Cell-free human papillomavirus DNA kinetics after surgery for human papillomavirus-associated oropharyngeal cancer

Abstract

Background: New ultrasensitive methods for detecting residual disease after surgery are needed in human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC).

Methods: To determine whether the clearance kinetics of circulating tumor human papillomavirus DNA (ctHPVDNA) is associated with postoperative disease status, a prospective observational study was conducted in 33 patients with HPV+OPSCC undergoing surgery. Blood was collected before surgery, postoperative days 1 (POD 1), 7, and 30 and with follow-up. A subcohort of 12 patients underwent frequent blood collections in the first 24 hours after surgery to define early clearance kinetics. Plasma was run on custom droplet digital polymerase chain reaction (ddPCR) assays for HPV genotypes 16, 18, 33, 35, and 45.

Results: In patients without pathologic risk factors for recurrence who were observed after surgery, ctHPVDNA rapidly decreased to <1 copy/mL by POD 1 (n = 8/8). In patients with risk factors for macroscopic residual disease, ctHPVDNA was markedly elevated on POD 1 (>350 copies/mL) and remained elevated until adjuvant treatment (n = 3/3). Patients with intermediate POD 1 ctHPVDNA levels (1.2-58.4 copies/mL) all possessed pathologic risk factors for microscopic residual disease (n = 9/9). POD 1 ctHPVDNA levels were higher in patients with known adverse pathologic risk factors such as extranodal extension >1 mm (P = .0481) and with increasing lymph nodes involved (P = .0453) and were further associated with adjuvant treatment received (P = .0076). One of 33 patients had a recurrence that was detected by ctHPVDNA 2 months earlier than clinical detection.

Conclusions: POD 1 ctHPVDNA levels are associated with the risk of residual disease in patients with HPV+OPSCC undergoing curative intent surgery and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future.

Lay summary: Human papillomavirus-associated oropharyngeal squamous cell carcinoma (HPV+OPSCC) is increasing at epidemic proportions and is commonly treated with surgery. This report describes results from a study examining the clearance kinetics of circulating tumor HPV DNA (circulating tumor human papillomavirus DNA [ctHPVDNA]) following surgical treatment of HPV+OPSCC. We found that ctHPVDNA levels 1 day after surgery are associated with the risk of residual disease in patients with HPV+OPSCC and thus could be used as a personalized biomarker for selecting adjuvant treatment in the future. These findings are the first to demonstrate the potential utility of ctHPVDNA in patients with HPV+OPSCC undergoing surgery.

Keywords: cell-free DNA; circulating tumor DNA; head and neck cancer; human papillomavirus (HPV); oropharyngeal cancer.

Figure 1.

Consolidated Standards of Reporting Trials diagram of patient cohorts. The red box is the primary surgical cohort. HPV indicates human papillomavirus.

Figure 2.

ctHPVDNA detection at the time of diagnosis and correlation with tumor stage and site. (A) ctHPVDNA detection at the time of diagnosis. The color of the shapes corresponds to genotype detected, whereas shape corresponds to the test used on biopsy to confirm HPV+OPSCC. All patients with direct HPV testing (RNA ISH and DNA PCR) also had p16 IHC. The red dotted line represents the test threshold for a positive versus negative test, demonstrating 1 patient below threshold. (B) T stage and (C) N stage were borderline associated with ctHPVDNA levels at presentation (P = .0453; P = .0506), whereas (D) the tumor anatomic site was not (P = .58). The median and interquartile range are shown for each graph (box). P values are based on a 2-sided Mann-Whitney U test. ctHPVDNA indicates circulating tumor human papillomavirus DNA; HPV, human papillomavirus; HPV+OPSCC, human papillomavirus (HPV)–associated oropharyngeal squamous cell carcinoma; IHC, immunohistochemistry; ISH, in situ hybridization; OPSCC, oropharyngeal squamous cell carcinoma; PCR, polymerase chain reaction.

Figure 3.

ctHPVDNA kinetics after surgery. (A) Early kinetics cohort demonstrating clear separation within 6 hours of patients with risk factors for persistent macroscopic disease (red lines) and remainder of cohort (blue lines). Samples were collected immediately before surgery, immediately after tumor removal, and every 6 hours for 24 hours. Total cohort size was 12 patients. Vertical red dotted line represents 6:00 AM postoperative day (POD) 1. (B) Patients undergoing early kinetics lacking all known clinicopathologic risk factors (n = 4) demonstrated a rapid decrease to <1 copy/mL in 1 to 18 hours after surgery. Data were normalized to the pretreatment level. (C) Patients with risk factors for macroscopic RD (red lines) and no risk factors (blue lines) demonstrated clear separation within 18 hours of surgery (POD 1) in full cohort. The black arrow indicates POD 1 values on the x-axis scale. ctHPVDNA indicates circulating tumor human papillomavirus DNA.

Figure 4.

Postoperative ctHPVDNA levels and clinicopathologic risk factors. Patients were stratified by postoperative day (POD) 1 ctHPVDNA: (A) lymph node burden, (B) ENE, and (C) surgical margins. The star represents patient with positive surgical margin on initial evaluation, but no tumor cells were identified on reresection; this suggests false-positive histopathology. (D) The percentage of patients with pathologic risk factors with >1 and >100 copies/mL on POD 1 demonstrating higher POD 1 ctHPVDNA with higher risk features. (E) POD 1 ctHPVDNA is predictive of the adjuvant treatment received. The adjuvant treatment received correlates with POD 1 ctHPVDNA levels. The median and interquartile range are shown for each graph. CRT indicates chemoradiation; ctHPVDNA, circulating tumor human papillomavirus DNA; ENE, extranodal extension; LN, lymph node; LVI, lymphovascular invasion; PNI, perineural invasion; XRT, radiation therapy.

Figure 5.

ctHPVDNA detects recurrence earlier than standard of care monitoring. This figure contrasts 2 patients with the same stage presentation prior to surgery (cT1N1) who had significant differences in postoperative circulating tumor human papillomavirus DNA (ctHPVDNA) levels indicating differences in their residual disease (RD) burden. Patient HL4 (blue line) had significant elevation on postoperative day (POD) 1, downtrending only after adjuvant chemoradiotherapy (blue vertical lines) and immediate re-elevation signifying persistence. Persistence was detected by ctHPVDNA 2 months before clinical detection. Patient 11 (red line) had an immediate decrease in ctHPVDNA to zero after surgery. ctHPVDNA remained undetectable for the remainder of the study with no clinical evidence of recurrence.