Loss Of Heterozygosity Data Research Articles

Value of the loss of heterozygosity to BRCA1 variant classification

At least 10% of the BRCA1/2 tests identify variants of uncertain significance (VUS) while the distinction between pathogenic variants (PV) and benign variants (BV) remains particularly challenging. As a typical tumor suppressor gene, the inactivation of the second wild-type (WT) BRCA1 allele is expected to trigger cancer initiation. Loss of heterozygosity (LOH) of the WT allele is the most frequent mechanism for the BRCA1 biallelic inactivation. To evaluate if LOH can be an effective predictor of BRCA1 variant pathogenicity, we carried out LOH analysis on DNA extracted from 90 breast and seven ovary tumors diagnosed in 27 benign and 55 pathogenic variant carriers. Further analyses were conducted in tumors with PVs yet without loss of the WT allele: BRCA1 promoter hypermethylation, next-generation sequencing (NGS) of BRCA1/2, and BRCAness score. Ninety-seven tumor samples were analyzed from 26 different BRCA1 variants. A relatively stable pattern of LOH (65.4%) of WT allele for PV tumors was observed, while the allelic balance (63%) or loss of variant allele (15%) was generally seen for carriers of BV. LOH data is a useful complementary argument for BRCA1 variant classification.

Loss of Heterozygosity and Base Mutation Rates Vary Among Saccharomyces cerevisiae Hybrid Strains.

A growing body of evidence suggests that mutation rates exhibit intra-species specific variation. We estimated genome-wide loss of heterozygosity (LOH), gross chromosomal changes, and single nucleotide mutation rates to determine intra-species specific differences in hybrid and homozygous strains of Saccharomyces cerevisiae. The mutation accumulation lines of the S. cerevisiae hybrid backgrounds - S288c/YJM789 (S/Y) and S288c/RM11-1a (S/R) were analyzed along with the homozygous diploids RM11, S288c, and YJM145. LOH was extensive in both S/Y and S/R hybrid backgrounds. The S/Y background also showed longer LOH tracts, gross chromosomal changes, and aneuploidy. Short copy number aberrations were observed in the S/R background. LOH data from the S/Y and S/R hybrids were used to construct a LOH map for S288c to identify hotspots. Further, we observe up to a sixfold difference in single nucleotide mutation rates among the S. cerevisiae S/Y and S/R genetic backgrounds. Our results demonstrate LOH is common during mitotic divisions in S. cerevisiae hybrids and also highlight genome-wide differences in LOH patterns and rates of single nucleotide mutations between commonly used S. cerevisiae hybrid genetic backgrounds.

Testing clonal relatedness of two tumors from the same patient based on their mutational profiles: update of the Clonality R package.

The Clonality R package is a practical tool to assess the clonal relatedness of two tumors from the same patient. We have previously presented its functionality for testing tumors using loss of heterozygosity data or copy number arrays. Since then somatic mutation data have been more widely available through next generation sequencing and we have developed new methodology for comparing the tumors' mutational profiles. We thus extended the package to include these two new methods for comparing tumors as well as the mutational frequency estimation from external data required for their implementation. The first method is a likelihood ratio test that is readily available on a patient by patient basis. The second method employs a random-effects model to estimate both the population and individual probabilities of clonal relatedness from a group of patients with pairs of tumors. The package is available on Bioconductor. Bioconductor (http://bioconductor.org/packages/release/bioc/html/Clonality.html). Supplementary data are available at Bioinformatics online.

Characterisation of the novel deleterious RAD51C p.Arg312Trp variant and prioritisation criteria for functional analysis of RAD51C missense changes

