Ramaswamy Anbazhagan

A genomic screen for genes upregulated by demethylation and histone deacetylase inhibition in human colorectal cancer

Aberrant hypermethylation of gene promoters is a major mechanism associated with inactivation of tumor-suppressor genes in cancer. We previously showed this transcriptional silencing to be mediated by both methylation and histone deacetylase activity, with methylation being dominant. Here, we have used cDNA microarray analysis to screen for genes that are epigenetically silenced in human colorectal cancer. By screening over 10,000 genes, we show that our approach can identify a substantial number of genes with promoter hypermethylation in a given cancer; these are distinct from genes with unmethylated promoters, for which increased expression is produced by histone deacetylase inhibition alone. Many of the hypermethylated genes we identified have high potential for roles in tumorigenesis by virtue of their predicted function and chromosome position.We also identified a group of genes that are preferentially hypermethylated in colorectal cancer and gastric cancer. One of these genes, SFRP1, belongs to a gene family; we show that hypermethylation of four genes in this family occurs very frequently in colorectal cancer, providing for (i) a unique potential mechanism for loss of tumor-suppressor gene function and (ii) construction of a molecular marker panel that could detect virtually all colorectal cancer.

A Cross-Study Comparison of Gene Expression Studies for the Molecular Classification of Lung Cancer

Purpose: Recent studies sought to refine lung cancer classification using gene expression microarrays. We evaluate the extent to which these studies agree and whether results can be integrated. Experimental Design: We developed a practical analysis plan for cross-study comparison, validation, and integration of cancer molecular classification studies using public data. We evaluated genes for cross-platform consistency of expression patterns, using integrative correlations, which quantify cross-study reproducibility without relying on direct assimilation of expression measurements across platforms. We then compared associations of gene expression levels to differential diagnosis of squamous cell carcinoma versus adenocarcinoma via reproducibility of the gene-specific t statistics and to survival via reproducibility of Cox coefficients. Results: Integrative correlation analysis revealed a large proportion of genes in which the patterns agreed across studies more than would be expected by chance. Correlation of t statistics for diagnosis of squamous cell carcinoma versus adenocarcinoma is high (0.85) and increases (0.925) when using only the most consistent genes identified by integrative correlation. Correlations of Cox coefficients ranged from 0.13 to 0.31 (0.33–0.49 with genes selected for consistency). Although we find genes that are significant in multiple studies but show discordant effects, their number is approximately that expected by chance. We report genes that are reproducible by integrative analysis, significant in all studies, and concordant in effect. Conclusions: Cross-study comparison revealed significant, albeit incomplete, agreement of gene expression patterns related to lung cancer biology and identified genes that reproducibly predict outcomes. This analysis approach is broadly applicable to cross-study comparisons of gene expression profiling projects.

A statistical framework for expression-based molecular classification in cancer

Genome-wide measurement of gene expression is a promising approach to the identification of subclasses of cancer that are currently not differentiable, but potentially biologically heterogeneous. This type of molecular classification gives hope for highly individualized and more effective prognosis and treatment of cancer. Statistically, the analysis of gene expression data from unclassified tumours is a complex hypothesis-generating activity, involving data exploration, modelling and expert elicitation. We propose a modelling framework that can be used to inform and organize the development of exploratory tools for classification. Our framework uses latent categories to provide both a statistical definition of differential expression and a precise, experiment-independent, definition of a molecular profile. It also generates natural similarity measures for traditional clustering and gives probabilistic statements about the assignment of tumours to molecular profiles. Copyright 2002 Royal Statistical Society.

