Molecular Testing of HPV / Detection in Cervical Cytology and Tissue

1. Introduction

2. Cytologic Detection - Pap Smear and Morphological Clues

• LSIL (Low-grade squamous intraepithelial lesion) - corresponds to CIN 1; shows koilocytic atypia (perinuclear halo, nuclear enlargement, nuclear irregularity) - the classic cytopathic effect (CPE) of productive HPV infection.
• HSIL (High-grade squamous intraepithelial lesion) - corresponds to CIN 2/3.
• ASC-US (Atypical squamous cells of undetermined significance) - does not fulfil all criteria for LSIL/HSIL; reflex HPV testing is indicated.
• ASC-H - cannot exclude HSIL.

• CIN 1: Viral CPE (koilocytosis) + dysplasia confined to lower 1/3 of epithelium.
• CIN 2: Dysplasia in lower 2/3 of epithelium.
• CIN 3 (CIS): Full-thickness dysplasia.

3. Molecular Tests for HPV Detection

• Nucleic acid target: DNA vs. mRNA (E6/E7 transcripts)
• Technology: nucleic acid amplification (PCR, TMA), signal amplification (hybrid capture, invader chemistry), or direct detection (ISH)
• Genotyping capability: broad high-risk group vs. individual genotype identification

A. Signal Amplification Methods

1. Hybrid Capture 2 (HC2) - Digene/Qiagen

• One of the earliest FDA-approved HPV assays.
• Principle: Uses RNA probes complementary to HPV DNA. RNA:DNA hybrids are captured by antibodies on a microplate and detected by chemiluminescence.
• Detects 18 high-risk genotypes as a group, without individual differentiation.
• Limitation: probe-based (not PCR-based), lacks an internal specimen adequacy control - inadequate specimens may falsely read as negative; slightly lower sensitivity compared to PCR-based assays.
• Approved collection media: SurePath and ThinPrep.

2. Cervista HPV HR / 16-18 (Hologic) - Invader Chemistry

• Uses the Third Wave Invader assay technology (isothermal, signal amplification).
• Cervista HR detects 14 hrHPV types without differentiation.
• Cervista 16/18 specifically reports genotypes 16 and 18 if present.
• Approved for ThinPrep specimens only.

B. Nucleic Acid Amplification Tests (NAATs) - DNA-Based

3. Cobas HPV Test (Roche) - Real-time PCR

• Targets HPV L1 gene and uses PCR with fluorescent probes.
• Detects 14 high-risk genotypes; specifically differentiates HPV 16 and HPV 18 (others reported as "HR HPV detected").
• Approved for primary screening (first FDA-approved for this indication in the USA).
• Approved for SurePath and ThinPrep.

4. BD Onclarity HPV Assay (BD Diagnostics) - Real-time PCR

• Targets E6 and E7 genes; detects 17 hrHPV genotypes.
• Specifically differentiates HPV 16, 18, and 45 - the three highest-risk genotypes.
• Also approved for primary screening.
• Approved for SurePath only.

C. mRNA-Based Assay - Transcription Mediated Amplification (TMA)

5. Aptima HPV Assay (Hologic)

• Detects E6/E7 oncogene mRNA transcripts rather than DNA.
• Key advantage: mRNA is produced only during active (transcriptionally active) infection. Large amounts of E6/E7 mRNA are not present during viral latency, so this assay is more specific for clinically relevant, biologically active infection.
• Detects 14 hrHPV types; does not differentiate them individually.
• A paired Aptima HPV 16 18/45 Genotype Assay is performed reflex on positive samples to differentiate HPV 16 and 18/45.
• Approved for ThinPrep specimens only.

Summary Table: FDA-Cleared HPV Molecular Assays

4. HPV Detection in Tissue Biopsies

A. In Situ Hybridization (ISH) - DNA ISH

• A labeled DNA probe with a sequence complementary to the target HPV gene (typically the L1 gene) is applied to formalin-fixed, paraffin-embedded (FFPE) tissue sections.
• The probe hybridizes to complementary HPV DNA within the tumor cell nucleus.
• The chromogen label allows visualization by light microscopy - a positive result appears as a blue/brown dot in the nucleus.
• Staining patterns:
  • Punctate (dot-like) pattern: suggests integrated HPV DNA.
  • Diffuse pattern: suggests episomal (non-integrated) HPV.
• Commercial kits are available for high-risk HPV genotypes in FFPE tissue.
• Allows precise localization of HPV DNA to the host cell nucleus.
• Limitation: does not prove transcriptional activity (biological activity) of the virus.

B. Polymerase Chain Reaction (PCR) on Tissue

• Highly sensitive; can amplify a single copy of the target sequence.
• After DNA denaturation, specific primers bind to the E6 or E7 gene (or the L1 gene for consensus PCR) and amplify the target.
• Consensus PCR: detects presence of HPV DNA broadly.
• Real-time PCR (qPCR): quantifies viral load.
• Can be performed on FFPE tissue, though best results are on fresh tissue.
• Limitation: detects DNA only - cannot confirm active viral transcription.

C. Tests to Determine Biological Activity (Transcriptional Activity)

1. Quantitative Reverse Transcription PCR (qRT-PCR)

• Directly detects E6 and E7 mRNA transcripts.
• cDNA is first synthesized from mRNA (reverse transcription), then amplified by PCR using E6/E7-specific primers.
• Current gold standard for confirming HPV biological activity (oncogenic transcription).
• Costly, labor-intensive, traditionally restricted to fresh/frozen tissue.

2. RNA In Situ Hybridization (RNA-ISH) / Chromogenic ISH (CISH)

• Detects E6/E7 mRNA transcripts directly in FFPE tissue without requiring reverse transcription.
• Commercial kits available with probes for E6/E7 mRNA of multiple hrHPV genotypes.
• Applied to FFPE sections - easily usable in routine practice.
• Sensitivity 97%, specificity 93% compared to qRT-PCR gold standard.
• Anticipated to become the routine standard for HPV testing in FFPE biopsies.

D. Surrogate Markers - p16INK4a Immunohistochemistry (IHC)

• Mechanism: HPV E7 oncoprotein degrades the retinoblastoma (RB) protein via the ubiquitin-proteasome system. Loss of RB leads to loss of E2F repression, which in turn drives overexpression of p16INK4a (a cyclin-dependent kinase inhibitor). Therefore, strong diffuse p16 overexpression is a surrogate marker for oncogenic HPV activity.
• Positive result: strong, diffuse block-positive nuclear and cytoplasmic staining in tumor cells.
• Highly sensitive, readily applicable to FFPE sections.
• Limitation: not 100% specific - p16 overexpression can occur via non-HPV mechanisms (e.g., methylation of RB).
• Used in conjunction with ISH for definitive HPV status determination (see image below).

5. Screening Algorithms (ASCCP Guidelines)

• Ages 21-65: Cytology alone every 3 years is acceptable.
• Ages 30-65 (preferred): "Co-testing" with cytology + hrHPV molecular test every 5 years.
• This is based on meta-analysis data supporting high sensitivity of molecular HPV testing for early carcinoma detection.
• HPV 16/18 positive + cytology negative: Immediate colposcopy (higher risk ~11.4% for CIN 2+).
• Other hrHPV positive + cytology negative: Retest at 1 year.
• Reflex testing: ASC-US cytology triggers immediate hrHPV testing; HPV positivity triggers colposcopy.
• Primary HPV screening: Cobas and BD Onclarity are FDA-approved; genotyping for 16/18 drives management.

6. HPV Serology

7. Key Points for MD Pathology Exam

Summary