Imbalances In Tumors Research Articles

Allele-specific gene expression in carcinogenesis

Recent large-scale genomic studies established the occurrence of multiple DNA sequence variants in genomes of healthy individuals that differ from the reference sequence. Among these variants mostly represented by germline single nucleotide polymorphisms disease-related alleles are detected including alleles which are associated with monogenic disorders, and putative deleterious genetic variants. Apart from functional significance of a particular variant and of a gene harboring it, the penetrance of these allelic variants depends on their expression level and can be determined by preferential expression of a particular allele, or allele-specific expression. It is estimated that 20–30 % of genes present in the human genome display allelic bias in a tissue-specific manner. Allele-specific expression is defined by a range of genetic and epigenetic mechanisms including cis-regulatory polymorphisms, allele-specific binding of transcription factors, allele-specific DNA methylation and regulation through non-coding RNA. Although the data on the issue are scarce, allele-specific expression has been reported to be implicated in several hereditary disorders including benign and malignant tumors of the large intestine. Recent studies that estimate allele-specific expression incidence in tumors and identify wide range of genes displaying allelic imbalance indicate that allele-specific expression might play a significant role in carcinogenesis. Eventually, estimation of transcriptional rate of allelic variants which cause dysfunction of oncogenes and tumor suppressors may prove to be essential for rational choice of antitumor therapeutic strategy. In this review, we outline the main concepts and mechanisms of allele-specific expression and the data on allelic imbalance in tumors.

Different patterns of allelic imbalance in sporadic tumors and tumors associated with long-term exposure to gamma-radiation

The study aimed to reveal cancer related mutations in DNA repair and cell cycle genes associated with chronic occupational exposure to gamma-radiation in personnel of the Siberian Group of Chemical Enterprises (SGCE). Mutations were analyzed by comparing genotypes of malignant tumors and matched normal tissues of 255 cancer patients including 98 exposed to external gamma-radiation (mean dose 128.1±150.5mSv). Also a genetic association analysis was carried out in a sample of 149 cancer patients and 908 healthy controls occupationally exposed to gamma-radiation (153.2±204.6mSv and 150.5±211.2mSv, respectively). Eight SNPs of genes of DNA excision repair were genotyped (rs13181, rs1052133, rs1042522, rs2305427, rs4244285, rs1045642, rs1805419 and rs1801133). The mutation profiles in heterozygous loci for selected SNP were different between sporadic tumors and tumors in patients exposed to radiation. In sporadic tumors, heterozygous genotype Arg/Pro of the rs1042522 SNP mutated into Arg/0 in 15 cases (9.6%) and 0/Pro in 14 cases (8.9%). The genotype Lys/Gln of the rs13181 SNP mutated into Lys/0 and 0/Gln in 9 and 4 cases, respectively. In tumors of patients exposed to low-level radiation, the rs1042522 Arg/0 mutated genotype was found in 12 cases (12.1%), while in 2 cases (2%) 0/Pro mutation was observed. Finally, the rs13181 0/Gln mutated genotype was observed in 15 cases (16,5%) . Thus, our study showed the difference in patterns of allelic imbalance in tumors appeared under low-level radiation exposure and spontaneous tumors for selected SNPs. This suggests different mechanisms of inactivation of heterozygous genotypes in sporadic and radiation-induced tumors.

ExomeAI: detection of recurrent allelic imbalance in tumors using whole-exome sequencing data

Whole-exome sequencing (WES) has extensively been used in cancer genome studies; however, the use of WES data in the study of loss of heterozygosity or more generally allelic imbalance (AI) has so far been very limited, which highlights the need for user-friendly and flexible software that can handle low-quality datasets. We have developed a statistical approach, ExomeAI, for the detection of recurrent AI events using WES datasets, specifically where matched normal samples are not available. ExomeAI is a web-based application, publicly available at: http://genomequebec.mcgill.ca/exomeai. JavadNadaf@gmail.com or somayyeh.fahiminiya@mcgill.ca Supplementary data are available at Bioinformatics online.

