Beatriz Carvalho

GATA4 and GATA5 are Potential Tumor Suppressors and Biomarkers in Colorectal Cancer

Purpose: The transcription factors GATA4 and GATA5 are involved in gastrointestinal development and are inactivated by promoter hypermethylation in colorectal cancer. Here, we evaluated GATA4/5 promoter methylation as potential biomarkers for noninvasive colorectal cancer detection, and investigated the role of GATA4/5 in colorectal cancer. Experimental Design: Promoter methylation of GATA4/5 was analyzed in colorectal tissue and fecal DNA from colorectal cancer patients and healthy controls using methylation-specific PCR. The potential function of GATA4/5 as tumor suppressors was studied by inducing GATA4/5 overexpression in human colorectal cancer cell lines. Results:GATA4/5 methylation was observed in 70% (63/90) and 79% (61/77) of colorectal carcinomas, respectively, and was independent of clinicopathologic features. Methylation frequencies in normal colon tissues from noncancerous controls were 6% (5 of 88, GATA4; P < 0.001) and 13% (13 of 100, GATA5; P < 0.001). GATA4/5 overexpression suppressed colony formation (P < 0.005), proliferation (P < 0.001), migration (P < 0.05), invasion (P < 0.05), and anchorage-independent growth (P < 0.0001) of colorectal cancer cells. Examination of GATA4 methylation in fecal DNA from two independent series of colorectal cancer patients and controls yielded a sensitivity of 71% [95% confidence interval (95% CI), 55-88%] and specificity of 84% (95% CI, 74–95%) for colorectal cancer detection in the training set, and a sensitivity of 51% (95% CI, 37–65%) and specificity of 93% (95% CI, 84-100%) in the validation set. Conclusions: Methylation of GATA4/5 is a common and specific event in colorectal carcinomas, and GATA4/5 exhibit tumor suppressive effects in colorectal cancer cells in vitro. GATA4 methylation in fecal DNA may be of interest for colorectal cancer detection.

Lack of microRNA-101 causes E-cadherin functional deregulation through EZH2 up-regulation in intestinal gastric cancer

E‐cadherin expression disruption is commonly observed in metastatic epithelial cancers and is a crucial step in gastric cancer (GC) initiation and progression. As aberrant expression of microRNAs often perturb the normal expression/function of pivotal cancer‐related genes, we characterized and dissected a pathway that causes E‐cadherin dysfunction via loss of microRNA‐101 and up‐regulation of EZH2 expression in GC. MicroRNA microarray expression profiling and array‐CGH were used to reinforce miR‐101 involvement in GC. By using quantitative real‐time PCR and quantitative SNaPshot genomic PCR, we confirmed that miR‐101 was significantly down‐regulated in GC (p < 0.0089) in comparison with normal gastric mucosas and, at least in 65% of the GC cases analysed, this down‐regulation was caused by deletions and/or microdeletions at miR‐101 genomic loci. Moreover, around 40% of cases showing miR‐101 down‐regulation displayed concomitant EZH2 over‐expression (at the RNA and protein levels), which, in turn, was associated with loss/aberrant expression of E‐cadherin. Interestingly, this occurred preferentially in intestinal‐type GCs, retaining allele(s) untargeted by classical CDH1‐inactivating mechanisms. We also demonstrated that miR‐101 gain of function or direct inhibition of EZH2 in Kato III GC cells led to a strong depletion of endogenous EZH2 and consequent rescue of E‐cadherin membranous localization, mimicking results obtained in clinical GC samples. In conclusion, we show that deletions and/or microdeletions at both miR‐101 genomic loci cause mature miR‐101 down‐regulation, subsequent EZH2 over‐expression and E‐cadherin dysfunction, specifically in intestinal‐type GC. Copyright

Somatic Mutations and Deletions of the E-Cadherin Gene Predict Poor Survival of Patients With Gastric Cancer

PURPOSE The prognosis of gastric cancer (GC) is poor, and the molecular pathogenesis players are vastly unknown. Surgery remains the primary option in GC treatment. The aim of this study was to investigate the impact of somatic CDH1 alterations in prognosis and survival of patients with GC. PATIENTS AND METHODS A series of patients with sporadic and familial GC (diffuse and intestinal; n = 246) were analyzed for somatic CDH1 mutations, promoter hypermethylation, and loss of heterozygosity (LOH) by polymerase chain reaction sequencing. E-cadherin protein expression was determined by immunohistochemistry. Associations between molecular, clinicopathologic, and survival data were analyzed. RESULTS CDH1 somatic alterations were found in approximately 30% of all patients with GC. Both histologic types of sporadic GC displayed LOH in 7.5%, mutations in 1.7%, and hypermethylation in 18.4% of patients. Primary tumors from hereditary diffuse GC, lacking germline CDH1 alterations, showed exclusively CDH1 promoter hypermethylation in 50% of patients. Familial intestinal GC (FIGC) tumors showed LOH in 9.4% and hypermethylation in 17.0%. CDH1 alterations did not associate with a particular pattern of E-cadherin expression. Importantly, the worst patient survival rate among all GCs analyzed was seen in patients with tumors carrying CDH1 structural alterations, preferentially those belonging to FIGC families. CONCLUSION CDH1 somatic alterations exist in all clinical settings and histotypes of GC and associate with different survival rates. Their screening at GC diagnosis may predict patient prognosis and is likely to improve management of patients with this disease.

