Interpreting Immunohistochemistry

1. Clinical question — Tumor typing, site-of-origin determination, prognostic marker assessment, predictive marker testing, or mismatch repair evaluation? Default: tumor classification.
2. Histologic context — What is the morphologic differential diagnosis from H&E evaluation? Default: undifferentiated malignancy, NOS.
3. Panel design — What antibodies are being ordered and why? Default: per institutional reflex panel for the differential.
4. Specimen type — FFPE biopsy, resection, cell block, cytology smear, or decalcified bone specimen? Default: FFPE biopsy.
5. Prior IHC — Are there previous IHC results from outside institutions? Default: none on file.
6. Tissue availability — Is tissue sufficient for the planned panel, or must the panel be prioritized? Default: sufficient tissue.
7. Scoring system — Which quantitative scoring system applies (Allred, H-score, percentage, 0-3+ scale)? Default: per marker-specific ASCO/CAP guidelines.

Documents to Request

• H&E stained sections with morphologic differential diagnosis
• Prior IHC and special stain results
• Clinical notes including primary site, treatment history, and imaging
• Antibody validation records for each marker in the panel
• External positive and negative control documentation
• Manufacturer package inserts for antibody clones used
• CAP/NordiQC proficiency testing results for each antibody

Step 1: Panel Design — Systematic Approach

Design the IHC panel based on the morphologic differential diagnosis. Use an evidence-based, stepwise approach:

Carcinoma of Unknown Primary (CUP) — First-Line Panel

Second-Line Organ-Specific Markers

Melanoma vs. Carcinoma vs. Lymphoma

Step 2: Predictive Marker Scoring — ASCO/CAP Guidelines

Score predictive markers using the ASCO/CAP-mandated systems:

ER and PR (Breast Cancer) — ASCO/CAP 2020

• Positive: >= 1% of tumor nuclei staining at any intensity
• Low positive: 1-10% (report separately; clinical benefit of endocrine therapy uncertain in this range)
• Negative: < 1% staining
• Report: Percentage of positive nuclei and predominant intensity (weak, moderate, strong)

HER2 (Breast Cancer) — ASCO/CAP 2023

HER2-low: IHC 1+ or IHC 2+/FISH non-amplified. Relevant for trastuzumab deruxtecan (T-DXd) eligibility.

PD-L1 Scoring

Step 3: Mismatch Repair (MMR) and Microsatellite Instability

Interpret the four-antibody MMR panel:

Scoring rule: Internal positive control (stromal cells, lymphocytes, normal epithelium) MUST show nuclear staining for the result to be valid. If internal controls are negative, the stain is invalid and must be repeated.

Step 4: Quality Control and Validation

Verify IHC quality before interpretation:

• External positive control: Tissue with known expression of the target antigen included on the same slide or in the same run.
• External negative control: Same tissue processed without primary antibody or with isotype control.
• Internal controls: Endogenous cell populations expected to express (positive) or not express (negative) the antigen serve as built-in quality indicators.
• Staining quality: Assess for background, edge artifact, necrosis artifact, and fixation artifact. Under-fixed tissue (< 6 hours in 10% NBF) may produce false-negative results for ER, PR, HER2.
• Fixation time: ASCO/CAP requires 6-72 hours in 10% neutral buffered formalin for breast biomarkers.

Step 5: Report Construction

Structure the IHC interpretation report:

1. Antibody panel: List each antibody with clone, vendor, and result.
2. Scoring: Apply appropriate scoring system for each predictive marker.
3. Interpretation: State the immunophenotypic profile and the favored diagnosis.
4. Differential diagnosis refinement: Explain how IHC results narrow or confirm the differential.
5. Discordance notes: If IHC results are unexpected for the morphologic diagnosis, discuss possible explanations (aberrant expression, fixation artifact, sampling).
6. Recommendations: Suggest additional markers if the panel is inconclusive.

Checkpoint B: Post-Draft Alignment (Mandatory)

1. Are all predictive markers (ER, PR, HER2, PD-L1) scored using the ASCO/CAP-mandated system?
2. Are external and internal controls valid for each antibody in the panel?
3. Does the IHC panel logically address the morphologic differential diagnosis?
4. Is the MMR panel result correctly interpreted with internal control validation?
5. Are fixation time requirements met for breast biomarkers (6-72 hours per ASCO/CAP)?

Quality Audit

• Each antibody validated per CAP IHC.25100 requirements
• External positive and negative controls included and valid
• Internal positive controls present and staining appropriately
• Antibody clone and vendor documented for each marker
• ER/PR scored per ASCO/CAP 2020 guidelines with percentage and intensity
• HER2 scored per ASCO/CAP 2023 four-tier system
• HER2 equivocal (2+) reflexed to FISH per protocol
• PD-L1 scored with correct system (TPS, CPS, IC) for the tumor type and assay
• MMR panel interpreted with internal control validation
• Fixation time documented and within ASCO/CAP requirements for predictive markers
• Proficiency testing participation current (CAP Surveys, NordiQC)
• Staining artifacts (background, edge, necrosis) addressed in interpretation
• Report includes antibody clone, scoring system, and interpretation for each marker

Guidelines

• Always design the IHC panel based on the morphologic differential diagnosis from H&E; avoid shotgun panels that waste tissue and introduce interpretive noise
• Score ER, PR, and HER2 strictly per the current ASCO/CAP guidelines; no institutional modifications to the scoring criteria are permitted for these predictive markers
• Report HER2-low status (IHC 1+ or 2+/FISH non-amplified) explicitly, as it now affects therapy eligibility for T-DXd
• Validate the internal positive control before interpreting any IHC stain as negative; if internal controls fail, repeat the stain before reporting
• For CUP workup, use a stepwise panel approach starting with CK7/CK20 and broad markers before adding organ-specific antibodies
• Never diagnose melanoma on a single marker; require positivity for at least two melanocytic markers (S100, SOX10, Melan-A, HMB-45)
• When MMR loss involves MLH1/PMS2, always recommend reflex testing for BRAF V600E and/or MLH1 promoter methylation to distinguish sporadic from Lynch syndrome
• Document the specific antibody clone used for every marker, as different clones may have different performance characteristics (e.g., SP1 vs. 1D5 for ER)