Candidate Cancer Genes Research Articles

Our changing view of the genomic landscape of cancer.

Sporadic tumours, which account for the majority of all human cancers, arise from the acquisition of somatic, genetic and epigenetic alterations leading to changes in gene sequence, structure, copy number and expression. Within the last decade, the availability of a complete sequence-based map of the human genome, coupled with significant technological advances, has revolutionized the search for somatic alterations in tumour genomes. Recent landmark studies, which resequenced all coding exons within breast, colorectal, brain and pancreatic cancers, have shed new light on the genomic landscape of cancer. Within a given tumour type there are many infrequently mutated genes and a few frequently mutated genes, resulting in incredible genetic heterogeneity. However, when the altered genes are placed into biological processes and biochemical pathways, this complexity is significantly reduced and shared pathways that are affected in significant numbers of tumours can be discerned. The advent of next-generation sequencing technologies has opened up the potential to resequence entire tumour genomes to interrogate protein-encoding genes, non-coding RNA genes, non-genic regions and the mitochondrial genome. During the next decade it is anticipated that the most common forms of human cancer will be systematically surveyed to identify the underlying somatic changes in gene copy number, sequence and expression. The resulting catalogues of somatic alterations will point to candidate cancer genes requiring further validation to determine whether they have a causal role in tumourigenesis. The hope is that this knowledge will fuel improvements in cancer diagnosis, prognosis and therapy, based on the specific molecular alterations that drive individual tumours. In this review, I will provide a historical perspective on the identification of somatic alterations in the pre- and post-genomic eras, with a particular emphasis on recent pioneering studies that have provided unprecedented insights into the genomic landscape of human cancer.

Elevated expression levels of testis-specific genesTEX101andSPATA19in basal cell carcinoma and their correlation with clinical and pathological features

Basal cell carcinoma (BCC) is characterized by a low rate of metastasis, slow growth and strong stroma dependency, with significant morbidity and public health burden. Cancer-testis (CT) genes are specifically expressed in normal testis, fetal ovary and different types of cancers. Testis immune privileged status makes CT genes promising candidates as cancer markers, vaccines and immunotherapy. To find new CT genes as cancer markers and candidate genes for immunotherapy and to correlate pathological and clinical features with their expression in patients with BCC. By means of digital differential display, seven testis-specific genes were selected. Their expression patterns were analysed in 78 BCC and 15 normal skin samples using semiquantitative reverse transcription-polymerase chain reaction. Pathological and clinical characteristics were determined using appropriate methods. SPATA19, TEX101, ODF1, ODF2 and ODF3 were expressed in 56.6%, 38.2%, 2.6%, 17.4% and 2.6% of BCCs but not in normal skin samples. ODF4 and PASD1 were not expressed in any BCC samples. TEX101 and SPATA19 expression in high-risk BCCs was higher than in low-risk tumours (P < 0.001). SPATA19 expression was correlated with a history of cancer radiotherapy (P < 0.001). Significant associations were found between expression of TEX101 with nodular subtype, ODF2 with infiltrating subtype, and ODF1 with tumours located on the neck. Among gene expressors, 42.1% co-expressed two genes and 5.3% co-expressed three genes. We report five new CT antigens, of which SPATA19 and TEX101 may be possible targets for cancer immunotherapy and novel markers for early detection of BCC.

Whole-body sleeping beauty mutagenesis can cause penetrant leukemia/lymphoma and rare high-grade glioma without associated embryonic lethality.

The Sleeping Beauty (SB) transposon system has been used as a somatic mutagen to identify candidate cancer genes. In previous studies, efficient leukemia/lymphoma formation on an otherwise wild-type genetic background occurred in mice undergoing whole-body mobilization of transposons, but was accompanied by high levels of embryonic lethality. To explore the utility of SB for large-scale cancer gene discovery projects, we have generated mice that carry combinations of different transposon and transposase transgenes. We have identified a transposon/transposase combination that promotes highly penetrant leukemia/lymphoma formation on an otherwise wild-type genetic background, yet does not cause embryonic lethality. Infiltrating gliomas also occurred at lower penetrance in these mice. SB-induced or accelerated tumors do not harbor large numbers of chromosomal amplifications or deletions, indicating that transposon mobilization likely promotes tumor formation by insertional mutagenesis of cancer genes, and not by promoting wide-scale genomic instability. Cloning of transposon insertions from lymphomas/leukemias identified common insertion sites at known and candidate novel cancer genes. These data indicate that a high mutagenesis rate can be achieved using SB without high levels of embryonic lethality or genomic instability. Furthermore, the SB system could be used to identify new genes involved in lymphomagenesis/leukemogenesis.

