Evidence-Based Liquid Biopsy Knowledge
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Cervical Cancer

HPV ctDNA for MRD Detection and Emerging Immunotherapy Targets

Clinical Overview

Cervical cancer, caused predominantly by persistent human papillomavirus (HPV) infection, represents a unique opportunity for ctDNA-based minimal residual disease detection. Unlike most solid tumors that require tracking of somatic mutations, cervical cancer monitoring utilizes HPV DNA sequences as the primary circulating tumor biomarker. This viral DNA provides highly specific detection with 100% specificity, though sensitivity varies by stage and HPV genotype.

Why HPV ctDNA Differs from Other Solid Tumors:

  • Viral Biomarker: HPV DNA sequences (not tumor mutations) serve as the primary MRD marker
  • Perfect Specificity: 100% specificity for tumor-derived signal (HPV DNA indicates malignant cells)
  • Strong Prognostic Value: HR 5.50-7.78 for recurrence when detected at 3 months post-treatment
  • Early Detection Window: Median 164 days (5-6 months) lead time before clinical recurrence
  • Genotype Dependency: HPV16 detected in 77% of cases vs 20% for HPV18 (p=0.002)
  • Immunotherapy Advances: Pembrolizumab + chemotherapy shows substantial survival benefit (HR 0.63-0.68)

Critical Distinction: Cervical cancer ctDNA testing primarily detects HPV viral DNA fragments released by dying tumor cells, not somatic tumor mutations. While molecular profiling can identify actionable mutations (PIK3CA, ERBB2 amplification, MSI-H), the core MRD application tracks HPV sequences. This provides exceptional specificity (100%) but introduces unique limitations including genotype-dependent sensitivity and inability to distinguish cervical from head/neck HPV-driven cancers.

ctDNA Testing Methodology

HPV DNA as Tumor-Specific Biomarker

Cervical cancer ctDNA detection utilizes a distinct approach compared to other solid tumors. Rather than identifying patient-specific somatic mutations through baseline profiling, HPV ctDNA testing directly detects viral DNA sequences known to drive malignant transformation.

HPV ctDNA Detection Approach:

  • Target Biomarker: HPV viral DNA sequences (primarily HPV16 and HPV18)
  • Testing Method: Targeted sequencing or PCR-based amplification of HPV sequences
  • Baseline Requirement: HPV genotyping from primary tumor (to identify which HPV type to track)
  • Detection Technology: Next-generation sequencing or digital PCR platforms
  • Sample Type: Plasma or serum collected at serial timepoints

Key Methodological Distinction: Unlike tumor-informed approaches that require baseline tissue sequencing to identify patient-specific mutations, HPV ctDNA testing leverages known viral sequences as the biomarker. However, baseline tumor HPV genotyping is still recommended to confirm which HPV type (16, 18, or other) to monitor, as detection sensitivity varies significantly by genotype.

Comparison to Traditional Biomarkers

Characteristic HPV ctDNA SCC Antigen (Squamous Cell Carcinoma Antigen)
Biomarker Type Viral DNA sequences Serum protein (tumor-associated antigen)
Specificity 100% (HPV DNA = tumor) Lower (can be elevated in benign conditions)
Sensitivity (Overall) 60.8-93% (stage-dependent) 30-60% (varies by stage)
Early-Stage Detection 42% (stage I-II) 20-30% (stage I-II)
Lead Time Median 164 days (5-6 months) Variable, typically shorter
Histology Restriction All histologies (requires HPV+) Squamous only (not adenocarcinoma)

Clinical Advantage: HPV ctDNA provides superior specificity compared to SCC antigen, with 100% specificity meaning any detectable HPV DNA unambiguously indicates presence of malignant cells. However, sensitivity varies substantially by disease stage and HPV genotype, with early-stage disease (I-II) detected in only 42% of cases.

MRD Detection: Clinical Utility

Strong Prognostic Value with Genotype-Dependent Sensitivity

HPV ctDNA detection demonstrates strong prognostic value for recurrence risk stratification following definitive chemoradiation therapy. Multiple studies establish detectable HPV ctDNA at post-treatment timepoints as a high-risk marker for disease recurrence.

