Molecular Pathology and Genetics
Overview
This domain tests the choice, interpretation and limitations of molecular tests in diagnosis, prognosis and therapy selection, and the standards used to classify variants.
Core rules
- Assay selection: PCR/qPCR for known hotspots; Sanger sequencing for single genes; FISH for translocations and copy number; next-generation sequencing for panels, exomes and genomes; MSI or mismatch repair immunohistochemistry as a surrogate for deficient mismatch repair.
- Variant classification (ACMG/AMP): pathogenic, likely pathogenic, variant of uncertain significance, likely benign, benign. Evidence tiers combine population frequency, computational prediction, functional data, segregation and de novo status.
- Somatic versus germline: tumor-only testing cannot reliably distinguish germline from somatic variants; paired normal tissue or blood is required.
- Actionable targets: EGFR exon 19 deletion and exon 21 L858R predict response to EGFR tyrosine kinase inhibitors in lung adenocarcinoma; KRAS G12C has a specific inhibitor; HER2 amplification and MSI-high status guide therapy across tumor types.
- Quality: every assay requires validated limits of detection, a defined specimen requirement (tumor cellularity, DNA input) and participation in external quality assessment.
Worked example
A lung adenocarcinoma resection shows an EGFR exon 19 deletion in a tumor-only panel with 40% variant allele frequency. Reasoning: a 40% allele frequency in a tumor with moderate cellularity may reflect germline or clonal hematopoiesis, so confirmation in blood or normal tissue is prudent, though EGFR exon 19 deletions in lung cancer are overwhelmingly somatic. The actionable conclusion is eligibility for an EGFR TKI.
Traps
Reporting a variant of uncertain significance as actionable. Ignoring tumor cellularity when a negative result is issued. Treating MSI by PCR and mismatch repair immunohistochemistry as unrelated assays when they are complementary.