Congenic mapping and allele-specific alteration analysis of Stmm1 locus conferring resistance to early-stage chemically induced skin papillomas.

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to early-stage chemically induced skin papillomas on chromosome 7 with a large number of [(FVB/N×MSM/Ms)×FVB/N] F1 backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 7. We used linkage analysis and congenic mouse strains to refine the location of Stmm1 (Skin tumor modifier of MSM 1) locus within a genetic interval of about 3 cM on proximal chromosome 7. In addition, we used patterns of allele-specific imbalances in tumors from F1 backcross and N10 congenic mice to narrow down further the region of Stmm1 locus to a physical distance of about 5.4 Mb. To gain the insight into the function of Stmm1 locus, we carried out a long term BrdU labelling experiments with congenic mice containing Stmm1 locus. Interestingly, we observed a decrease of BrdU-LRCs (Label Retaining Cells) in a congenic strain heterozygous or homozygous for MSM allele of Stmm1. These results suggest that Stmm1 responsible genes may have an influence on papillomagenesis in the two-stage skin carcinogenesis by regulating epidermal quiescent stem cells.

Mapping of actionable mutations to histological subtype domains in lung adenocarcinoma: implications for precision medicine.

Precision medicine depends on the accurate identification of actionable mutations in a tumor sample. It is unknown how heterogeneous the distribution of such mutations can be in a tumor. Morphological (i.e. histopathological) heterogeneity is well described in lung adenocarcinoma and has been specifically recognized in the most recent official clinico-pathological classification. The most predominant subtype present is now used to classify each lung adenocarcinoma. No molecular profile exists to explain the intratumoral differences in lung adenocarcinoma morphology, despite the consistently observed association between specific predominant subtypes and poorer survival. Given a recent proposal stratifying lung adenocarcinoma into subtypes of differing metastatic potential, we questioned the assumption that major mutations are present uniformly throughout tumors; especially those showing discrete different subtypes. We selected formalin-fixed paraffin embedded lung adenocarcinoma specimens that showed discrete areas of different subtypes, extracted subtype DNA samples from those areas and screened for mutations in hotspot regions of the EGFR, KRAS and BRAF genes using high resolution melting. Sanger sequencing was used to confirm all identified mutations. Chromogenic in situ hybridization (CISH) was used to identify mutant allele specific imbalances in tumors with EGFR mutations. Interestingly, we found that KRAS and BRAF mutations could be confined to morphological domains of higher grade. On the other hand, EGFR mutations were found through all histological subtypes in each tumor consistent with the driver status of this mutation. Intratumoral heterogeneity has major implications for tumorigenesis, chemoresistance and the role of histopathology in molecular screening for precision medicine. This study not only confirms that intratumoral mutational heterogeneity does occur, but also that it is associated with morphologically distinct regions in some tumors. From a practical perspective, small biopsies may not adequately represent a tumor's full mutational profile, particularly for later arising but prognostically important mutations such as those in the KRAS and BRAF genes.

Identification of <i>Stmm3</i> locus Conferring Resistance to Late-stage Chemically Induced Skin Papillomas on Mouse Chromosome 4 by Congenic Mappingand Allele-specific Alteration Analysis

Genome-wide association studies have revealed that many low-penetrance cancer susceptibility loci are located throughout the genome; however, a very limited number of genes have been identified so far. Using a forward genetics approach to map such loci in a mouse skin cancer model, we previously identified strong genetic loci conferring resistance to chemically induced skin papillomas on chromosome 4 and 7 with a large number of [(FVB/N × MSM/Ms) F1 × FVB/N] backcross mice. In this report, we describe a combination of congenic mapping and allele-specific alteration analysis of the loci on chromosome 4. We used linkage analysis and a congenic mouse strain, FVB.MSM-Stmm3 to refine the location of Stmm3 (Skin tumor modifier of MSM 3) locus within a physical interval of about 34 Mb on distal chromosome 4. In addition, we used patterns of allele-specific imbalances in tumors from N2 and N10 congenic mice to narrow down further the region of Stmm3 locus to a physical distance of about 25 Mb. Furthermore, immunohistochemical analysis showed papillomas from congenic mice had less proliferative activity. These results suggest that Stmm3 responsible genes may have an influence on papilloma formation in the two-stage skin carcinogenesis by regulating papilloma growth rather than development.

