Margaret Schnitzler

The identification of false positive responses to the pentagastrin stimulation test in RET mutation negative members of MEN 2A families

OBJECTIVE The pentagastrin stimulation test is the traditional test used for the identification of asymptomatic individuals in multiple endocrine neoplasia type 2A (MEN 2A) and familial medullary thyroid carcinoma (FMTC). The identification of mutations in the RET proto‐oncogene segregating with the disease phenotype in MEN 2A and FMTC families has made it possible to re‐examine the validity of using this test for the identification of affected family members.

Cancer and inflammatory bowel disease: bias, epidemiology, surveillance, and treatment.

Abstract. Individuals with chronic ulcerative colitis are at increased risk of developing colorectal carcinoma, particularly if there is long-standing disease or extensive colitis. It is generally accepted that the risk of colorectal cancer does not begin until 8 to 10 years after the time of diagnosis of ulcerative colitis. Thereafter it increases by approximately 0.5% to 1.0% per year. In patients with Crohn’s disease, the risk of malignancy is smaller and less well defined. The most significant predictor of the risk of malignancy in patients with inflammatory bowel disease is the presence of dysplasia in colonic biopsies. There is considerable controversy in the literature regarding the efficacy of colonoscopic surveillance programs and the role of prophylactic surgery to prevent colorectal cancer. Surveillance certainly fails to detect carcinoma in some patients who are having regular colonoscopy. Concerns have also been raised as to the cost-benefit of colonoscopic surveillance in patients with colitis. Randomized controlled trials of surveillance programs are highly unlikely in view of the low prevalence of IBD in the population, the long period of observation required, and the probability of contamination of surveillance programs by colonoscopy for assessment of disease activity. Despite the lack of clear guidelines, surveillance colonoscopy and biopsy continues to be widely practiced. Research is proceeding to identify genetic and biochemical markers that may prove clinically useful for predicting cancer risk. At present, however, surveillance programs are likely to continue according to institutional practice. It is important for those participating in such programs to be aware of the limitations of colonoscopy and biopsy as a means of reducing the risk of cancer in inflammatory bowel disease.

The role of chemotherapy in microsatellite unstable (MSI-H) colorectal cancer

IntroductionHigh-frequency microsatellite instability (MSI-H) is an alternate pathway of colorectal carcinogenesis, which accounts for 15% of all sporadic colorectal cancers. These tumours arise from mutations in the DNA mismatch repair system and thus have different responses to chemotherapeutic agents compared to microsatellite stable (MSS) cancers.ObjectiveThis review aims to summarise the available literature on the responses to chemotherapy in MSI-H colorectal cancer (CRC).Results and discussion5 Fluorouracil (5FU) is commonly used as a chemotherapeutic agent in colon cancer and in vitro evidence shows reduced response to 5FU in MSI-H CRC. The clinical evidence is conflicting but favours a reduced response to 5FU in MSI-H CRC. Several newer agents such as COX-2 inhibitors and irinotecan are also reviewed.ConclusionAvailable evidence suggests that MSI-H CRC have different behaviour patterns and response to chemotherapy compared with MSS CRC.

Mutations associated with microsatellite unstable colorectal carcinomas exhibit widespread intratumoral heterogeneity

Although microsatellite instability (MSI) has been shown to be present in 15% of sporadic colorectal carcinomas, the genetic events underlying the development of these tumors have not been well described. By investigating intratumoral heterogeneity, this study attempts to elucidate whether MSI‐positive colorectal carcinomas develop as the result of a random accumulation of mutations or as an ordered, stepwise sequence of genetic alterations. Eighty‐six regions from 16 MSI‐positive sporadic colorectal carcinomas were examined for mutations in repeat nucleotide sequences of the tumour suppressor genes transforming growth factor β type II receptor (TGFBRII), insulin‐like growth factor II receptor (IGFIIR), and BAX, and the mismatch repair genes MSH3 and MSH6. At least 2 and up to 5 of these genes were mutated in each tumour, and widespread intratumoral heterogeneity was observed for each gene. Regions of tumour with TGFBRII mutations were correlated with a poorly differentiated histology. Unlike the situation in microsatellite stable colorectal carcinomas, the findings of the present study did not suggest that a particular sequence of tumour suppressor and mismatch repair genes are mutated during colorectal tumorigenesis. It seems likely that a random accumulation of mutations, as a result of a defect in the mismatch repair pathway, drives tumour progression in this type of colorectal carcinoma.

Breast cancer immunohistochemistry can be useful in triage of some HNPCC families

Immunohistochemistry of tumour samples is increasingly used in the triage of families where hereditary non-polyposis colorectal cancer (HNPCC) due to mismatch repair defects is suspected. Usually, this is undertaken in tumours that are a recognised part of the spectrum of HNPCC-related cancers e.g. colon or endometrial cancers. Although breast cancers are not classed as part of this spectrum, this study examined the extent to which some breast tumours do arise by the mismatch repair pathway in these families. This may have clinical utility in families where an individual with a ‘classic HNPPC-related’ tumour is not available for evaluation. Immunohistochemistry of a breast tumour may identify an individual in whom germline mutation testing is worthwhile.

