Colorectal cancers with DNA mismatch repair (MMR) gene mutations characteristically display a high rate of replication errors in simple repetitive sequences detectable as microsatellite instability (MSI). Most are the result of somatic MMR dysfunction; however, a subset are caused by germline mutations. The availability of commercial antibodies for MSH1 and MLH2 offers an alternative strategy to molecular methods for identifying MMR deficient cancers. To evaluate immunohistochemistry, MLH1 and MSH2 expression was studied using monoclonal antibodies in formalin fixed, paraffin wax embedded cancers. The immunohistochemical staining patterns of 23 cancers displaying MSI, including four cases with germline mutations, were compared with 23 microsatellite stable (MSS) cancers. All MSS cancers exhibited staining with both antibodies. Twenty two of the MSI cases showed absent MMR expression with either anti-MSH1 or anti-MLH2. The high sensitivity and predictive value of immunohistochemistry in detecting MMR deficiency offers a method of discriminating between MSI and MSS cancers caused by MSH1 and MLH2 dysfunction. The application and suitability of immunohistochemistry for the detection of MSI and as a strategy for prioritising the mutational analysis of MMR genes in routine clinical practice is discussed.
- colorectal cancer
- mismatch repair
- hereditary non-polyposis colorectal cancer
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The human DNA mismatch repair (MMR) system, responsible for correcting DNA mismatches arising during replication, comprises at least six genes, with the principal ones being MSH1 and MLH2. Inherited deficiency of MMR function underlies the hereditary non-polyposis colorectal cancer (HNPCC) syndrome.1 Approximately 80% of HNPCC can be ascribed to germline mutations in either MHL1 or MSH2.1
The study of MMR deficient cancers from individuals affected with HNPCC has led to the recognition that MMR inactivation provides an alternative pathway for colorectal carcinogenesis. MMR deficient cancers are not typified by chromosomal instability and exhibit low rates of allele loss. Morphologically they are often polypoidal, mucinous, and undifferentiated and display extensive necrosis.2 DNA mismatch repair deficiency is also a feature of approximately 15% of sporadic colorectal cancers; in general, as a result of hypermethylation of the promoter sequence, but sometimes as a result of somatic mutations.3, 4
The biological behaviour of colorectal cancers with MMR deficiency appears to be distinct from those with intact MMR.2 Paradoxically, despite a high risk of metachronous disease in patients harbouring germline mutations, the prognosis appears to be better than for patients with MMR competent tumours.5 This also appears to be a feature of sporadic colorectal cancers that are MMR deficient. In addition, there is experimental evidence indicating that tumours deficient for MMR respond differently to chemotherapy.6 MMR deficient cells are highly tolerant to methylating drugs such as streptozocin and temozolomide and, to a lesser extent, to cis-platin and doxorubicin.6 Although there is little direct evidence from trials in humans, it seems plausible that the MMR status of tumours may become an important determinant in the choice of chemotherapeutic intervention.
Distinguishing colorectal cancers that exhibit MMR deficiency provides a method of refining the identification of individuals harbouring germline mutations. Furthermore, it provides a means of identifying patients with colorectal cancer in whom the behaviour of the disease will be different. The increased rate of spontaneous point mutations and high frequency of deletion/insertion mutations in short repetitive DNA that characterises these MMR deficient cancers offers a method of identifying cases through the detection of microsatellite instability (MSI). However, establishing MSI is not simple, and requires access to molecular diagnostic facilities. The recent availability of commercial antibodies for MSH1 and MLH2 potentially offers a more straightforward approach to identifying MMR deficient colorectal cancers. To assess the usefulness of this approach we have undertaken a study of 46 colorectal cancers.
Materials and methods
PATIENTS AND TUMOUR SAMPLES
For our study, we made use of an ongoing survey of the frequency of MMR germline mutations in early onset colorectal cancer (patientsless than 55 years old at diagnosis) systematically ascertained through three regional UK cancer registries. Formalin fixed, paraffin wax embedded blocks of colorectal cancers and EDTA venous blood samples were obtained from each patient. Our study was undertaken with approval from the relevant local ethics committee.
MSI was assessed using a fluorescent polymerase chain reaction (PCR) based assay at the following loci: D1S508, D2S123, D3S1561, D5S346, D11S29, TGFBIIR, D15S970, DCC, D19S565, BAT25, and BAT26. Microsatellite instability was defined as the presence of altered allele sizes in the PCR amplified product of tumour DNA compared with normal DNA. Tumours were designated as MSI positive if altered bands were seen in two or more microsatellites. The full coding sequences and splice junctions of human MLH1 (hMLH1) and hMSH2 were amplified using the PCR. Both PCR primers were end labelled with γ[32P] ATP using T4 polynucleotide kinase and the amplified fragments were analysed by conformation sensitive gel electrophoresis.7 PCR products from samples that showed migration shifts were directly sequenced in forward and reverse directions and analysed on ABI377 DNA sequencers. Mutations were numbered according to accepted conventions.