Discovering cancer genes by integrating network and functional properties

BackgroundIdentification of novel cancer-causing genes is one of the main goals in cancer research. The rapid accumulation of genome-wide protein-protein interaction (PPI) data in humans has provided a new basis for studying the topological features of cancer genes in cellular networks. It is important to integrate multiple genomic data sources, including PPI networks, protein domains and Gene Ontology (GO) annotations, to facilitate the identification of cancer genes.MethodsTopological features of the PPI network, as well as protein domain compositions, enrichment of gene ontology categories, sequence and evolutionary conservation features were extracted and compared between cancer genes and other genes. The predictive power of various classifiers for identification of cancer genes was evaluated by cross validation. Experimental validation of a subset of the prediction results was conducted using siRNA knockdown and viability assays in human colon cancer cell line DLD-1.ResultsCross validation demonstrated advantageous performance of classifiers based on support vector machines (SVMs) with the inclusion of the topological features from the PPI network, protein domain compositions and GO annotations. We then applied the trained SVM classifier to human genes to prioritize putative cancer genes. siRNA knock-down of several SVM predicted cancer genes displayed greatly reduced cell viability in human colon cancer cell line DLD-1.ConclusionTopological features of PPI networks, protein domain compositions and GO annotations are good predictors of cancer genes. The SVM classifier integrates multiple features and as such is useful for prioritizing candidate cancer genes for experimental validations.

Distinct High-Profile Methylated Genes in Colorectal Cancer

BackgroundMutations and promoters' methylation of a set of candidate cancer genes (CAN genes) are associated with progression of colorectal cancer (CRC). We hypothesized that these genes' promoters are inactivated through epigenetic silencing and may show a different profile in high-risk populations. We investigated the status of CAN gene methylation and CHD5 protein expression in African American CRC tissue microarrays (TMA) using immunohistochemical staining.Methodology/Principal FindingsThe promoter methylation status of the CAN genes was studied by methylation-specific PCR (MSP) in 51 Iranians (a white population) and 51 African Americans (AA). Microsatellite instability (MSI) was analyzed as well. The differential frequency of methylation for each gene was tested by chi-square analysis between the two groups based on matched age and sex. CHD5 protein expression was evaluated in moderate to well differentiated and poorly differentiated carcinomas compared to matched normal tissue using TMA. In addition, the correlation between these epigenetic biomarkers and various clinicopathological factors, including, age, location, and stage of the disease were analyzed.Seventy-seven and 34% of tumors were distal in Iranian and African American patients, respectively. In both populations, the percentage of methylation was >65% for SYNE1, MMP2, APC2, GPNMB, EVL, PTPRD, and STARD8, whereas methylation was <50% for LGR6, RET, CD109, and RNF. The difference in methylation between the two populations was statistically significant for CHD5, ICAM5 and GPNMB. Thirty-one percent AA tumors showed MSI-H, compared to 28% in Iranians.Conclusions/SignificanceA significantly higher methylation rate was found for GPNMB, ICAM5, and CHD5 genes in AA patients compared to Iranians. These genes might play a role in the high incidence and aggressiveness of CRC in the AA population. The hypermethylation of the CAN genes can be considered as a marker of colon carcinogenesis.

Mutational analysis of FANCL , FANCM and the recently identified FANCI suggests that among the 13 known Fanconi Anemia genes, only FANCD1/BRCA2 plays a major role in high-risk breast cancer predisposition

