DNA microsatellite instability and mismatch repair protein loss in adenomas presenting in hereditary non-polyposis colorectal cancer

Abstract

Background and aim: Hereditary non-polyposis colorectal cancer (HNPCC), as its name implies, is associated with few adenomas, and the early evolution of colorectal neoplasia is poorly understood. In this study our aim was to clarify the genetic profiles of benign polyps in subjects with HNPCC using a combined molecular and immunohistochemical approach.

Methods: Thirty adenomas and 17 hyperplastic polyps were obtained from 24 affected HNPCC subjects. DNA was extracted from paraffin embedded tissue by microdissection and analysed for the presence of microsatellite instability (MSI) and mutations in five genes known to be targets in mismatch repair deficiency (TGFbetaRII, IGF2R, BAX, hMSH3, and hMSH6). Serial sections were stained by immunohistochemistry for hMLH1 and hMSH2.

Results: Twenty four (80%) of 30 adenomas showed MSI. Of MSI positive adenomas, 66.7% showed MSI at more than 40% of markers (high level of MSI (MSI-H)). Two of 17 hyperplastic polyps revealed MSI at one marker (low level of MSI (MSI-L)). A significant association was found between MSI-H and high grade dysplasia in adenomas (p=0.004). Eight of nine adenomas with mutations of coding sequences revealed high grade dysplasia and all nine were MSI-H. Four of the nine ranged in size from 2 to 5 mm. The presence of the hMSH6 mutation was significantly correlated with high levels of MSI (80% of markers) (p<0.02). Twenty four adenomas gave evaluable results with immunohistochemistry. One of six (17%) microsatellite stable, six of seven (86%) MSI-L, and 11 of 11 (100%) MSI-H adenomas showed loss of either hMLH1 or hMSH2.

Conclusions: Most adenomas in subjects with a definite diagnosis of HNPCC show MSI (80%). The finding of MSI-L is usually associated with loss of expression of hMLH1 or hMSH2, unlike the situation in MSI-L sporadic colorectal cancer. The transition from MSI-L to MSI-H correlated with the finding of high grade dysplasia and mutation of coding sequences and may be driven by mutation of secondary mutators such as hMSH3 and hMSH6. Advanced genetic changes may be present in adenomas of minute size.

Figure 1

Sample of four loci showing bandshifts, including three microsatellite loci (D2S123, BAT40, and c-myb) and the poly(A)8 tract of hMSH3. DNA was extracted from a 2 mm tubular adenoma from a patient carrying a germline mutation of hMSH2. Despite the small size of the adenoma, all nine microsatellite loci showed instability, as did hMSH6 (not shown). N, normal; T, tumour DNA.

Figure 2

Frequency of bandshifts in microsatellite markers used to distinguish MSI-H and MSI-L adenomas. All markers are sensitive for MSI-H adenomas whereas dinucleotide markers (particularly D5S346, D10S197, and D18S58) are sensitive for MSI-L adenomas.

Figure 3

Loss of hMSH2 (A) and retention of hMLH1 (B) in a 3 mm tubular adenoma that was MSI-H. The subject had a known germline mutation of hMSH2. Immunohistochemical staining with anti-hMSH2 and anti-MLH1.