Promoter DNA methylation of Oncostatin M receptor-β as a novel diagnostic and therapeutic marker in colon cancer

In addition to genetic changes, the occurrence of epigenetic alterations is associated with accumulation of both genetic and epigenetic events that promote the development and progression of human cancer. Previously, we reported a set of candidate genes that comprise part of the emerging “cancer methylome”. In the present study, we first tested 23 candidate genes for promoter methylation in a small number of primary colon tumor tissues and controls. Based on these results, we then examined the methylation frequency of Oncostatin M receptor-β (OSMR) in a larger number of tissue and stool DNA samples collected from colon cancer patients and controls. We found that OSMR was frequently methylated in primary colon cancer tissues (80%, 80/100), but not in normal tissues (4%, 4/100). Methylation of OSMR was also detected in stool DNA from colorectal cancer patients (38%, 26/69) (cut-off in TaqMan-MSP, 4). Detection of other methylated markers in stool DNA improved sensitivity with little effect on specificity. Promoter methylation mediated silencing of OSMR in cell lines, and CRC cells with low OSMR expression were resistant to growth inhibition by Oncostatin M. Our data provide a biologic rationale for silencing of OSMR in colon cancer progression and highlight a new therapeutic target in this disease. Moreover, detection and quantification of OSMR promoter methylation in fecal DNA is a highly specific diagnostic biomarker for CRC.

A novel classification of colorectal tumors based on microsatellite instability, the CpG island methylator phenotype and chromosomal instability: implications for prognosis

BACKGROUND We studied the overlap between the major (epi)genomic events microsatellite instability (MSI), the CpG island methylator phenotype (CIMP) and chromosomal instability (CIN) in colorectal cancer (CRC), and whether specific (epi)genotypes were associated with CRC-related deaths. PATIENTS AND METHODS Molecular analyses using tumor DNA were successful in 509 CRC cases identified within the Netherlands Cohort Study in the period 1989-1993. Follow-up for the vital status until May 2005 was 100%. RESULTS MSI (12.6%), CIMP-only (5.3%), CIMP + CIN (13.4%), CIN-only (58.2%) and triple-negative tumors (10.6%) differed significantly regarding tumor localization, differentiation grade, initial adjuvant therapy (AT) use and genetic characteristics (P ≤ 0.03). CIMP-only, CIMP + CIN and triple-negative tumors, compared with CIN-only tumors, were significantly associated with a 3.67, 2.44 and 3.78-fold risk of CRC-related deaths after 2-year follow-up (95% confidence intervals, CIs, 1.70-7.91, 1.35-4.41 and 1.97-7.25, respectively), but not after late follow-up. MSI tumors were borderline significantly associated with a 0.40-fold risk of CRC-related deaths after late follow-up (95% CI 0.15-1.03). CONCLUSION(S) This is the first study to show that specific (epi)genotypes may hold a differential prognostic value that may vary over time. Although no specific treatment data were available, an explanation for the differential findings over time might be that (epi)genotypes modify therapy response.

Cross-platform array comparative genomic hybridization meta-analysis separates hematopoietic and mesenchymal from epithelial tumors

A series of studies have been published that evaluate the chromosomal copy number changes of different tumor classes using array comparative genomic hybridization (array CGH); however, the chromosomal aberrations that distinguish the different tumor classes have not been fully characterized. Therefore, we performed a meta-analysis of different array CGH data sets in an attempt to classify samples tested across different platforms. As opposed to RNA expression, a common reference is used in dual channel CGH arrays: normal human DNA, theoretically facilitating cross-platform analysis. To this aim, cell line and primary cancer data sets from three different dual channel array CGH platforms obtained by four different institutes were integrated. The cell line data were used to develop preprocessing methods, which performed noise reduction and transformed samples into a common format. The transformed array CGH profiles allowed perfect clustering by cell line, but importantly not by platform or institute. The same preprocessing procedures used for the cell line data were applied to data from 373 primary tumors profiled by array CGH, including controls. Results indicated that there is no apparent feature related to the institute or platform and that array CGH allows for unambiguous cross-platform meta-analysis. Major clusters with common tissue origin were identified. Interestingly, tumors of hematopoietic and mesenchymal origins cluster separately from tumors of epithelial origin. Therefore, it can be concluded that chromosomal aberrations of tumors from hematopoietic and mesenchymal origin versus tumors of epithelial origin are distinct, and these differences can be picked up by meta-analysis of array CGH data. This suggests the possibility of prospectively using combined analysis of diverse copy number data sets for cancer subtype classification.