Fanconi Anemia (FA) is a rare recessive syndrome characterized by cellular hypersensitivity to DNA-cross-linking agents. To date, 13 FA complementation groups have been described and all 13 genes associated to each of these groups have been currently identified. Three of the known FA genes are also high-risk (FANCD1/BRCA2) or moderate-risk (FANCN/PALB2 and FANCJ/BRIP1) breast cancer susceptibility genes, which makes all members of the FA pathway particularly attractive breast cancer candidate genes. Most FA genes have been screened for mutations in breast cancer families negative for BRCA1/2 mutations but the role of FANCL, FANCM and the recently identified FANCI has not been evaluated to date. This fact and novel data sustaining greater functional relevance of the three genes within the FA pathway prompted us to scrutinize all coding sequences and splicing sites of FANCI, FANCL and FANCM in 95 BRCA1/2-negative index cases from Spanish high-risk breast cancer families. We identified 68 sequence variants of which 24 were coding and 44 non-coding. Six exonic and 26 non-coding variants had not been described previously. None of the coding changes caused clearly pathogenic changes and computational analysis of all non-described intronic variants did not revealed major impact in splicing. With the present study, all known FA genes have been evaluated within the context of breast cancer high-risk predisposition. Our results rule out a major role of FANCI, FANCL and FANCM in familial breast cancer susceptibility, suggesting that among the 13 known FA genes, only FANCD1/BRCA2 plays a major role in high-risk breast cancer predisposition.

Candidate pathways and genes for prostate cancer: a meta-analysis of gene expression data

BackgoundThe genetic mechanisms of prostate tumorigenesis remain poorly understood, but with the advent of gene expression array capabilities, we can now produce a large amount of data that can be used to explore the molecular and genetic mechanisms of prostate tumorigenesis.MethodsWe conducted a meta-analysis of gene expression data from 18 gene array datasets targeting transition from normal to localized prostate cancer and from localized to metastatic prostate cancer. We functionally annotated the top 500 differentially expressed genes and identified several candidate pathways associated with prostate tumorigeneses.ResultsWe found the top differentially expressed genes to be clustered in pathways involving integrin-based cell adhesion: integrin signaling, the actin cytoskeleton, cell death, and cell motility pathways. We also found integrins themselves to be downregulated in the transition from normal prostate tissue to primary localized prostate cancer. Based on the results of this study, we developed a collagen hypothesis of prostate tumorigenesis. According to this hypothesis, the initiating event in prostate tumorigenesis is the age-related decrease in the expression of collagen genes and other genes encoding integrin ligands. This concomitant depletion of integrin ligands leads to the accumulation of ligandless integrin and activation of integrin-associated cell death. To escape integrin-associated death, cells suppress the expression of integrins, which in turn alters the actin cytoskeleton, elevates cell motility and proliferation, and disorganizes prostate histology, contributing to the histologic progression of prostate cancer and its increased metastasizing potential.ConclusionThe results of this study suggest that prostate tumor progression is associated with the suppression of integrin-based cell adhesion. Suppression of integrin expression driven by integrin-mediated cell death leads to increased cell proliferation and motility and increased tumor malignancy.

GWAS Meets Microarray: Are the Results of Genome-Wide Association Studies and Gene-Expression Profiling Consistent? Prostate Cancer as an Example

BackgroundGenome-wide association studies (GWASs) and global profiling of gene expression (microarrays) are two major technological breakthroughs that allow hypothesis-free identification of candidate genes associated with tumorigenesis. It is not obvious whether there is a consistency between the candidate genes identified by GWAS (GWAS genes) and those identified by profiling gene expression (microarray genes).Methodology/Principal FindingsWe used the Cancer Genetic Markers Susceptibility database to retrieve single nucleotide polymorphisms from candidate genes for prostate cancer. In addition, we conducted a large meta-analysis of gene expression data in normal prostate and prostate tumor tissue. We identified 13,905 genes that were interrogated by both GWASs and microarrays. On the basis of P values from GWASs, we selected 1,649 most significantly associated genes for functional annotation by the Database for Annotation, Visualization and Integrated Discovery. We also conducted functional annotation analysis using same number of the top genes identified in the meta-analysis of the gene expression data. We found that genes involved in cell adhesion were overrepresented among both the GWAS and microarray genes.Conclusions/SignificanceWe conclude that the results of these analyses suggest that combining GWAS and microarray data would be a more effective approach than analyzing individual datasets and can help to refine the identification of candidate genes and functions associated with tumor development.

