MSK Investigators Create a Highly Accurate Liquid Biopsy for Identifying Early-Stage HPV-Associated Oral and Anal Cancers

By Daniel Higginson, MD Tuesday, December 10, 2019

Together with my colleagues at Memorial Sloan Kettering Cancer Center, we developed a highly accurate assay for identifying early-stage oral and anal cancers associated with human papillomavirus (HPV) using liquid biopsy. This low-cost technique could be advantageous in early detection screening trials and disease response monitoring.

The assay uses droplet digital polymerase chain reaction (PCR), the most sensitive circulating tumor DNA (ctDNA) method available. It detected HPV 16 and HPV 33, the two most common subtypes of HPV, with an overall sensitivity of 96 percent and a specificity of 100 percent in banked plasma specimens from patients with HPV who had been treated at MSK. The assay identified these HPV subtypes in a similar distribution, as reported in major genomic studies. ⁽¹⁾

We also found that HPV 16 ctDNA levels corresponded directly to tumor responses to chemoradiation and surgery in a subset of 68 patients with HPV-positive oropharyngeal squamous cell carcinoma, demonstrating the tool’s high sensitivity. ⁽¹⁾

We are excited that our low-cost innovation, published recently in JCO Precision Oncology, has the potential to revolutionize clinical screening and disease monitoring for HPV-associated cancers. In practice, low-cost liquid biopsy could be a first step for screening purposes, followed by additional tests, such as Papanicolaou (Pap) smears and next-generation sequencing of additional plasma specimens.

We are excited that [this test] has the potential to revolutionize clinical screening and disease monitoring for HPV-associated cancers.

Daniel S. Higginson Assistant Attending Radiation Oncologist

Human Papillomavirus Detection

HPV-associated cancers constitute 4.5 percent of all cancers worldwide. ⁽²⁾ They include squamous cell carcinomas of the oropharynx, cervix, vulva, vagina, anal canal, and penis. Pelvic exams and Pap smears are widely used screening methods for the early detection of HPV-associated cervical cancers, but there are no effective screening tools for other disease sites.

HPV-associated oropharyngeal squamous cell carcinoma is an emerging epidemic with rapidly increasing incidence. ⁽³⁾ Previous studies of head and neck cancers using low-cost ctDNA PCR-based methods have shown a modest sensitivity of only 19 to 79 percent in patients with gross disease. ⁽⁴⁾^, ⁽⁵⁾^, ⁽⁶⁾^, ⁽⁷⁾^, ⁽⁸⁾^, ⁽⁹⁾ The HPV 16 and HPV 33 subtypes account for more than 95 percent of all oropharyngeal ⁽¹⁰⁾^, ⁽¹¹⁾ and anal ⁽¹²⁾ HPV-associated squamous cell carcinomas.

Assay Design

A major challenge for HPV screening for early cancers is specificity, since the prevalence of oral, penile, cervical, and anal HPV 16 infection is only 1 to 4 percent in the general population. ⁽¹³⁾^, ⁽¹⁴⁾^, ⁽¹⁵⁾^, ⁽¹⁶⁾

To develop the best possible primer-probe set for the droplet digital PCR, we chose primers and probes within the E6 and E7 oncogenes, since these sequences are the most highly amplified in tumor genomes. ⁽¹⁷⁾ We also looked for a smaller amplicon size to increase the probability of amplifying the view of highly fragmented circulating free DNA, as well as primers or probes that perfectly matched both European and non-European HPV 16 isolates.

Using the Papillomavirus Episteme knowledge resource, ⁽¹⁸⁾ we identified ten sequences representative of HPV 16 sublineages, spanning European, Asian, and African isolates. Then we matched exactly the ten most common sequences and compared those sequences with an additional 455 HPV 16 isolates found in GenBank, the genetic sequence database of the National Institutes of Health. A primer-probe within E6 containing 97 base pairs demonstrated the best PCR efficiency and sensitivity down to a single molecule of template HPV 16 DNA. ⁽¹⁾

In a challenge test, our assay outperformed two other HPV DNA identification platforms, detecting a 16-fold and five-fold higher number of ctDNA signals compared to the Roche Cobas HPV Test (Roche, Indianapolis, Indiana) and quantitative PCR, respectively. ⁽¹⁾

Study Design

To test the clinical utility of the droplet digital PCR to detect HPV via ctDNA, we used banked plasma specimens from 97 HPV-positive patients with locoregionally confined oropharyngeal squamous cell carcinoma and eight HPV-positive patients with anal squamous cell carcinoma. HPV positivity was defined as p16 overexpression by immunohistochemistry with greater than 70 percent diffuse nuclear or cytoplasmic staining or a clinically reported positive DNA- or RNA-based in situ hybridization test for HPV. ⁽¹⁾

We included negative controls as follows: seven patients with HPV-negative head and neck cancer, eight patients with HPV-positive oropharyngeal squamous cell carcinoma who had already undergone surgical resection, and 20 patients without cancer. Gross tumor volume data was determined from radiation plans. ⁽¹⁾

We determined the HPV 16 copy number in genomic DNA in pathological samples from a subset of patients who had received targeted next-generation sequencing with the MSK-IMPACT^™ sequencing platform. ⁽¹⁹⁾We also examined the ability of the assay to measure patient responses to chemoradiation and surgery in a subset of 68 patients. ⁽¹⁾

Study Results

Our droplet digital PCR assay detected HPV 16 in the ctDNA of 90 of 97 patients with HPV-positive oropharyngeal squamous cell carcinoma, demonstrating a sensitivity of 93 percent. It also detected HPV 16 in seven of eight patients with HPV-positive anal cancers. ⁽¹⁾

Among the control samples, which included 20 people without cancer, seven patients with HPV-negative head and neck cancer, and eight patients who had undergone surgery, no HPV 16-positive droplets were detected, demonstrating a specificity of 100 percent. ⁽¹⁾

Then we developed a droplet digital PCR assay for HPV 33, the next most common type of HPV associated with oropharyngeal squamous cell carcinoma. ⁽²⁰⁾ Among seven specimens that tested negative for HPV 16, three were HPV 33-positive. Thus, across both HPV 16 and HPV 33 subtypes, the droplet digital PCR assay detected cancer in 93 of 97 patients, demonstrating an overall sensitivity of 96 percent. ⁽¹⁾

Our novel tool also readily detected HPV ctDNA levels in all 19 patients with low-volume tumor burden and identified HPV 16 and HPV 33 in a similar distribution as reported in major genomic profiling studies. ⁽¹⁾

For a subset of 68 patients with HPV-positive oropharyngeal squamous cell carcinoma, we examined samples obtained every week during and after chemoradiation and observed rapid declines in HPV in all patients. HPV 16 ctDNA was generally cleared by week seven after the start of chemoradiation, except for three patients who still had detectable levels at ten weeks. ⁽¹⁾

Advancing Early Detection of HPV

Another approach to detect HPV-associated cancers is next-generation sequencing, which can capture multiple subtypes in one test. A recent study using next-generation sequencing to determine circulating HPV in the plasma DNA of 55 patients with locally advanced head and neck squamous cell carcinoma showed 100 percent sensitivity. ⁽²¹⁾ However, it is a much more costly method.

To detect cervical cancer with our method in a single test, we would need to add an HPV 18 droplet digital PCR test to our assay, which is reasonably easy to execute.

At MSK, we are dedicated to finding new ways to detect HPV-associated cancers — including oropharyngeal cancer, anal cancer, cervical cancer, and penile cancer — at the earliest stages, to improve outcomes for patients. Our multidisciplinary surgeons, radiation oncologists, and medical oncologists collaborate to determine the best treatment approach for each patient.

This study was supported by the James and Judith K. Dimon Foundation, and a research grant from the Society of Memorial Sloan Kettering Cancer Center. It was also supported in part by a National Institutes of Health/National Cancer Institute Cancer Center Support Grant (P30CA008748), Cycle for Survival, and the Marie-Josée and Henry R. Kravis Center for Molecular Oncology at MSK.

Dr. Higginson and R. R. Damerla are inventors on a provisional patent application filed by MSK.

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