Molecular Pathology Diagnostics
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Direct answer
A BCR-ABL1 transcript number now steers chronic myeloid leukaemia therapy the way a haemoglobin once steered transfusion — molecular pathology quantifies disease, predicts drug response, and detects residual clones below any microscope's threshold. The toolkit is ordered by question: PCR (including quantitative RT-PCR) quantifies fusion transcripts and mutations; FISH localises targets in tissue — break-apart probes for sarcoma translocations, dual-fusion for leukaemia; next-generation sequencing screens many genes at once; Sanger sequencing confirms single variants. Companion diagnostics have made molecular testing mandatory before prescribing — ATRA-defining PML-RARA in acute promyelocytic leukaemia, EGFR, ALK and ROS1 testing in lung adenocarcinoma, HER2 in breast and gastric cancer, BRAF in melanoma, and mismatch-repair deficiency with microsatellite instability for immunotherapy response.
What you must remember
- Translocation-transcript pairs to memorise: BCR-ABL1 p210 in CML (p190 in some Ph-positive ALL), PML-RARA t(15;17) in APL, RUNX1-RUNX1T1 t(8;21) and CBFB-MYH11 inv(16) in core-binding-factor AML, EWSR1-FLI1 t(11;22) in Ewing sarcoma, SYT-SS18 in synovial sarcoma, and ETV6-RUNX1 in childhood ALL.
- Quantitative monitoring: RT-PCR for BCR-ABL1 on the international scale defines milestones (10 per cent or lower at 3 months; 0.1 per cent, MR3, deeper), detects resistance — prompting kinase-domain testing, T315I the feared exit — and tracks molecular relapse before haematologic relapse.
- Companion diagnostic set: EGFR-sensitising mutations (exon 19 deletion, L858R) and ALK/ROS1 rearrangements before targeted therapy in lung adenocarcinoma; HER2 amplification (IHC 3+ or FISH) for trastuzumab; BRAF V600E for BRAF inhibitors in melanoma; KRAS G12C mutations now targetable in lung and colorectal cancer; JAK2 V617F calibrating myeloproliferative neoplasms.
- Immunotherapy predictors: mismatch repair protein loss (MLH1, MSH2, MSH6, PMS2) with microsatellite instability predicts checkpoint-inhibitor response; PD-L1 tumour proportionality scoring in non-small cell lung cancer, with 50 per cent the first-line monotherapy threshold.
- Infectious-disease molecular armamentarium: CBNAAT (Xpert MTB/RIF, and the more sensitive Ultra platform) detects Mycobacterium tuberculosis and rifampicin resistance within about two hours and is the first-line test under India's NTEP; viral load PCR guides HIV and hepatitis B and C management under the national programme.
- Technique-trait matching: FISH for structural rearrangements in archival tissue (ALK, HER2), PCR for known point mutations and fusions fast and cheap, NGS panels for broad tumour profiling, and array CGH for copy-number change.
- Liquid biopsy: circulating tumour DNA measured in plasma for EGFR T790M detection at progression, minimal residual disease assessment, and non-invasive tumour genotyping — a rising NEET-PG theme.
- Quality caveats: assays demand validated pre-analytics (fixation, tumour percentage); molecular classification of gliomas (1p/19q codeletion, MGMT methylation, IDH mutation) is now part of WHO grading itself.
Choosing the right molecular test
Match the clinical question to the platform. A young patient with a pelvic small blue cell tumour: EWSR1 break-apart FISH on the biopsy, which works even in decalcified or archived material. In metastatic lung adenocarcinoma, an NGS panel for EGFR, ALK, ROS1, BRAF, MET, RET and KRAS — each result rewrites the prescription. Monitoring CML on imatinib: quantitative PCR on the international scale every three months; a rising transcript triggers ABL kinase-domain sequencing to find the mutation dictating second-generation inhibitor choice. Managing APL: t(15;17) at diagnosis, then PCR for PML-RARA to document molecular clearance — the original molecular-cure story, with ATRA and arsenic driving the clone to extinction. Screening a colorectal cancer family: MMR-protein IHC first, then MLH1 promoter methylation to separate sporadic from Lynch, with germline sequencing to confirm.
How the exam frames it
One-liners test the pairing: drug with alteration — imatinib with BCR-ABL1, ATRA with PML-RARA, trastuzumab with HER2, vemurafenib with BRAF V600E, osimertinib with EGFR (including T790M), sotorasib with KRAS G12C. Monitoring questions ask what a rising BCR-ABL1 transcript means — loss of response, adherence problem, or resistance mutation — with kinase-domain sequencing as the next step. Technique questions contrast FISH and PCR: FISH sees structure in situ and tolerates poor tissue; PCR is faster and quantitative but needs known sequence and contamination control (amplicon carryover, dedicated areas, dUTP-UNG systems — a favourite). The Indian fixture is CBNAAT under NTEP — TB plus rifampicin resistance in about two hours; expect it whenever "rapid, molecular" appears.
Frequently asked questions
What is a major molecular response in CML and why does it matter?
A BCR-ABL1 transcript of 0.1 per cent or lower on the international scale, indicating deep suppression of the leukaemic clone and excellent long-term prognosis without progression.
Which molecular test is first-line for suspected tuberculosis under NTEP?
Cartridge-based nucleic acid amplification testing (Xpert MTB/RIF or Ultra), which simultaneously detects M. tuberculosis complex and rifampicin resistance within about two hours.
How do EGFR and ALK results change lung cancer treatment?
Sensitising EGFR mutations and ALK rearrangements mandate first-line tyrosine kinase inhibitors (erlotinib/osimertinib or crizotinib/alectinib generations) instead of chemotherapy — the archetype of companion diagnostics.
What is liquid biopsy and its main applications?
Analysis of circulating tumour DNA from a blood draw, used for genotyping when tissue is unavailable (EGFR T790M at progression), minimal residual disease monitoring, and tracking resistance mutations.
Why test mismatch repair proteins in colorectal carcinoma?
Loss of MLH1, MSH2, MSH6 or PMS2 identifies microsatellite-instability-high tumours — screening for Lynch syndrome and predicting strong response to PD-1 checkpoint immunotherapy.