Systems-level cancer gene identification from protein interaction network topology applied to melanogenesis-related functional genomics data

Many real-world phenomena have been described in terms of large networks. Networks have been invaluable models for the understanding of biological systems. Since proteins carry out most biological processes, we focus on analysing protein-protein interaction (PPI) networks. Proteins interact to perform a function. Thus, PPI networks reflect the interconnected nature of biological processes and analysing their structural properties could provide insights into biological function and disease. We have already demonstrated, by using a sensitive graph theoretic method for comparing topologies of node neighbourhoods called 'graphlet degree signatures', that proteins with similar surroundings in PPI networks tend to perform the same functions. Here, we explore whether the involvement of genes in cancer suggests the similarity of their topological 'signatures' as well. By applying a series of clustering methods to proteins' topological signature similarities, we demonstrate that the obtained clusters are significantly enriched with cancer genes. We apply this methodology to identify novel cancer gene candidates, validating 80 per cent of our predictions in the literature. We also validate predictions biologically by identifying cancer-related negative regulators of melanogenesis identified in our siRNA screen. This is encouraging, since we have done this solely from PPI network topology. We provide clear evidence that PPI network structure around cancer genes is different from the structure around non-cancer genes. Understanding the underlying principles of this phenomenon is an open question, with a potential for increasing our understanding of complex diseases.

Transposon-mediated mutagenesis of somatic cells in the mouse for cancer gene identification

To successfully treat cancer we will likely need a much more detailed understanding of the genes and pathways meaningfully altered in individual cancer cases. One method for achieving this goal is to derive cancers in model organisms using unbiased forward genetic screens that allow cancer gene candidate discovery. We have developed a method using a “cut-and-paste” DNA transposon system called Sleeping Beauty (SB) to perform forward genetic screens for cancer genes in mice. Although the approach is conceptually similar to the use of replication competent retroviruses for cancer gene identification, the SB system promises to allow such screens in tissues previously not amenable to forward genetic screens such as the gastrointestinal tract, brain, and liver. This article describes the strains useful for SB-based screens for cancer genes in mice and how they are deployed in an experiment.

Molecular Profiling of Breast Cancer Cell Lines Defines Relevant Tumor Models and Provides a Resource for Cancer Gene Discovery

BackgroundBreast cancer cell lines have been used widely to investigate breast cancer pathobiology and new therapies. Breast cancer is a molecularly heterogeneous disease, and it is important to understand how well and which cell lines best model that diversity. In particular, microarray studies have identified molecular subtypes–luminal A, luminal B, ERBB2-associated, basal-like and normal-like–with characteristic gene-expression patterns and underlying DNA copy number alterations (CNAs). Here, we studied a collection of breast cancer cell lines to catalog molecular profiles and to assess their relation to breast cancer subtypes.MethodsWhole-genome DNA microarrays were used to profile gene expression and CNAs in a collection of 52 widely-used breast cancer cell lines, and comparisons were made to existing profiles of primary breast tumors. Hierarchical clustering was used to identify gene-expression subtypes, and Gene Set Enrichment Analysis (GSEA) to discover biological features of those subtypes. Genomic and transcriptional profiles were integrated to discover within high-amplitude CNAs candidate cancer genes with coordinately altered gene copy number and expression.FindingsTranscriptional profiling of breast cancer cell lines identified one luminal and two basal-like (A and B) subtypes. Luminal lines displayed an estrogen receptor (ER) signature and resembled luminal-A/B tumors, basal-A lines were associated with ETS-pathway and BRCA1 signatures and resembled basal-like tumors, and basal-B lines displayed mesenchymal and stem/progenitor-cell characteristics. Compared to tumors, cell lines exhibited similar patterns of CNA, but an overall higher complexity of CNA (genetically simple luminal-A tumors were not represented), and only partial conservation of subtype-specific CNAs. We identified 80 high-level DNA amplifications and 13 multi-copy deletions, and the resident genes with concomitantly altered gene-expression, highlighting known and novel candidate breast cancer genes.ConclusionsOverall, breast cancer cell lines were genetically more complex than tumors, but retained expression patterns with relevance to the luminal-basal subtype distinction. The compendium of molecular profiles defines cell lines suitable for investigations of subtype-specific pathobiology, cancer stem cell biology, biomarkers and therapies, and provides a resource for discovery of new breast cancer genes.