Nuclear genes involved in mitochondria-to-nucleus communication in breast cancer cells

BackgroundThe interaction of nuclear and mitochondrial genes is an essential feature in maintenance of normal cellular function. Of 82 structural subunits that make up the oxidative phosphorylation system in the mitochondria, mitochondrial DNA (mtDNA) encodes 13 subunits and rest of the subunits are encoded by nuclear DNA. Mutations in mitochondrial genes encoding the 13 subunits have been reported in a variety of cancers. However, little is known about the nuclear response to impairment of mitochondrial function in human cells.ResultsWe isolated a Rho0 (devoid of mtDNA) derivative of a breast cancer cell line. Our study suggests that depletion of mtDNA results in oxidative stress, causing increased lipid peroxidation in breast cancer cells. Using a cDNA microarray we compared differences in the nuclear gene expression profile between a breast cancer cell line (parental Rho+) and its Rho0 derivative impaired in mitochondrial function. Expression of several nuclear genes involved in cell signaling, cell architecture, energy metabolism, cell growth, apoptosis including general transcription factor TFIIH, v-maf, AML1, was induced in Rho0 cells. Expression of several genes was also down regulated. These include phospholipase C, agouti related protein, PKC gamma, protein tyrosine phosphatase C, phosphodiestarase 1A (cell signaling), PIBF1, cytochrome p450, (metabolism) and cyclin dependent kinase inhibitor p19, and GAP43 (cell growth and differentiation).ConclusionsMitochondrial impairment in breast cancer cells results in altered expression of nuclear genes involved in signaling, cellular architecture, metabolism, cell growth and differentiation, and apoptosis. These genes may mediate the cross talk between mitochondria and the nucleus.

Mucinous cancers have fewer genomic alterations than more common classes of breast cancer.

Mucinous cancers of the breast are distinguished histologically by their abundant pools of mucin and low degree of nuclear pleomorphism. Relative to the more common breast cancers of no distinctive type (ductal carcinoma), mucinous cancers have a relatively favorable prognosis. In a study of chromosomal changes in mucinous cancers, we evaluated the extent of loss of heterozygosity (LOH) at chromosomal regions commonly deleted in usual infiltrating ductal carcinoma, including markers on chromosomal arms 1p, 1q, 3p, 6q, 8p, 9p, 11p, 11q, 13q, 16q, 17p, and 17q. Remarkably, we found an average frequency of LOH of only 1.9 of these 12 chromosomal arms in 18 cases of mucinous carcinoma, compared to an average frequency of LOH of 6.4 of these same chromosomal arms in cases of infiltrating ductal cancer. In three of the 18 cases of mucinous carcinoma studied, including one case with regional lymph node metastases, no LOH was seen at any of the 12 chromosomal regions studied. We considered the possibility of other chromosomal loci being more commonly affected in mucinous cancers and conducted comparative genomic hybridization on six of the cases. These studies demonstrated a low overall frequency of genomic copy number changes (mean of 3.1 changes per case) and failed to reveal any other chromosomal locus with frequent losses that had not been evaluated by microsatellite analysis. Together, these data indicate that mucinous cancers of the breast do not have the extensive genomic alterations that are typically found in more common variants of breast cancer. Thus, mucinous cancers most likely have less genetic instability than most other forms of breast cancer and the molecular pathogenesis of this form of breast cancer is likely to be substantially different than that of usual ductal breast cancer.

Functional Genomics, Gene Arrays, and the Future of Pathology

The human genome project has attracted a great deal of attention in recent years among the general public as well as the scientific community. Although it is likely to be a number of years before many of the expected benefits of the genomics revolution are realized, the impact of these scientific breakthroughs on diagnostic pathology is likely to become apparent relatively quickly. In particular, gene array technology, which allows gene expression measurements of thousands of genes in parallel, provides a powerful tool for pathologists seeking new markers for diagnosis. Several recent studies demonstrate how the gene array approach can not only recognize markers for known categories of neoplasia but also lead to recognition of different categories not previously appreciated. Although this approach shows great potential, the successful application of gene arrays to diagnostic problems will require thoughtful interpretation, just as immunochemical technologies require careful planning and analysis.

