Study Notes: Quick Reference Handbook for Surgical Pathologists

General Introduction to Immunohistochemistry (IHC)

  • Definition & Fundamental Principles: IHC is the process of detecting antigens (e.g., proteins) in cells of a tissue section by exploiting the principle of antibodies binding specifically to antigens in biological tissues.
  • Applications in Anatomic Pathology:
    • Tumor Diagnosis: Categorizing poorly differentiated tumors into broad categories. Carcinoma (Cytokeratin+), Lymphoma (CD45+), Melanoma (SOX10/S100+).
    • Carcinoma of Unknown Primary (CUP): Identifying the origin site. Example: Lung (TTF-1+), Prostate (PSA+), Colon (CDX2+).
    • Diagnosis of Invasion: Identifying the loss of specific cell layers. Breast cancer shows a loss of myoepithelial cells; prostate cancer shows a loss of basal cells.
    • Prognostication: Markers like Ki67/MIB1 (proliferation) and HER2 (adverse prognosis in breast/gastric cancer).
    • Predictive Markers for Therapy: Markers that dictate specific treatments. Example: ER/PR for Tamoxifen; HER2 for Herceptin; PD-L1 for checkpoint inhibitors.
    • Detection of Micrometastases: Using CK for breast cancer or melanocytic markers for melanoma in lymph nodes.
    • Infectious Diseases: Identifying viruses (CMV, HSV, Adenovirus), bacteria (H. pylori, Syphilis), or protozoa (Toxoplasma).

Antigen Localization

  • Nuclear Reactivity: Typical for transcription factors and steroid hormone receptors. Examples include TTF-1, CDX2, Myogenin, PAX8, WT1, p53, p40/p63, ER, PR, and AR. Proliferation marker Ki67 is also nuclear.
  • Cytoplasmic Reactivity: Most common localization. Includes cytoskeletal/intermediate filaments (CK, Desmin, Vimentin), contractile proteins (Actins), and secretory products (ACTH, Trypsin).
  • Membranous Reactivity: Expected for surface receptors and adhesion molecules. Includes HER2, EGFR, E-cadherin, and CD antigens (CD3, CD20, CD34).
  • Granular Reactivity: Indicates localization to organelles. Granular cytoplasmic reactivity seen in AMACR (mitochondria), HepPar1 (mitochondria), and Napsin A (lysosomal).
  • Nuclear and Cytoplasmic: Rare. Includes S100, p16, and Calretinin. β\beta-catenin moves from membranous to nuclear in specific WNT-pathway mutated tumors (e.g., desmoid tumor).
  • Punctate/Perinuclear Dot-like: Characteristic of Keratins in neuroendocrine carcinomas (Small cell and Merkel cell).

Epithelial Markers and Cytokeratins (CK)

  • Moll’s Catalogue: System for designating the 2020 types of cytokeratins based on migration in 2D gel electrophoresis.
  • Categories:
    • Type II (Basic): CK1 through CK8.
    • Type I (Acidic): CK9 through CK20.
  • Weight-Based Categorization:
    • High Molecular Weight (HMWCK): Expressed in squamous epithelia and basal cells (CK1, 5, 10, 14). Antibodies include 34β\betaE12 and CK5/6.
    • Low Molecular Weight (LMWCK): Found in simple glandular epithelia and visceral organs (CK7, 8, 18, 19). Main antibody: Cam5.2.
  • Diagnostic Cocktails:
    • AE1/AE3 (Pan-CK): Broad-spectrum, detects most acidic and basic keratins. Exceptions: HCC and some high-grade NE carcinomas are often negative.
    • Cam5.2: Excellent for LMWCK; often labels HCC when AE1/AE3 fails.
  • Major Specific Markers:
    • CK7 and CK20 Patterns:
      • CK7+/CK20-: Lung, Breast, Thyroid, GYN tract (Ovary/Uterus).
      • CK7-/CK20+: Colorectum, Merkel cell carcinoma.
      • CK7+/CK20+: Pancreaticobiliary, Stomach, Urothelium.
      • CK7-/CK20-: Liver (HCC), Kidney (RCC), Prostate.
    • EMA (MUC1): Labels most non-squamous carcinomas; helpful in identifying RCC and Meningioma.
    • Ber-EP4 & MOC31: Distinguishes adenocarcinoma (+) from mesothelioma (-).
    • Claudin-4: Currently considered the best pan-carcinoma marker to distinguish from mesothelioma.