Identification of PDE4D as a Proliferation Promoting Factor in Prostate Cancer Using a Sleeping Beauty Transposon-Based Somatic Mutagenesis Screen

Retroviral and transposon-based mutagenesis screens in mice have been useful for identifying candidate cancer genes for some tumor types. However, many of the organs that exhibit the highest cancer rates in humans, including the prostate, have not previously been amenable to these approaches. This study shows for the first time that the Sleeping Beauty transposon system can be used to identify candidate prostate cancer genes in mice. Somatic mobilization of a mutagenic transposon resulted in focal epithelial proliferation and hyperplasia in the prostate. Efficient methods were established to identify transposon insertion sites in these lesions, and analysis of transposon insertions identified candidate prostate cancer genes at common insertion sites, including Pde4d. PDE4D was also overexpressed in human prostate cancer patient samples and cell lines, and changes in PDE4D mRNA isoform expression were observed in human prostate cancers. Furthermore, knockdown of PDE4D reduced the growth and migration of prostate cancer cells in vitro, and knockdown of PDE4D reduced the growth and proliferation rate of prostate cancer xenografts in vivo. These data indicate that PDE4D functions as a proliferation promoting factor in prostate cancer, and the Sleeping Beauty transposon system is a useful tool for identifying candidate prostate cancer genes.

Preparing a re-sequencing DNA library of 2 cancer candidate genes using the ligation-by-amplification protocol by two PCR reactions

To meet the needs of large-scale genomic/genetic studies, the next-generation massively parallelized sequencing technologies provide high throughput, low cost and low labor-intensive sequencing service, with subsequent bioinformatic software and laboratory methods developed to expand their applications in various types of research. PCR-based genomic/genetic studies, which have significant usage in association studies like cancer research, haven't benefited much from those next-generation sequencing technologies, because the shortgun re-sequencing strategy used by such sequencing machines as the Illumina/Solexa Genome Analyzer may not be applied to direct re-sequencing of short-length target regions like those in PCR-based genomic/genetic studies. Although several methods have been proposed to solve this problem, including microarray-based genomic selections and selector-based technologies, they require advanced equipment and procedures which limit their applications in many laboratories. By contrast, we overcame such potential drawbacks by utilizing a ligation by amplification (LBA) protocol, a method using a pair of Universal Adapters to randomly ligate target regions in a two-step-PCR procedure, whose Long LBA products were easily fragmented and sequenced on the next-generation sequencing machine. In this concept-proven study, we chose the consensus coding sequences of two human cancer genes: BRCA1 and BRCA2 as target regions, specifically designed LBA primer pairs to amplify and randomly ligate them. 70 target sequences were successfully amplified and ligated into Long LBA products, which were then fragmented to construct DNA libraries for sequencing on both a conventional Sanger sequencer ABI 3730xl DNA Analyzer and the next-generation 'synthesis by sequencing technology' Illumina/Solexa Genome Analyzer. Bioinformatic analysis demonstrated the utility and efficiency (including the coverage and depth of each target sequence and the SNPs detection effectiveness) of using the LBA protocol in facilitating PCR-based re-sequencing and genetic-variant-detection studies on the next-generation sequencing machine, raising the prospect of various PCR-based genomic/genetic studies using this strategy.

Recurrent and nonrandom DNA copy number and chromosome alterations in Myc transgenic mouse model for hepatocellular carcinogenesis: implications for human disease

Mouse models for hepatocellular carcinoma (HCC) provide an experimental ground for dissecting the genetic and biological complexities of human liver cancer and contribute to our ability to gain insights into the relevance of candidate cancer genes. We examined, using spectral karyotyping (SKY) and array-based CGH (aCGH), seven cell lines derived from HCC spontaneously developed in transgenic Myc mice (Myc), and four cell lines established from tumors induced in nude mice by inoculation with the original Myc cells (nuMyc). All the cell lines exhibited gain of material from chromosomes 5, 6, 8, 10, 11, 15, and 19 and DNA copy-number loss from chromosomes 2, 4, 7, 9, 12, 14, and X. In addition, several recurrent chromosome reorganizations were found, including del(3), t(3;8), del(4), t(4;11), t(6;5), del(7), del(8), del(9), t(10;14), del(11), and del(16). Chromosome breakpoints underlying rearrangements clustered in the regions previously identified as important for the early stages of Myc-induced hepatocarcinogenesis. The results strongly suggest the importance of recurrent breakage and loss of chromosomes 4, 9, and 14 and gain of chromosomes 15 and 19 in mouse liver neoplasia. Genomic changes observed in Myc HCC cell lines are also recurrent in HCC developed in other transgenic mouse models, in mouse spontaneous HCC and derivative cell lines, and in preneoplastic liver lesions induced with chemical carcinogens. Overall, the present results document selective, nonrandom genomic changes involving chromosomal regions homologous to those implicated in human HCC.

