Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer

Abstract

Purpose: To identify a profile of circulating tumor human papilloma virus (HPV) DNA (ctHPVDNA) clearance kinetics that is associated with disease control after chemoradiotherapy (CRT) for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC).

Experimental design: A multi-institutional prospective biomarker trial was conducted in 103 patients with (i) p16-positive OPSCC, (ii) M0 disease, and (iii) receipt of definitive CRT. Blood specimens were collected at baseline, weekly during CRT, and at follow-up visits. Optimized multianalyte digital PCR assays were used to quantify ctHPVDNA (types 16/18/31/33/35) in plasma. A control cohort of 55 healthy volunteers and 60 patients with non-HPV-associated malignancy was also analyzed.

Results: Baseline plasma ctHPVDNA had high specificity (97%) and high sensitivity (89%) for detecting newly diagnosed HPV-associated OPSCC. Pretreatment ctHPV16DNA copy number correlated with disease burden, tumor HPV copy number, and HPV integration status. We define a ctHPV16DNA favorable clearance profile as having high baseline copy number (>200 copies/mL) and >95% clearance of ctHPV16DNA by day 28 of CRT. Nineteen of 67 evaluable patients had a ctHPV16DNA favorable clearance profile, and none had persistent or recurrent regional disease after CRT. In contrast, patients with adverse clinical risk factors (T4 or >10 pack years) and an unfavorable ctHPV16DNA clearance profile had a 35% actuarial rate of persistent or recurrent regional disease after CRT (P = 0.0049).

Conclusions: A rapid clearance profile of ctHPVDNA may predict likelihood of disease control in patients with HPV-associated OPSCC patients treated with definitive CRT and may be useful in selecting patients for deintensified therapy.

Conflict of interest statement

Conflict of Interest: B.S.C, S.K., and G.P.G. have filed a U.S. patent application on aspects of the technology described in this manuscript (Application No. 62/732,117). B.S.C. and G.P.G. serve on the advisory board of Naveris, Inc, and hold equity interest. All other authors have no pertinent conflicts of interest to declare.

©2019 American Association for Cancer Research.

Figures

Figure 1:

REMARK diagram of patient cohorts analyzed in this study.

Figure 2.. Detection of plasma ctHPVDNA in HPV-associated oropharyngeal cancer patients.

(A) Measurement of ctHPVDNA copies/mL plasma in 55 healthy volunteers, 60 patients with non-HPV associated cancers, and 103 patients with HPV-associated OPSCC. Two-toned (red-black) circles denote patients who were negative for ctHPV16DNA but positive for ctHPVDNA from an alternative high-risk HPV strain (-18/31/33/35). (B-D) Baseline levels of ctHPV16DNA stratified by (B) tumor stage, (C) nodal stage, and (D) clinical risk factors. Median and inter-quartile range is shown for each graph. *, p

Figure 3.. Variable kinetics of ctHPV16DNA clearance during chemoradiotherapy.

(A) Normalized ctHPV16DNA abundance (relative to the highest measured value for each patient) at baseline, peak value between week 1–3 of CRT, and nadir after treatment (within 6 months of completing therapy) for 54 evaluable patients with detectable ctHPV16DNA. In one subgroup (red lines, N = 19) ctHPV16DNA levels decrease immediately after initiating CRT, whereas in the second subgroup (blue lines, N = 35) ctHPV16DNA levels initially spike followed by a subsequent decrease. (B) Percentage of patients with ctHPV16DNA elimination at different time points after initiating CRT. (C) Percentage clearance of ctHPV16DNA at week 4 in 40 patients with baseline ctHPV16DNA levels >200 copies/mL. Red dots indicate patients that later developed residual or recurrent disease. (D) Rapid ctHPV16DNA clearance kinetics in patients with >95% clearance of baseline ctHPV16DNA levels by week 4 of CRT. (E) Delayed ctHPV16DNA clearance kinetics in patients with 95% or less clearance of baseline ctHPV16DNA levels by week 4 of CRT.

Figure 4.. Correlation between ctHPV16DNA and tumor HPV genomic features.

(A) Spearman correlation between copies of HPV16 detected in tumor biopsy samples by dPCR and NGS (B) Tumor HPV16 copies per genome equivalent (GE) in patients grouped by baseline ctHPV16DNA levels. Mean ± SEM is shown. P value calculated using a two-tailed, unpaired t-test with unequal variances. (C) Normalized NGS counts for hybrid HPV-chromosomal reads (indicative of HPV integration) relative to HPV-HPV fusion reads (positive control). (D) Circos plots illustrating the detecting HPV and chromosomal rearrangements in a tumor with purely episomal HPV (left panel) and HPV integration into a locus on chromosome 8 (right panel). (E-F) Number of tumor samples with integrated versus episomal HPV after grouping patients by (E) tumor HPV copy number (per GE) or (F) baseline ctHPV16DNA (copies/mL). P values calculated by a two-tailed Fisher’s exact test.

Figure 5.. A favorable ctHPV16DNA clearance profile correlates with disease control in OPSCC patients treated with CRT.

(A) Percentages of clinical low-risk and clinical high-risk patients with a favorable ctHPV16DNA profile, which is defined as baseline ctHPV16DNA >200 copies/mL and >95% clearance by week 4. (B) Percentage of patients within each subgroup who experience a positive neck dissection, regional recurrence, or distant metastasis. (C) Kaplan-Meier analysis of regional disease-free survival stratified by clinical risk and ctHPV16DNA profile. P value calculated using a two-tailed log-rank test for trend.