Background:Despite a high prevalence of deleterious missense variants, most studies of RAD51C ovarian cancer susceptibility gene only provide in silico pathogenicity predictions of missense changes. We identified a novel deleterious RAD51C missense variant (p.Arg312Trp) in a high-risk family, and propose a criteria to prioritise RAD51C missense changes qualifying for functional analysis.Methods:To evaluate pathogenicity of p.Arg312Trp variant we used sequence homology, loss of heterozygosity (LOH) and segregation analysis, and a comprehensive functional characterisation. To define a functional-analysis prioritisation criteria, we used outputs for the known functionally confirmed deleterious and benign RAD51C missense changes from nine pathogenicity prediction algorithms.Results:The p.Arg312Trp variant failed to correct mitomycin and olaparib hypersensitivity and to complement abnormal RAD51C foci formation according to functional assays, which altogether with LOH and segregation data demonstrated deleteriousness. Prioritisation criteria were based on the number of predictors providing a deleterious output, with a minimum of 5 to qualify for testing and a PredictProtein score greater than 33 to assign high-priority indication.Conclusions:Our study points to a non-negligible number of RAD51C missense variants likely to impair protein function, provides a guideline to prioritise and encourage their selection for functional analysis and anticipates that reference laboratories should have available resources to conduct such assays.

Loss of heterozygosity of chromosome 13q33-34 region and molecular analysis of ING1 and p53 genes in bladder carcinoma.

Cancer is a consequence of accumulation of genetic and epigenetic alterations in the cell which can lead to activation of oncogenes or inactivation of tumor suppressor genes (TSG). Since members of ING family were discovered as TSGs in different cancer types, it was aimed to analyze the chromosome 13q33-34 region, ING1 and p53 genes in bladder cancer. 30 paired normal and tumor tissues were investigated in terms of microdeletion of chromosome 13q33-34 region, ING1 expression and mutation status of ING1 and p53 genes. Because there is no data available about the transcription factors which bind to ING1 promoter, the promoter sequence was analyzed via Genomatix-MatInspector and TFSEARCH softwares. Used DS markers were D13S285, D13S1315, D13S796, D13S278, D13S158, and D13S779 where loss of heterozygosity (LOH) results were as 23.3, 20, 6.7, 3.3, 6.7, and 0 %, respectively. The highest LOH scores were obtained with markers D13S285 and D13S1315 which are flanking the ING1. Seven of 30 cases showed alteration in expression (p > 0.05). However, no mutation was detected in the exons of ING1. One patient showed a two-nucleotide deletion in p53 gene. However no significant TSG activity of ING1 was observed while higher activity was reported in different cancer types. As for the LOH data 13q33-34 region may contain different candidate TSGs like COL4A1, COL4A2 and SOX1. As a result of computational promoter analysis, some factors like ABL, E2F, HIF1, SOX, P53, BPTF, NRSF, c-Rel and c-ETS were associated with the promoter region. Molecular analysis of ING1 promoter warrants further analysis.

Fine Mapping of the 15q21 Region Implicates TP53BP1 and B2M in the Lymphomagenesis of Follicular and Diffuse Large B-Cell Lymphomas

Background and aim: Recent studies focusing on the discovery of common alterations across cases suffering from follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL) have reported loss of heterozygosity (LOH) and deletions events targeting the 15q21 region, indicating the relevance of this region in the lymphomagenesis of FL and DLBCL. Herein, we investigated the genetic structure of this region by studying identified LOH and copy-loss events and examined this region in the lymphomagenesis of FL and DLBCL. Methods: Fine mapping of the genomic region between the 15q15.1 and 15q21.1 loci was performed using data from copy number variation (CNV) and high resolution LOH analyses of FL (n=21) and DLBCL (n=21) cases. Validation of LOH of this region was performed using microsatellites followed by quantitative-PCR (qPCR) to measure the transcriptional abundance of TP53BP1 and B2M. Also, direct sequencing of exons 1 and 2 of B2M was performed on tumor DNA from 24 FL and 23 DLBCL samples. Results: The integration of LOH and CNV data identified copy-loss alterations at the 15q21 loci spanning a 7.5 Mb region, covering two LOH regions, termed LOH-1 and LOH-2. The LOH-1 region spans 3.4 Mb and contains 53 genes, from which TP53BP1 (tumor-protein-p53-binding-protein-1) and B2M (Beta-2-Microglobulin) were identified as the most likely target genes due to their roles in DNA double strand break (DSB) repair and immune recognition, respectively. Expression analyses revealed a significant up-regulation of TP53BP1 in NHL with LOH, but no significant changes in B2M expression were observed. Direct sequencing of exons 1 and 2 in B2M in FL and DLBCL identified two monoallelic microdeletions associated with DLBCL. Conclusion: This study identified that deletion mapping to the 15q21 locus cover two LOH regions. LOH of the TP53BP1 and B2M genes appear to be common alterations in FL and DLBCL tumorigenesis.