Abstract 4857: Allelic-specific imbalance mapping identifies HDAC9 as a candidate susceptibility gene for cutaneous squamous cell carcinoma

Abstract Non-melanoma skin cancer (NMSC) is the most common cancer in the world. More than 3.5 million NMSCs were treated in 2006; of those, 700,000 were cutaneous squamous cell carcinomas (cSCC). There are several predisposing factors to cSCC; however, little is known about the genetic risk factors. Identification of genetic factors contributing to cSCC will enable the identification of those at risk and will lead to improved therapeutic options. A cSCC susceptibility locus, Skts5, was identified on mouse chromosome 12 by linkage analysis of F1 backcrosses between resistant Mus Spretus (Spret/GS) and susceptible Mus Musculus (NIH/Ola) mice. Other susceptibility loci identified in these crosses show preferential allelic imbalance in skin tumors, indicating that allele-specific somatic genetic alterations in these regions may be markers for cancer susceptibility loci. Skts5 spans a 14 megabase region with 65 coding elements. Based on sequence variations and differential gene expression between Spret/GS and NIH/Ola we were able to identify 11 candidate genes. The orthologous locus to Skts5 in humans maps to 7p21 and 7q31. Previous studies conducted in our lab show gains on 7p and 7q31 in 10% of cSCC tumors. An additional 3% of cSCC tumors were found to have copy-neutral loss of heterozygosity. Further analysis revealed that microsatellite markers on 7p21 and 7q31 demonstrated preferential allelic imbalance in cSCC tumors. The question of this study is whether genetic variations at SKTS5 are playing a role in human cSCC susceptibility. We hypothesize that human cSCC tumors will show allele-specific somatic genetic changes at SKTS5 and that these alterations contribute to cSCC risk. In order to identify candidate genes at SKTS5, we performed genotyping of 70 single nucleotide polymorphisms (SNPs) encompassing our top mouse candidate genes using Sequenom MassARRAY. Three SNPs at SKTS5, two in HDAC9 (rs6959028 and rs12540872) and one in IFRD1 (rs2074796) showed statistically significant evidence of preferential allelic imbalance in cSCC tumors. Conversely, when SNPs were tested in two cSCC case/control collections no significant association for risk was seen. Because our study analyzed 40 SNPs in HDAC9 and two showed preferential allelic imbalance in tumors, we hypothesized that there might be HDAC9 haplotypes demonstrating preferential imbalance. We generated 4,5,6 and 7 marker haplotypes. Following multiple comparisons testing, significant haplotypes of each size containing rs6959028 and/or rs12540872 were identified with differential frequencies between normal blood and tumor DNA. From these studies our data identifies HDAC9 as a candidate gene for cSCC tumorigenesis. Risk with this locus needs to be further evaluated. Future studies will be needed to determine which HDAC9 SNPs are causal, as well as to identify the molecular mechanism(s) by which the variants contribute to cSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4857. doi:1538-7445.AM2012-4857

Allele-Specific Deletions in Mouse Tumors Identify Fbxw7 as Germline Modifier of Tumor Susceptibility

