When loss of MLH1 and PMS2 expression is observed, which additional molecular tests are required to determine hereditary status?

Additional Testing for MLH1/PMS2 Loss to Confirm Hereditary Status

When MLH1 and PMS2 loss is detected on immunohistochemistry, you must first perform MLH1 promoter hypermethylation testing and/or BRAF V600E mutation analysis on the tumor tissue to distinguish sporadic cancer from Lynch syndrome before proceeding to germline genetic testing. 1, 2, 3

Testing Algorithm

Step 1: Initial Tumor Tissue Testing

When loss of MLH1 expression (with or without concurrent PMS2 loss) is observed:

• MLH1 promoter hypermethylation analysis on tumor tissue 1, 2, 4
• BRAF V600E mutation testing (for colorectal cancer only - not applicable to endometrial or other cancers) 1, 2, 3

Rationale: Approximately 10-15% of colorectal cancers show MLH1/PMS2 loss due to sporadic somatic hypermethylation of the MLH1 promoter, often associated with BRAF V600E mutation. This is NOT hereditary Lynch syndrome 1, 5.

Step 2: Interpretation and Next Steps

If MLH1 promoter is hypermethylated OR BRAF V600E mutation is present:

• This indicates sporadic cancer, not Lynch syndrome
• No germline genetic testing needed
• Manage based on personal/family history 1, 3

If MLH1 promoter is NOT hypermethylated AND BRAF is negative (or not tested):

• Proceed to germline genetic testing for MLH1 and PMS2 genes 1, 3
• Include both DNA sequencing AND large rearrangement/deletion analysis 1, 2
• Consider multi-gene panel testing (MLH1, MSH2, MSH6, PMS2, EPCAM) 3

Step 3: Additional Considerations

If germline testing is negative despite abnormal IHC:

• Consider somatic tumor testing for double somatic mutations in MMR genes 1, 3
• Evaluate for constitutional MLH1 epimutation (rare) 2, 3
• Confirm that large rearrangement analysis was performed 3
• Some patients may have "Lynch-like syndrome" - manage based on personal/family history 3

Important Caveats

Tissue-Specific Differences:

• BRAF testing is only useful for colorectal cancer - it cannot be used as a proxy for MLH1 hypermethylation in gynecological cancers 4
• For endometrial cancer with MLH1/PMS2 loss, proceed directly to MLH1 methylation testing or germline analysis 4

Heterogeneous Staining Patterns:

• Isolated PMS2 loss can occasionally be caused by MLH1 promoter hypermethylation (heterogeneous pattern) 6
• MLH1 germline mutations can sometimes present with isolated PMS2 loss (24% in one series) 7
• If isolated PMS2 loss is seen, still perform MLH1 methylation analysis before proceeding to PMS2 genetic testing 6

Genetic Testing Requirements:

• Full germline testing must include both sequencing and large deletion/rearrangement analysis, as large deletions are common 1, 2
• Consider EPCAM deletion testing, which can cause MSH2 silencing through hypermethylation 2, 5

Clinical Context Matters:

• Even with MLH1 hypermethylation present, evaluate family and personal cancer history - occasional patients may harbor germline variants in other MMR genes 8
• Strong family history (Amsterdam criteria) or young age (<50 years) may warrant germline testing regardless of methylation status 1, 4