Molecular heterogeneity in malignant peripheral nerve sheath tumors associated with neurofibromatosis type 1

Neurofibromatosis type-1 (NF1), resulting from NF1 gene loss of function, is characterized by an increased risk of developing benign and malignant peripheral nerve sheath tumors (MPNSTs). Whereas the cellular heterogeneity of NF1-associated tumors has been well studied, the molecular heterogeneity of MPNSTs is still poorly understood. Mutational heterogeneity within these malignant tumors greatly complicates the study of the underlying mechanisms of tumorigenesis. We have explored this molecular heterogeneity by performing loss of heterozygosity (LOH) analysis of the NF1, TP53, RB1, PTEN, and CDKN2A genes on sections of 10 MPNSTs derived from 10 unrelated NF1 patients. LOH data for the TP53 gene was found to correlate with the results of p53 immunohistochemical analysis in the same tumor sections. Further, approximately 70% of MPNSTs were found to display intra-tumoral molecular heterogeneity as evidenced by differences in the level of LOH between different sections of the same tumor samples. This study constitutes the first systematic analysis of molecular heterogeneity within MPNSTs derived from NF1 patients. Appreciation of the existence of molecular heterogeneity in NF1-associated tumors is important not only for optimizing somatic mutation detection, but also for understanding the mechanisms of NF1 tumorigenesis, a prerequisite for the development of specifically targeted cancer therapeutics.

Allele Loss and Down-Regulation of Heparanase Gene Are Associated with the Progression and Poor Prognosis of Hepatocellular Carcinoma

ObjectivesThe role of heparanase (HPSE) gene in cancers including hepatocellular carcinoma (HCC) is currently controversial. This study was aimed at investigating the impact of genetic alteration and expression change of HPSE on the progression and prognosis of HCC.MethodsThe HPSE gene was studied in three different aspects: (1) loss of heterozygosity (LOH) by a custom SNP microarray and DNA copy number by real-time PCR; (2) mRNA level by qRT-PCR; and (3) protein expression by immunohistochemistry. The clinical significances of allele loss and expression change of HPSE were analyzed.ResultsMicroarray analysis showed that the average LOH frequency for 10 SNPs located within HPSE gene was 31.6%, three of which were significantly correlated with tumor grade, serum HBV-DNA level, and AFP concentration. In agreement with SNP LOH data, DNA copy number loss of HPSE was observed in 38.74% (43/111) of HCC cases. HPSE mRNA level was notably reduced in 74.1% (83/112) of tumor tissues compared with non-tumor liver tissues, which was significantly associated with DNA copy number loss, increased tumor size, and post-operative metastasis. HPSE protein level was also remarkably reduced in 66.3% (53/80) of tumor tissues, which was correlated with tumor grade. Patients with lower expression level of HPSE mRNA or protein had a significantly lower survival rate than those with higher expression. Cox regression analysis suggested that HPSE protein was an independent predictor of overall survival in HCC patients.ConclusionsThe results in this study demonstrate that genetic alteration and reduction of HPSE expression are associated with tumor progression and poor prognosis of HCCs, suggesting that HPSE behaves like a tumor suppressor gene and is a potential prognostic marker for HCC patients.

OC-100 The clonal origins of gastric adenocarcinoma

IntroductionWe have previously shown that entire fields of dysplasia in the human stomach are derived from a single mutated, metaplastic gland.1 This suggests that intestinal metaplasia (IM) can be considered...