Genome-wide association studies (GWAS) have been successful in finding associations between specific genetic variants and cancer susceptibility in human populations. These studies have identified a range of highly statistically significant associations between single nucleotide polymorphisms (SNPs) and susceptibility to development of a range of human tumors. However, the effect of each SNP in isolation is very small, and all of the SNPs combined only account for a relatively minor proportion of the total genetic risk (5–10%). There is therefore a major requirement for alternative routes to the discovery of genetic risk factors for cancer. We have previously shown using mouse models that chromosomal regions harboring susceptibility genes identified by linkage analysis frequently exhibit allele-specific genetic alterations in tumors. We demonstrate here that the Fbxw7 gene, a commonly mutated gene in a wide range of mouse and human cancers, shows allele-specific deletions in mouse lymphomas and skin tumors. Lymphomas from three different F1 hybrids show 100% allele-specificity in the patterns of allelic loss. Parental alleles from 129/Sv or Spretus/Gla mice are lost in tumors from F1 hybrids with C57BL/6 animals, due to the presence of a specific non-synonymous coding sequence polymorphism at the N-terminal portion of the gene. A specific genetic test of association between this SNP and lymphoma susceptibility in interspecific backcross mice showed a significant linkage (p = 0.001), but only in animals with a functional p53 gene. These data therefore identify Fbxw7 as a p53-dependent tumor susceptibility gene. Increased p53-dependent tumor susceptibility and allele-specific losses were also seen in a mouse skin model of skin tumor development. We propose that analysis of preferential allelic imbalances in tumors may provide an efficient means of uncovering genetic variants that affect mouse and human tumor susceptibility.

Thymidylate synthetase allelic imbalance in clear cell renal carcinoma

To investigate the allelic status of the thymidylate synthetase (TYMS) gene, located at chromosome band 18p11.32, in renal cell carcinoma (RCC). TYMS is a key target of the 5-fluorouracil (5-FU)-based class of drugs, frequently considered in combination therapies in advanced RCC. TYMS variants, such as the TYMS polymorphic 5'-untranslated region variable number tandem repeat sequence (VNTR), are under investigation to guide 5-FU treatment. Yet, no information is available with regard to changes in TYMS allele frequencies in RCC malignances. Blood and matched tumor samples were collected from 41 histological proven clear cell RCC affected patients (30 males, 11 females.). TYMS VNTR genotype was first determined in blood to identify heterozygotes employing PCR techniques. To evaluate for allelic imbalance, fragment analysis was performed both in blood and matched tumor DNA of the heterozygote patients. Microsatellite analysis, employing the markers D18S59 and D18S476 mapping, respectively, at the TYMS locus (18p11.32) and 1.5 Mb downstream of the TYMS gene sequence (18p11.31), was performed to confirm TYMS allelic imbalance in tumors. Germ-line TYMS VNTR distribution was: 2R/2R (19.5%), TYMS 2R/3R (36.6%) and TYMS 3R/3R (43.9%). Allelic imbalance for the TYMS tandem repeat region was detected in 26.6% of the heterozygote patients. Microsatellite analysis confirmed the allelic imbalance detected by TYMS VNTR analysis and revealed that the overall frequence of allelic imbalance of chromosome band 18p11.32 was 35%, while the overall allelic imbalance of chromosome band 18p11.31 was 28%. By focusing on the TYMS polymorphic variants in renal cancer, we here provide evidence, to our knowledge, for the first time showing loss of 18p11.32 and 18p11.31 in renal cell carcinomas. As allelic imbalances involving TYMS locus may be an important variable affecting 5-FU responsiveness, this study may contribute to explain different responses of advanced RCC in combined chemotherapeutic regimens incorporating fluoropyridines.

Array Comparative Genomic Hybridization Copy Number Profiling: A New Tool for Translational Research in Solid Malignancies