Molecular Diagnostics for Identifying an Increased Likelihood of Recurrence of Breast Cancer

Existing prognostic factors based on histological and clinical parameters result in under‐treating some patients at high risk of recurrence and over‐treating others that may not benefit from chemotherapies. Current gene expression tests are based on gross tissue biopsies exhibiting cellular heterogeneity. Our goal was to develop a subset of genes (5‐15) based on UniGene (NCBI) analysis and published data using whole tissue, while exploiting larger gene sets derived from cancer cells collected by LCM (e.g., Ma et al., Breast Cancer Res Treat 82:S15, 2003). Applying the UniGene database, comparisons of molecular signatures reporting &gt;2500 sequences were reduced to 5‐15 gene subsets of candidate cancer and stromal cell genes. Each gene was validated by qPCR and correlated with clinical outcome. A molecular signature from cancer and stromal cell candidate genes predicted early breast cancer recurrence. A separate 13 gene signature in ER+ breast cancer correlated with disease‐free and overall survival. Gene candidates were analyzed on the Ziplex® Automated Workstation (Xceed Molecular) with a TipChip array. Our results suggest a molecular diagnostic test based on expression of gene subsets predicts clinical outcome. Supported in part by grants from Phi Beta Psi Charity Trust, University of Louisville's Office of Technology Transfer and Xceed Molecular Corp. SAA &amp; DAK are recipients of IPIBS Graduate Fellowships.

Genetic sequence variations of BRCA1-interacting genes AURKA, BAP1, BARD1 and DHX9 in French Canadian Families with high risk of breast cancer

Breast cancer is a heterogeneous disease displaying some degree of familial clustering. Highly penetrant breast cancer susceptibility genes represent approximately 20-25% of the familial aggregation of breast cancer. A significant proportion of this familial aggregation of breast cancer is thus yet to be explained by other breast cancer susceptibility genes. Given the high susceptibility conferred by the two major breast cancer predisposition genes, BRCA1 and BRCA2 and the implication of these genes in many key cellular processes, assessment of genes encoding BRCA1-interacting proteins as plausible breast cancer candidate genes is thus attractive. In this study, four genes encoding BRCA1-interacting proteins were analyzed in a cohort of 96 breast cancer individuals from high-risk non-BRCA1/BRCA2 French Canadian families. Although no deleterious truncating germline mutations or aberrant spliced mRNA species were identified, a total of 10, 4, 11 and 6 variants were found in the AURKA, BAP1, BARD1 and DHX9 genes, respectively. The allele frequency of each variant was further ascertained in a cohort of 98 healthy French Canadian unrelated women and a difference in allele frequency was observed for one BARD1 variant based on single-marker analysis. Haplotype estimation, haplotype blocks and tagging SNPs identification were then performed for each gene, providing a valuable tool for further searches of common disease-associated variants in these genes and therefore further analyses on these genes in larger cohorts is warranted in the search of low-to-moderate penetrance breast cancer susceptibility alleles.

Genome-Wide Association Studies and Aggressive Surgery Toward Individualized Prevention, and Improved Local Control and Overall Survival for Gastric Cancer

