Development of a high-performance multi-probe droplet digital PCR assay for high-sensitivity detection of human papillomavirus circulating tumor DNA from plasma.

To develop a high-performance droplet digital PCR (ddPCR) assay capable of enhancing the detection of human papillomavirus (HPV) circulating tumor DNA (ctDNA) in plasma from patients with HPV-associated oropharyngeal squamous cell carcinoma (HPV+ OPSCC). Plasma samples from subjects with HPV+ OPSCC were collected. We developed a high-performance ddPCR assay designed to simultaneously target nine regions of the HPV16 genome. The new assay termed 'ctDNA HPV16 Assessment using Multiple Probes' (CHAMP- 16) yielded significantly higher HPV16 counts compared to our previously validated 'Single-Probe' (SP) assay and a commercially available NavDx® assay. Analytical validation demonstrated that the CHAMP-16 assay had a limit of detection (LoD) of 4.1 copies per reaction, corresponding to < 1 genome equivalent (GE) of HPV16. When tested on plasma ctDNA from 21 patients with early-stage HPV+ OPSCC and known HPV16 ctDNA using the SP assay, all patients were positive for HPV16 ctDNA in both assays and the CHAMP-16 assay displayed 6.6-fold higher HPV16 signal on average. Finally, in a longitudinal analysis of samples from a patient with recurrent disease, the CHAMP-16 assay detected HPV16 ctDNA signal ∼ 20 months prior to the conventional SP assay. Increased HPV16 signal detection using the CHAMP-16 assay suggests the potential for detection of recurrences significantly earlier than with conventional ddPCR assays in patients with HPV16+ OPSCC. Critically, this multi-probe approach maintains the cost-benefit advantage of ddPCR over next generation sequencing (NGS) approaches, supporting the cost-effectiveness of this assay for both large population screening and routine post-treatment surveillance.

Bhambhani C ，Sandford E ，Haring CT ，Brummel C ，Tuck KL ，Olesnavich M ，Bhangale AD ，Walline HM ，Dermody SM ，Spector ME ，Chinn SB ，Casper K ，Mierzwa M ，Swiecicki PL ，Chad Brenner J ，Tewari M ... -  《-》

HPV circulating tumoral DNA quantification by droplet-based digital PCR: A promising predictive and prognostic biomarker for HPV-associated oropharyngeal cancers.

We aimed to determine whether pretherapeutic assessment of HPV circulating tumoral DNA (HPV ctDNA) by droplet-based digital PCR (ddPCR) could constitute a predictive and prognostic biomarker for HPV-associated oropharyngeal squamous cell carcinoma (OPSCC). A mono-institutional prospective biomarker study on 66 patients with p16+/HPV16-positive oropharyngeal squamous cell carcinoma (OPSCC) was conducted in European Georges Pompidou Hospital, Paris, France. Blood samples were collected at the time of diagnosis before any treatment. Optimized digital PCR assays were used to quantify HPV16 ctDNA. Forty-seven (71%) patients showed a positive pretherapeutic HPV ctDNA at time of diagnosis. Interestingly, the quantity of HPV16 ctDNA at baseline, as assessed by ddPCR, was significantly correlated with the T/N/M status or OPSCC stages according to the 2018 new staging criteria for high-risk human papillomavirus (HR HPV) related OPSCC from American Joint Committee on Cancer (AJCC). Moreover, all recurrences and the majority (83%) of death reported events occurred in patients with positive HPV16 ctDNA at baseline. Finally, when posttreatment blood samples were available (n = 6), the kinetic of pretreatment/posttreatment HPV16 ctDNA was clearly associated with treatment success or failure. HPV ctDNA monitoring by ddPCR could constitute a useful and noninvasive dynamic biomarker to select HR HPV-related OPSCC patients eligible for potential treatment de-escalation and to monitor treatment response.

Veyer D ，Wack M ，Mandavit M ，Garrigou S ，Hans S ，Bonfils P ，Tartour E ，Bélec L ，Wang-Renault SF ，Laurent-Puig P ，Mirghani H ，Rance B ，Taly V ，Badoual C ，Péré H ... -  《-》

Detection of Circulating HPV16 DNA as a Biomarker for Cervical Cancer by a Bead-Based HPV Genotyping Assay.

Galati L ，Combes JD ，Le Calvez-Kelm F ，McKay-Chopin S ，Forey N ，Ratel M ，McKay J ，Waterboer T ，Schroeder L ，Clifford G ，Tommasino M ，Gheit T ... -  《Microbiology Spectrum》

Detection of human papillomavirus type 16 in oropharyngeal squamous cell carcinoma using droplet digital polymerase chain reaction.

Biron VL ，Kostiuk M ，Isaac A ，Puttagunta L ，O'Connell DA ，Harris J ，Côté DW ，Seikaly H ... -  《-》

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

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

O'Boyle CJ ，Siravegna G ，Varmeh S ，Queenan N ，Michel A ，Pang KCS ，Stein J ，Thierauf JC ，Sadow PM ，Faquin WC ，Wang W ，Deschler DG ，Emerick KS ，Varvares MA ，Park JC ，Clark JR ，Chan AW ，Busse PM ，Corcoran RB ，Wirth LJ ，Lin DT ，Iafrate AJ ，Richmon JD ，Faden DL ... -  《-》