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IHC in Pathology: Recent Advances - A Comprehensive Review

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1. IHC for Carcinoma of Unknown Primary (CUP)

The CK7/CK20 Framework (First Step)

Expanded IHC Panel by Suspected Primary (Table 97-2, Harrison's 22E, 2025)

• GATA3 is a newer, highly sensitive marker for breast and urothelial carcinomas
• NKX3-1 has replaced or supplements PSA for prostate - more specific
• PSMA (prostate-specific membrane antigen) is increasingly used for prostate CUP
• INSM1 is a newer neuroendocrine marker (nuclear transcription factor), more sensitive than chromogranin in poorly preserved specimens
• Molecular profiling (RNA-seq/CGP) is now recommended alongside IHC per the 2025 SEOM-GECOD guidelines; the CUPISCO trial (Lancet Oncol 2024) showed survival benefit when NGS guides site-specific treatment in CUP
• IHC alone is insufficient to guide site-specific treatment - results are "suggestive" not "definitive"

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2. IHC for Lymphoma

Step 1: Lineage Determination

Step 2: B-Cell Lymphoma Subclassification

• GCB type: CD10+ (>30%) OR CD10-/BCL6+/MUM1- → better prognosis
• Non-GCB: MUM1+ → worse prognosis, responds differently to treatment

Step 3: T-Cell/NK Lymphoma Subclassification

• SOX11: Useful marker for cyclin D1-negative MCL; SOX11-negative MCL tends to be more indolent
• BRAF V600E IHC: Now used as a surrogate for mutation testing in hairy cell leukemia and some follicular lymphomas
• PD-L1 IHC: Increasingly important for DLBCL, primary mediastinal B-cell lymphoma, and Hodgkin lymphoma - guides checkpoint inhibitor eligibility
• MYC/BCL2/BCL6 double/triple-hit: IHC for MYC (>40%), BCL2 (>50%) triggers FISH confirmation; "double expressor" lymphomas have poorer prognosis
• EBER in situ hybridization (not strictly IHC but part of the panel) is mandatory for NK/T-cell and EBV-associated lymphomas
• IRF4/MUM1: Distinguishes GCB from non-GCB subtypes of DLBCL

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3. IHC for Metastatic Tumors

General Principles

• No single marker is 100% sensitive or specific - panels outperform individual markers
• TTF-1 stains ~75% of pulmonary adenocarcinomas but also stains thyroid carcinoma and occasionally ovarian tumors
• Organ-specific markers (PSA for prostate, thyroglobulin for thyroid) are highly specific but these rarely present as CUP

Common Differential Diagnoses and IHC

• PAX8: One of the most useful newer markers - positive in renal, thyroid, ovarian/mullerian, and thymic tumors
• CAIX (carbonic anhydrase IX): Highly specific for clear cell RCC metastases
• SATB2: Highly specific for colorectal and appendiceal origin; supplements CDX-2
• NKX3-1: Now the preferred prostate-specific marker - more sensitive than PSA in poorly differentiated metastatic prostate carcinoma
• INSM1: Better neuroendocrine marker in small crushed biopsies vs chromogranin/synaptophysin
• Claudin-18.2: Emerging marker for gastric/GEJ adenocarcinoma metastases - also a therapeutic target (zolbetuximab)
• TRPS1: A newer, highly sensitive marker for breast origin (more sensitive than GATA3 in some studies)

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4. IHC for Breast Carcinoma

Routine Therapeutic Markers (Mandatory in all invasive breast carcinoma)

Molecular Subtype Classification by IHC Surrogates

Additional Diagnostic/Subclassification Markers

• HER2-low (IHC 1+ or 2+/ISH-) is now a distinct therapeutic category - eligible for trastuzumab deruxtecan (T-DXd); this has redefined HER2 reporting
• HER2-ultralow (IHC >0 and <1+): Emerging category with potential T-DXd benefit (under investigation)
• TRPS1: Newer breast marker with high sensitivity, even in triple-negative cases
• BIRD3/LRRC15: Emerging markers for stromal biology in TNBC
• PIK3CA IHC surrogates: Being investigated as predictors of alpelisib response

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5. IHC for Lung Tumors

Adenocarcinoma vs. Squamous Cell Carcinoma (Core Panel)

• Adenocarcinoma → molecular testing for EGFR, ALK, ROS1, KRAS, BRAF, MET, RET, NTRK, PD-L1
• SCC → immunotherapy assessment; bevacizumab is contraindicated

Neuroendocrine Tumors (NET) Panel

• INSM1 is now recommended as a supplementary neuroendocrine marker; it is nuclear, works well in crushed small biopsies where cytoplasmic markers are lost
• Ki-67 is used as a surrogate for mitotic counting in small biopsies

Other Lung Tumor IHC

• PD-L1 (22C3 clone): Mandatory for all advanced NSCLC; TPS ≥50% = pembrolizumab monotherapy eligible; TPS ≥1% = combination eligible
• ALK (D5F3 clone): IHC now accepted as a standalone test without mandatory FISH confirmation in many centers
• ROS1 IHC (D4D6 clone): Screening marker - positive cases confirmed by FISH/NGS
• INSM1: Added to 2021 WHO-recommended neuroendocrine panel
• SMARCA4/SMARCB1 loss: IHC markers for rare undifferentiated thoracic malignancies
• Claudin-18: In lung adenocarcinoma subtyping and as a potential therapeutic target
• HER2 IHC: Being validated for lung adenocarcinoma (as in breast) - trastuzumab deruxtecan now approved for HER2-mutant NSCLC

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6. IHC for Undifferentiated Tumors

Step 1: The "Big Five" Lineage Screen

Initial screen → broad keratin (AE1/AE3) + LCA (CD45) + S100 + vimentin
                ↓                    ↓              ↓
           Carcinoma            Lymphoma         Melanoma/sarcoma
             panel                panel              panel

Step 2: After Lineage Confirmation

Recent Advances for Undifferentiated Tumors

• INI1 (SMARCB1) loss: Key marker for malignant rhabdoid tumor, epithelioid sarcoma, poorly differentiated chordoma, some renal medullary carcinoma - nuclear loss is highly specific
• SMARCA4 (BRG1) loss: SMARCA4-deficient undifferentiated carcinoma, thoracic sarcoma; increasingly recognized entity
• NUT IHC: Pathognomonic speckled nuclear pattern for NUT (NUTM1-rearranged) carcinoma - any undifferentiated midline carcinoma in young patients
• INSM1: Superior neuroendocrine marker for crush artifact specimens; better retained in undifferentiated neuroendocrine carcinomas
• TFE3 IHC: For alveolar soft part sarcoma and Xp11 translocation RCC in young patients
• TRPS1: Emerging breast lineage marker
• Claudin-4: Pan-epithelial marker useful when AE1/AE3 is equivocal
• p40 vs p63: p40 (ΔNp63) is now preferred over p63 for squamous identification as it is more specific (p63 is positive in myoepithelial, prostate basal cells)
• SOX10: More specific than S100 for melanoma; also positive in nerve sheath tumors

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Summary Table: "Signature" IHC Markers by Context (2025)

Key principle across all 6 categories: IHC results indicate a "suggestive primary profile" - no panel is 100% sensitive or specific. Pattern recognition using coexpression of multiple markers is more reliable than any single marker. Per the 2025 SEOM-GECOD and NCCN guidelines, tissue conservation for NGS/molecular profiling is now a priority alongside IHC, particularly in CUP and undifferentiated tumors.