Mismatch repair (MMR) proteins
In human, nine genes with MMR function have been identified, of which five have particular clinical interest because they may be mutated in families with hereditary non-polyposis colorectal cancer (HNPCC) (the relative frequency in parenthesis): MLH1 (49%), PMS1 (0.3%), PMS2 (2%), MSH2 (38%), and MSH6 (9%). More than 300 different mutations have been identified. Persons carrying a mutation have normal MMR proteins, but in case of a DNA damage involving the nonmutated allel, the protein production stops (loss of heterozygosity).
When forming complexes MLH1 heterodimerizes with PMS2, PMS1 or MLH3 (another MMR protein, for which a mutation has not been detected), while MSH2 heterodimerizes with MSH6. In case of MSH2 deficiency, MSH6 protein is also lost, possibly because of protein instability.
Carriers of an MLH1 or MSH2 mutation has a more than 70% lifetime risk of developing a colorectal carcinoma and also markedly increased risk of developing endometrial carcinomas (50%), as well as an less increased risk of developing carcinomas of stomach, biliary tract, ovary and urinary tract, as well as brain tumours and sebaceous skin tumours. The distribution of extraintestinal tumours is somewhat dependent on the type of mutation. About 5% of colorectal carcinomas are associated with a germline mutation.
In about 15% of colorectal carcinomas loss of MLH1 protein occurs without a mutation but because of a hypermethylation of the MLH1 promoter.
MSI-H (MMR protein deficient) colorectal carcinomas are associated with significant survival advantage independently of grade and stage, compared to MSS colorectal carcinomas (which are characterized by chromosomal instability). MSI-H carcinomas usually display high prevalence of activated intraepithelial T-lymphocytes (which is suspected to increase tumour cell apoptosis and, hence, to impede the growth potential). They are more often localized in the right hemicolon, and show a slightly deviant morphologic spectrum, as there is an overrepresentation of mucinous adenocarcinomas and medullary carcinomas (inappropriately designated low differentiated [adeno-]carcinomas).
It appears that among colorectal carcinomas the response to chemotherapy is different in MSI-H and MSS cases, but the data are conflicting.
Compared to molecular biological techniques, immunohistochemical analysis of MMR protein expression is much simpler and cheaper. Immunohistochemical analysis helps to pinpoint the affected gene and should be readily accessible in a pathology laboratory. Staining for MLH1, MSH2 and MSH6 in colorectal carcinomas should be carried out in patients < 55 years-of-age or with a family history of these tumours. Staining may also be carried out prior to planned chemotherapy. At some hospitals, all colorectal carcinomas are analyzed because of the prognostic implications.
Frequently used well functioning Abs are:
MLH1: mAb clones G168-15 and ES05 (new, tested by NordiQC lab but not yet included in an assessment).
MSH2: mAb clones 25D12, FE-11, G219-1129, and 27 (the latter, however, is now withdrawn from production),
MSH6: mAb clone 44.
The general sensitivity* of immunohistochemicalcal staining for MLH1 and MSH2 in predicting MSI-H or MMR protein gene mutation is about 90% (however, for MLH1 in series with germ line mutation it has been as low as 74%) . The general specificity** is close to 100% (however, for MLH1 in series with germ line mutation it has been as low as 81%). The causes for the discrepancies are multiple and only partly explained, but include preanalytical (i.e., biological), analytical (i.e., technical) and postanalytical (i.e., interpretation related) causes.
For instance, weak or focal expression in neoplastic cells has usually been regarded as intact protein expression but can be associated with gene defects: MLH1 genes frequently show a missense mutation that may result in functionally inactive but antigenetically more or less intact proteins. In sporadic MSI-H cases the MLH1 gene may be partly methylated, which results in decreased expression.
The main reason for technically inappropriate stains is insufficient antigen retrieval. However, staining difficulties have also emerged from using older tissue blocks and slides stored at room temperature (the latter particularly with MLH6). Occasional cytoplasmic staining, which is at present unexplained, may hamper the interpretation.
MLH1 or MSH2 is involved in more than 90% of tumours displaying high levels of MSI (MSI-H), while MLH1 and MSH2 are basically never lost in tumours that are microsatellite stable (MSS) or only show low levels of MSI (MSI-L). However, a few studies have shown negative staining for MMR-proteins in tumours classified as MSS or MSI-L. The cause for this is unknown but it should be emphasised that the sensitivity and specificity of analyses for MSI-H and mutations of MLH1, MSH2 and MSH6 are also less than 100% in HNPCC patients.
Control tissue: The slide should include non-neoplastic tissue (such as normal adjacent mucosa) from the patient.
* The sensitivity indicates how often the staining of a tumour is true negative for MMR proteins in case of proven MSI-H or mutation. A percentage lower than 100 means that a number of tumours show false positive staining of the nuclei.
** The specificity indicates how often the staining of a tumour is true positive for MMR proteins in case of no MSI-H or mutation found. A percentage lower than 100 means that a number of tumours show false negative staining of the nuclei.
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www.nfdth.nl (mutation database).