Oral Cavity and Oropharyngeal Cancer

Circulating Tumor DNA for Minimal Residual Disease Detection & HPV-Stratified Molecular Profiling

Clinical Overview

Oral cavity and oropharyngeal cancers are part of the head and neck squamous cell carcinoma (HNSCC) spectrum. HPV status fundamentally divides these cancers into distinct molecular and clinical entities, with critical implications for prognosis and ctDNA detection rates. HPV-positive oropharyngeal cancers (primarily tonsil and base of tongue) show rising incidence, better prognosis, and higher ctDNA shedding rates compared to HPV-negative disease.

HPV-Positive Oropharyngeal Cancer

Sites: Tonsil, base of tongue
Incidence: Rising prevalence in developed countries
Molecular profile: PIK3CA alterations (20-30%); rare TP53 mutations
Prognosis: Significantly better than HPV-negative disease
ctDNA shedding: Higher detection rates (70-90% MRD sensitivity)
5-year survival: 70-85% (stage-dependent)

HPV-Negative Oral/Oropharyngeal Cancer

Risk factors: Tobacco, alcohol use
Molecular profile: TP53 mutations (70%), CDKN2A inactivation
Prognosis: Worse outcomes; median OS ~1 year in R/M disease
ctDNA shedding: Lower detection rates (60-70% MRD sensitivity)
5-year survival: 40-60% (stage-dependent)

Critical Clinical Limitation: While HPV DNA can be detected in plasma, it cannot distinguish anatomic site of origin. HPV DNA may originate from oropharyngeal cancer, cervical cancer, or other HPV-associated malignancies. Somatic mutation tracking (not viral DNA) provides site-specific disease monitoring.

Understanding ctDNA Testing Methodology

What is Circulating Tumor DNA (ctDNA)?

Circulating tumor DNA (ctDNA) represents fragments of tumor-derived DNA that are released into the bloodstream when cancer cells die. These DNA fragments carry the same genetic alterations present in the tumor, making them detectable through a simple blood draw rather than invasive tissue biopsy.

How ctDNA Testing Differs from Other Methods

ctDNA vs Tissue Biopsy

Sample collection: Blood draw vs surgical/needle biopsy
Risk profile: Minimal risk vs procedural complications
Tumor heterogeneity: Captures DNA from all tumor sites vs single location
Serial monitoring: Easy repeat testing vs limited repeat procedures
HNSCC consideration: Anatomically challenging biopsies in post-radiation fields

ctDNA vs Cellular Blood Tests

Target: Cell-free DNA fragments vs intact circulating tumor cells
Sensitivity: Detects 0.01-0.001% tumor fraction vs 0.1-1% requirement
Processing: Plasma separation vs cell isolation techniques
Clinical applications: MRD detection and monitoring vs enumeration only

ctDNA vs HPV DNA Testing

Target: Somatic tumor mutations vs viral DNA sequences
Specificity: Site-specific disease tracking vs cannot distinguish anatomic origin
Detection: Both can identify HPV+ disease
Limitation: HPV DNA alone cannot differentiate oropharyngeal from other HPV+ cancers

Testing Approaches: Tumor-Informed vs Tumor-Agnostic

Tumor-Informed (Baseline-Based) Approach

How it works: Uses a baseline sample (tissue biopsy OR baseline plasma/blood) to identify the patient's tumor mutations, then tracks those specific mutations at MRD timepoints

Advantages:

Ultra-high sensitivity (0.001-0.01% tumor fraction)
Tracks patient's known mutations from baseline
Higher sensitivity in HPV+ disease (70-90%)
Moderate sensitivity in HPV- disease (60-70%)

Best used for:

Post-surgical MRD detection
Monitoring during definitive chemoradiation
Early relapse detection in surveillance (2-5 month lead time)

Requirements: Baseline sample (tissue from surgery/biopsy or blood sample) for initial mutation identification

Tumor-Agnostic (No Baseline) Approach

How it works: Tests directly at MRD timepoint without prior baseline profiling, using panels covering common HNSCC mutations (TP53, PIK3CA, CDKN2A, KRAS, NRAS, BRAF)

Advantages:

