This pilot study provided data on the prevalence of MMR gene defects in colorectal cancers and to extrapolate the frequency of cases suggestive of HNPCC in Malaysia. Immunohistochemistry was used to evaluate abnormal MMR gene protein expression as it has been shown to have a high specificity and sensitivity in predicting microsatellite instability status3,4,12–17
. It also has several advantages over microsatellite instability testing as a first-line screening tool for identifying HNPCC. It is easily available as part of a routine technique used in pathology laboratories, is inexpensive13
, rapid and is performed on paraffin embedded tissue sampled at time of colectomy. It also aids in identification of which particular MMR
gene may be defective and therefore, enables more efficient mutation analysis. The sensitivity of immunohistochemistry is enhanced with all four antibodies as part of the antibody panel.
The study sample comprised a mixture of both sporadic and hereditary colorectal cancers. In general, about 85 per cent of sporadic colorectal cancers possess normal MMR function, whereas 15 per cent have defective MMR function. The cancer in this group is often associated with hypermethylation of the promoter region of the MLH1
gene resulting in transcriptional silencing of MLH1
gene and absent protein expression7,8
. In the present study, three subjects had isolated absent MLH1 expression, a characteristic of sporadic CRC.
HNPCC accounts for 2-5 per cent of all colorectal cancers, in which there is a germline mutation in one of the MMR genes, usually MLH1 (40-45% of cases), MSH2 (40-45%), MSH6 (5-10%) and PMS2 (1%)22
. The loss of expression of MSH2, MSH6 or PMS2 in isolation or in combination, provides reasonably strong evidence of a germline mutation in the respective gene and therefore, highly suggestive of HNPCC8,11,22
. The presence of synchronous or metachronous colorectal or other HNPCC-associated tumours is a criterion for MSI testing4
. In our study two subjects showed synchronous tumours associated with absence of at least two MMR
gene proteins expression, thus reflecting a high probability of HNPCC.
About 15 per cent subjects in this study did not show MSH2, MSH6 and PMS2 protein expression in isolation or in combination with other MMR
genes. This appears to be a rather high frequency of subjects likely to have MMR germline mutation and hence HNPCC. This may be due to the use of a panel of four antibodies against MLH1, MSH2, MSH6 and PMS2 instead of the usual two i.e
., MLH1 and MSH2 used in screening. Though the majority of germline mutations in HNPCC occur in MLH1
genes, the additional screening of PMS2 and MSH6 proteins is able to identify more such cases17
The formation of heterodimers between MLH1 and PMS2 proteins accounts for the frequent absence of these proteins in combination in immunohistochemistry. Since MLH1 often produces weak and heterogeneic immunohistochemical staining pattern, the addition of PMS2 antibody improves the predictive value of germline mutation2,13,15,17,23
. Fourteen subjects in the current study had loss of MLH1 and PMS2 proteins in contrast to only 3 subjects with loss of MLH1 staining alone. In a similar manner, there is often a concurrent loss of MSH2 and MSH6 proteins, though our study did not show a significant increase in number of case detection using these two antibodies.
Fifteen of the 26 subjects with absence of MMR
gene protein expression fulfilled at least one of the Modified Bethesda Guidelines22 i.e.
, being diagnosed with colorectal cancer below the age of 50 yr (6 subjects), having synchronous colorectal cancers (2 subjects) or histological features of microsatellite instability tumours which include mucinous (6 cases), signet ring (2 cases) or poorly differentiated carcinoma (4 cases).
In concordance with other studies, our results showed that abnormal MMR
gene protein expression was associated with MSI-H histopathological features which include right sided tumours, mucinous, signet ring and poorly differentiated morphology4,8–10
. Though it is almost impossible to distinguish sporadic and HNPCC tumours by histology, presence of tumour-infiltrating lymphocytes appears to be more evident in HNPCC while mucinous histology, poor differentiation and co-existing serrated polyps are features suggestive of sporadic cancer8,22
. In the current study, only data on tumour differentiation and existence of colorectal polyps were obtained from the pathology reports.
Our results did not show any significant association between abnormal MMR protein expression and patients’ age, gender or ethnic group. This was expected as there were no specific selection criteria for subjects other than having a diagnosis of colorectal cancer. Therefore, the majority of cancers would be of the sporadic type, with 78.4 per cent of subjects in this study being older than 50 yr. The male to female ratio was comparable to the Malaysian cancer statistics of 1.1: 11
. However, it was interesting to note a higher incidence of colorectal cancer in Malays compared to Chinese and Indians in the present study, unlike the national cancer statistics which quoted the highest incidence in the Chinese community1
. A major limitation is that our study was confined to specific regions of Peninsular Malaysia and hence not reflective of the true ethnic ratio.
Age at onset of colorectal cancer, family history and tumour morphology are important data that aid in distinguishing sporadic CRC and HNPCC8
. In clinically suspected HNPCC patients, immunohistochemistry is often used as the first line screening tool which is followed up by microsatellite instability testing if there is no immunohistochemical abnormality. Patients with cancers showing lack of any MMR
gene proteins will be confirmed by the germline mutation studies for that specific gene14,20
. Sporadic CRC is associated with a lower frequency of APC
mutation, and higher frequency of BRAF
mutation and DNA methylation8,11,22
In conclusion, there appears to be a high frequency of abnormal MMR gene protein expression in this heterogenous population of colorectal cancers in Malaysia. These patients need to be further evaluated in terms of clinical and family history, and a diagnosis of HNPCC confirmed by germline mutation analysis in clinically suspected cases. It is essential that appropriate clinical management and genetic counselling are in place for patients and their families.