Terence O'Connor

Microsatellite instability and the clinicopathological features of sporadic colorectal cancer

BACKGROUND AND AIMS In this study, we prospectively examined the clinical significance of the microsatellite instability (MSI) phenotype in sporadic colorectal cancer, and investigated methods for effective identification of these tumours in routine pathology practice. METHODS DNA was extracted from 310 tumours collected from 302 consecutive individuals undergoing curative surgery for sporadic colorectal cancer. Microsatellite status was determined by polymerase chain reaction amplification using standard markers, while immunostaining was used to examine expression of MLH1, MSH2, and p53. RESULTS Eleven per cent of tumours showed high level instability (MSI-H), 6.8% had low level instability (MSI-L), and the remainder were stable. MSI-H tumours were significantly more likely to be of high histopathological grade, have a mucinous phenotype, and to harbour increased numbers of intraepithelial lymphocytes. They were also more likely to be right sided, occur in women, and be associated with improved overall survival. In total, 25 (8%) tumours showed loss of staining for MLH1 and a further three tumours showed absence of staining for MSH2. The positive and negative predictive value of immunohistochemistry in the detection of MSI-H tumours was greater than 95%. CONCLUSIONS We conclude that the MSI-H phenotype constitutes a pathologically and clinically distinct subtype of sporadic colorectal cancer. Immunohistochemical staining for MLH1 and MSH2 represents an inexpensive and accurate means of identifying such tumours.

Restriction Endonuclease-Mediated Selective Polymerase Chain Reaction : A Novel Assay for the Detection of K-ras Mutations in Clinical Samples

The enriched polymerase chain reaction (PCR) assay has been used extensively in the detection of ras gene mutations in many types of human malignancies. Although it is very sensitive, it has a number of features that limit its use in the routine diagnostic laboratory. The aim of this study was to develop a novel enriched PCR strategy, in which the concurrent activity of the restriction enzyme BstNI and Taq polymerase allowed the amplification of mutant K-ras while inhibiting the formation of wild-type product. This restriction endonuclease-mediated selective PCR assay uses three sets of primers, together with BstNI, in the reaction mix, and the amplification products are analyzed by gel electrophoresis. The reliability of the restriction endonuclease-mediated selective PCR assay to detect activated K-ras was determined in a variety of clinical samples, including 139 fresh colorectal carcinomas and 113 paraffin-embedded blocks from 80 separate tumors of the colon and rectum, pancreas, breast, or kidney. Codon 12 mutations of the K-ras oncogene were identified in DNA from both fresh and paraffin-embedded tumors in a rapid, sensitive, and reproducible manner. Mutations were detected in 33 (24%) of the fresh colorectal cancers and 16 (20%) of the paraffin-embedded tumors. These results were 97% concordant in cases in which paraffin blocks and fresh specimens from the same tumor were available for analysis. We conclude that restriction endonuclease-mediated selective PCR is a sensitive, rapid, and robust assay for the detection of point mutations in a variety of clinical samples. Importantly, there is no need for manipulation of the sample once the PCR has been set up, and therefore, the chance of contamination is significantly reduced. In contrast to previous assays, restriction endonuclease-mediated selective PCR is not labor intensive, and its format is suitable for use in routine diagnostic laboratory.

Activation of the K-ras oncogene in colorectal neoplasms is Associated with decreased apoptosis†

Recent in vitro data indicate that the oncogenic effects of activated ras genes may be mediated, at least in part, through inhibition of apoptotic cell death. To examine this proposition in vivo, the relationship between mutations of the K‐ras gene and the frequency of apoptosis was studied in a series of 69 sporadic colorectal neoplasms (11 adenomas and 58 carcinomas).

Routine testing for mismatch repair deficiency in sporadic colorectal cancer is justified.

