Cell Free HPV DNA: A new diagnostic and predictive test for HPV oropharynx cancer

Written by Marshall Posner, MD & Eric Genden, MD, MHCA, FACS

A large shift in the causes and natural history of head and neck cancer has radically changed the prognosis and survival of a significant fraction of patients in the last two decades. This has been driven by the identification of human papilloma virus (HPV) as the major causative factor in oropharynx cancer (OPC). HPV-related OPC is now so common and the biology and prognosis are so different from environmentally and smoking related head and neck cancers that it has a specific designation – HPVOPC. HPV is a transmissible virus which is passed among people by exchange of bodily fluids usually associated with sexual activity. Living virus does not enter into the blood stream and cannot be transmitted by blood transfusion or exposure. Many people are infected in their teenage years or in young adulthood by sexual or intimate acts that transmit bodily fluids. The virus is highly infectious and is likely transmitted by kissing or exchange of saliva in teenagers and adults. Fortunately, the development of an HPV vaccine may reduce the incidence of this cancer. The impact of preventitive HPV vaccines on later cancer development will not be realized for about 30 more years as the vaccinated population matures. While HPV infection is nearly ubiquitous, very few people develop an HPV-related cancer. In fact, the vast majority of those infected will not develop HPV-associated malignancy and may asymptomatically carry the virus. Nonetheless, HPV- associated OPC now represents about 60-70% of all head and neck oropharynx cancer seen in the United States and it is expected that there will be more than 30,000 cases a year by 2025. We have also found that HPV is implicated in significant fraction of sinus cancers and a small fraction of nasopharynx cancers. Outside of the head and neck HPV is associated with anal, cervical, vulvar, and penile cancers. 

Unlike tobacco-related head and neck cancer, HPV OPC is very responsive to radiation or chemotherapy and cure rates for patients range between 65% and 95% – as opposed to 30-45% for tobacco-related head and neck cancer. While biomarkers such as invasion of blood vessels, nerves, and lymphatics are considered poor prognostic factors in tobacco-related disease, these biomarkers are less indicative of a poor outcome in HPV-associated cancer. These findings support that HPV-associated disease is fundamentally different than tobacco-associated disease.

Because cure rates are high in HPV associated oropharyngeal cancer, more emphasis has been placed on treatment toxicity. This is especially important because, in general, the patients develop this disease at a younger age than patients afflicted with tobacco associated disease. They will survive for decades after treatment. The long-term consequences of therapy have become an important consideration in this population of patients. Therefore, the goal of reducing aggressive therapy and thus the significant late toxic effects of chemo-radiation and surgery have to enter into the equation of management and decision making. Radiation carries the greatest burden of acute toxicity effects and significant long-term consequential toxicity. Reducing the dose of radiation, while preserving high survival rates would have a significant impact on quality of life in this highly curable disease. Additionally, surveillance and follow-up are critically important to identify recurrent disease early in the process so that salvage treatment can be instituted. There is a need to develop better diagnostic and prognostic tests that are accurate and cost effective. With increasing numbers of survivors, there is an increasing need for noninvasive surveillance. Positron emission tomography (PET) scan is one of the best diagnostic tests available for this disease. Unfortunately, it is costly and is associated with a relative high radiation dose compared to other radiologic methods and may be associated with unexpected toxicity and consequences over time. Nonetheless, finding recurrences or identifying persistent disease early after initial treatment can lead to successful salvage treatments. As more patients achieve cure, surveillance testing becomes increasingly necessary and expensive. Second, a pre-therapy prognostic test that identifies patients who will most likely achieve cure with a reduced dose of radiotherapy or chemotherapy would help to facilitate risk based reduction in these treatments. Reducing primary treatment, especially radiation therapy, will improve long-term consequences and morbidity as well as improve survival for patients. Third, a post radiation or postoperative test that accurately identified patients who need more therapy because of persistence or residual disease would allow early intervention with additional therapies including vaccines and immune modulators. Evaluation of patients in the immediate postoperative or post radiation setting is very complicated by tissue inflammation and swelling. Having a highly sensitive or specific test would improve identification of persistent disease where additional treatment may be beneficial. 

Recently, a new test has been developed which has some characteristics that would appear to meet some or all of these needs. This biomarker test is based on the fact that in HPV OPC, human papilloma virus is present in all HPV OPC tumors and that measuring virus in blood may serve as a highly specific biomarker of HPV cancer. Because the virus is necessary for the tumors to maintain a malignant state it is both a target for therapy and a diagnostic biomarker test for the cancer and for prognosis. It has now been shown by academic laboratories (Dana Farber Cancer Institute; Mount Sinai Medical Center; Johns Hopkins University School of Medicine; Case Western Reserve; MD Anderson Cancer Center; University of North Carolina) that it is possible to detect cell free fragments of HPV viral DNA in the blood of patients who have active HPV OPC 1-6. There is also least 1 commercial entity (Naveris) which provides commercial clinical testing. This test is in the early stages of development and the technology is relatively expensive and not routinely available. It is, however, very promising. The concept makes good rational sense and is currently being investigated to see if the promise of a specific and usable plasma test can be realized. We now know that cell free HPV DNA (cfHPVDNA) as a blood test is highly specific to HPV caused cancers. Thus, it may be an excellent test to screen patients who might be at high risk for developing a cancer, predict who might do well with surgery, chemotherapy or radiation and identify recurrences in patients who have been treated for cure and who are in surveillance. 