No baseline sample required
Faster turnaround (5-7 days vs 2-3 weeks)
Can detect mutations emerging during treatment
Useful for identifying actionable mutations (PIK3CA, KRAS/NRAS/BRAF)

Best used for:

Recurrent/metastatic disease monitoring
Treatment selection (immunotherapy, targeted therapy)
Cases where baseline profiling was not performed

Limitations: Lower sensitivity for MRD (0.1-0.5% tumor fraction)

Clinical Decision Points

When to Use Each Approach

Clinical Scenario	Recommended Approach	Rationale
Post-surgical MRD (resectable disease)	Tumor-informed	Maximum sensitivity; uses baseline profiling from surgery
Post-chemoradiation MRD	Tumor-informed	Higher sensitivity using baseline mutation tracking
Recurrent/metastatic genotyping	Either approach	Focus on actionable mutations (PIK3CA, PD-L1, resistance markers)
No baseline available	Tumor-agnostic	Tests directly at MRD timepoint
HPV+ disease monitoring	Tumor-informed preferred	Higher sensitivity (70-90%) with baseline-guided approach
Surveillance (years 0-2)	Tumor-informed	Highest sensitivity; 2-5 month lead time before imaging

MRD Detection Clinical Utility

Prognostic Performance by HPV Status

HPV-Positive Disease: Superior ctDNA Detection

Detection Performance:

MRD Sensitivity: 70-90% (higher ctDNA shedding)
Lead Time: 2-5 months before imaging detection
Hazard Ratio: 8.5-15 for ctDNA+ vs ctDNA-
Clinical Advantage: Better prognostic discrimination

Biological Rationale: HPV+ tumors demonstrate higher ctDNA shedding rates, likely related to increased apoptosis and immune infiltration. This results in superior MRD detection sensitivity compared to HPV- disease.

HPV-Negative Disease: Moderate Detection Rates

Detection Performance:

MRD Sensitivity: 60-70% (lower ctDNA shedding)
Lead Time: 2-4 months before imaging detection
Hazard Ratio: 15-35 for ctDNA+ vs ctDNA-
Clinical Challenge: Higher false-negative rate requires clinical correlation

Biological Rationale: HPV- tumors shed less ctDNA, resulting in lower sensitivity. When detected, ctDNA positivity remains highly specific for recurrence with very high hazard ratios.

Detection Timing and Lead Time

                    Post-Treatment MRD Kinetics
                    
                                Timepoint
                                ctDNA Detection Rate
                                Clinical Significance
                            
                                4-8 weeks post-surgery
                                15-25% (persistence)
                                High-risk MRD; consider adjuvant therapy intensification
                            
                                3 months post-chemoRT
                                10-20% (residual)
                                Incomplete response; consider salvage surgery or clinical trials
                            
                                6-12 months surveillance
                                5-15% (relapse)
                                Early recurrence detection; 2-5 month lead time before imaging
                            
                                12-24 months surveillance
                                3-8% (late relapse)
                                Ongoing recurrence risk; continued monitoring indicated

Timepoint	ctDNA Detection Rate	Clinical Significance
4-8 weeks post-surgery	15-25% (persistence)	High-risk MRD; consider adjuvant therapy intensification
3 months post-chemoRT	10-20% (residual)	Incomplete response; consider salvage surgery or clinical trials
6-12 months surveillance	5-15% (relapse)	Early recurrence detection; 2-5 month lead time before imaging
12-24 months surveillance	3-8% (late relapse)	Ongoing recurrence risk; continued monitoring indicated

Prognostic Value: Recurrence Risk Stratification

Risk Stratification by ctDNA Status

ctDNA-Positive Patients:

2-year recurrence risk: 60-80% (high-risk population)
Hazard ratio vs ctDNA-: 8.5-35 depending on HPV status
Median time to recurrence: 4-8 months
Clinical action: Intensified surveillance, adjuvant therapy consideration

ctDNA-Negative Patients:

2-year recurrence risk: 5-15% (favorable prognosis)
Recurrence pattern: Late relapses (>12 months) more common
Clinical consideration: Standard surveillance may be sufficient
Important caveat: 10-30% false negatives (higher in HPV- disease)

Clinical Application Algorithm

                    Evidence-Based MRD Testing Strategy
                    Baseline Profiling:
                            Obtain tissue or baseline blood sample for mutation identification
Document HPV status (critical for interpretation)
Identify somatic mutations for tracking (TP53, PIK3CA, CDKN2A)