Integrated analysis of copy number and loss of heterozygosity in primary breast carcinomas using high-density SNP array

Breast cancer is a heterogeneous disease, marked by extensive chromosomal aberrations. In this study, we aimed to explicate the underlying chromosomal copy number (CN) alterations and loss of heterozygosity (LOH) implicated in a cohort of Malaysian hospital-based primary breast carcinoma samples using a single nucleotide polymorphism (SNP) array platform. The analysis was conducted by hybridizing the extracted DNA of 70 primary breast carcinomas and 37 normal peripheral blood samples to the Affymetrix 250K Sty SNP arrays. Locus-specific CN aberrations and LOH were statistically summarized using the binary segmentation algorithm and hidden Markov model. Selected genes from the SNP array analysis were also validated using quantitative real-time PCR. The merging of CN and LOH data fabricated distinctive integrated alteration profiles, which were comprised of finely demarcated minimal sites of aberrations. The most prevalent gains (≥ 30%) were detected at the 8q arm: 8q23.1, 8q23.3, 8q24.11, 8q24.13, 8q24.21, 8q24.22, 8q24.23 and 8q24.3, whilst the most ubiquitous losses (≥ 20%) were noted at the 8p12, 8p21.1, 8p21.2, 8p21.1-p21.2, 8p21.3, 8p22, 8p23.1, 8p23.1‑p23.2, 8p23.3, 17p11.2, 17p12, 17p11.2-p12, 17p13.1 and 17p13.2 regions. Copy-neutral LOH was characterized as the most prevailing LOH event, in which the most frequent distributions (≥ 30%) were revealed at 3p21.31, 5q33.2, 12q24.12, 12q24.12‑q24.13 and 14q23.1. These findings offer compre-hensive genome-wide views on breast cancer genomic changes, where the most recurrent gain, loss and copy-neutral LOH events were harboured within the 8q24.21, 8p21.1 and 14q23.1 loci, respectively. This will facilitate the uncovering of true driver genes pertinent to breast cancer biology and the develop-ment of prospective therapeutics.

Loss of heterozygosity (LOH) as a measure of whole-genome instability in ovarian cancer correlates with clinical outcomes.

5027 Background: The aim of the study was to assay the amount of loss of heterozygosity (LOH) across the whole genome as a measure of genomic instability in ovarian tumors and to determine the correlation between the fraction of the ovarian cancer genome with LOH and the clinical outcomes of ovarian cancer patients. Methods: We assessed the prevalence of LOH in 100 unselected ovarian cancer tumors using whole genome 500K Single Nucleotide Polymorphism (SNP) arrays (Affymetrix, Inc.). Allele dosage, copy number, and degree of contamination with benign tissue were used to estimate the likelihood of various genotypes in tumor tissue. Regions with the copy number of more than 3 were eliminated from the analysis since LOH data are unreliable when copy number exceeds 3. Results: We found that copy number changes of 1, 2 and 3 composed 12.6%, 45.7% and 21.5% of the genome in ovarian cancer, respectively. These changes contributed to 42.4%, 43.8% and 13.8% of LOH in ovarian cancer, respectively. The average fraction of the whole genome with LOH in ovarian cancer was 35%. The fraction of the genome with LOH was significantly correlated with tumor grade. The fraction of the ovarian cancer genome with LOH was statistically highly correlated with both overall survival (OS;p=3.4x10-6) and progression-free survival (PFS; p=0.0016) after debulking surgery and platinum/taxane-based chemotherapy. In multivariate analyses with clinical variables, only the fraction of the genome with LOH and residual tumor size (RTS) after debulking surgery were independent predictive factors for OS and PFS. Conclusions: Increased fraction of the genome with LOH as a measure of genomic instability in ovarian cancer is a significant and independent negative prognostic factor for both OS and PFS. The validation of the fraction of the ovarian cancer genome with LOH as an independent predictor of RFS and OS in a second ovarian cancer sample set will also be presented at the meeting.

