Center for Human Genetics and Laboratory Diagnostics, Dr. Klein, Dr. Rost and Colleagues

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Colorectal Carcinoma (CRC) – Analysis of Microsatellite Instability (MSI) [C18.-, C19, C20, C21.-] [Z80.0)

Omim number: 120435

Dr. biol. hum. Stefanie Kühner,
Prof. Dr. med. Barbara Dockhorn-Dworniczak

Scientific Background

Hereditary non-polyposis colorectal cancer (HNPCC) is inherited in an autosomal dominant pattern as a result of mutations in the DNA mismatch repair genes (MMR) MLH1, MSH2, MSH6 and PMS2. Usually inherited from one parent, the germline mutation is present in every cell of the body although the second, functional allele is sufficient for an intact repair system. Through a random mutation (somatic mutation) the previously intact allele is nonfunctional and the cell shows a repair defect (two-hit hypothesis by Knudson).

Inactivating point mutations in MLH1 and MSH2 are found in about 60% and 30%, respectively, of the MMR gene mutations, 7-10% are MSH6 mutations and less than 5% PMS2 mutations. The MMR system recognizes and corrects mistakes during DNA replication. As a result of mutations in MMR genes faulty DNA replications occur during cell division in the tumor tissue which can be detected by the variable length of microsatellite DNA (microsatellite instability, MSI).

HNPCC is diagnosed in stages. If HNPCC is suspected (according to Amsterdam, or rather revised Bethesda Guidelines), a microsatellite (MSI) analysis and immunohistochemical analysis (IHC) is first performed from tumor material. Tumors can be categorized into two groups on the basis of their MSI status:

1) Tumors without MSI, also known as MSI-stable tumors (MSS),

2) Tumors with a high level of MSI (>29%) (MSI-H)

MSI-H can be associated either with Lynch syndrome due to inheritance of a MMR gene mutation, or a sporadic MSI due to hypermethylation of the MLH1 promoters. Patients with MSI-H have a better prognosis than patients with MSS tumors. Further, it can be shown that patients with MMR deficient cells are more resistant to 5-Fluorouracil (5-FU) than patients with intact MMR.

Immunohistochemical analysis can provide an indication of the affected geneby the lack of staining of the gene products of MLH1, MSH2, MSH6 or PMS2. MLH1 and PMS2 together, as well as MSH2 and MSH6 together, form protein complexes. Therefore, a germline mutation in MLH1 causes loss of expression of both the MLH1 and PMS2 proteins and a MSH2 mutation leads to loss of expression of both the MSH2 and the MSH6 proteins. The reverse is not true.

If IHC shows loss of MLH1 and PMS2 proteins, it should be determined whether it is due to a loss of protein expression caused by MLH1 promotor methylation in sporadic cancer. To this end a BRAF analysis or analysis of MLH1 promotor methylation is performed on tumor material, since the promotor methylation may be caused by somatic mutations in BRAF. According to current knowledge BRAF mutations (in particular the V600E mutation) do not occur in HNPCC patients, but only in approximately 40% of sporadic colorectal MSI-H tumors with MLH1 loss. In the case of apparent immunohistochemistry without indication of a sporadic tumor, the molecular genetic analysis of genes MLH1, MSH2, MSH6 and PMS2 from EDTA blood for a germline mutation depending on IHC loss, is performed. The detection of a germline mutation in one of the MMR gene is diagnostic for HNPCC or Lynch syndrome.