Which genetic and tumor tests should be ordered for each of the recommended genes (MLH1, MSH2, MSH6, PMS2, EPCAM, BRCA1, BRCA2, PTEN, POLD1, POLE) to assess hereditary cancer risk?

Genetic and Tumor Testing for Hereditary Cancer Risk Genes

For the genes you've listed (MLH1, MSH2, MSH6, PMS2, EPCAM, BRCA1, BRCA2, PTEN, POLD1, POLE), order germline multigene panel testing that includes all these genes simultaneously using next-generation sequencing, as this is the standard approach recommended by current guidelines rather than testing each gene individually 1, 2.

Testing Approach by Gene Category

Lynch Syndrome Genes (MLH1, MSH2, MSH6, PMS2, EPCAM)

Germline Testing:

• Order a multigene panel that includes all five Lynch syndrome genes simultaneously 1, 2
• These genes should be included in any hereditary cancer panel given their importance and prevalence 1
• Testing uses next-generation sequencing to detect pathogenic variants 2, 3

Tumor Testing (when applicable):

• Perform immunohistochemistry (IHC) on tumor tissue to assess protein expression of MLH1, PMS2, MSH2, and MSH6 2, 4, 5
• If MLH1/PMS2 loss is detected on IHC, perform MLH1 promoter hypermethylation testing to distinguish sporadic from hereditary cases 5
• Microsatellite instability (MSI) testing can be performed as an alternative or complementary approach 2, 6
• Critical caveat: MLH1 hypermethylation typically indicates sporadic cancer, but germline testing may still be warranted if personal/family history is concerning for Lynch syndrome 5

BRCA Genes (BRCA1, BRCA2)

Germline Testing:

• Include BRCA1 and BRCA2 in multigene panel testing 1, 2
• These genes are considered "more strongly recommended" and should be included in panels for any patient with cancer undergoing germline genetic testing 1
• Testing detects pathogenic variants using next-generation sequencing 2, 3

Tumor Testing:

• Tumor sequencing panels (e.g., TruSight Oncology 500) can detect somatic BRCA variants 7
• If tumor testing shows BRCA variant with variant allele frequency (VAF) ≥40%, germline confirmation testing is recommended 7

PTEN Gene

Germline Testing:

• Include PTEN in multigene panels, particularly for endometrial cancer risk assessment 2
• PTEN is associated with Cowden syndrome and carries high cancer risk 1, 2
• Testing uses next-generation sequencing 2

Polymerase Proofreading Genes (POLD1, POLE)

Germline Testing:

• Include POLD1 and POLE in multigene panels for colorectal cancer risk 1, 8
• These genes are associated with polymerase proofreading-associated polyposis 9
• Testing uses next-generation sequencing to detect pathogenic variants 6

Tumor Testing:

• Next-generation sequencing of tumor tissue can detect POLE mutations 6
• Variant interpretation should follow ACMG/AMP guidelines 6
• Important note: POLE mutations in tumors can create complex molecular phenotypes (e.g., POLEmut + MMRd + p53abn) 6

Practical Testing Algorithm

1. Order a comprehensive multigene panel that includes all genes of interest (MLH1, MSH2, MSH6, PMS2, EPCAM, BRCA1, BRCA2, PTEN, POLD1, POLE) rather than individual gene tests 1, 2

2. For colorectal or endometrial cancers, perform tumor testing first:

   • IHC for mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) 2, 4
   • MSI testing 2, 6
   • If MLH1/PMS2 loss detected, add MLH1 hypermethylation testing 5

3. Proceed to germline testing when:

   • Tumor testing suggests hereditary syndrome 2, 4
   • Personal/family history meets criteria regardless of tumor results 1, 2
   • MLH1 hypermethylation is present but family history is concerning 5

4. For other cancer types, proceed directly to germline multigene panel testing based on cancer type and family history 1

Critical Pitfalls to Avoid

• Do not skip germline testing in patients with MLH1-hypermethylated tumors if family history suggests Lynch syndrome—these patients may harbor germline variants in other mismatch repair genes 5
• Do not rely solely on tumor testing to rule out hereditary syndromes—studies show 34.5% of Lynch syndrome patients would be missed without germline testing 2
• Do not test genes individually—multigene panel testing is more efficient and cost-effective 2, 3
• Ensure genetic counseling is provided, as genetic counselors are most involved in result interpretation and family communication (71.5% involvement rate) 10