Cindy Postma

MiR-17-92 cluster is associated with 13q gain and c-myc expression during colorectal adenoma to adenocarcinoma progression

Background:MicroRNAs are small non-coding RNA molecules, which regulate central mechanisms of tumorigenesis. In colorectal tumours, the combination of gain of 8q and 13q is one of the major factors associated with colorectal adenoma to adenocarcinoma progression. Functional studies on the miR-17-92 cluster localised on 13q31 have shown that its transcription is activated by c-myc, located on 8q, and that it has oncogenic activities. We investigated the contribution of the miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression.Methods:Expression levels of the miR-17-92 cluster were determined in 55 colorectal tumours and in 10 controls by real-time RT–PCR. Messenger RNA c-myc expression was also determined by real-time RT–PCR in 48 tumours with array comparative genomic hybridisation (aCGH) data available.Results:From the six members of the miR-17-92 cluster, all except miR-18a, showed significant increased expression in colorectal tumours with miR-17-92 locus gain compared with tumours without miR-17-92 locus gain. Unsupervised cluster analysis clustered the tumours based on the presence of miR-17-92 locus gain. Significant correlation between the expression of c-myc and the six miRNAs was also found.Conclusion:Increased expression of miR-17-92 cluster during colorectal adenoma to adenocarcinoma progression is associated to DNA copy number gain of miR17-92 locus on 13q31 and c-myc expression.

Multiple putative oncogenes at the chromosome 20q amplicon contribute to colorectal adenoma to carcinoma progression

Objective: This study aimed to identify the oncogenes at 20q involved in colorectal adenoma to carcinoma progression by measuring the effect of 20q gain on mRNA expression of genes in this amplicon. Methods: Segmentation of DNA copy number changes on 20q was performed by array CGH (comparative genomic hybridisation) in 34 non-progressed colorectal adenomas, 41 progressed adenomas (ie, adenomas that present a focus of cancer) and 33 adenocarcinomas. Moreover, a robust analysis of altered expression of genes in these segments was performed by microarray analysis in 37 adenomas and 31 adenocarcinomas. Protein expression was evaluated by immunohistochemistry on tissue microarrays. Results: The genes C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5, mapping at 20q, were significantly overexpressed in carcinomas compared with adenomas as a consequence of copy number gain of 20q. Conclusion: This approach revealed C20orf24, AURKA, RNPC1, TH1L, ADRM1, C20orf20 and TCFL5 genes to be important in chromosomal instability-related adenoma to carcinoma progression. These genes therefore may serve as highly specific biomarkers for colorectal cancer with potential clinical applications.

Genomic Alterations in Primary Gastric Adenocarcinomas Correlate with Clinicopathological Characteristics and Survival

Background & aims: Pathogenesis of gastric cancer is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. In order to investigate the patterns of chromosomal aberrations in gastric carcinomas, we performed genome‐wide microarray based comparative genomic hybridisation (microarray CGH). With this recently developed technique chromosomal aberrations can be studied with high resolution and sensitivity. Methods: Array CGH was applied to a series of 35 gastric adenocarcinomas using a genome‐wide scanning array with 2275 BAC and P1 clones spotted in triplicate. Each clone contains at least one STS for linkage to the sequence of the human genome. These arrays provide an average resolution of 1.4 Mb across the genome. DNA copy number changes were correlated with clinicopathological tumour characteristics as well as survival. Results: All thirty‐five cancers showed chromosomal aberrations and 16 of the 35 tumours showed one or more amplifications. The most frequent aberrations are gains of 8q24.2, 8q24.1, 20q13.12, 20q13.2, 7p11.2, 1q32.3, 8p23.1–p23.3, losses of 5q14.1, 18q22.1, 19p13.12–p13.3, 9p21.3–p24.3, 17p13.1–p13.3, 13q31.1, 16q22.1, 21q21.3, and amplifications of 7q21–q22, and 12q14.1–q21.1. These aberrations were correlated to clinicopathological characteristics and survival. Gain of 1q32.3 was significantly correlated with lymph node status (p=0.007). Tumours with loss of 18q22.1, as well as tumours with amplifications were associated with poor survival (p=0.02, both). Conclusions: Microarray CGH has revealed several chromosomal regions that have not been described before in gastric cancer at this frequency and resolution, such as amplification of at 7q21–q22 and 12q14.1–q21.1, as well gains at 1q32.3, 7p11.2, and losses at 13q13.1. Interestingly, gain of 1q32.3 and loss of 18q22.1 are associated with a bad prognosis indicating that these regions could harbour gene(s) that may determine aggressive tumour behaviour and poor clinical outcome.

