10.3 Specialty Laboratory Testing Methods: IHC, FISH, PCR/RT-PCR, NGS, Flow Cytometry & Cytogenetics

Why lab methods are tested

The ODS outline lists "specialty lab testing methods (e.g., RT-PCR, FISH)." Registrars do not perform the tests, but they must recognize what a report means. For example, "HER2 IHC 2+, reflex FISH ratio 2.4" means HER2-positive. "BCR::ABL1 detected by RT-PCR" confirms CML. They must also know which registry item the result belongs in.

Tissue-based protein and DNA tests

Immunohistochemistry (IHC)

• How it works: antibodies tagged with a colored label bind to specific proteins in tissue sections, and the pathologist scores the percentage and intensity of stained cells.
• Uses:
  • Breast: ER and PR (reported as percent of nuclei stained; 1% or more is positive, and 1–10% is "low positive" under ASCO/CAP guidance), HER2 (scored 0, 1+, 2+ or 3+), Ki-67 proliferation index.
  • Lineage and site of origin: TTF-1 (lung and thyroid), CDX2 and CK20 (colorectal), CK7, PAX8 (kidney and Müllerian), GATA3 (breast and urothelial), PSA and NKX3.1 (prostate), S100 and SOX10 (melanoma), chromogranin and synaptophysin (neuroendocrine).
  • Mismatch repair (MMR) proteins MLH1, PMS2, MSH2 and MSH6: loss of staining means deficient MMR (dMMR).
  • PD-L1 (tumor proportion score or combined positive score) to predict response to immunotherapy.

In situ hybridization (ISH), including FISH

• How it works: labeled DNA probes bind matching DNA sequences in place in cells. FISH uses fluorescent probes; CISH and SISH use chromogenic or silver probes that can be read with a regular microscope.
• Uses: gene amplification, such as HER2 (ERBB2) in breast and gastric cancer, which is usually reported as a HER2/CEP17 ratio plus the average HER2 copy number. Gene rearrangements such as ALK and ROS1 in lung cancer, and MYC, BCL2 and BCL6 in lymphoma. Deletions such as 1p/19q codeletion in oligodendroglioma, and del(17p) in CLL and myeloma.
• Typical workflow: HER2 IHC 2+ (equivocal) leads to reflex ISH, and the ISH result decides the final HER2 status. IHC 3+ is positive, and 0 or 1+ is negative. HER2-low (1+, or 2+ with negative ISH) is a clinical category used for newer therapies.

Nucleic acid amplification and sequencing

PCR and RT-PCR

• Polymerase chain reaction (PCR) makes millions of copies of a targeted DNA sequence, so small mutations can be detected (for example, KRAS codon 12 or 13, BRAF V600E, EGFR exon 19 deletion or L858R). Quantitative (real-time) PCR also measures how much target is present.
• Reverse transcription PCR (RT-PCR) first converts RNA into complementary DNA with reverse transcriptase, then amplifies it. It detects gene expression or fusion transcripts. Examples:
  • BCR::ABL1 transcripts confirm CML (with the t(9;22) Philadelphia chromosome), and quantitative RT-PCR monitors treatment response.
  • The 21-gene recurrence score (Oncotype DX) measures RNA expression by RT-PCR to estimate breast cancer recurrence risk. AJCC uses it in prognostic staging.
  • PML::RARA in acute promyelocytic leukemia.
• MSI testing by PCR compares microsatellite lengths in tumor and normal tissue: MSI-H (high), MSI-L (low) or MSS (stable).

Next-generation sequencing (NGS)

• NGS sequences many genes, or whole exomes or genomes, at once. Panels detect point mutations, insertions and deletions, copy-number changes, fusions (with RNA-based NGS), tumor mutational burden (TMB) and MSI.
• Uses: lung cancer driver panels (EGFR, ALK, ROS1, BRAF, KRAS G12C, MET, RET, NTRK), colorectal (KRAS, NRAS, BRAF, MSI), and hereditary cancer germline panels (BRCA1/2, Lynch genes).
• Germline vs. somatic: germline testing of blood or saliva identifies inherited risk. Somatic testing of the tumor identifies acquired changes that guide treatment.

Liquid biopsy

• Circulating tumor DNA (ctDNA) in blood can reveal targetable mutations or residual disease. It is a biomarker test, not tissue confirmation for Diagnostic Confirmation.

Gene-expression profiling

• Multigene assays estimate prognosis. Examples are the 21-gene recurrence score (RT-PCR) and the 70-gene signature (MammaPrint, microarray) in breast cancer, and genomic classifiers in prostate cancer. These appear in the breast multigene signature SSDIs.

Cell-based and chromosome tests (mainly hematologic)

Where the results go in the registry

Quick contrasts

• IHC measures protein; FISH looks at DNA copies or rearrangements; RT-PCR detects RNA.
• Flow cytometry works on cells in suspension (blood, marrow, fluid); IHC works on fixed tissue sections.
• MMR IHC looks for missing proteins, MSI PCR or NGS looks for the DNA effect, and both identify the same biology (dMMR/MSI-H).