Microsatelite Instability by Immunohistochemistry
MSI seen in 15% of sporadic colon Ca and 85% of Lynch (HNPCC). Most patients with MSI did not have family history of HNPCC. In Lynch mutations are not repaired and persist. Confirmation of HNPCC requires identify germline mutations in DNA from blood cells (blood test that require patient consent) for families with suspected syndrome, even if result of IHC is normal. MSI occur in loci with 100-200 base pairs with repeated dinucleotide CA. Why pt with high frequncy of MSI good px ? they have fewer mutations of APC and p53. Mutations in DNA mismatch repair genes (MMR) or MSI may be tested by PCR in paraffin blocks or resulting proteins by IHC (nuclear expression). IHC or PCR may be performed as first line tests, or PCR requested as a reflex. PCR compares 5 different loci or bands in tumoral and non tumoral areas: BAT-26, NR-21, BAT-25, MONO-27 and NR-24. PCR reports: Microsatellite Instability (MSI) if shifted bands (bases) vs Microsatelite stability (MSS) if identical bands (bases).
IHC detects loss of DNA MMR proteins. Based in 4 microsatellite markers report is : MSI-High if 2-4 markers affected, MSI-Low if only one marker affected and Microsatellite stable if none. 40% show mutations of MLH1 and MSH2 with high sensitivity and specificity, 10% MSH6 and 5% PMS2. MSI High is present in many sporadic colonic AdenoCa, commonly with loss of MLH1 (instable BAT26 by PCR) and px is better than MSS colon (fewer metastatic nodes although increased resistance to 5Fu).
Indications for MSI:
1) Colon Ca < 50
2) other HNPCC related tumors like stomach, bowel, endometrium, ovary, ureter, pancreatobiliar, glioblastoma etc.
3) MSI histology: crohn like lymphocytic reaction, mucinous or signet ring, medullary pattern or poorly differentated Ca in < 60s.
4) Ca in first or second degree relatives . 5) young patients with large colonic adenomatous polyps showing high grade dysplasia may be indicated.
If MLH1 mutation: MLH1 and PMS2 loss and the other 2 preserved.
MSH2 mutation: MSH2 and MSH6 loss and the other 2 preserved.
MSH6 mutation: loss in MSH6 and the other 3 preserved. 10% of cases of Lynch.
PMS2 mutation: PMS2 loss and the other 3 preserved. Rare cases of Lynch.
Summary of interpretation of the Immunohistochemistry: Suspect Lynch in patients with family history, young (<50), crohn like reaction, right side cancer and mucinous changes. If loss of expression in MSH2, MSH6 or PMS2, most likely the patient has Lynch and should be refered to sequencing. 90% of Lynch cases has MLH1 and MSH2 mutation, 10% only have MSH6 mutation. If patient only has loss of expression in MLH1, may be sporadic case or Lynch. In this case, you have to order BRAF V600 and if the test is positive for the mutation is a sporadic colorectal cancer with MSI. But if the test is normal, patient may have Lynch and requires sequencing.
Summary of interpretation of MSI by PCR: No instability = No Lynch. Instability in 1 marker risk is intermediate and require counseling and probably germline testing with sequencing. Presence of 2 to 5 markers is HIGH and requires counseling and germline testing with sequencing.
Typical diagnosis template for negative MSI:
No loss of expression of the MMR proteins detected by IHC. These findings are typically seen in Ca result of chromosomal rather than MSI instability, arguing against HNPCC. 60% of patients with HNPCC have at least defect in one of the MMR genes. Sometimes nonsense mutations may only be detected by PCR. PCR may be indicated to exclude MSI only if HNPCC is strongly suspected and not confirmed with IHC. Molecular mutational analysis for BRAF V600 by PCR is in process and results will follow.
Typical diagnosis template for the most common scenario in positive cases: Loss of expression detected in MLH-1 and PMS2. These findings are most probably related with MLH-1 sporadic mutation. Molecular studies for BRAF and hMSH1 are in progress and reports will follow. Also, MSI reflex test by PCR was requested to confirm. If the BRAF test and hMLH1 are both positive, it will confirm that this is probaly a sporadic case of MSI mutation. If both tests are negative (BRAF and hMLH1), further genetic evaluation with DNA sequencing may be required to rule out germline mutation (Lynch Syndrome).
For more information please see Case of the month from February 2013 and Podcast No 6 about MSI Interpretation.