                        
Post-Treatment MRD (4-8 weeks):
                            First ctDNA timepoint after surgery or definitive chemoRT
Persistent positivity: 15-25% detection rate
Consider adjuvant therapy or intensified surveillance

                        
Surveillance Monitoring:
                            Every 3 months for years 0-2 (highest recurrence risk)
Every 6 months for years 3-5
Earlier imaging if ctDNA becomes positive

                        
ctDNA Conversion to Positive:
                            Obtain confirmatory sample within 2-4 weeks
Initiate imaging workup (PET/CT preferred)
Median lead time: 2-5 months before radiographic detection
Consider biopsy confirmation if oligometastatic disease amenable to salvage

                        

                

Clinical Limitations of MRD Testing

⚠️ Important Limitations

Sensitivity varies by tumor site: Oropharynx >80%, oral cavity 60-75%, larynx 60-70%
HPV DNA cannot distinguish anatomic origin: May originate from cervical, anal, or other HPV+ cancers
False negatives in HPV- disease: 30-40% of recurrences may be ctDNA-negative
Low tumor burden limitation: Small volume disease (<1 cm) may be undetectable
No prospective interventional trials: No RCT data demonstrating treatment benefit from ctDNA-guided decisions
Cannot replace imaging: ctDNA should complement, not replace, standard surveillance imaging

Genotyping for Treatment Selection

HPV-Positive Disease: Molecular Profile

PIK3CA Alterations: Emerging Therapeutic Target

Prevalence:

PIK3CA mutations: 20-30% of HPV+ oropharyngeal cancers
Common hotspots: E542K, E545K, H1047R
PIK3CA amplification: Additional 10-15% of cases

Clinical Significance:

Prognostic value: Associated with better outcomes in HPV+ disease
Therapeutic targeting: Alpelisib (PI3K inhibitor) under investigation
Trial enrollment: Multiple trials recruiting PIK3CA-mutated HNSCC
Detection method: Can be identified via tissue NGS or ctDNA profiling

HPV-Negative Disease: TP53 and Tumor Suppressor Mutations

TP53: Dominant Driver Mutation

Prevalence and Impact:

TP53 mutations: 70% of HPV- oral/oropharyngeal cancers
Mutation spectrum: Diverse hotspots; loss of function predominates
Prognostic significance: Associated with worse outcomes, aggressive disease
Therapeutic implications: No direct targeted therapy; clinical trial consideration

CDKN2A Inactivation:

Mechanism: Deletion, mutation, or methylation
Prevalence: 50-60% of HPV- disease
Co-occurrence: Often found with TP53 mutations
Clinical impact: Poor prognosis marker

Immunotherapy: PD-L1 and Treatment Selection

KEYNOTE-048: Pembrolizumab-Based First-Line Therapy

Pembrolizumab alone or with chemotherapy vs cetuximab + chemotherapy for recurrent/metastatic HNSCC

Study Design:

Population: Recurrent/metastatic HNSCC (oral, oropharyngeal, laryngeal, hypopharyngeal)
Arms: Pembrolizumab monotherapy vs pembrolizumab + chemotherapy vs cetuximab + chemotherapy
Biomarker stratification: PD-L1 Combined Positive Score (CPS)

Primary Results:

PD-L1 CPS ≥1 (pembrolizumab monotherapy):
- Overall survival: 12.3 vs 10.3 months (HR 0.78, p=0.0007)
- Improved tolerability vs chemotherapy
- Lower toxicity profile
Total population (pembrolizumab + chemotherapy):
- Overall survival: 13.0 vs 10.7 months (HR 0.77, p=0.0004)
- Progression-free survival: 4.9 vs 5.1 months (HR 0.84)
- Benefit regardless of PD-L1 status

Current Treatment Paradigm:

PD-L1 CPS ≥1: Pembrolizumab monotherapy is reasonable first-line option
PD-L1 CPS <1 or need for rapid response: Pembrolizumab + platinum/5-FU preferred
PD-L1 testing: Should be performed on tissue biopsy; ctDNA cannot assess PD-L1 expression