The molecular genetic investigation of cancer is rapidly evolving because of ever-improving technology. Insights into cancer disease mechanisms are being elucidated using new chromosome-based biomarkers. Until recently, diagnostic and prognostic assessment of diseased tissues and tumors relied heavily on histologic indicators that permitted only general classifications into morphologic subtypes and did not take into account the alterations in individual gene expression or copy number. Genomic and expression profiling now allow the simultaneous interrogation of thousands of genes and offer unprecedented opportunities to obtain global molecular signatures of neoplastic cells in patient samples. One limitation of global profiling at the expression level is that acquisition and optimal transport of high-quality RNA is problematic because of its inherent instability in vitro. In contrast, tumor DNA is stable, relatively easy to transport, and can be obtained from archival paraffin tissue blocks. Thus, there is now a tremendous opportunity to globally profile copy number imbalances in tumors using array comparative genomic hybridization (CGH), which can identify at high resolution the presence of genomic copy number changes in constitutional or tumor DNA samples. Array CGH profiling has already allowed a deeper insight into the biology of a variety of tumor types and in the near future will undoubtedly prove to be a key technology leading to better cancer classification, prognosis, and outcome prediction.

22q11.2 Distal Deletion: A Recurrent Genomic Disorder Distinct from DiGeorge Syndrome and Velocardiofacial Syndrome

Microdeletions within chromosome 22q11.2 cause a variable phenotype, including DiGeorge syndrome (DGS) and velocardiofacial syndrome (VCFS). About 97% of patients with DGS/VCFS have either a common recurrent approximately 3 Mb deletion or a smaller, less common, approximately 1.5 Mb nested deletion. Both deletions apparently occur as a result of homologous recombination between nonallelic flanking low-copy repeat (LCR) sequences located in 22q11.2. Interestingly, although eight different LCRs are located in proximal 22q, only a few cases of atypical deletions utilizing alternative LCRs have been described. Using array-based comparative genomic hybridization (CGH) analysis, we have detected six unrelated cases of deletions that are within 22q11.2 and are located distal to the approximately 3 Mb common deletion region. Further analyses revealed that the rearrangements had clustered breakpoints and either a approximately 1.4 Mb or approximately 2.1 Mb recurrent deletion flanked proximally by LCR22-4 and distally by either LCR22-5 or LCR22-6, respectively. Parental fluorescence in situ hybridization (FISH) analyses revealed that none of the available parents (11 out of 12 were available) had the deletion, indicating de novo events. All patients presented with characteristic facial dysmorphic features. A history of prematurity, prenatal and postnatal growth delay, developmental delay, and mild skeletal abnormalities was prevalent among the patients. Two patients were found to have a cardiovascular malformation, one had truncus arteriosus, and another had a bicuspid aortic valve. A single patient had a cleft palate. We conclude that distal deletions of chromosome 22q11.2 between LCR22-4 and LCR22-6, although they share some characteristic features with DGS/VCFS, represent a novel genomic disorder distinct genomically and clinically from the well-known DGS/VCF deletion syndromes.

Defective Chromosome Segregation and Telomere Dysfunction in Aggressive Wilms' Tumors

In many childhood neoplasms, prognostic subgroups have been defined based on specific chromosome changes. In Wilms' tumor (WT), such subclassification has been hampered by the diverse and relatively unspecific pattern of chromosomal imbalances present in these tumors. Unspecific patterns of cytogenetic imbalances in tumors are often caused by mitotic segregation errors due to short dysfunctional telomeres. As an alternative to cytogenetic classification, we therefore have evaluated whether the rate of telomere-dependent chromosomal instability could influence the clinical course in WT patients. Telomere function and mitotic segregation errors were assessed in 12 cultured tumors and in tumor tissue sections from 41 WT patients. Abnormal telomere shortening was found in cultured cells and in tissue sections from highly aggressive tumors. In vitro, dysfunctional telomeres were associated to specific cell division abnormalities, including anaphase bridges and multipolar mitoses. Assessment of mitotic figures in tissue sections revealed that anaphase bridges and multipolar mitoses were predominantly, but not exclusively, present in high-risk tumors and were predictors of poor event-free and overall survival. Telomere-dependent mitotic instability is present in a subgroup of WT, predominantly consisting of high-risk tumors.

