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Molecular pathology — PCR, FISH, next-generation sequencing and liquid biopsy
The molecular test is chosen by the type of change sought — point mutation, amplification, translocation or instability — and by the type of specimen; and specimen quality (tumour-cell percentage, fixation, decalcification) decides whether the result will be reliable.
Why it matters in the exam
The bank asks which method suits which question and when RNA is preferred. PCR amplifies a defined sequence and suits known mutations and clonality testing (rearrangement of IGH and TCR genes when lymphoma is suspected). RT-PCR starts from RNA and suits detection and quantification of fusion transcripts, such as BCR-ABL1 in monitoring chronic myeloid leukaemia; RNA is also preferred for detecting fusions in genes with many breakpoints and for measuring expression. FISH shows amplification (HER2) and gene break-apart (ALK, MYC, BCL2) in intact cells, and is also used for 1p/19q codeletion in oligodendroglioma.
Next-generation sequencing (NGS) tests at once point mutations, small deletions and insertions, copy-number changes, fusions, tumour mutational burden and MSI, so it is the choice in advanced lung cancer, colon cancer and rare tumours. Liquid biopsy measures cell-free tumour DNA in blood (ctDNA): an EGFR mutation in the blood of a former smoker with a lung mass suggests lung cancer, and the method is also used to follow resistance (T790M); a negative result does not exclude, because the tumour may not shed enough DNA.
Quality: the pathologist marks an area with a sufficient percentage of tumour cells; prolonged formalin fixation fragments DNA; decalcification with strong acid destroys nucleic acids, so EDTA is used for bone. It is important to distinguish a somatic change (in the tumour only) from an inherited (germline) change, which requires genetic counselling. In inherited testing there are situations in which a deletion test is negative and the diagnosis comes from methylation testing — for example Prader-Willi syndrome from uniparental disomy.
Key points
- The test is chosen by type of change and specimen.
- RT-PCR from RNA for fusion transcripts and quantification.
- FISH for amplification and gene break-apart.
- NGS for a broad panel; ctDNA for blood.
- Tumour-cell percentage, fixation and EDTA determine quality.
Summary table
| Method | What it detects | Example | Limitation |
|---|---|---|---|
| PCR | Known mutation, clonality | KRAS, IGH and TCR rearrangement | Only what is sought |
| RT-PCR (from RNA) | Fusion transcripts, expression | BCR-ABL1 quantification | RNA degrades easily |
| FISH | Amplification, break-apart, deletion | HER2, ALK, 1p/19q | Few genes per test |
| NGS | Mutations, fusions, mutational burden, MSI | Advanced lung cancer | Needs quantity and quality |
| Liquid biopsy | ctDNA in blood | EGFR, T790M | Negative does not exclude |
A free exam question
מוטציה מסוג MDM2 :אופיינית לגידול (the question as examined, in Hebrew)
- א. Myxoid liposarcoma
- ב. Pleomorphic liposarcoma
- ג. Well differentiated liposarcoma
- ד. Lipomatosis
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Sources
- Association for Molecular Pathology / CAP / ASCO: Standards and guidelines for interpretation and reporting of sequence variants in cancer, 2017 — Somatic variant tiers
- CAP/IASLC/AMP Guideline: Molecular testing for selection of patients with lung cancer for targeted therapy, 2018 — Specimen requirements and methods
- Robbins and Cotran Pathologic Basis of Disease, 10th edition (2021) — Genetic and pediatric diseases — molecular diagnosis
Sources are given for further reading; the text is written afresh and not copied from them.
Reviewed by the Examqo AI Professor · 2026-11-06. The text may contain errors; when in doubt, check the source.