Prognostic Performance at 3 Months Post-Treatment

Recurrence Risk Stratification:

  • Hazard Ratio (Multivariable): HR 5.50-7.78 for recurrence when HPV ctDNA detectable at 3 months post-CRT
  • Interpretation: Patients with detectable HPV ctDNA have 5.5 to 7.8-fold higher risk of recurrence
  • Negative Predictive Value: 95% when HPV ctDNA undetectable at end of treatment
  • Clinical Implication: Undetectable HPV ctDNA indicates very low recurrence risk; detectable HPV ctDNA identifies high-risk population requiring intensified surveillance

Early Detection Lead Time

Detection Before Clinical Recurrence:

  • Median Lead Time: 164 days (approximately 5-6 months)
  • Clinical Opportunity: HPV ctDNA rises 5-6 months before imaging or clinical evidence of recurrence
  • Actionability Window: Provides sufficient time for salvage therapy planning or clinical trial enrollment

Sensitivity and Specificity by Clinical Context

Clinical Setting Sensitivity Specificity Clinical Notes
Overall (All Stages) 60.8-93% 100% Wide range reflects stage and genotype variation
Early-Stage (I-II) 42% 100% Limited sensitivity in early disease
Advanced-Stage (III-IV) 80-93% 100% Improved sensitivity with higher tumor burden
End of Treatment Variable 100% NPV 95% when undetectable

Stage-Dependent Performance: HPV ctDNA sensitivity increases with disease stage, ranging from 42% in early-stage (I-II) to 80-93% in advanced-stage (III-IV) disease. This stage dependency reflects the relationship between tumor burden and circulating DNA shedding, with larger tumor volumes producing more detectable circulating HPV DNA.

HPV Genotype-Dependent Detection

A critical limitation of HPV ctDNA monitoring is significantly reduced sensitivity for HPV18-driven tumors compared to HPV16.

Genotype Detection Rates:

  • HPV16: 77% detection rate
  • HPV18: 20% detection rate
  • Statistical Significance: p=0.002
  • Clinical Impact: HPV18-positive patients have nearly 4-fold lower detection sensitivity
  • Implication: Negative HPV ctDNA results less reassuring in HPV18-positive disease

Mechanistic Hypothesis: The poor HPV18 detection may reflect biological differences in viral DNA integration patterns, tumor shedding characteristics, or circulating DNA fragment size. Regardless of mechanism, clinicians must interpret negative HPV ctDNA results with caution in patients with HPV18-positive tumors.

Clinical Application: HPV ctDNA provides strong prognostic stratification following definitive therapy, with HR 5.50-7.78 for recurrence when detectable at 3 months post-treatment. The 5-6 month lead time enables early identification of treatment failures. However, stage-dependent sensitivity (42% in stage I-II) and poor HPV18 detection (20% vs 77% for HPV16) limit utility in certain populations. Perfect specificity (100%) means any detectable HPV ctDNA unambiguously indicates residual or recurrent disease.

Genotyping: Clinical Utility

Actionable Mutations Beyond HPV Status

While HPV DNA serves as the primary MRD biomarker, comprehensive molecular profiling via ctDNA can identify actionable mutations that inform therapeutic decisions, particularly in recurrent or metastatic disease.

PIK3CA Mutations

PIK3CA Mutation Profile:

  • Prevalence: 13.6-27.1% of cervical cancers
  • Mechanism: Activating mutations in PI3K pathway driving cell growth and survival
  • Hotspot Mutations: E542K, E545K (helical domain); H1047R (kinase domain)
  • Therapeutic Target: Alpelisib (PI3K inhibitor approved in breast cancer)
  • Clinical Trial Evidence: PI3K inhibitors under investigation in PIK3CA-mutant cervical cancer
  • ctDNA Detection: Readily detectable in plasma via targeted sequencing

Therapeutic Potential: PIK3CA mutations represent a rational therapeutic target given the pathway's role in tumor growth and survival. Alpelisib, an alpha-specific PI3K inhibitor approved for PIK3CA-mutant breast cancer, is being evaluated in basket trials including cervical cancer cohorts. ctDNA profiling enables identification of PIK3CA-mutant patients without requiring repeat tissue biopsy.

ERBB2 (HER2) Amplification

ERBB2 Amplification Profile:

  • Prevalence: 3.5-15% of cervical cancers
  • Mechanism: HER2 receptor overexpression driving proliferation and survival signaling
  • Detection Method: Copy number analysis via ctDNA sequencing or tissue IHC/FISH
  • Therapeutic Target: Trastuzumab deruxtecan (antibody-drug conjugate)
  • Clinical Evidence: Basket trials show activity in HER2-positive solid tumors including cervical cancer
  • Indication: Trastuzumab deruxtecan approved for HER2-positive solid tumors (tumor-agnostic)

Clinical Application: ERBB2 amplification identifies candidates for trastuzumab deruxtecan, which demonstrated clinically meaningful activity in HER2-positive solid tumors across multiple histologies. While prevalence is modest (3.5-15%), identification of this subgroup provides access to effective targeted therapy in otherwise treatment-refractory disease.

