Immunohistochemistry and Molecular Pathology
Immunohistochemistry
IHC depends on preanalytical factors: fixation time, antigen retrieval, antibody clone, and titration. Always run positive and negative controls. Antibodies are used in panels, not singly. Cytokeratins mark epithelial cells: CK7/CK20 patterns help localize carcinomas - CK7+/CK20− suggests lung, breast, or thyroid; CK7−/CK20+ suggests colorectal. TTF-1 marks lung adenocarcinoma and thyroid; p63/p40 marks squamous differentiation; synaptophysin and chromogranin mark neuroendocrine tumors; CD45 marks leukocytes; CD20 B cells; CD3 T cells; CD30 Reed–Sternberg cells; CD138 plasma cells.
Therapeutic markers: ER and PR in breast cancer (nuclear, percentage and intensity), HER2 (IHC 0, 1+, 2+, 3+; 2+ requires in situ hybridization), Ki-67 proliferation index, mismatch repair proteins (MLH1, PMS2, MSH2, MSH6) with loss indicating MSI-high, and PD-L1 scoring.
Molecular pathology
Techniques include PCR, Sanger sequencing, FISH, and next-generation sequencing. Key targets: EGFR, ALK, ROS1, BRAF V600E, KRAS, HER2 amplification, BCR-ABL1, JAK2 V617F. Preanalytical variables (necrosis, decalcification, low tumor cellularity) cause false negatives.
Traps
Questions ask "most sensitive" versus "most specific," or which marker confirms origin. Remember HER2 2+ is equivocal, not positive; ER positivity is not all-or-none.
Rule of thumb: build a panel around the differential, not a single stain.
Worked example: a metastatic adenocarcinoma of unknown primary with CK7+/CK20− and TTF-1+ favors a lung primary.