Absence of intragenic mismatch mutations in small cell lung cancers with microsatellite instability

Small deletions or expansions of the simple nucleotide repeats of microsatellites ( i.e., microsatellite instability [MI]) are well described in cancers from patients with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome and result from mutations of mismatch repair enzyme genes such as hMSH2, hMLH1, hPMS1, hPMS2or GTBP (reviewed by Speicher, 1995). The potential significance of mismatch mutations in carcinogenesis became apparent when HNPCC cancers were also found to have frequent frameshift mutations of simple nucleotide repeats within the coding regions of several key genes involved in the regulation of cell growth or apoptosis. These intragenic repeats include a poly (A) 10 tract in the transforming growth factorb type II receptor ( RII) gene (Parsonset al.,1995), a poly (G) 8 tract in the insulin-like growth factor type II receptor ( IGFIIR) gene (Souza et al.,1996), a poly (G)8 tract in BAX (Rampinoet al.,1997) and a poly (AGC) 13 tract in theE2F-4gene (Souzaet al.,1997). Although MI is considered to be most characteristic of cancers associated with the hereditary non-polyposis colorectal cancer (HNPCC) syndrome, MI has also been described in a variety of sporadic human cancers (Speicher, 1995). For example, we have previously recognized frequent microsatellite instability in primary small cell lung cancer (SCLC), with 15 of 33 cancers displaying alterations of (CA) n repeats (Merloet al., 1994). MI in these cases of SCLC affected only 4 –44% of all tested microsatellite loci, representing a distinctly lower frequency than that found in HNPCC cancers with MI. However, the frequency of MI in at least a subset of SCLCs appears to be greater than that for many other sporadic cancers (including non-small cell lung cancer [NSCLC]), both in the number of identified cases with MI and in the frequency of alleles affected (Abeet al.,1996; Maoet al.,1994). The significance of mismatch-induced frameshift mutations in the pathogenesis of lung cancers, including SCLCs, is unknown. Previous studies of frameshift mutations in lung cancer have been limited to evaluation of the RII gene. In one study, only 1 of 203 NSCLCs was found to have MI and this tumor did not have a mutation of the RII gene (Abeet al.,1996). More recently, a set of 33 sporadic lung cancers of unspecified histology from individuals with rareH-ras1alleles (which is frequently associated with MI [Ryberg et al., 1995]) were evaluated and no mutations of RII were found (Takenoshita et al.,1997). These previous investigations have not addressed the issues of frameshift mutations specifically in SCLC, which has a greater frequency of MI than NSCLC. Furthermore, these studies have not investigated possible frameshift mutations in g nes other thanRII and it is well recognized that different t pes of human cancers frequently have a diverse array of genetic alterations. For example, RER1 endometrial cancers frequently have mutations of IGFRII and only rarely mutations of RII, whereasRER1 colorectal cancers frequently have mutations ofRII and only rarely mutations of IGFRII (Myeroff et al.,1995). The purpose of the present study, therefore, was to evaluate the 4 genes found to be commonly affected by frameshift mutations in HNPCC tumors (the RII, IGFIIR, BAX andE2F-4 genes) in cases of SCLC with MI. Adequate quantities of DNA were available from 13 of the 15 cases of SCLC previously reported to have MI. To test for mutations, polymerase chain r action (PCR) amplification of tumor DNA was performed using the primers and conditions specified in Table I. Reactions were radiolabelled using 0.2μCi of [32P]CTP in each 10μl reaction mixture and products were electrophoresed on 6% polyacrylamide denaturing gels and visualized by autoradiography. Frameshift mutation was determined by visualization of ands in tumor DNA that were not present in corresponding normal. We examined all repetitive sequences in the RII, IGFIIR, BAX andE2F-4genes for each of the 13 cases of SCLC, and found no evidence of frameshift mutations in any of these cases. Figure 1 demonstrates representative amplifications of repeats segments of the RII andIGFIIR genes. Our results demonstrate that although frameshift mutations of microsatellites are relatively common in a subset of SCLC, frameshift mutations of genes commonly mutated in HNPCC cancers ( i.e., RII, IGFIIR, BAX andE2F-4) are uncommon in this type of cancer. Mutations of these four genes examined may