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.

Deletion of chromosome 4q predicts outcome in Stage II colon cancer patients

BackgroundAround 30% of all stage II colon cancer patients will relapse and die of their disease. At present no objective parameters to identify high-risk stage II colon cancer patients, who will benefit from adjuvant chemotherapy, have been established. With traditional histopathological features definition of high-risk stage II colon cancer patients is inaccurate. Therefore more objective and robust markers for prediction of relapse are needed. DNA copy number aberrations have proven to be robust prognostic markers, but have not yet been investigated for this specific group of patients. The aim of the present study was to identify chromosomal aberrations that can predict relapse of tumor in patients with stage II colon cancer.Materials and methodsDNA was isolated from 40 formaldehyde fixed paraffin embedded stage II colon cancer samples with extensive clinicopathological data. Samples were hybridized using Comparative Genomic Hybridization (CGH) arrays to determine DNA copy number changes and microsatellite stability was determined by PCR. To analyze differences between stage II colon cancer patients with and without relapse of tumor a Wilcoxon rank-sum test was implemented with multiple testing correction.ResultsStage II colon cancers of patients who had relapse of disease showed significantly more losses on chromosomes 4, 5, 15q, 17q and 18q. In the microsatellite stable (MSS) subgroup (n = 28), only loss of chromosome 4q22.1-4q35.2 was significantly associated with disease relapse (P < 0.05, FDR < 0.15). No differences in clinicopathological characteristics between patients with and without relapse were observed.ConclusionIn the present series of MSS stage II colon cancer patients losses on 4q22.1-4q35.2 were associated with worse outcome and these genomic alterations may aid in selecting patients for adjuvant therapy.

Candidate driver genes in focal chromosomal aberrations of stage II colon cancer

Chromosomal instable colorectal cancer is marked by specific large chromosomal copy number aberrations. Recently, focal aberrations of 3 Mb or smaller have been identified as a common phenomenon in cancer. Inherent to their limited size, these aberrations harbour one or few genes. The aim of this study was to identify recurrent focal chromosomal aberrations and their candidate driver genes in a well‐defined series of stage II colon cancers and assess their potential clinical relevance. High‐resolution DNA copy number profiles were obtained from 38 formalin‐fixed, paraffin‐embedded colon cancer samples with matched normal mucosa as a reference using array comparative genomic hybridization. In total, 81 focal chromosomal aberrations were identified that harboured 177 genes. Statistical validation of focal aberrations and identification of candidate driver genes were performed by enrichment analysis and mapping copy number and mutation data of colorectal, breast, and pancreatic cancer and glioblastomas to loci of focal aberrations in stage II colon cancer. This analysis demonstrated a significant overlap with previously identified focal amplifications in colorectal cancer, but not with cancers from other sites. In contrast, focal deletions seemed less tumour type‐specific since they also showed significant overlap with focal deletions of other sites. Focal deletions detected were significantly enriched for cancer genes and genes frequently mutated in colorectal cancer. The mRNA expression of these genes was significantly correlated with DNA copy number status, supporting the relevance of focal aberrations. Loss of 5q34 and gain of 13q22.1 were identified as independent prognostic factors of survival in this series of patients. In conclusion, focal chromosomal copy number aberrations in stage II colon cancer are enriched in cancer genes that contribute to and drive the process of colorectal cancer development. DNA copy number status of these genes correlates with mRNA expression and some are associated with clinical outcome. Copyright

Aurora kinase A (AURKA) expression in colorectal cancer liver metastasis is associated with poor prognosis

Background:Five-year survival after resection of colorectal cancer liver metastasis (CRLCM) is <30%. We recently found that aurora kinase A (AURKA) drives 20q gain-associated tumour progression and is associated with disease recurrence. This study evaluates the prognostic value of AURKA expression in CRCLM of patients who underwent liver resection.Methods:Tissue microarrays (TMAs) were generated using formalin-fixed paraffin-embedded CRCLM and matched primary tumour from a multi-institutional cohort of patients with CRCLM who underwent liver resection between 1990 and 2010. Tissue microarrays were stained for AURKA using immunohistochemistry, and a hazard rate ratio (HRR) for the association between overall survival (OS) and nuclear AURKA expression in CRCLM was calculated. Results were validated by 500-fold cross-validation.Results:The expression of AURKA was evaluated in CRCLM of 343 patients. High AURKA expression was associated with poor OS (HRR 1.55, P<0.01), with a cross-validated average HRR of 1.57 (P=0.02). Average HRR was adjusted for the established prognostic clinicopathological variables in a multivariate analysis (average HRR 1.66; P=0.02). The expression of AURKA in CRCLM was correlated to its expression in corresponding primary tumour (P<0.01).Conclusion:The expression of AURKA protein is a molecular biomarker with prognostic value for patients with CRCLM, independent of established clinicopathological variables.