Prevention and improved treatment can dramatically improve the poor prognosis of gastric cancer. Two pivotal studies in biomedical sciences and clinical research for the identification of genetic-factors-based primary prevention and surgical treatment of stomach cancer have most recently been reported from Japan. What are the clinical implications of these studies? In the field of risk-stratification-based personalized prevention, a novel gastric cancer candidate gene has been identified. A new genome-wide association study (GWAS) published in the June issue of Nature Genetics has demonstrated that genetic variants in the common, lowpenetrance PSCA gene are associated with increased risk of sporadic diffuse-type gastric cancer. Could genetic testing for these risk variants, or other new variants which will be identified by GWAS in the future, be used in the clinic? In the field of treatment, the results of a phase III randomized trial comparing D2 gastrectomy alone or D2 plus para-aortic lymph nodal dissection (PAND) in patients with advanced resectable gastric cancer have been reported in the July 31 issue of NEJM. The overall 5-year survival rate was around 70%, similar in both groups. This excellent result has been achieved by surgery alone. Given the poor prognosis of the disease in the West, despite the addition of modern adjuvant treatment to surgery, what are the applications of these Japanese results in the USA and Europe? Worldwide, gastric cancer, despite its declining incidence, still remains an important health problem, being the fourth most common cancer and the second most common cause of death. There is wide geographical variation. The annual age-standardized incidence rate in Japan ranges from 50 to 80 per 100,000 among men and from 20 to 30 per 100,000 among women while in the USA the figures are about one-tenth of those: 5.9 per 100,000 for men and 2.5 per 100,000 for women. We need effective prevention, early detection, and treatment strategies to reduce morbidity and mortality of the disease.

Identification of new breast cancer candidate genes associated with stromal invasion.

Abstract Abstract #4036 Background: We recently reported our results of a study identifying a number of DNA copy number changes that differentiated low grade ductal carcinoma in situ (DCIS) lesions with and without associated stromal invasion. We now describe a series of validation experiments that support the role of four genes in early breast cancer progression, most of which were not previously implicated in mammary neoplasia.&amp;#x2028; Materials and Methods: DNA was extracted from microdissected paraffin sections of formalin fixed low grade DCIS lesions with an invasive component (n=25), as well as morphologically similar DCIS lesions without associated invasion within at least 5 years (n=20). The DNA was used for comparative PCR, with GAPDH as the reference gene. Specific primers were designed for fourteen candiate genes. Amplicon sizes ranged from 80 to 90 base pairs. Using log2 ratio cutoffs of +1.0 and -0.5, respectively, the frequency of copy number gains and losses was determined for each candidate gene. Additional validation studies employed genomic DNA samples from an independent cohort of 35 microdissected DCIS lesions with (n=17) and without (n=18) an associated invasive component.&amp;#x2028; Results: Our previous whole genome profiling study using high density BAC arrays revealed that DNA copy number changes on 1q, 3p, 5q, 6p, 9p, 10q, 11q, 12q, 16p, 16q, 18q, 20q, and 22q may occur at different rates in low grade DCIS lesions with and without associated stromal invasion. From the differentially amplified or deleted chromosomal sites, thus far we have selected fourteen candidate genes for validation by comparative PCR, utilizing aliquots of the same DNA samples that had previously been used for array CGH analysis. Four genes were confirmed to have differential copy number changes. NCOR2 (on 12q24.31), DYNLRB2 (on 16q23.2), CELSR1 and UPK3A (both on 22q13.31) were more frequently amplified in DCIS lesions that had no invasive component. Moreover, NCOR2 and DYNLRB2 showed more frequent copy number losses in DCIS lesions that had progressed to invasive carcinoma. For NCOR2, these observations held true in an independent cohort of microdissected low grade DCIS cases. The other ten candidate genes revealed significant frequencies of copy number changes that were not differentially distributed, however. TEME9, CALN1, and CENTA1 were amplified in 30-40% of all cases. IRF8, THAP7, and FIGNL1 were deleted in 20-50% of all cases. FBXO31, CDH13, IKZF1, and MAPK9 revealed both gains and losses in &amp;gt;20% of all cases.&amp;#x2028; Discussion: Our validation experiments have provided additional evidence for the role of several novel genes in early breast cancer progression. Four of the candidate genes we assayed so far may function as invasion suppressor genes. Ten additional genes showed significant copy number changes but did not discriminate DCIS lesions with and without the propensity to invade the stroma. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4036.

Exclusion of ABCB8 and ABCB10 as cancer candidate genes in acute myeloid leukemia

