Molecular Pathology: Assays and Clinical Meaning
Molecular Pathology tests nucleic-acid methods and their clinical use: PCR, reverse-transcription PCR, Sanger and next-generation sequencing, FISH, microsatellite instability and clonality. The exam emphasizes analytic validity and the difference between somatic and germline findings.
Core rules
- PCR amplifies DNA; contamination can cause false positives. RT-PCR detects RNA, including fusion transcripts and viral RNA.
- Sanger sequencing is useful for single genes; next-generation sequencing panels allow broad testing but require adequate tumor cellularity.
- FISH detects translocations, amplifications and deletions at chromosome level.
- Somatic mutations occur in tumor tissue; germline variants are inherited and may require genetic counseling.
- A variant of uncertain significance is not automatically pathogenic.
- Microsatellite instability reflects mismatch-repair deficiency and can predict immunotherapy response.
Worked example
A lung adenocarcinoma from a nonsmoking woman is tested by next-generation sequencing and shows an EGFR exon 19 deletion. This is a somatic activating mutation that predicts response to EGFR tyrosine kinase inhibitors. Reasoning: the result is actionable, but tumor cellularity and assay limits must be adequate. A germline EGFR variant would have different family implications and should not be assumed from tumor testing alone.
Traps
Do not treat a variant of uncertain significance as pathogenic. Low tumor cellularity can cause false-negative mutation results. PCR contamination can mimic mutation. A negative FISH result does not exclude a rearrangement outside the probe. Tumor-only testing may miss germline disease.