Specialized Differentiation Markers

  • Muscle Markers:
    • Pan-Muscle: Desmin and Muscle Specific Actin (MSA).
    • Smooth Muscle: SMA, Calponin, h-Caldesmon, SMMHC.
    • Striated (Skeletal) Muscle: Myogenin, MyoD1 (both nuclear), α\alpha-sarcomeric actin.
  • Neuroendocrine (NE) Markers:
    • Synaptophysin (SYN): Most sensitive granular cytoplasmic marker.
    • Chromogranin (CHR): Most specific; marks neurosecretory granules.
    • CD56 (NCAM): Membrane marker; useful in Small Cell Carcinoma (SmCC) when others are negative.
    • INSM1: Modern nuclear marker for neuroendocrine differentiation.
  • Melanocytic Markers:
    • SOX10: Highly sensitive for all melanoma types including desmoplastic; nuclear.
    • S100: "Stains 100 things"; very sensitive but low specificity.
    • HMB45 & Melan-A: Specific for epithelioid melanoma; usually negative in desmoplastic melanoma.
  • Vascular Markers:
    • ERG: Modern nuclear marker; high sensitivity and specificity for endothelial differentiation.
    • CD31 & CD34: Membrane/cytoplasmic markers. CD34 is non-specific (stains SFT, GIST, DFSP).

Predictive and Prognostic Indices

  • Proliferation (Ki67/MIB1): Assessed as a percentage of positive nuclei. Crucial for grading neuroendocrine tumors and breast cancer.
  • p53 Interpretation:
    • Wild-type: Scattered, weak nuclear positivity.
    • Mutation-type: Diffuse/strong "block" staining or complete "null" phenotype (loss of all staining).
  • HER2 (c-erbB-2):
    • Breast Score 3+ (Positive): Strong, complete membranous staining in >10%>10\% of cells.
    • Breast Score 2+ (Equivocal): Weak to moderate complete membranous staining in >10%>10\% of cells; requires FISH.
    • Gastroesophageal: Criteria vary between biopsy and resection; focuses on cluster staining.
  • DNA Mismatch Repair (MMR):
    • Proteins: MLH1, PMS2, MSH2, MSH6.
    • Screening: Used for Lynch Syndrome. Loss of MLH1/PMS2 usually implies sporadic hypermethylation (check BRAF in CRCs), whereas loss of MSH2/MSH6 or isolated PMS2/MSH6 strongly implies germline mutation.

Tumor Grading Systems (Solid Tumors)

  • Adenocarcinoma (GI): Based on gland formation. Grade 1 (Well): >95%>95\% glands; Grade 2 (Mod): 50−95%50-95\%; Grade 3 (Poor): <50%<50\%.
  • Nottingham (Elston-Ellis) Grade for Breast Cancer:
    • Sum of scores from 1-3 for: (1) Tubule formation, (2) Nuclear pleomorphism, (3) Mitotic rate.
    • Grade I: 3−53-5 pts; Grade II: 6−76-7 pts; Grade III: 8−98-9 pts.
  • Gleason Grading for Prostate Cancer:
    • Pattern 3: Distinct, separate glands.
    • Pattern 4: Fused, ill-defined, or cribriform glands.
    • Pattern 5: Sheets, cords, or necrosis.
    • Prognostic Groups: Group 1 (GS ≤6\le 6), Group 2 (GS 3+4=73+4=7), Group 3 (GS 4+3=74+3=7), Group 4 (GS 88), Group 5 (GS 9−109-10).
  • Fuhrman/ISUP Grading for RCC: Based on nucleolar prominence (at 10x10x, 20x20x, or 40x40x magnification).
  • Sarcoma Grading (FNCLCC): Based on (1) Differentiation (1−31-3), (2) Mitotic count (1−31-3), (3) Necrosis (0−20-2).

Hematopathology IHC and Classification

  • B-cell Markers: CD19, CD20, PAX5, CD79a.
  • T-cell Markers: CD2, CD3 (most specific), CD5, CD7, CD43.
  • Germinal Center Markers: CD10, BCL6.
  • Plasma Cell Markers: CD138, CD38, MUM1, Light chains (κ/λ\kappa/\lambda).
  • Blast Markers: CD34, TdT.
  • Key Classification Criteria (WHO 2016 Update):
    • Follicular Lymphoma: Graded by centroblast count per HPF (G1:0−5G1: 0-5; G2:6−15G2: 6-15; G3:>15G3: >15).
    • Plasma Cell Myeloma: Requires ≥10%\ge 10\% plasma cells and CRAB criteria (Calcium elevation, Renal insufficiency, Anemia, Bone lesions).
    • MDS: Classified by the number of dysplastic lineages and blast percentage (<5%<5\% vs 5−9%5-9\% vs 10−19%10-19\%).

Tumor Syndromes and Metastases

  • Hereditary Syndromes Clues: Early age of onset, multifocal/bilateral disease, synchronous tumors, unusual histology (e.g., medullary thyroid CA in MEN2).
  • Metastasis Rules: Carcinomas usually spread via lymphatics (Exc: RCC, Follicular Thyroid). Sarcomas spread hematogenously (Exc: Clear cell sarcoma, Angiosarcoma, Synovial Sarcoma).
  • Bone Metastases Mnemonic: "BLT and Kosher Pickle" - Breast, Lung, Thyroid, Kidney (RCC), Prostate.