Genomic profiling of gastric cancer predicts lymph node status and survival

Gastric carcinogenesis is driven by an accumulation of genetic changes that to a large extent occur at the chromosomal level. We analysed the patterns of chromosomal instability in 35 gastric carcinomas and their clinical correlations. With microarray competitive genomic hybridization, genomewide chromosomal copy number changes can be studied with high resolution and sensitivity. A genomewide scanning array with 2275 BAC and P1 clones spotted in triplicate was used. This array provided an average resolution of 1.4 Mb across the genome. Patterns of chromosomal aberrations were analysed by hierarchical cluster analysis of the normalized log2 tumour to normal fluorescence ratios of all clones, and cluster membership was correlated to clinicopathological data including survival. Hierarchical cluster analysis revealed three groups with different genomic profiles that correlated significantly with lymph node status (P=0.02). Moreover, gastric cancer cases from cluster 3 showed a significantly better prognosis than those from clusters 1 and 2 (P=0.02). Genomic profiling of gastric adenocarcinomas based on microarray analysis of chromosomal copy number changes predicted lymph node status and survival. The possibility to discriminate between patients with a high risk of lymph node metastasis could clinically be helpful for selecting patients for extended lymph node resection.

LOH of PTPRJ occurs early in colorectal cancer and is associated with chromosomal loss of 18q12–21

Recently, the gene PTPRJ (protein tyrosine phosphatase receptor type J) was identified as the candidate gene for the mouse colon cancer susceptibility locus Scc1. Its human homologue PTPRJ is frequently deleted in several cancer types, including colorectal cancer. To elucidate the role of PTPRJ loss in different stages of colorectal cancer and in its pathways of progression, we expanded the previously published comparative genomic hybridization results with novel data on loss of heterozygosity (LOH) at the PTPRJ locus. We identified a strong association between the LOH of PTPRJ and the loss of chromosomal region 18q12–21 (P=0.009). This observation is specific for progressed colorectal adenomas, suggesting that an interaction between LOH of PTPRJ and loss of 18q12–21 may be involved in the development of a more progressed form of adenomas.

Identification of key genes for carcinogenic pathways associated with colorectal adenoma-to-carcinoma progression

Colorectal adenomas form a biologically and clinically distinct intermediate stage in development of colorectal cancer (CRC) from normal colon epithelium. Only 5% of adenomas progress into adenocarcinomas, indicating that malignant transformation requires other biological alterations than those involved in adenoma formation. The present study aimed to explore which cancer-related biological processes are affected during colorectal adenoma-to-carcinoma progression and to identify key genes within these pathways that can serve as tumor markers for malignant transformation. The activity of 12 cancer-related biological processes was compared between 37 colorectal adenomas and 31 adenocarcinomas, using the pathway analysis tool Gene Set Enrichment Analysis. Expression of six gene sets was significantly increased in CRCs compared to adenomas, representing chromosomal instability, proliferation, differentiation, invasion, stroma activation, and angiogenesis. In addition, 18 key genes were identified for these processes based on their significantly increased expression levels. For AURKA and PDGFRB, increased mRNA expression levels were verified at the protein level by immunohistochemical analysis of a series of adenomas and CRCs. This study revealed cancer-related biological processes whose activities are increased during malignant transformation and identified key genes which may be used as tumor markers to improve molecular characterization of colorectal tumors.

Promoter methylation precedes chromosomal alterations in colorectal cancer development.

Background: Colorectal cancers are characterized by genetic and epigenetic alterations. This study aimed to explore the timing of promoter methylation and relationship with mutations and chromosomal alterations in colorectal carcinogenesis. Methods: In a series of 47 nonprogressed adenomas, 41 progressed adenomas (malignant polyps), 38 colorectal carcinomas and 18 paired normal tissues, we evaluated promoter methylation status of hMLH1, O6MGMT, APC, p14ARF, p16INK4A, RASSF1A, GATA-4, GATA-5, and CHFR using methylation-specific PCR. Mutation status of TP53, APC and KRAS were studied by p53 immunohistochemistry and sequencing of the APC and KRAS mutation cluster regions. Chromosomal alterations were evaluated by comparative genomic hybridization. Results: Our data demonstrate that nonprogressed adenomas, progressed adenomas and carcinomas show similar frequencies of promoter methylation for the majority of the genes. Normal tissues showed significantly lower frequencies of promoter methylation of APC, p16INK4A, GATA-4, and GATA-5 (P-values: 0.02, 0.02, 1.1×10−5 and 0.008 respectively). P53 immunopositivity and chromosomal abnormalities occur predominantly in carcinomas (P values: 1.1×10−5 and 4.1×10−10). Conclusions: Since promoter methylation was already present in nonprogressed adenomas without chromosomal alterations, we conclude that promoter methylation can be regarded as an early event preceding TP53 mutation and chromosomal abnormalities in colorectal cancer development.