This study prospectively examines the accuracy of immunohistochemical staining in the identification of mismatch repair defective (MMRD) colorectal cancer in routine clinical practice. The potential impact of this information on decisions regarding adjuvant treatment and germline testing were quantified. A consecutive series of fresh tissue (836 cancers) was obtained from 786 individuals undergoing curative surgery for colorectal cancer at one institution. As part of normal practice, each tumour was screened for the expression of MLH1 and MSH2 by immunohistochemical staining (IHC) and relevant clinicopathological details were documented. Microsatellite instability (MSI) was assessed using standard markers. Overall, 108 (13%) tumours showed loss of staining for either MLH1 (92 tumours) or MSH2 (16 tumours). The positive predictive value of mismatch repair IHC when used alone in the detection of MSI tumours was 88%, and the negative predictive value was 97%. Specificity and positive predictive value were improved by correlation with microsatellite status. Tumour stage (HR 3.5, 95% CI 2.0–6.0), vascular space invasion (HR 1.9, 95% CI 1.2–3.0) and mismatch repair deficiency (HR 0.2, 95% CI 0.05–0.87) were independent prognostic factors in stages II and III disease. Screening by mismatch repair IHC could reasonably have been expected to prevent ineffective treatment in 3.6% of stage II and 7.6% of stage III patients. The frequency of germline mismatch repair mutations was 0.8%, representing six unsuspected hereditary non‐polyposis colorectal cancer (HNPCC) cases. Routine screening of colorectal cancers by mismatch repair IHC identifies individuals at low risk of relapse, and can prevent unnecessary adjuvant treatments in a significant number of individuals. Abnormal immunohistochemistry should be confirmed by microsatellite testing to ensure that false‐positive results do not adversely impact on treatment decisions. Copyright

The role of hMLH1 methylation in the development of synchronous sporadic colorectal carcinomas.

AbstractPURPOSE: AB. B. subset of sporadic colorectal carcinomas show microsatellite instability, usually as a result of biallelic hMLH1 gene promoter methylation. Synchronous tumors occur in up to 5 percent of patients with colorectal cancer, but their cause is poorly understood. We hypothesized that in the setting of sporadic microsatellite instability cancers, synchronicity may reflect a global predisposition of colorectal epithelium toward tumor development because of gene hypermethylation. METHODS: We identified 14 individuals with 33 synchronous cancers from a series of 362 patients with 381 sporadic colorectal cancers. We then analyzed the synchronous lesions for microsatellite status, hMLH1 protein expression, and hMLH1 promoter methylation. RESULTS: Seven of 33 synchronous tumors (21 percent) showed microsatellite instability, compared with 36 of 348 solitary tumors (10.3 percent, P = 0.06). The 14 patients with synchronous tumors were significantly older than those with solitary tumors (mean age 79.4 vs. 68.2 years, P = 0.01), and 5 of these patients had at least one microsatellite instability tumor. However, only one patient harbored synchronous tumors that were all of the microsatellite instability type. Methylation of the hMLH1 promoter was seen in 9 synchronous cancers from 27 assessable lesions in 7 patients and was associated with microsatellite instability (P = 0.01), right-sidedness (P = 0.01), and loss of expression of hMLH1 (P = 0.03). Only one case showed methylation in all synchronous tumors, whereas in five cases synchronous tumors showed different methylation status within the one individual. CONCLUSION: Our data suggest that synchronous tumors arise as independent events and that the slightly greater frequency of synchronous tumors in individuals with microsatellite instability cancers is likely to be a chance event reflecting the older age of these individuals rather than arising from a predisposition toward cancer as a result of global hypermethylation of colorectal epithelium.

A rapid PCR ELISA for the detection of activated K-ras in colorectal cancer.

Aims—To develop a rapid PCR ELISA procedure for the detection of mutations in K-ras in a microtitre plate format, and to evaluate the assay for the detection of these mutations in human colorectal cancer. Methods—An enriched PCR method was used with labelled primers, and PCR product was captured on GCN4 coated immunoassay plates. Detection of biotinylated mutant product was performed by colorimetric assay with streptavidin-horseradish peroxidase. The assay was used to determine K-ras status in a series of 60 human colorectal neoplasms, together with paired normal colonic mucosa. Results from gel electrophoretic analysis were compared with ELISA results. Results—The assay proved reliable in detecting K-ras mutations in DNA extracted from both fresh and paraffin embedded colorectal tumours. ELISA results were comparable with results from gel electrophoresis. Mutations of K-ras were detected in 16 of 48 adenocarcinomas and five of 12 adenomas but no mutations were detected in normal mucosa. There was a highly significant difference (p<0·0005) between optical density values for carcinomas with mutant K-ras and their paired normal data. Adenomas did not show the clear distinction between positive and negative results seen with carcinomas. Conclusions—This assay provides a rapid and reliable means of detecting mutations in codon 12 of the K-ras oncogene. The single tube format colorimetric analysis in microtitre plates and clear discrimination between mutant and wild type genes makes the assay suitable for automation. The occurrence of intermediate results in the case of adenomas provides support for the hypothesis that mutations of K-ras occur early in the course of colorectal carcinogenesis.