Cetuximab Resistance: KRAS/NRAS/BRAF Mutations

EGFR Pathway Alterations

Prevalence:

KRAS mutations: 3-5% of HNSCC (rare)
NRAS mutations: 2-3% of HNSCC (rare)
BRAF mutations: 1-2% of HNSCC (very rare)

Clinical Significance:

Cetuximab resistance: RAS/BRAF mutations confer primary resistance to EGFR inhibitors
Treatment selection: Avoid cetuximab in RAS/BRAF-mutated disease
Alternative approaches: Immunotherapy or chemotherapy preferred
Detection: Can be identified via tissue NGS or ctDNA profiling

BRAF V600E-Specific Considerations:

Targeted therapy: Dabrafenib + trametinib combination (extrapolated from melanoma/other cancers)
Response rates: Case series showing 30-50% response in BRAF V600E+ HNSCC
Clinical trial enrollment: Basket trials enrolling BRAF V600E+ solid tumors

Clinical Genotyping Recommendations

                    When to Perform Molecular Profiling
                    
                                Clinical Scenario
                                Recommended Testing
                                Actionable Findings
                            
                                Recurrent/metastatic disease
                                Comprehensive NGS panel (tissue or ctDNA)
                                PIK3CA, BRAF V600E, KRAS/NRAS, PD-L1 CPS
                            
                                HPV+ oropharyngeal (R/M)
                                PIK3CA mutation testing + PD-L1
                                Alpelisib trials, immunotherapy eligibility
                            
                                HPV- disease (R/M)
                                TP53, CDKN2A, PD-L1, RAS/BRAF
                                Immunotherapy, clinical trial matching
                            
                                Considering cetuximab
                                KRAS, NRAS, BRAF mutation testing
                                Avoid cetuximab if RAS/BRAF mutated
                            
                                Immunotherapy candidacy
                                PD-L1 CPS (tissue IHC)
                                CPS ≥1: pembrolizumab monotherapy option
                            
                                Newly diagnosed locally advanced
                                HPV status, consider baseline profiling for MRD
                                Prognostic stratification, MRD tracking

Clinical Scenario	Recommended Testing	Actionable Findings
Recurrent/metastatic disease	Comprehensive NGS panel (tissue or ctDNA)	PIK3CA, BRAF V600E, KRAS/NRAS, PD-L1 CPS
HPV+ oropharyngeal (R/M)	PIK3CA mutation testing + PD-L1	Alpelisib trials, immunotherapy eligibility
HPV- disease (R/M)	TP53, CDKN2A, PD-L1, RAS/BRAF	Immunotherapy, clinical trial matching
Considering cetuximab	KRAS, NRAS, BRAF mutation testing	Avoid cetuximab if RAS/BRAF mutated
Immunotherapy candidacy	PD-L1 CPS (tissue IHC)	CPS ≥1: pembrolizumab monotherapy option
Newly diagnosed locally advanced	HPV status, consider baseline profiling for MRD	Prognostic stratification, MRD tracking

References

Cancer Genome Atlas Network. Comprehensive genomic characterization of head and neck squamous cell carcinomas. Nature 2015;517:576-582.
Burtness B, Harrington KJ, Greil R, et al. Pembrolizumab alone or with chemotherapy versus cetuximab with chemotherapy for recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-048). Lancet 2019;394:1915-1928.
Chaudhuri AA, Chabon JJ, Lovejoy AF, et al. Early detection of molecular residual disease in localized lung cancer by circulating tumor DNA profiling. Cancer Discov 2017;7:1394-1403.
Mazurek AM, Rutkowski T, Fiszer-Kierzkowska A, et al. Detection of circulating tumor DNA in blood of patients with head and neck squamous cell carcinoma. Transl Oncol 2023;27:101577.
Seiwert TY, Burtness B, Mehra R, et al. Safety and clinical activity of pembrolizumab for treatment of recurrent or metastatic squamous cell carcinoma of the head and neck (KEYNOTE-012). Lancet Oncol 2016;17:956-965.
André F, Ciruelos E, Rubovszky G, et al. Alpelisib for PIK3CA-mutated, hormone receptor-positive advanced breast cancer. N Engl J Med 2019;380:1929-1940.

Evidence summary as of January 2026 | Document Version: 2.0