Colorectal adenoma to carcinoma progression is accompanied by changes in gene expression associated with ageing, chromosomal instability, and fatty acid metabolism

BackgroundColorectal cancer develops in a multi-step manner from normal epithelium, through a pre-malignant lesion (so-called adenoma), into a malignant lesion (carcinoma), which invades surrounding tissues and eventually can spread systemically (metastasis). It is estimated that only about 5% of adenomas do progress to a carcinoma.AimThe present study aimed to unravel the biology of adenoma to carcinoma progression by mRNA expression profiling, and to identify candidate biomarkers for adenomas that are truly at high risk of progression.MethodsGenome-wide mRNA expression profiles were obtained from a series of 37 colorectal adenomas and 31 colorectal carcinomas using oligonucleotide microarrays. Differentially expressed genes were validated in an independent colorectal gene expression data set. Gene Set Enrichment Analysis (GSEA) was used to identify altered expression of sets of genes associated with specific biological processes, in order to better understand the biology of colorectal adenoma to carcinoma progression.ResultsmRNA expression of 248 genes was significantly different, of which 96 were upregulated and 152 downregulated in carcinomas compared to adenomas. Classification of adenomas and carcinomas using the expression of these genes showed to be very accurate, also when tested in an independent expression data set. Gene-sets associated with ageing (which is related to senescence) and chromosomal instability were upregulated, and a gene-set associated with fatty acid metabolism was downregulated in carcinomas compared to adenomas. Moreover, gene-sets associated with chromosomal location revealed chromosome 4q22 loss and chromosome 20q gain of gene-set expression as being relevant in this progression.Concluding remarkThese data are consistent with the notion that adenomas and carcinomas are distinct biological entities. Disruption of specific biological processes like senescence (ageing), maintenance of chromosomal instability and altered metabolism, are key factors in the progression from adenoma to carcinoma.

Genome wide array comparative genomic hybridisation analysis of premalignant lesions of the stomach

Background: Gastric cancer is one of the most frequent malignancies in the world, ranking fifth in the Netherlands as a cause of cancer death. Surgery is the only curative treatment for advanced cases, but results of gastrectomy largely depend on the stage of the disease. A better understanding of the mechanisms of progression from a preneoplastic condition through intraepithelial neoplasia to invasive cancer may provide information relevant to designing focused prevention strategies. Methods: Because the pattern of chromosomal aberrations in precursors of gastric cancer is unclear, 11 gastric polyps with intraepithelial neoplasia (three hyperplastic polyps and eight adenomas) were analysed by microarray comparative genomic hybridisation to study chromosomal instability in precursors of gastric cancer. Results: Chromosomal aberrations were detected in all specimens. Adenomas showed no more chromosomal aberrations than did the hyperplastic polyps. The most frequent aberrations were gain of 7q36 and 20q12, and loss of 5q14–q21 in the adenomas, and loss of 15q11–14, 1p21–31, and 21q11–21.2 in the hyperplastic polyps. The most frequent chromosomal aberration in common to both types was loss of 9p21.3. Conclusion: Hyperplastic polyps showed many chromosomal aberrations, confirming that neoplastic transformation can occur in these lesions. These observations are consistent with the existence of two morphologically and genetically distinct pathways to gastric cancer—the hyperplastic polyp pathway and the (intestinal type) adenoma pathway. The relative contribution of each to gastric carcinogenesis in general, and how they compare to patterns of chromosomal aberrations in the more prevalent flat foci of intraepithelial neoplasia remain to be determined.

Intratumor Heterogeneity of K-Ras and p53 Mutations among Human Colorectal Adenomas Containing Early Cancer

The molecular pathways and the timing of genetic events during human colorectal carcinogenesis are still not fully understood. We have addressed the intratumor heterogeneity of the mutational status of the k-ras oncogene and of the p53 oncosuppressor gene during the adenoma-carcinoma sequence by investigating 26 human colorectal adenomas containing early cancer. An intratumor comparative analysis was obtained among the adenomatous and carcinomatous component pairs. Additionally, we have analyzed 17 adenomas having cancer in the near vicinity. The adenomatous components of the adenomas containing early cancer and the adenomas having cancer in the near vicinity had comparable frequencies for k-ras mutations (28 and 47%) but different for p53 mutations (52 and 7%, p-value = 0.01). Interestingly, the adenomatous and carcinomatous components of the adenomas containing early cancer were rarely heterogeneous for the k-ras mutational status (only in 13% of the cases) but were characterized by heterogeneity of the p53 status in 59% of the cases (p-value < 0.01). In addition, the mutations of p53 for the adenomatous components of the adenomas containing early cancer were statistically significantly associated with severe dysplasia (p-value = 0.01). Intratumor homogeneity of k-ras status during the human colorectal adenoma-carcinoma sequence suggests that the role of k-ras is more related to tumor initiation than to tumor progression. On the contrary, intratumor heterogeneity of p53 mutations indicates that the type of the p53 mutations may also be relevant for selection and expansion of new subclones leading to tumor progression.