we used the reference panel of five pairs of microsatellite primers recommended for colorectal cancer specimens. These include the microsatellite markers BAT25, BAT26, D5S346 (APC), D2S123 (hMSH2), and D17S250 (P53). In addition, we used microsatellite markers for loss of heterozygosity analyses that were tightly linked to tumor suppressor genes. These included D3S1611 (hMLH1), D17S513 (P53), D18S46 and 18qTA (DCC/SMAD4), D5S107 (APC), and CA5 (hMSH2). There were two HIV-positive and two HIV-negative patients in each group. Three HIV-positive patients (one in the chemoradiotherapy group and two in the nonchemoradiotherapy group) demonstrated loss of heterozygosity. In the chemoradiotherapy group, one HIV-positive patient demonstrated loss of heterozygosity at the hMLH1 locus. In the nonchemoradiotherapy group, two HIV-positive patients exhibited a total of four instances of loss of heterozygosity. One tumor had loss of heterozygosity at hMSH2 and DCC/SMAD4; another tumor demonstrated loss of heterozygosity at hMSH2 and APC. Microsatellite instability-low was found in two HIV-positive patients. No instances of loss of heterozygosity and microsatellite instability were detected in HIV-negative patients. Loss of heterozygosity and microsatellite instability, which reflect inactivation of tumor-suppressor genes and genomic instability, occur with increased frequency in HIV-associated squamous-cell carcinoma. These data demonstrate for the first time evidence of loss of heterozygosity at the APC and DCC/SMAD4 gene loci in anal carcinoma. Although the findings presented here need to be expanded in a larger study, the recurrent loss of heterozygosity at D2S123, which was demonstrated in HIV-positive patients with radio-resistant squamous-cell carcinoma of the anus, is notable.

Analysis of loss of heterozygosity in lymphoma and leukaemia arising in F1 hybrid mice locates a common region of chromosome 4 loss.

Previous studies have identified five lymphoma-related tumour suppressor gene regions on murine chromosome 4. Using detailed allelotype analysis on a range of lympho-haematopoietic tumour types arising in F1 hybrid mice, we now show a consistent pattern of loss of heterozygosity (LOH) which identifies a common region of loss delineated by microsatellites D4Mit21 and D4Mit53 on proximal chromosome 4. This critical segment corresponds to the thymic lymphoma tumour suppressor region 5 (TLSR5) identified in an earlier study. Tumours of this type have also been reported as showing allelic loss from the Trp53 and Ikaros regions on chromosome 11. In the present study, only a small fraction of tumours showed LOH in the Ikaros region, while a minority of lymphomas, but not acute myeloid leukaemias, showed allelic loss of the chromosome 11 segment encoding Trp53. These and other data indicate strongly that the genomic regions identified as showing recurrent LOH depend on the genetic background of the mice. Overall, the results indicate a key role for a tumour suppressor gene(s) encoded in an approximately 3 cM segment on proximal chromosome 4 and provide an experimental basis for the further investigation of the functional role of candidate genes which include Pax5 and Tgfbr1.