Pathology of Oropharyngeal Squamous Cell Carcinoma (OPSCC)

Two Distinct Disease Entities:
- OPSCC is now classified as two biologically and prognostically distinct diseases:
  - Based on high-risk HPV status:
    - HPV-associated (p16-positive) and HPV-independent (p16-negative) carcinoma:
      - A division formalized in the WHO classification and the AJCC 8th edition staging system
- HPV-associated tumors:
  - Carry a markedly better prognosis:
    - With 8-year overall survival of 70.9% versus 30.2% for HPV-negative disease in RTOG 0129:
      - HR 0.30; 95% CI 0.21–0.42
Histopathology:
- HPV-Associated (
- ) OPSCC:
  - These tumors arise from the specialized reticulated epithelium lining the tonsillar crypts rather than the surface mucosa
  - Characteristic features include:
    - Nonkeratinizing growth:
      - Lobules and sheets of immature basaloid cells with high nuclear-to-cytoplasmic ratios and hyperchromatic nuclei; absent or minimal keratinization
    - Basaloid cytology:
      - Resembling deep crypt epithelium, with lobular / endophytic growth and smooth pushing borders
    - Permeating tumor-infiltrating lymphocytes:
      - Within and around tumor nests
    - Absence of surface dysplasia:
      - Dysplasia is confined to crypts
    - Cystic nodal metastases:
      - Which can mimic branchial cleft cysts
  - In a 4-year prospective study of 435 OPSCCs:
    - Strictly defined nonkeratinizing morphology had a 99.1% positive predictive value for p16 positivity and 100% PPV for HR-HPV mRNA positivity
    - A “nonkeratinizing with maturation” (hybrid) intermediate pattern is p16-positive in ~92% of cases
- HPV-Independent (Keratinizing) OPSCC:
  - These resemble conventional keratinizing SCC of the upper aerodigestive tract:
    - With keratin pearl formation
    - Individual cell keratinization
    - Intercellular bridges
    - Progressive squamous maturation
    - Origin from dysplastic surface epithelium
  - Molecularly they are characterized by high rates of:
    - TP53 mutation
    - CCND1 (cyclin D1) copy-number gains
    - 9p21 / CDKN2A loss
A Note on Grading:
- Although HPV-associated tumors appear “poorly differentiated” by conventional criteria:
  - They paradoxically carry the best prognosis:
    - Westra has argued they may be best viewed as well-differentiated given their resemblance to native crypt epithelium
- Both the 2018 CAP guideline and its 2025 update recommend against providing traditional grade / differentiation status for HPV-associated OPSCC:
  - Instead recommend reporting the WHO histologic subtype
- Conventional grading (G1 to G4) still applies to HPV-independent OPSCC
- Certain morphologic features retain prognostic value even within p16-positive disease:
  - Tumor cell anaplasia (nuclei ≥ 5 lymphocyte diameters) and multinucleation:
    - Independently predicted worse disease-specific survival (HR 9.9 and 11.9, respectively), and “non-classic” HPV-positive morphology was associated with poorer 5-year survival (58.4% vs 83.9%)
  - Keratinization:
    - Independently predicts worse survival

The 2025 CAP update enumerates the recognized morphologic subtypes.

Histologic Variants:
- Basaloid SCC:
  - Is a mixed entity resolvable by HPV status:
    - HPV-positive:
      - Younger patients
      - p16+ / p53−
      - Prognosis – favorable
    - HPV-negative:
      - Older patients
      - Tobacco / alcohol-related
      - p53 overexpression
      - Biology – aggressive
- Lymphoepithelial-like carcinoma:
  - Requires mandatory dual HPV and EBV testing:
    - As it closely mimics metastatic nasopharyngeal carcinoma
HPV / p16 Testing:
- Who to Test:
  - All patients with newly diagnosed OPSCC should undergo HR-HPV testing, regardless of histologic subtype:
    - A strong CAP recommendation, endorsed by ASCO and listed as required in NCCN workup
  - Testing may be performed on the:
    - Primary tumor, a nodal metastasis, or FNA of a cervical node
  - Routine testing is not recommended for:
    - Nonsquamous oropharyngeal or non-oropharyngeal head and neck carcinomas (except sinonasal)
  - p16 IHC — the First-Line Test:
    - p16 immunohistochemistry is the recommended primary test:
      - Positivity requires:
        
        ≥ 70% nuclear AND cytoplasmic expression with at least moderate-to-strong intensity:
        
        Typically in a confluent / block-like pattern
    - The E6H4 clone is most widely used and validated
    - With these criteria:
      - Sensitivity for transcriptionally active HR-HPV approaches 100% with specificity of ~ 85% to 95% in the oropharynx
    - Cases with 50% to 70% staining are equivocal and warrant HPV-specific testing
    - p16 and HPV may be used interchangeably only within the oropharynx
  - HPV-Specific (Direct) Testing:
    - The 2025 CAP update expanded indications for confirmatory HPV-specific testing beyond p16 IHC:

HPV-specific testing should be performed in:
- Low-prevalence geographic regions
- Equivocal p16 staining
- Discrepancy between p16 and morphology (e.g., p16+ but keratinizing)
- Large multisite tumors
- Non-tonsillar / non-base-of-tongue subsites
- Clinical trials
- SCC of unknown primary
Discordant Results:
- p16+ / HPV− discordance occurs in ~ 4% to 20% of cases:
  - In the multinational HNCIG-EPIC-OPC analysis (n=7,654):
    - Discordant patients had intermediate prognosis:
      - Significantly worse than double-positive and better than double-negative: