Sensitive PCR Test Predicts Treatment Response in Metastatic Head and Neck Cancers

A recently developed droplet digital PCR (ddPCR)-based assay for circulating tumor DNA (ctDNA) from human papillomavirus HPV16 (which is responsible for almost 90% of HPV-positive oropharyngeal cancers) predicts treatment response in metastatic human papillomavirus related (HPV+) oropharyngeal squamous cell carcinoma (OPSCC).

Head and neck squamous cell carcinomas (HNSCC) constitute 3–5% of all malignancies worldwide and there are approximately 600,000 newly diagnosed cases annually. The majority of patients with HNSCC present with locally and/or regionally advanced disease at diagnosis. Despite the rising incidence of HPV + OPSCC, treatment of metastatic disease remains palliative rather than curative. Even with new treatments such as immunotherapy, response rates are low and can be delayed, while even mild tumor progression in the face of an ineffective therapy can lead to rapid death. Real-time biomarkers of response to therapy could improve outcomes by guiding early change of therapy in the metastatic setting.


Investigators at the University of Michigan (Ann Arbor, USA) turned to a sensitive liquid biopsy-based PCR test for such biomarkers. The classical PCR test carries out one reaction per single sample. The digital PCR (dPCR) method also carries out a single reaction within a sample, however the sample is separated into a large number of partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid amounts. The dPCR method has been demonstrated as useful for studying variations in gene sequences - such as copy number variants and point mutations - and it is routinely used for clonal amplification of samples for next-generation sequencing.

Droplet digital PCR (ddPCR) is a variation of dPCR in which a 20 microliter sample reaction including assay primers and either Taqman probes or an intercalating dye, is divided into about 20,000 nanoliter-sized oil droplets through a water-oil emulsion technique, thermocycled to endpoint in a 96-well PCR plate, and fluorescence amplitude read for all droplets in each sample well in a droplet flow cytometer.

In initiating the current study, the investigators hypothesized that: 1) a HPV16 ctDNA test would offer a precise assay for detection of HPV+ OPSCC and 2) the assay would predict progressive disease prior to radiographic imaging in patients undergoing treatment for recurrent or metastatic HPV+ OPSCC. Thus, they developed and analytically validated a highly sensitive and specific ddPCR assay for absolute quantification of HPV ctDNA from plasma specimens and performed an evaluation of clinical utility in a prospective, longitudinal patient cohort.

For this study the investigators analyzed more than 100 samples taken over nearly two years from 16 patients with advanced cancers that were positive for HPV16. Seven of the patients were treated with immunotherapy regimens. Eleven patients underwent treatment that included chemotherapy. Results revealed that increasing levels of HPV-positive circulating tumor DNA after a course of treatment were a strong indicator that the cancer was not responding to the treatment. Furthermore, it was found that the tumor DNA could be detected months earlier than tumor growth that could be measured on imaging scans.

"Currently, the only way doctors know if a treatment is working is for the patient to get an imaging scan every few months to see whether their tumors are shrinking," said contributing author Dr. Paul Swiecicki, assistant professor of medical oncology at the University of Michigan. "And this is not fully accurate since some cancers show what we call pseudoprogression, where a successful treatment actually makes the tumors bigger before it shrinks them. Our goal was to develop a test that could tell us whether a treatment is likely to work after a single cycle."

The study was published in the June 22, 2021, online edition of the journal Oncotarget.

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