Convergence of congenic mapping and allele-specific alterations in tumors for the resolution of the Skts1 skin tumor susceptibility locus

Although several familial cancer genes with high-penetrance mutations have been identified, the major genetic component of susceptibility to sporadic cancers is attributable to low-penetrance alleles. These 'weak' tumor susceptibility genes do not segregate as single Mendelian traits and are therefore difficult to find in studies of human populations. Previously, we have proposed that a combination of germline mapping and analysis of allele-specific imbalance in tumors may be used to refine the locations of susceptibility genes using mouse models of cancer. Here, we have used linkage analysis and congenic mouse strains to map the major skin tumor susceptibility locus Skts1 within a genetic interval of 0.9 cM on proximal chromosome 7. This interval lies in an apparent recombination cold spot, and corresponds to a physical distance of about 15 Mb. We therefore, used patterns of allele-specific imbalances in tumors from backcross and congenic mice to refine the location of Skts1. We demonstrate that this single tumor modifier locus has a dramatic effect on the allelic preference for imbalance on chromosome 7, with at least 90% of tumors from the congenics showing preferential gain of markers on the chromosome carrying the susceptibility variant. Importantly, these alterations enabled us to refine the location of Skts1 at higher resolution than that attained using the congenic mice. We conclude that low-penetrance susceptibility genes can have strong effects on patterns of allele-specific somatic genetic changes in tumors, and that analysis of the directionality of these somatic events provides an important and rapid route to identification of germline genetic variants that confer increased cancer risk.

Aneuploidy Arises at Early Stages of Apc-Driven Intestinal Tumorigenesis and Pinpoints Conserved Chromosomal Loci of Allelic Imbalance between Mouse and Human

Although chromosomal instability characterizes the majority of human colorectal cancers, the contribution of genes such as adenomatous polyposis coli (APC), KRAS, and p53 to this form of genetic instability is still under debate. Here, we have assessed chromosomal imbalances in tumors from mouse models of intestinal cancer, namely Apc(+/1638N), Apc(+/1638N)/KRAS(V12G), and Apc(+/1638N)/Tp53-/-, by array comparative genomic hybridization. All intestinal adenomas from Apc(+/1638N) mice displayed chromosomal alterations, thus confirming the presence of a chromosomal instability defect at early stages of the adenoma-carcinoma sequence. Moreover, loss of the Tp53 tumor suppressor gene, but not KRAS oncogenic activation, results in an increase of gains and losses of whole chromosomes in the Apc-mutant genetic background. Comparative analysis of the overall genomic alterations found in mouse intestinal tumors allowed us to identify a subset of loci syntenic with human chromosomal regions (eg, 1p34-p36, 12q24, 9q34, and 22q) frequently gained or lost in familial adenomas and sporadic colorectal cancers. The latter indicate that, during intestinal tumor development, the genetic mechanisms and the underlying functional defects are conserved across species. Hence, our array comparative genomic hybridization analysis of Apc-mutant intestinal tumors allows the definition of minimal aneuploidy regions conserved between mouse and human and likely to encompass rate-limiting genes for intestinal tumor initiation and progression.

Gains of 13q are correlated with a poor prognosis in liposarcoma

Liposarcomas are a phenotypical heterogenous group of tumors divided into four main subtypes: well-differentiated, dedifferentiated, myxoid/round cell, and pleomorphic. The aim of this study was to compare DNA sequence copy number changes of these subtypes as investigated by comparative genomic hybridization in 36 patients. Comparative genomic hybridization revealed genomic imbalances in tumors of 27 patients (mean 5.6 imbalances per tumor). The most frequent gains were within single regions of 1q, 12q, and 13q. We found a significant correlation of poor overall survival and gain of 13q21 (P=0.0221), 13q22 (P=0.0341), 13q31 (P=0.0410), and 13q32 (P=0.0074). The univariate Cox regression analysis revealed an increased risk of tumor-related death for patients whose liposarcomas possess with gains of 13q21 and 13q32 simultaneously (P=0.010; RR=7.1; 95% CI 1.6–31.7). Furthermore, 12 high-level amplifications were found in tumors of seven patients. In four cases 12q14–q15 and in two cases 13q32–q33 were amplified. We identified in different liposarcoma subtypes characteristic genomic changes: Gains and high-level amplifications of 12q occurred in all 11 investigated well-differentiated liposarcomas, and these changes were often present simultaneously with gains of 1q (mean 5.5 changes). In the two dedifferentiated liposarcomas, gains of 1q in both liposarcomas, and a high-level amplification of 13q were striking. Only eight of the 17 patients with myxoid/round cell liposarcomas showed changes in DNA copy number (mean 3.4 imbalances). In four of these eight cases gains of 13q occurred. The six pleomorphic liposarcomas possessed the most frequent genomic imbalances (mean number 16.3) of all liposarcoma subtypes investigated. These imbalances were in almost all chromosomal regions detected predominantly as over-representations of chromosomes 1, 5p, 13q, and 22q. Summarizing, all subtypes but well-differentiated liposarcomas showed gains of 13q, which were associated with a poor prognosis.

