What are immunohistochemistry (IHC), cytoflowmetry (Flow Cytometry), and Fluorescence In Situ Hybridization (FISH) used for in cancer diagnosis?

Diagnostic Tests in Cancer: Immunohistochemistry, Flow Cytometry, and FISH

Immunohistochemistry (IHC)

Immunohistochemistry detects specific protein expression in tissue samples and serves as a primary screening tool for cancer diagnosis, classification, and treatment selection. 1

Core Applications

• Cancer classification and subtyping: IHC uses antibody panels to distinguish between different cancer types (e.g., p40 and TTF-1 to differentiate squamous cell carcinoma from adenocarcinoma in lung cancer) 2

• Biomarker assessment for targeted therapy: IHC detects protein overexpression that guides treatment decisions, most notably HER2 protein in breast cancer where 3+ staining (uniform intense membrane staining of >30% of invasive tumor cells) indicates eligibility for trastuzumab therapy 1, 3

• Hormone receptor status: IHC quantifies estrogen receptor (ER) and progesterone receptor (PR) expression to determine endocrine therapy eligibility in breast cancer 1

• Screening for genetic abnormalities: Novel IHC markers like panTRK antibodies can screen for gene fusions with 95-97% sensitivity and 98-100% specificity, serving as a rapid, cost-effective initial test before molecular confirmation 1, 4

Technical Interpretation

• Staining patterns reveal underlying biology: Membranous staining typically indicates receptor overexpression, nuclear staining suggests transcription factor activity or gene rearrangements, and cytoplasmic staining may indicate protein accumulation 4

• Scoring systems are standardized: HER2 IHC uses a 0-3+ scale where 0-1+ is negative, 2+ is equivocal (requires FISH confirmation), and 3+ is positive 1

Critical Limitations

• Interlaboratory variability is substantial: HER2 IHC concordance between laboratories ranges from 74-92%, with discordance rates of 8-26% 5

• Equivocal results (2+ staining) require molecular confirmation: Only 35% of IHC 2+ cases correlate with FISH positivity, making FISH mandatory for these cases before initiating targeted therapy 1, 6

• False results occur: IHC can show false positives (5-7%) and false negatives (<1%) even in quality-assured programs 1

Flow Cytometry (Cytoflowmetry)

Flow cytometry analyzes cell surface and intracellular markers on individual cells in suspension, making it essential for diagnosing hematologic malignancies and establishing clonality. 1

Primary Uses in Cancer Diagnosis

• Establishing clonality in lymphoproliferative disorders: Flow cytometry detects monoclonal B-cell populations by demonstrating kappa/lambda light chain restriction, which is diagnostic for chronic lymphocytic leukemia (CLL) when ≥5000 monoclonal B lymphocytes/mcL are present 1

• Immunophenotyping panels for classification: A standard CLL/SLL panel includes CD19, CD20, CD5, CD23, CD10, kappa/lambda, with the typical CLL phenotype being CD5+, CD10−, CD19+, CD20 dim, surface immunoglobulin dim, CD23+ 1

• Distinguishing between similar malignancies: Flow cytometry differentiates CLL from mantle cell lymphoma by demonstrating CD23 positivity and cyclin D1 negativity in CLL 1

• Detecting aberrant T-cell populations: Flow cytometry identifies immunophenotypically abnormal T-cells in lymphocyte-variant hypereosinophilic syndrome 7

Advantages Over Other Methods

• Rapid turnaround: Flow cytometry provides results within hours compared to days for tissue-based methods 1

• Requires minimal tissue: Peripheral blood or fine needle aspirate samples are often sufficient, avoiding invasive biopsies 1

• Quantitative analysis: Flow cytometry provides precise percentages of cell populations and marker expression intensity 1

Fluorescence In Situ Hybridization (FISH)

FISH detects specific DNA sequences and chromosomal abnormalities in both dividing and non-dividing cells, serving as the gold standard for confirming gene amplifications and translocations that guide cancer treatment. 1

Core Diagnostic Applications

• Confirming HER2 gene amplification: FISH is the definitive test for HER2 status in breast cancer, with positive defined as HER2/CEP17 ratio ≥2.2 or average HER2 gene copy number ≥6 signals/nucleus 1

• Detecting prognostic chromosomal abnormalities: In CLL, FISH identifies del(11q), del(13q), trisomy 12, and del(17p), which have major prognostic implications and guide treatment selection 1

• Confirming gene rearrangements: FISH detects specific translocations like ETV6-NTRK3 in infantile fibrosarcoma and congenital mesoblastic nephroma with high specificity 1

• Resolving equivocal IHC results: FISH is mandatory for all HER2 IHC 2+ breast cancers to determine true amplification status before initiating trastuzumab therapy 1

Technical Advantages

• Works on archived tissue: FISH can be performed on formalin-fixed, paraffin-embedded (FFPE) tissue, allowing retrospective analysis 1

• Analyzes non-dividing cells: Unlike conventional cytogenetics, FISH detects abnormalities in interphase nuclei, which is critical since many cancer cells have low proliferative activity 1, 8

• Higher concordance than IHC: FISH shows 97% concordance between central laboratories compared to 96% for IHC, with less variation in borderline cases 1

Important Limitations

• Cannot detect unknown fusion partners: Break-apart FISH probes (e.g., ETV6 FISH) confirm gene rearrangement but don't identify the fusion partner, which may be clinically relevant 1

• Variant translocation sites cause false negatives: Approximately 20% of NTRK fusions may be missed by FISH due to atypical breakpoints where probes fail to anneal 1

• Higher cost and longer turnaround: FISH is more expensive and takes longer than IHC, making it impractical as a first-line screening test 6

• Requires specialized expertise: Only approximately 500 laboratories in the United States perform HER2 FISH compared to 2,000 performing IHC, concentrating expertise in high-volume centers 1

Integrated Testing Algorithm

The optimal approach uses IHC for initial screening, followed by FISH for confirmation of equivocal results or when treatment decisions require definitive molecular evidence. 1

Recommended Testing Sequence

• Start with IHC for protein expression: IHC provides rapid, cost-effective screening with high negative predictive value for clearly negative (0-1+) and positive (3+) cases 1, 6

• Proceed to FISH for all equivocal IHC results: Any IHC 2+ result or cases with discordant clinical features require FISH confirmation before treatment decisions 1

• Use FISH as primary test in specific contexts: For HER2 testing in metastatic breast cancer or when IHC quality is questionable, FISH should be the initial test 1, 3

• Consider next-generation sequencing for comprehensive analysis: When multiple genetic alterations need evaluation (e.g., adenocarcinoma requiring EGFR, ALK, ROS1, BRAF testing), NGS may be more efficient than sequential single-gene FISH tests 1, 2

Critical Quality Control Considerations

• Retesting is necessary when results are discordant: Discordance between primary tumor and metastasis occurs in up to 50% of cases for HER2, mandating retesting of metastatic lesions 5

• Pre-analytical factors affect accuracy: Tissue fixation time and type significantly impact both IHC and FISH results; specimens should be fixed in neutral buffered formalin for 6-72 hours 1

• Heterogeneity requires adequate sampling: At least 20 non-overlapping cells in two separate areas should be counted for FISH, and IHC should evaluate multiple tumor areas since 4-5% of breast cancers show heterogeneous HER2 amplification 1, 5