Chapter 11 Answers to discussion questions

Microsatellite instability (MSI) and/or mismatch repair proteins (MMR) deficiency is used for screening of Lynch syndrome, but also for prognostic purposes as well as a biomarker for response to immunotherapy. Which techniques could you use to assess MSI or MMR? What are the advantages or disadvantages?

MSI is assessed by a panel of five mononucleotide markers (NR-21, BAT-25, MONO-27, NR-24 and BAT-26) by fluorescence multiplex PCR-based method. It requires normal and neoplastic tissue. MSI high is defined as an instability in ≥2 of the five markers, whilst low-frequency MSI (MSI-L) requires instability in only one marker. All five mononucleotide markers can be performed in a single reaction and the size of the products amplified are easily visualised using capillary electrophoresis.

Immunocytochemistry using a panel of four antibodies (MLH1, MSH2, MSH6 and PMS2) is used to define mismatch repair protein deficiency (at least 1 out of 4 protein expression lost) of mismatch repair protein proficiency (all four proteins are expressed in normal tissue).

Microsatellite instability:

• Advantages: 1) single reaction, the test can be performed in a single tumour sections (but also requires normal / non – neoplastic tissue); 2) can identify tumours with defective DNA MMR but with “normal” immunocytochemical staining (e.g., defects in other genes other than MLH1, PMS2, MSH2 or MSH6).
• Disadvantages: MSI is not a specific diagnostic test for Lynch syndrome (MSI is detected in 10-15% of sporadic CRC); 2) some tumour with germline mutations (e.g., in MSH6) usually show low-frequency of MSI; 3) the test is laborious and more expensive than ICC.

MMR – immunocytochemistry

• Advantages: 1) ICC is reliable for detecting mutations leading to truncation or degradation of the protein; 2) ICC is easily available and inexpensive; 3) ICC helps to identify the mutated gene (particularly loss of expression of PMS2, MSH6 and MSH2 very often correlates with the presence of germline mutations in these genes); 3) Can identify cases missed by MSI.
• Disadvantages: 1) May not be reliable in small biopsies (MMR protein staining might be patchy, particularly regarding MSH6); 2) ICC depends on tissue fixation and antibodies used; 3) cases with inconclusive ICC or normal staining but with clinical suspicion of Lynch will benefit from MSI also.

In the last few years large Next Generation Sequencing panels (300+ cancer-related genes) have allowed the assessment of tumour mutation burden (TMB) based on the number of mutations per coding area or, in other words, the number of mutations within a tumour genome. Mutation thresholds to distinguish high-mutation from low mutation samples have yet to be agreed.