An integrated Bayesian analysis of LOH and copy number data

BackgroundCancer and other disorders are due to genomic lesions. SNP-microarrays are able to measure simultaneously both genotype and copy number (CN) at several Single Nucleotide Polymorphisms (SNPs) along the genome. CN is defined as the number of DNA copies, and the normal is two, since we have two copies of each chromosome. The genotype of a SNP is the status given by the nucleotides (alleles) which are present on the two copies of DNA. It is defined homozygous or heterozygous if the two alleles are the same or if they differ, respectively. Loss of heterozygosity (LOH) is the loss of the heterozygous status due to genomic events.Combining CN and LOH data, it is possible to better identify different types of genomic aberrations. For example, a long sequence of homozygous SNPs might be caused by either the physical loss of one copy or a uniparental disomy event (UPD), i.e. each SNP has two identical nucleotides both derived from only one parent. In this situation, the knowledge of the CN can help in distinguishing between these two events.ResultsTo better identify genomic aberrations, we propose a method (called gBPCR) which infers the type of aberration occurred, taking into account all the possible influence in the microarray detection of the homozygosity status of the SNPs, resulting from an altered CN level. Namely, we model the distributions of the detected genotype, given a specific genomic alteration and we estimate the parameters involved on public reference datasets. The estimation is performed similarly to the modified Bayesian Piecewise Constant Regression, but with improved estimators for the detection of the breakpoints.Using artificial and real data, we evaluate the quality of the estimation of gBPCR and we also show that it outperforms other well-known methods for LOH estimation.ConclusionsWe propose a method (gBPCR) for the estimation of both LOH and CN aberrations, improving their estimation by integrating both types of data and accounting for their relationships. Moreover, gBPCR performed very well in comparison with other methods for LOH estimation and the estimated CN lesions on real data have been validated with another technique.

Clonal Mutations in the Cancer-Associated Fibroblasts: The Case against Genetic Coevolution

It is well established that carcinoma-associated fibroblasts (CAFs) differ phenotypically from fibroblasts associated with normal tissue, but the mechanisms underlying these differences remain controversial. Because CAFs can be propagated in vitro for extended periods and still maintain their cancer promoting phenotype, it has been proposed that they might have acquired somatic genetic alterations analogous to those observed in malignant epithelium. Whereas some investigators have reported frequent and profound genomic alterations in CAFs, other groups have found no such evidence. One striking common trait of those studies reporting frequent clonal somatic alterations in CAFs is the use of tissues and techniques which are well known to be highly prone to generating artefacts, such as limiting and poor quality DNA followed by highly multiplexed PCR-based analysis. We conclude that reported frequent clonal somatic mutations in CAFs are likely to be artefacts and are not the biological basis of the cancer promoting attributes of CAFs. [corrected]

Deletion of IKZF1 (Ikaros) Predicts Poor Outcome and Impaired Maturation in B-Progenitor Acute Lymphoblastic Leukemia