Assessment of allele dosage at polymorphic microsatellite loci displaying allelic imbalance in tumors by means of quantitative competitive-polymerase chain reaction

Analysis of allelic imbalance at polymorphic marker loci is usually employed to identify chromosomal regions affected by recurrent aberrations in tumor genomes. Such regions are likely to harbor genes involved in the onset and/or progression of cancer. Although often used to identify regions of loss of heterozygosity caused by deletions/rearrangements near tumor suppressor gene loci, allelic imbalance can also reflect regional amplification, indicating the presence of oncogenes. It is difficult to tell these two situations apart after ordinary polymerase chain reaction (PCR), but here we describe a method that distinguishes allelic loss from allelic gain. The level of allelic imbalance was determined by quantitative PCR (QPCR) in the presence of an internal control DNA that displayed a third allele at the locus studied. To validate the efficiency of allele quantitation, we analyzed an amplified region in a set of rat fibrosarcomas. In four tumor samples with amplification of the Met oncogene, we could show with QPCR that there was amplification of one of the alleles at a microsatellite marker located close to Met. QPCR may be useful for cancer studies because experiments may be predesigned for using either suitable microsatellite markers or the abundant and polymorphic poly-A tails of rodent identifier sequences.

Characterization of 10 vulvar carcinoma cell lines by karyotyping, comparative genomic hybridization and flow cytometry

Objective and methods. Ten vulvar squamous cell carcinoma cell lines established at the University of Michigan (UM-SCV-1A, -1B, -2, -3, -4, -6, -7) and at the University of Turku (UT-SCV-1, -2, -3) were characterized by G-banding karyotyping, comparative genomic hybridization (CGH), and deoxyribonucleic acid (DNA) flow cytometry. Results. All cell lines had hyperdiploid DNA content as measured by flow cytometry. The DNA index (DI) remained relatively stable through different passages in 9 of 10 cases. DIs of UM-SCV-3 and UT-SCV-2 were near-diploid, as were the corresponding karyotypes. The 10 SCVs showed remarkable genetic similarities with respect to consistent chromosome rearrangements. Loss of 3p, noted in 8/10 SCVs, was narrowed to the smallest common region at 3p11–3p13. Loss of 8pter-p11 was observed in 10/10 cell lines. Loss of 11qter-q23 was present in UM-SCV-1 and -2, and in all four recently karyotyped SCVs. Other consistent losses include Xpter-p11 in 6/10, and 18qter-q11 in 7/10 cell lines. Common gains included gain of 8q in 8/10 and 3q in 6/10. Consistent copy number imbalances were confirmed by CGH; concerning loss of 3p, in 63%, to loss of 8p in 70%, to gain of 3q in 83%, and to gain of 8q in 75% of the cell lines. Conclusions. CGH and karyotyping showed concordance in defining copy number imbalances, thus supporting the accuracy of CGH to detect chromosome imbalances in tumors that cannot be karyotyped.

The genetics of cancer susceptibility: from mouse to man.