Microsatellite Instability and Mismatch Repair Deficiency

MSI-H/dMMR Profile:

  • Prevalence: 2-4% of cervical cancers
  • Mechanism: Defective DNA mismatch repair leading to high mutational burden and neoantigen load
  • Detection: MSI testing via tumor tissue or ctDNA sequencing
  • Immunotherapy Response: Pembrolizumab overall response rate approximately 30% in MSI-H solid tumors
  • Indication: Pembrolizumab approved for MSI-H/dMMR solid tumors (tumor-agnostic)
  • Clinical Implication: MSI-H tumors show enhanced immunotherapy sensitivity compared to microsatellite-stable tumors

PD-L1 Expression

PD-L1 Testing:

  • Biomarker: PD-L1 expression measured by Combined Positive Score (CPS)
  • Threshold: CPS ≥1 (any detectable PD-L1 expression)
  • Testing Method: Immunohistochemistry on tumor tissue (not ctDNA)
  • Clinical Context: Pembrolizumab approved in combination with chemotherapy regardless of PD-L1 status
  • Prognostic Value: Higher PD-L1 expression (CPS ≥10) associated with greater pembrolizumab benefit
  • Testing Requirement: Not required for treatment decision (benefit seen across all CPS levels)

Important Note: While PD-L1 is a relevant biomarker in cervical cancer, it requires tissue-based immunohistochemistry and is not assessed via ctDNA. PD-L1 testing is not required for pembrolizumab treatment decisions in first-line metastatic disease given consistent benefit across PD-L1 expression levels.

Comprehensive Profiling Strategy: In recurrent or metastatic cervical cancer, ctDNA-based molecular profiling can identify actionable alterations including PIK3CA mutations (13.6-27.1%, alpelisib target), ERBB2 amplification (3.5-15%, trastuzumab deruxtecan), and MSI-H/dMMR status (2-4%, enhanced pembrolizumab response). While prevalence of individual alterations is modest, comprehensive profiling maximizes likelihood of identifying targetable vulnerabilities in treatment-refractory disease.

Immunotherapy Clinical Trials

Level 1 Evidence for Pembrolizumab in Advanced Disease

Cervical cancer has witnessed a transformation in treatment options with the integration of immune checkpoint inhibition. Three major phase III randomized controlled trials establish pembrolizumab-based regimens as standard of care in both first-line and locally advanced settings.

KEYNOTE-826: First-Line Metastatic Disease

Study Design: Phase III randomized trial evaluating pembrolizumab + chemotherapy (with or without bevacizumab) vs placebo + chemotherapy (with or without bevacizumab) in treatment-naive persistent, recurrent, or metastatic cervical cancer.

Trial Structure:

  • Patient Population: Treatment-naive persistent, recurrent, or metastatic cervical cancer (n=617)
  • Intervention Arm: Pembrolizumab + paclitaxel + platinum (± bevacizumab)
  • Control Arm: Placebo + paclitaxel + platinum (± bevacizumab)
  • Primary Endpoints: Progression-free survival (PFS) and overall survival (OS)
  • PD-L1 Stratification: Enrolled regardless of PD-L1 status

KEYNOTE-826 Results:

  • Median Overall Survival:
    • Pembrolizumab + chemotherapy: 26.4 months
    • Placebo + chemotherapy: 16.8 months
    • Absolute Benefit: 9.6 months
  • Hazard Ratio for Death: HR 0.63 (37% reduction in risk of death, p<0.001)
  • Progression-Free Survival: Significantly improved (data mature at time of OS analysis)
  • Benefit Consistency: Observed across all PD-L1 expression levels (CPS ≥1, CPS ≥10, and CPS <1)
  • Current Status: Established as standard of care for first-line metastatic disease

Clinical Significance: KEYNOTE-826 established pembrolizumab + chemotherapy as the preferred first-line regimen for persistent, recurrent, or metastatic cervical cancer. The 9.6-month median survival improvement represents a substantial clinical benefit, and the consistency across PD-L1 subgroups supports universal pembrolizumab use without requiring biomarker selection.

KEYNOTE-A18: Locally Advanced Disease with Definitive CRT

Study Design: Phase III randomized trial evaluating pembrolizumab + concurrent chemoradiation + brachytherapy vs placebo + concurrent chemoradiation + brachytherapy in high-risk locally advanced cervical cancer.