In genome-wide analyses of DNA copy number abnormalities (CNAs) and loss of heterozygosity (LOH) using single nucleotide polymorphism (SNP) microarrays, we previously reported a high frequency of genetic alterations of regulators of B lymphoid development and cell cycle in B-progenitor acute lymphoblastic leukemia (B-ALL), and a near-obligate deletion of the transcription factor Ikaros (IKZF1) in BCR-ABL1 ALL (Nature 2007; 446:758 and Nature 2008; 453:110). To assess the prognostic significance of genetic alterations and their effects on the leukemic cell transcriptome, we have now extended these studies to a cohort of 221 NCI high risk pediatric ALL cases treated on the Children's Oncology Group (COG) P9906 trial. The majority of these cases (N=170) lacked known cytogenetic abnormalities, and none were BCR-ABL1 positive. CNA and LOH data were generated using Affymetrix 500K SNP arrays, and gene expression data was available for 207 cases. Genomic resequencing was performed for PAX5 and IKZF1. These analyses revealed mutations in genes regulating B lymphoid development in 57.5% of cases, including PAX5 CNAs (31.7%), PAX5 sequence mutations (8.1%), IKZF1 deletions (24.9%) and IKZF1 sequence mutations (2.6%). In addition, recurring CNAs were detected in a number of other genes known to play roles in transformation and drug response including CDKN2A/B (45.7%), RB1 (11.3%), BTG1 (10.4%), IL3RA (6.8%), KRAS (6.3%), NRAS (2%), NR3C1 (2%), NR3C2 (3%), and ERG (6.3%). To examine associations between genetic alterations and outcome in a genome-wide fashion, we used a semi-supervised principal components approach and identified a predictor of outcome driven by deletion/mutation of IKZF1, EBF, and BTLA (hazard ratio 3.74, P=8.1×10−5). IKZF1 lesions were most strongly associated with poor outcome (cumulative incidence of relapse 68.8% in IKZF1-mutated ALL vs. 29.1% for IKZF1 wild type cases, P=0.002). This copy number predictor also predicted outcome in an independent cohort of 258 B-progenitor ALL cases treated at St Jude Children's Research Hospital (HR 3.76, P=0.00023). Importantly, the St Jude cohort had a much higher proportion of cases with conventional recurring cytogenetic abnormalities including hyperdiploidy and translocations than the COG P9906 cohort (60.4% versus 23%, respectively), including BCR-ABL1 (N=21; 8.1%). Notably, the association of IKZF1 mutation and poor outcome was also observed in the cases lacking BCR-ABL1. The presence of an IKZF1 mutation was also associated with a high-level of minimal residual disease following induction therapy in both cohorts. We next defined a gene expression profile of high risk (IKZF1-mutated) ALL in each cohort. Using gene set enrichment analysis (GSEA), we found the high risk gene expression signature to be positively enriched for hematopoietic stem cell genes and genes associated with minimal residual disease, and negatively enriched for genes regulating cell cycle progression and genes upregulated during B cell maturation. Moreover, GSEA also demonstrated significant similarity of the expression signatures of high risk COG and St Jude ALL, a striking finding given the marked differences in sample composition of the two groups. As IKZF1 mutation is a near obligate lesion in BCR-ABL1 lymphoid leukemia (Nature 2008; 453:110) and in this study is a strong independent predictor of poor outcome in BCR-ABL1 negative ALL, we next examined similarity of the gene expression signatures of high risk (IKZF1-mutated) BCR-ABL1 negative ALL and BCR-ABL1 positive ALL. Using GSEA and direct comparison of differentially expressed genes, there was highly significant similarity between the two signatures. These findings indicate that IKZF1 mutation is a key determinant of the gene expression signature of both BCR-ABL1 positive and negative ALL, and is thus likely to be central to the pathogenesis and poor outcome of both leukemia subtypes. Thus, IKZF1 deletion is a new prognostic factor in ALL, and is associated with a gene expression profile indicative of impaired maturation of the leukemic blasts, an observation compatible with the known role of IKZF1 in regulating early lymphoid development. These findings have important implications for risk stratification of ALL.

Clonal relationships between epidermotropic metastatic melanomas and their primary lesions: a loss of heterozygosity and X-chromosome inactivation-based analysis

Loss of heterozygosity (LOH) has previously been demonstrated at multiple chromosome microsatellites in primary and metastatic melanomas. Epidermotropic metastases of melanoma are unique in their varied histopathologic appearance, which can mimic a primary lesion. Our objective was to compare LOH profiles in primary and epidermotropic metastatic melanoma to delineate their clonal relationship. We examined the pattern of allelic loss in the primary melanomas of nine patients in addition to the 21 corresponding epidermotropic metastatic melanomas (average 2.3 metastases per patient). DNA samples were prepared from formalin-fixed, paraffin-embedded tissue sections using laser capture microdissection. Eight DNA microsatellite markers on six different chromosomes were analyzed: D1S214 (1p), D6S305 (6q), D9S171 (9p), D9S157 (9p), IFNA (9p), D10S212 (10q), D11S258 (11q), D18S70 (18q). In addition, X-chromosome inactivation analysis was performed in tumors from four women. LOH was seen in 67% (6/9) of primary melanomas and 81% (17/21) of epidermotropic metastatic melanomas. The most frequent allelic losses in informative cases occurred at 10q (33%), 9p (22%), and 11q (22%) in primary melanomas, and at 10q (50%), 1p (44%), and 6q (39%) in epidermotropic metastatic melanomas. Primary lesions demonstrating LOH had concordant allelic loss in at least one locus in a corresponding epidermotropic metastatic melanoma in 83% (5/6) of cases. X-chromosome analysis showed nonrandom inactivation in 75% (3/4) and 71% (5/7) of primary melanoma and epidermotropic metastatic melanoma cases, respectively. Our LOH and X-chromosome inactivation analysis data suggest that epidermotropically metastatic melanomas are clonally related to their primary lesion in many cases. Our data also indicated that some cases diagnosed as epidermotropic metastatic melanoma might be divergent clones or new primaries rather than metastatic disease.