For each colorectal cancer, eight sections (3–4 μm thick) were cut and mounted on to glass slides. After dewaxing and rehydration of sections, antigenic site retrieval was accomplished by microwaving each slide for five minutes in 0.01 M citric acid buffer (pH 6.0). Endogenous peroxidase activity was blocked by incubation with 2% hydrogen peroxide for 20 minutes and non-specific binding prevented by incubation with 1% bovine serum albumin (BSA) in phosphate buffered saline (PBS). Sections were subsequently incubated with either monoclonal anti-MSH2 or anti-MLH1 antibodies (Oncogene, Cambridge, Massachusetts, USA) for two hours at room temperature. Antibody binding was detected using the Elite Vectastain ABC kit (Vector Laboratories Ltd, Peterborough, UK), which is based on the biotin–avidin system, using the manufacturer's protocol. The reaction was visualised using a VIP substrate kit for peroxidase (Vector Laboratories Ltd). Sections were then dehydrated and mounted. Normal colorectal tissue adjacent to the carcinoma was used as the positive control. Loss of expression was recorded when nuclear staining was observed in normal tissue but not in adjacent malignant cells.
Table 1 shows the MSI and germline MSH1 and MLH2 status of the 46 colorectal cancers studied. Also shown are the immunohistochemical results for the anti-MSH1 and anti-MLH2 antibodies. The scoring of both antibodies was essentially straightforward. Intact nuclear staining of tumour cells with antibodies to both MSH1 and MLH2 was seen in all of the 23 MSI negative colorectal cancers, concordant with the molecular analysis indicating that these tumours had no evidence of MMR deficiency. In contrast, 22 of the 23 cancers displaying MSI showed no nuclear staining for one of the MMR proteins, permitting the underlying gene inactivation to be inferred (table 1).
The four colorectal cancers harbouring germline mutations in MLH1 were correctly identified. Of the MSI positive colorectal cancer cases that did not harbour a germline MMR mutation there was a preponderance of MLH1 deficiency compared with MSH2 (2.8 : 1). No tumour showed loss of staining with both anti-MLH1 and anti-MSH2 antibodies. These observations of MSH1 and MLH2 staining suggest that immunohistochemistry identifies MMR deficiency with 96% sensitivity (95% confidence limits (CL), 90% to 100%). The negative predictive value of 96% (95% CL, 90% to 100%) reflects the fact that one tumour exhibited MSI but showed staining for both antibodies. Table 2 shows summary statistics for our study and other recently published studies that have evaluated the use of immunohistochemistry as a method of defining the MMR status of colorectal cancers.
Colorectal cancers displaying MMR deficiency are characterised by a distinctive morphological and clinical phenotype. Testing cancers for MMR deficiency provides a method of delineating a subset of sporadic cancers with a different clinical course and possible difference in response to chemotherapy.5, 6 Furthermore, and more importantly, testing colorectal cancers for this phenotype offers a means of refining the identification of patients harbouring germline mutations. The high risk of cancer conferred by constitutional mutations in the MMR genes (an approximate 70% risk of colorectal cancer and a pronounced increase in the risk of uterine and other adenocarcinomas)1 necessitates the long term follow up and screening of carriers.
The screening of individuals with colorectal cancer for germline MMR mutations is currently undertaken in cases compatible with a hereditary basis (young onset, right sided, dominant inheritance of colorectal cancer). However, the analysis of constitutional DNA for MSH1 and MLH2 mutations is not straightforward because both genes are relatively large and mutations are scattered throughout each gene; hence, identification of MSI in tumours provides a practical method of identifying those patients in whom mutational analysis is appropriate. The detection of MSI in colorectal cancers classically requires microdissection and access to molecular biological facilities. Thus, it is a relatively complex procedure, expensive, and labour intensive, and is therefore not ideally suited to routine clinical practice. Furthermore, it does not circumvent the issue of screening more than one MMR gene for mutations.
In our study, we have evaluated the usefulness of immunohistochemistry as a method of identifying colorectal cancers with MMR deficiency as a result of the inactivation of the MSH1 or MLH2 gene. We found the sensitivity of this approach to be over 90%, which supports the findings of other recently published studies (table 2). Therefore, immunohistochemistry appears to be a good surrogate test for the detection of MSI caused by MSH2 and MLH1 dysfunction. One caveat to this is that, because of the interdependency between MMR genes, absent or reduced protein expression may be a consequence of the disruption of an interacting MMR gene. The other is that the demonstration of normal protein expression does not entirely preclude gene disruption because mutations that have pathological consequences through RNA decay or other similar mechanisms will go undetected. Therefore, testing cancers for MSI will still be required in cases where there is a high probability of HNPCC but protein expression is present.
Although immunohistochemistry alone cannot be relied upon to distinguish MSI colorectal cancers, it offers an additional method of prioritising mutation analyses suited to routine clinical laboratory practice. Because a small number of MSI positive colorectal cancers are caused by mutations in MMR genes other than MSH1 and MLH2,1 the usefulness of immunohistochemistry as an initial screening tool will be extended by the use of antibodies against MSH6, PMS1, and PMS2.
This work was supported by research grants from the National Health Service and the Cancer Research Campaign. Part of the work was conducted in the Jean Rook Sequencing Laboratory, which is supported by BREAKTHROUGH Breast Cancer, UK charity 328323.
In the text the MSH2 and MLH1 genes were sometimes mistakenly written as MSH1 and MHL2, respectively. The authors apologise for this error.
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