Trial Structure:

  • Patient Population: High-risk locally advanced cervical cancer (FIGO 2014 stage IB2-IIB node-positive or stage III-IVA any node status)
  • Intervention Arm: Pembrolizumab + concurrent cisplatin + radiation + brachytherapy, followed by pembrolizumab maintenance
  • Control Arm: Placebo + concurrent cisplatin + radiation + brachytherapy, followed by placebo maintenance
  • Primary Endpoint: Progression-free survival (PFS)
  • Key Secondary Endpoint: Overall survival (OS)

KEYNOTE-A18 Results:

  • 36-Month Progression-Free Survival:
    • Pembrolizumab + CRT: 69.3%
    • Placebo + CRT: 56.9%
    • Absolute Benefit: 12.4 percentage points
  • Hazard Ratio for Progression or Death: HR 0.68 (32% reduction, p<0.001)
  • Overall Survival: Data immature at time of primary PFS analysis
  • Benefit Consistency: Consistent across PD-L1 subgroups and stage subgroups
  • Emerging Standard: Positioned to become standard of care for high-risk locally advanced disease

Clinical Significance: KEYNOTE-A18 demonstrates that adding pembrolizumab to definitive concurrent chemoradiation significantly improves progression-free survival in high-risk locally advanced cervical cancer. The 12.4 percentage point improvement in 36-month PFS translates to meaningful reduction in recurrence risk, with overall survival data awaited.

BEATcc: Alternative First-Line Regimen

Study Design: Phase III randomized trial evaluating pembrolizumab + chemotherapy vs chemotherapy alone (without bevacizumab) in first-line recurrent or metastatic cervical cancer.

BEATcc Results:

  • Median Overall Survival:
    • Pembrolizumab + chemotherapy: 32.1 months
    • Chemotherapy alone: 22.8 months
    • Absolute Benefit: 9.3 months
  • Hazard Ratio for Death: HR 0.68 (32% reduction in risk of death)
  • Consistency with KEYNOTE-826: Similar magnitude of benefit across both trials
  • Clinical Context: Provides alternative to bevacizumab-containing regimens

Clinical Significance: BEATcc corroborates the KEYNOTE-826 findings, demonstrating substantial survival benefit with pembrolizumab + chemotherapy. The similar HR (0.68) and absolute survival gain (9.3 months) provide consistent evidence for pembrolizumab as a cornerstone of first-line therapy.

Summary of Immunotherapy Evidence

Trial Setting Primary Endpoint Hazard Ratio Absolute Benefit Status
KEYNOTE-826 First-line metastatic OS: 26.4 vs 16.8 months HR 0.63 +9.6 months Standard of care
KEYNOTE-A18 Locally advanced + CRT 36-mo PFS: 69.3% vs 56.9% HR 0.68 +12.4% at 36 months Emerging standard
BEATcc First-line metastatic OS: 32.1 vs 22.8 months HR 0.68 +9.3 months Alternative regimen

Immunotherapy Transformation: Three phase III trials establish pembrolizumab-based regimens as standard of care across multiple clinical settings in cervical cancer. KEYNOTE-826 and BEATcc demonstrate approximately 9-10 month overall survival improvements (HR 0.63-0.68) in first-line metastatic disease, while KEYNOTE-A18 shows significant progression-free survival benefit (HR 0.68) when added to definitive chemoradiation in locally advanced disease. These consistent results across trials and settings represent a fundamental shift in cervical cancer treatment paradigms.

Clinical Summary

HPV ctDNA in Cervical Cancer Management

Cervical cancer represents a unique ctDNA application where viral DNA (HPV sequences) rather than somatic mutations serves as the primary biomarker. This provides exceptional specificity (100%) but introduces genotype-dependent and stage-dependent sensitivity limitations.

MRD Detection (HPV ctDNA):

  • Biomarker: HPV viral DNA sequences (HPV16, HPV18, others) - not tumor somatic mutations
  • Sensitivity: 60.8-93% overall (stage-dependent); 42% in stage I-II; 80-93% in stage III-IV
  • Specificity: 100% (any detectable HPV DNA indicates tumor)
  • Negative Predictive Value: 95% when undetectable at end of treatment
  • Prognostic Value: HR 5.50-7.78 for recurrence when detectable at 3 months post-CRT (multivariable)
  • Lead Time: Median 164 days (5-6 months) before clinical/imaging recurrence
  • Genotype Dependency: HPV16 detected in 77%; HPV18 detected in only 20% (p=0.002)