ACTuDB, a new database for the integrated analysis of array-CGH and clinical data for tumors

In recent years, an increasing number of projects have investigated tumor genome structure, using microarray-based techniques like array comparative genomic hybridization (array-CGH) or single nucleotide polymorphism (SNP) arrays. The forthcoming studies have to integrate these former results and compare their findings to the existing sets of copy number data for validation. These sets also form the basis from which many comparative retrospective analyses can be carried out. Nevertheless, exploitation of this mass of data relies on a homogeneous preparation of copy number data, which will make it possible to compare them together, and their integration into a unified bioinformatics environment with ad hoc analysis tools and interfaces. To our knowledge, no such data integration has been proposed yet. Therefore the biologists and clinicians involved in cancer research urgently need such an integrative tool, which motivated us to undertake the construction of a database for array-CGH and other DNA copy number data for tumors (ACTuDB). When available, the associated clinical, transcriptome and loss of heterozygosity data were also integrated into ACTuDB. ACTuDB contains currently about 1500 genomic profiles for tumors and cell lines for the bladder, brain, breast, colon, liver, lymphoma, neuroblastoma, mouth and pancreas, together with data for replication timing experiments. The CGH array data were processed, using ad hoc algorithms (probe mapping, breakpoint detection, gain or loss status assignment and visualization) developed at Institut Curie. The database is available from http://bioinfo.curie.fr/actudb/ and can be browsed with a user-friendly interface. This database will be a useful resource for the genomic profiling of tumors, a field of highly active research. We invite research groups involved in tumor genome profiling to submit their data to ACTuDB.

Frequent occurrence of uniparental disomy in colorectal cancer

We used SNP arrays to identify and characterize genomic alterations associated with colorectal cancer (CRC). Laser microdissected cancer cells from 15 adenocarinomas were investigated by Affymetrix Mapping 10K SNP arrays. Analysis of the data extracted from the SNP arrays revealed multiple regions with copy number alterations and loss of heterozygosity (LOH). Novel LOH areas were identified at chromosomes 13, 14 and 15. Combined analysis of the LOH and copy number data revealed genomic structures that could not have been identified analyzing either data type alone. Half of the identified LOH regions showed no evidence of a reduced copy number, indicating the presence of uniparental structures. The distribution of these structures was non-random, primarily involving 8q, 13q and 20q. This finding was supported by analysis of an independent set of array-based transcriptional profiles, consisting of 17 normal mucosa and 66 adenocarcinoma samples. The transcriptional analysis revealed an unchanged expression level in areas with intact copy number, including regions with uniparental disomy, and a reduced expression level in the LOH regions representing factual losses (including 5q, 8p and 17p). The analysis also showed that genes in regions with increased copy number (including 7p and 20q) were predominantly upregulated. Further analyses of the SNP data revealed a subset of the identified alterations to be specifically associated with TP53 inactivation (including 8q gain and 17p loss) and lymph node metastasis status (gain of 7q and 13q). Another subset of the identified alterations was shown to represent intratumor heterogeneity. In conclusion, we demonstrate that uniparental disomy is frequent in CRC, and identify genomic alterations associated with TP53 inactivation and lymph node status.

Allele-specific loss of heterozygosity in multiple colorectal adenomas: toward an integrated molecular cytogenetic map II