Cancer affects approximately 1 in 3 individuals. An individual's susceptibility to cancer is partly determined by environmental exposures and by the combination of inherited cancer susceptibility and resistance genes. Initial mapping of these low penetrance cancer susceptibility genes has been done in the mouse because human low penetrance genes are extremely difficult to find using traditional methods due to heterogeneity and interacting factors. Also, the choice of candidate genes for human association studies can miss the unknown or unexpected. Mouse models also have limitations; it can be difficult to identify causal polymorphisms in the mouse because linkage disequilibrium often extends across several genes. To exploit the strengths of both systems, we outline a cross-species strategy to identify human variants associated with increased cancer risk. This approach uses linkage analysis and haplotyping, allelic imbalance in tumors, and gene expression studies in the mouse, combined with association studies and tumor imbalance studies in humans to identify causal cancer susceptibility variants. Allelic variants in both mouse and human can then be used to better understand the mechanisms behind cancer risk and as targets for intervention.

CHK2-decreased protein expression and infrequent genetic alterations mainly occur in aggressive types of non-Hodgkin lymphomas

The CHK2 gene codifies for a serine/threonine kinase that plays a central role in DNA damage response pathways. To determine the potential role of CHK2 alterations in the pathogenesis of lymphoid neoplasms we have examined the gene status, protein, and mRNA expression in a series of tumors and nonneoplastic lymphoid samples. A heterozygous Ile157Thr substitution, also present in the germ line of the patient, was detected in a blastoid mantle cell lymphoma (MCL). CHK2 protein and mRNA expression levels were similar in all types of lymphomas and reactive samples, and these levels were independent of the proliferative activity of the tumors. However, 5 tumors, one typical MCL, 2 blastoid MCLs, and 2 large cell lymphomas, showed marked loss of protein expression, including 2 samples with complete absence of CHK2 protein. These 2 lymphomas showed the highest number of chromosomal imbalances detected by comparative genomic hybridization in the whole series of cases. However, no mutations, deletions, or hypermethylation of the promoter region were identified in any of these tumors. mRNA levels were similar in cases with low and normal protein expression, suggesting a posttranscriptional regulation of the protein in these tumors. CHK2 gene and protein alterations were not related to p53 and ATM gene status. In conclusion, CHK2 alterations are uncommon in malignant lymphomas but occur in a subset of aggressive tumors independently of p53 or ATM alterations. The high number of chromosomal imbalances in tumors with complete absence of CHK2 protein suggests a role of this gene in chromosomal instability in human lymphomas.

Comparative genomic hybridization in glioma: a meta-analysis of 509 cases

Much data about genetic imbalances in tumors have been accumulated by comparative genomic hybridization (CGH). In order to distinguish between significantly and coincidentally involved regions in glioma by means of a meta-analysis, we summarized and analyzed the CGH results of 509 cases published in 26 reports between 1992 and 2001. The expansion of all aberrations to the 850-band level impressively visualized distinct patterns in astrocytoma, oligodendroglioma, and ependymoma as well as loci of frequent aberrations. For example, in astrocytoma the frequency of gains culminated at 7p12, 8q24.1, and 12q13∼q15 (the loci of EGF-R, C-MYC and CDK4, respectively) and losses at 9p21 (the locus of p15 and p16) and 10q23.3 where PTEN resides. Most chromosomes were variably prone to copy number changes at different scales of aberrations. At the whole chromosome level the analysis showed +7, −10 in astrocytoma and +9, +18 in ependymoma, but +20q, −9p in astrocytoma and +1q, −22q in ependymoma at the p-q arm level. Furthermore, we could confirm the correlation between the average number of copy alterations per patient (average number of copy alterations [ANCA] index) and malignancy for astrocytoma in a refined graduation as well as for oligodendroglioma. As a new parameter, the average number of affected GTG-bands per patient (average number of affected GTG bands [ANAG] index) showed an even more striking correlation with the World Health Organization grade for gains and losses.