Several studies have shown that the detection of cfHPVDNA can predict recurrence in patients with HPVOPC who have been treated for cure after surgery and chemoradiotherapy. These results are very promising although preliminary. In one study 100% of recurrences were identified by a first-time positive test. Many of those patients were initially negative after curative therapy and became positive during repeated testing in follow up. A large fraction of patients, 43%, had an initial false-positive that on repeat testing became negative and only 6% with 2 positive tests did not have recurrence identified. However, the remaining patients, all with 2 positive tests (94%), were found to have a recurrence2. These early results are very exciting. There are many details that have to be worked out for physicians to know how to use cfHPVDNA and for routine testing to become a standard of care6. One clear conclusion from the data is that many patients were negative after definitive treatment and then developed a positive test indicating that there is a minimum volume threshold necessary to identify persistent or recurrent disease. This leads to a number of questions on how to utilize the test: for example, how often to do testing. Should it be every month for the first 6 months or year after surgery or radiation therapy or perhaps only in the first 6 months and then quarterly after that? How truly sensitive and accurate is the test? Does this become a confirmatory test for a positive scan or the primary diagnostic test? Are we finding tumors early enough in recurrence and how much of a threshold of volume is necessary to be positive – and might they be found by physical exam or appear on PET scans earlier when they are still small and below a cfHPVDNA threshold? There may not be a direct correlation of cfHPVDNA to volume. The biology of each cancer in each person might be unique and might impact on the sensitivity of the assay to recurrence. A positive cfHPVDNA does not tell the physician if this is a local or metastatic spread. This may confound decisions about regional versus systemic salvage treatment. Finally, in our unpublished experience we found that some HPVOPC patients developed late second cancers that were HPV negative and may have been secondary to life style causes such a smoking or to prior radiation therapy. These second cancers can be shown to be molecularly distinct and not HPV related by molecular diagnostic studies. These latter events support lifelong follow-up in patients who smoked or were treated with radiation.

One potential use of the test would be to select patients after surgery who would benefit from postoperative radiotherapy or immunotherapy. This testing could take place in the 6 week window in which postoperative chemoradiotherapy is optimally started. It might also be possible for those patients getting primary chemoradiotherapy to do a midcourse test and evaluate whether they are having a sufficient response such that more or less radiation would be indicated. Pretreatment testing will also be important. There is some very preliminary data that suggests lower levels of cfHPVDNA fragments prior to therapy may be predictive of a poor prognosis biology and early recurrence although this is not consistent6. It is possible that molecular characteristics of the fragments might give information on the prognosis as well. These changes could suggest a more resistant form of HPV in the cancers. In patients with locally advanced disease who receive chemotherapy prior to chemoradiotherapy or primary chemo radiotherapy evaluating the velocity of decrease – the rate at which the levels of cfHPVDNA decrease or if they reach 0 during therapy- might help select patients for even further reductions in chemoradiation therapy. 

Many of these questions have not been addressed as of yet. However, this is a very exciting new technology which holds great promise for helping improve the outcome in patients. Getting sufficient data through clinical trials to optimally use this test will take some time. However, as our understanding and utilization of the test unfold we will hopefully be able to apply it to improve the quality of life, cost of treatment, treatment toxicity, and anxiety of our patients.

Selected Bibliography:

1. Chera BS, Kumar S, Beaty BT, et al: Rapid Clearance Profile of Plasma Circulating Tumor HPV Type 16 DNA during Chemoradiotherapy Correlates with Disease Control in HPV-Associated Oropharyngeal Cancer. Clin Cancer Res 25:4682-4690, 2019

2. Chera BS, Kumar S, Shen C, et al: Plasma Circulating Tumor HPV DNA for the Surveillance of Cancer Recurrence in HPV-Associated Oropharyngeal Cancer. J Clin Oncol:JCO1902444, 2020

3. Hanna GJ, Supplee JG, Kuang Y, et al: Plasma HPV cell-free DNA monitoring in advanced HPV-associated oropharyngeal cancer. Ann Oncol 29:1980-1986, 2018

4. Dahlstrom KR, Li G, Hussey CS, et al: Circulating human papillomavirus DNA as a marker for disease extent and recurrence among patients with oropharyngeal cancer. Cancer 121:3455-64, 2015

5. Wang Y, Springer S, Mulvey CL, et al: Detection of somatic mutations and HPV in the saliva and plasma of patients with head and neck squamous cell carcinomas. Sci Transl Med 7:293ra104, 2015

6. Chatfield-Reed K, Roche VP, Pan Q: cfDNA detection for HPV+ squamous cell carcinomas. Oral Oncol 115:104958, 2021

Editors Note: Dr. Marshall Posner is Professor of Medicine Icahn School of Medicine at Mount Sinai, NY. He is Medical Director of the Head and Neck Oncology Program, Associate Director of the Center for Personalized Cancer Therapeutics, and co-Director of the Cancer Clinical Investigation Program for the Tisch Cancer Institute. Dr. Posner has published over 275 peer-reviewed laboratory and clinical studies. He has been principal investigator of NIH grants in immunology and has been principal investigator of multiple Phase 1, 2 and 3 clinical trials. He has a strong clinical and clinical research focus on Immunotherapy, HPV oropharynx cancer and salivary gland cancers.

Dr. Eric Genden is a member of the Icahn School of Medicine at Mount Sinai faculty since 1988. Dr. Genden is an internationally recognized expert and innovator in the management of head and neck cancer, microvascular reconstruction of the head and neck, and transplantation. He is the Dr. Isidore Friesner Endowed Professor and System Chairman of the Department of Otolaryngology – Head and Neck Surgery and a Professor of Neurosurgery and Immunology. His many leadership roles at Mount Sinai include Senior Associate Dean for Clinical Affairs at the Icahn School of Medicine at Mount Sinai. Dr. Genden plays a leadership role with the Mount Sinai Doctors Faculty Practice and a he is a Senior Vice-President for Ambulatory Surgery Development. 


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