Colorectal cancer (CRC) remains a significant public health challenge despite our increased understanding of the genetic defects underlying the pathogenesis of this common disease. It has been thought that multiple mechanisms lead to the malignant phenotype, with familial predisposition syndromes accounting for only a small proportion of all CRC cases. To identify additional loci likely involved in CRC and to test the hypothesis of allele-specific loss of heterozygosity (LOH) for the localization of CRC susceptibility genes, we initially conducted a genome-wide allelotyping analysis of 48 adenomas from a patient with familial adenomatous polyposis coli (FAP) and 63 adenomas from 7 patients with sporadic CRC using 79 fluorescently tagged oligonucleotide primers amplifying microsatellite loci covering the human genome. Frequent allelic losses were identified at D17S802 (41%), D7S518 (40%), D18S53 (38%), D10S249 (32%), D2S391 (29%), D16S419 (27%), D15S1005 and D15S120 (24%), D9S274 and D11S1318 (23%), D14S65 (20%), D14S274 and D17S953 (19%), D19S424 (18%), D5S346 and D1S397 (15%), and D6S468 (13%) in multiple FAP adenomas. Common LOH was also detected at D4S1584 (42%), D11S968 (31%), D17S953 (28%), D5S394, D9S286 and D10S249 (24%), D8S511 (23%), D13S158 (21%), D7S669 (20%), D18S58 (19%), D2S162 and D16S432 (16%), D2S206 (15%), D7S496 and D17S946 (14%), D6S292 (13%), D4S1586 and D8S283 (11%), and D1S2766 (10%) in multiple CRC adenomas. In addition, allele-specific LOH at D5S346, D15S1005, and D15S120 was observed in multiple FAP adenomas ( P < 0.01) and at D2S206 and D16S423 in multiple CRC ( P < 0.05). To compare our data to previous reports, we determined the band-specific frequency of chromosomal imbalances in CRC karyotypes reported in the Mitelman database, and from the CGH results of cases accessible through the PROGENETIX website. Furthermore, published genome-wide allelotyping analysis of CRC and other allele-specific LOH studies were compiled and collated with our LOH data. The combined results not only provide a comprehensive view of genetic losses in CRC, indicating the comparability of these different techniques, but they also reveal different novel loci in multiple adenomas from FAP and sporadic CRC patients, suggesting that they represent a distinct subtype of CRC in terms of allelic losses. Allele-specific LOH is an alternative approach for cancer gene mapping.

Feature‐Specific Penalized Latent Class Analysis for Genomic Data

Genomic data are often characterized by a moderate to large number of categorical variables observed for relatively few subjects. Some of the variables may be missing or noninformative. An example of such data is loss of heterozygosity (LOH), a dichotomous variable, observed on a moderate number of genetic markers. We first consider a latent class model where, conditional on unobserved membership in one of k classes, the variables are independent with probabilities determined by a regression model of low dimension q. Using a family of penalties including the ridge and LASSO, we extend this model to address higher-dimensional problems. Finally, we present an orthogonal map that transforms marker space to a space of "features" for which the constrained model has better predictive power. We demonstrate these methods on LOH data collected at 19 markers from 93 brain tumor patients. For this data set, the existing unpenalized latent class methodology does not produce estimates. Additionally, we show that posterior classes obtained from this method are associated with survival for these patients.

Somatic deletion of the NF1 gene in a neurofibromatosis type 1-associated malignant melanoma demonstrated by digital PCR

BackgroundNeurofibromatosis type 1 (NF1) is the most common hereditary neurocutaneous disorder and it is associated with an elevated risk for malignant tumors of tissues derived from neural crest cells. The NF1 gene is considered a tumor suppressor gene and inactivation of both copies can be found in NF1-associated benign and malignant tumors. Melanocytes also derive from neural crest cells but melanoma incidence is not markedly elevated in NF1. In this study we could analyze a typical superficial spreading melanoma of a 15-year-old boy with NF1 for loss of heterozygosity (LOH) within the NF1 gene. Neurofibromatosis in this patient was transmitted by the boy's farther who carried the mutation NF1 c. 5546 G/A.ResultsMelanoma cells were isolated from formalin-fixed tissue by liquid coverslip laser microdissection. In order to obtain statistically significant LOH data, digital PCR was performed at the intragenic microsatellite IVS27AC28 with DNA of approx. 3500 melanoma cells. Digital PCR detected 23 paternal alleles and one maternal allele. Statistical analysis by SPRT confirmed significance of the maternal allele loss.ConclusionTo our knowledge, this is the first molecular evidence of inactivation of both copies of the NF1 gene in a typical superficial spreading melanoma of a patient with NF1. The classical double-hit inactivation of the NF1 gene suggests that the NF1 genetic background promoted melanoma genesis in this patient.