5-Fluorouracil (5FU) treatment does not influence invasion and metastasis in microsatellite unstable (MSI-H) colorectal cancer

Microsatellite instability is a recognised pathway of colorectal carcinogenesis responsible for about 15% of all sporadic colorectal cancers. Recent evidence has suggested that these tumours may not have the same response as microsatellite stable colon cancers to 5-fluorouracil (5FU)-based chemotherapy. The response to 5FU in four microsatellite unstable (MSI-H) cell lines was examined by cell viability assays and invasion assays. Flow cytometry was used to assess the effect of 5FU on MSI-H cell lines. In vivo response to 5FU was assessed by intraperitoneal injection of 5FU or control to 80 nude mice that had received intrasplenic injections of an MSI-H cell line KM12C prior to commencing treatment. There was inhibition of cell growth in MSI-H cell lines when treated with 5FU. There was no difference in invasiveness in the MSI-H cell lines when treated with 5FU. Primary tumours formed in 27 of the untreated and 25 of the 5FU treated mice (p=NS). There was a 36% reduction in splenic weight in those mice treated with 5FU (p<0.03). Metastases formed in 5 of the untreated and 9 of the treated mice (p=0.12). 5FU treatment of MSI-H tumours results in a reduction in growth but does not result in a reduction in invasion or metastasis.

Effective strategy to guide pathology test ordering in surgical patients

Background:  Ordering of pathology testing by junior medical staff is often a haphazard process with little regard to the appropriateness of test ordering. The aim of the present study was to reduce ordering of inappropriate pathology tests in surgical patients attending the pre‐admission clinic (PAC) through the introduction of a protocol‐based test ordering system and to create an environment where such improvement can be sustained.

Genetic analysis of multiple sporadic colon carcinomas from a single patient

Abstract At least two separate genetic pathways of carcinogenesis in sporadic colon cancer involving the accumulation of mutations at various genetic loci have been described. About 15% of sporadic colorectal carcinomas arise via a mechanism associated with microsatellite instability (MSI) and mutations in transforminggrowth factorβ receptor II (TGFβRII), insulin-like growth factor II receptor (IGFIIR) and BAX, whilst the remaining 85% are associated with aneuploidy and gross chromosomal rearrangements. An 81-year-old woman had a sigmoid colon carcinoma resected and 18 months later developed two additional carcinomas of the caecum and transverse colon. To investigate whether there was a common genetic mechanism of carcinogenesis for the three lesions, MSI status was assessed, TGFβRII, IGFIIR and BAX were analysed for mutations and protein expression of transforming growth factor β1 (TGFβ1) and p53 were studied using immunohistochemistry. The caecal and transverse colonic carcinomas were both MSI positive but different mutations were identified in each lesion. No genetic abnormalities were identified in the sigmoid colonic carcinoma. This suggests that each carcinoma arose via a separate genetic mechanism of carcinogenesis.

There is no increase in frequency of somatic mutations in metastases compared with primary colorectal carcinomas with microsatellite instability.

This study investigates the molecular features of metastasis in sporadic colon carcinomas with high‐level microsatellite instability (MSI‐H). DNA from 51 regions from 10 MSI‐H metastatic carcinomas and 26 corresponding metastases was analyzed for mutations in TGFBRII, IGFIIR, BAX, MSH3, MSH6, and TCF4, which are associated with MSI‐H carcinomas. In addition, 10 metastatic and 10 non‐metastatic MSI‐H carcinomas and 10 metastatic microsatellite‐stable (MSS) carcinomas were examined for expression of vascular endothelial growth factor (VEGF) and mutant TP53. The frequency of microsatellite instability and somatic mutations was not significantly increased in the metastases compared with the that of primary carcinomas. Although significantly fewer MSI‐H carcinomas expressed VEGF (P < 0.01) and mutant TP53 (P < 0.005) than MSS carcinomas, there was no difference in VEGF and mutant TP53 expression in metastatic and non‐metastatic MSI‐H carcinomas. In conclusion, metastasis does not appear to be associated with an increase in somatic mutation rate in any of the genes examined in MSI‐H colon carcinomas. Furthermore, VEGF and TP53 expression did not appear to be involved in metastasis in MSI‐H colon carcinomas.

A further investigation of combined mismatch repair and BRAFV600E mutation specific immunohistochemistry as a predictor of overall survival in colorectal carcinoma.

Mutation specific immunohistochemistry (IHC) is a promising new technique to detect the presence of the BRAFV600E mutation in colorectal carcinoma (CRC). When performed in conjunction with mismatch repair (MMR) IHC, BRAFV600E IHC can help to further triage genetic testing for Lynch Syndrome. In a cohort of 1426 patients undergoing surgery from 2004 to 2009 we recently demonstrated that the combination of MMR and BRAFV600E IHC holds promise as a prognostic marker in CRC, particularly because of its ability to identify the poor prognosis MMR proficient (MMRp) BRAFV600E mutant subgroup. We attempted to validate combined MMR and BRAFV600E IHC as a prognostic indicator in a separate cohort comprising consecutive CRC patients undergoing surgery from 1998 to 2003. IHC was performed on a tissue microarray containing tissue from 1109 patients with CRC. The 5 year survivals stratified by staining patterns were: MMRd/BRAFwt 64%, MMRd/BRAFV600E 64%, MMRp/BRAFwt 60% and MMRp/BRAFV600E 53%. Using the poor prognosis MMRp/BRAFV600E phenotype as baseline, univariate Cox regression modelling demonstrated the following hazard ratios for death: MMRd/BRAFwt HR = 0.71 (95%CI = 0.40–1.27), p = 0.31; MMRd/BRAFV600E HR = 0.74 (95%CI = 0.51–1.07), p = 0.11 and MMRp/BRAFwt HR = 0.79 (95%CI = 0.60–1.04), p = 0.09. Although the findings did not reach statistical significance, this study supports the potential role of combined MMR and BRAF IHC as prognostic markers in CRC.