Acute myeloid leukemia (AML) exhibits great heterogeneity in manifestation, sensitivity to therapy and genetic basis of pathology. Numerous studies have implied that AML arises from the sequential accumulation of mutations at the level of the hematopoietic stem/progenitor cell, resulting in a disturbed balance of proliferation, differentiation and apoptosis of immature myeloid progenitors.1 To gain more insight into somatic mutations that affect biological pathways in tumorigenesis, a systematic mutation analysis of virtually all annotated human protein-coding genes in eleven breast and eleven colorectal cancers has been performed,2 leading to the identification of 280 genes as candidate cancer (CAN) genes. Importantly, several of the identified CAN genes are known to be mutated in hematological malignancies, including CBFA2T3 (ETO2), FBXW7, KRAS, NF1, PTEN, RUNX1T1 and TP53.1, 2 Several genes newly identified to be mutated in breast and colorectal cancers belong to the class of ATP-binding cassette (ABC) transporters,2 which are known to affect the response to therapy of AML and other cancers by conferring chemoresistance. Two of the ABC transporters discovered to harbor novel somatic mutations in breast cancer, the mitochondrial transporters ABCB8 and ABCB10, are highly expressed in both normal and leukemic hematopoietic stem cells.3 ABCB8 and ABCB10 are structurally closely related and are evolutionary highly conserved, suggesting a fundamental function in cellular physiology. Although their functions have not been completely elucidated, some clues regarding their biological roles can be postulated on the basis of homologues of ABCB10 in other species. Firstly, multi-drug resistance like 1 (MDL1), the ABCB10 homologue from Saccharomyces cerevisiae, regulates cellular responses to oxidative stress, such as reactive oxygen species (ROS).4 Reactive Oxygen Species signals stimulate proliferation and induce genetic instability in malignant transformed cells, conferring increased basal oxidative stress in cancer cells.5 ABCB10 might represent an important factor in mediating the response to oxidative stress in cancer cells. Secondly, the murine ABCB10 homologue, ABC-mitochondria erythroid (ABC-me), is a direct target of the transcription factor GATA1 and overexpression of murine ABCB10 enhances hemoglobin synthesis.6 Furthermore, a recent study identified ABCB10 to be essential for proliferation of human mammary epithelial cells.7 Thus, mutations in ABCB10 may cause resistance to oxidative stress, thereby contributing to cancer development. In this context, we pursued to investigate whether ABCB8 and ABCB10 are CAN genes in AML. To this end, the coding regions of ABCB8 and ABCB10 were screened by heteroduplex analysis (HA) using genomic DNA isolated from diagnostic bone marrow or peripheral blood samples from a panel of 94 AML patients who were referred to the Department of Hematology at the Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands, between 1991 and 2004. To identify potential mutations that affect splicing, the first 15 intronic nucleotides surrounding the intron–exon junctions were analyzed as well. Amplicons covering relevant regions were generated by a two-step polymerase chain reaction (PCR) followed by HA (for details see Supplementary Methods and Supplementary Figure S1 and 2; for primer sequences see Supplementary Table S1). Fragments exhibiting altered peak patterns in HA were subsequently sequenced to identify the nucleotide changes. Exons with multiple known single nucleotide polymorphisms (SNPs) were subjected to direct sequencing. A limitation of the HA method is that homozygous mutations or SNPs will not be detected. As all reported somatic mutations in ABCB8 and ABCB10 are heterogeneous,2 we reasoned that most point mutations in the cohort would be detected by HA.

Mutational profiling of cancer candidate genes in glioblastoma, melanoma and pancreatic carcinoma reveals a snapshot of their genomic landscapes

A recent systematic analysis of 18.191 well annotated coding sequences (RefSeq) in breast and colorectal cancers has led to the identification of somatic mutations in 1.718 genes (Wood et al., 2007). Based on statistical parameters 280 of these have been denominated candidate cancer (CAN) genes. This analysis has provided an interesting snapshot of the landscape of tumor genomes by showing that they contain a few frequently mutated genes (denominated 'mountains'). On the contrary, the large majority of CAN genes are altered at low frequency (designated 'hills'). Whether 'hill' type CAN genes are tumor specific is largely unknown. To address this question we evaluated the mutational profiles of 27 'hill' CAN genes in glioblastoma, melanoma and pancreatic carcinoma by sequencing the exons previously found mutated by Wood and colleagues. Only 4 of the breast/colorectal 'hill' type CAN genes (SMAD4, MYO18B, NAV3 and MMP2) were also mutated in melanoma and pancreatic carcinoma, while none was altered in glioblastoma. These results suggest that 'hill' type CAN genes are not frequently shared by different tumor types and that their mutation patterns are tissue specific. Tumor-specific genome wide mutational profiling will be required to identify 'hill' type CAN genes that characterize the genomic landscapes of each cancer lineage.