Genotyping (Actionable Mutations):

  • PIK3CA Mutations: 13.6-27.1% prevalence; potential target for alpelisib (PI3K inhibitor)
  • ERBB2 Amplification: 3.5-15% prevalence; target for trastuzumab deruxtecan
  • MSI-H/dMMR: 2-4% prevalence; pembrolizumab ORR approximately 30%
  • PD-L1 Expression: CPS ≥1 threshold; pembrolizumab benefit across all expression levels
  • Clinical Context: Comprehensive profiling identifies therapeutic targets in recurrent/metastatic disease

Immunotherapy Evidence (Level 1 RCTs):

  • KEYNOTE-826: First-line metastatic - median OS 26.4 vs 16.8 months; HR 0.63 (p<0.001)
  • KEYNOTE-A18: Locally advanced + CRT - 36-month PFS 69.3% vs 56.9%; HR 0.68 (p<0.001)
  • BEATcc: First-line metastatic - median OS 32.1 vs 22.8 months; HR 0.68
  • Current Standard: Pembrolizumab + chemotherapy established as first-line for metastatic disease
  • Emerging Standard: Pembrolizumab + CRT for high-risk locally advanced disease

Clinical Limitations:

  • Source Ambiguity: Cannot distinguish cervical from head/neck HPV source
  • HPV18 Detection Failure: Only 20% detection vs 77% for HPV16 (p=0.002)
  • Early-Stage Insensitivity: 42% sensitivity in stage I-II disease
  • Guideline Recommendation: Not recommended for routine surveillance (ESMO 2022)
  • Appropriate Use: Advanced-stage HPV16-positive disease, post-CRT risk stratification, clinical trials

Bottom Line: Cervical cancer ctDNA monitoring utilizes HPV viral DNA as a tumor-specific biomarker, providing 100% specificity and strong prognostic value (HR 5.50-7.78 at 3 months post-CRT) with 5-6 month lead time before clinical recurrence. However, genotype-dependent sensitivity (20% for HPV18 vs 77% for HPV16), stage-dependent performance (42% in early-stage), and inability to distinguish cervical from head/neck HPV sources limit clinical utility. ESMO 2022 guidelines do not recommend routine surveillance use. Cervical cancer treatment has been transformed by pembrolizumab-based regimens (KEYNOTE-826, KEYNOTE-A18, BEATcc) showing 9-10 month survival improvements (HR 0.63-0.68). Comprehensive molecular profiling via ctDNA identifies actionable mutations (PIK3CA, ERBB2, MSI-H) providing therapeutic targets in recurrent/metastatic disease.

References

  1. Han K et al. Prognostic value of circulating tumor DNA in patients with HPV-positive cervical cancer undergoing concurrent chemoradiotherapy. Oncotarget 2017;8:106572-106584
  2. Jeannot E et al. Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma. J Pathol Clin Res 2016;2:201-209
  3. Cabel L et al. HPV circulating tumor DNA to monitor the efficacy of anti-PD-1 therapy in metastatic squamous cell carcinoma of the cervix. Gynecol Oncol Rep 2018;25:22-25
  4. Colombo N et al. Pembrolizumab for persistent, recurrent, or metastatic cervical cancer. N Engl J Med 2021;385:1856-1867
  5. Monk BJ et al. Pembrolizumab plus chemotherapy versus placebo plus chemotherapy for persistent, recurrent, or metastatic cervical cancer (KEYNOTE-826): final overall survival results. Ann Oncol 2023;34:S1279-S1280
  6. Lorusso D et al. Pembrolizumab plus chemoradiotherapy for high-risk locally advanced cervical cancer: a randomised, double-blind, phase 3 trial (KEYNOTE-A18/ENGOT-cx11/GOG-3047). Lancet 2024;403:1341-1350
  7. Oaknin A et al. Atezolizumab plus bevacizumab and chemotherapy for metastatic, persistent, or recurrent cervical cancer (BEATcc): a randomised, open-label, phase 3 trial. Lancet 2023;403:31-43
  8. Cancer Genome Atlas Research Network. Integrated genomic and molecular characterization of cervical cancer. Nature 2017;543:378-384
  9. Chung HC et al. Efficacy and safety of pembrolizumab in previously treated advanced cervical cancer. Oncologist 2019;24:259
  10. Marth C et al. Cervical cancer: ESMO Clinical Practice Guidelines for diagnosis, treatment and follow-up. Ann Oncol 2022;33:216-231

Evidence summary as of January 2026 | Educational Resource