Advances in biomarkers and treatment strategies for HPV-associated head and neck cancer

Understanding the etiology of human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) is becoming a matter of increasing necessity and urgency as the incidence of HPV(+) OPSCC continues to rise and now exceeds that of HPV-associated cervical cancers [1]. HPV(+) head and neck cancers, over 90% of which are caused by HPV type 16, are clinically and epidemiologically distinct from those not associated with HPV. Moreover, HPV(+) and HPV(-) OPSCCs display distinct mutation and methylation landscapes and different protein expression profiles [2, 3]. Regardless of treatment strategy, HPV-positivity confers a favorable prognosis with 5-year survival rates of 75-80% versus 45-50% among HPV-negative patients [4]. Despite these differences, treatment guidelines recommend similar treatment regardless of HPV status. Efforts to de-escalate therapy for HPV(+) patients are now not only being prioritized to maintain cure rates, but also to decrease treatment-related side effects. Treatment for advanced head and neck cancer includes radiation and platinum-based chemotherapy, which are associated with dose-related adverse side effects including acute toxicities like mucositis and loss of taste, as well as long-term problems of dysphagia, osteoradionecrosis, xerostomia, muscle fibrosis, and lymphedema. These side effects can lead to downstream infections, difficulty eating, and increased hospitalizations, which decrease a patient’s quality of life. Limiting these side effects is especially important in HPV(+) patients, who present at a younger age. Despite their improved cure rates, 20-30% of HPV(+) tumors will recur and upon recurrence, effective treatment options are limited. Given toxicities associated with aggressive therapy, recent studies have focused to improve treatment of HPV(+) OPSCC. The most common strategies include limiting platinum-based chemotherapies and decreasing radiation fields or dosage. At best, these strategies can minimize therapeutic side effects, but at the risk of decreased cure rates. Our group has taken a different approach with two major points of focus. The first has been to identify vulnerabilities of HPV-associated OPSCC that may lead to more targeted toward HPV(+) tumors treatments. The second has been to identify reliable biomarkers that predict which patients can benefit from and safely undergo treatment de-escalation. We hope that these research directions will not only decrease side effects while maintaining efficacy, but also provide treatment for those with recurrent or resistant HPV(+) OPSCC. Recently, we became interested in 5-azacytidine (5-azaC), a DNA-demethylating agent, following the discovery that HPV(+) tumors have a hypermethylated genome [3]. Previous literature has shown that demethylation decreased expression of the major HPV oncogenes E6 and E7, which have been linked to hypermethylation and invasiveness in cervical cancer [5, 6]. In our preclinical studies and in a window clinical trial at the Yale Cancer Center, 5-azaC markedly downregulated expression of all HPV genes. 5-azaC treatment of cultured cells inhibited growth and caused cell death that was dependent on stabilization of p53 (Figure 1) [7]. Increased p53 protein levels following 5-azaC correlated with decreased expression of the HPV E6, providing a mechanism for this effect. We also examined the ability of 5-azaC to inhibit tumor metastasis. Using cell culture, xenograft mouse model, and samples from the window clinical trial, we found that 5-azaC decreased expression of matrix metalloproteinases (MMPs) 1 and 10 that degrade the extracellular matrix to allow cancer cells to Editorial


HPV-associated head and neck cancer Cassie Pan, Wendell G. Yarbrough and Natalia Issaeva
Understanding the etiology of human papillomavirus (HPV)-driven oropharyngeal squamous cell carcinoma (OPSCC) is becoming a matter of increasing necessity and urgency as the incidence of HPV(+) OPSCC continues to rise and now exceeds that of HPV-associated cervical cancers [1]. HPV(+) head and neck cancers, over 90% of which are caused by HPV type 16, are clinically and epidemiologically distinct from those not associated with HPV. Moreover, HPV(+) and HPV(-) OPSCCs display distinct mutation and methylation landscapes and different protein expression profiles [2,3]. Regardless of treatment strategy, HPV-positivity confers a favorable prognosis with 5-year survival rates of 75-80% versus 45-50% among HPV-negative patients [4]. Despite these differences, treatment guidelines recommend similar treatment regardless of HPV status. Efforts to de-escalate therapy for HPV(+) patients are now not only being prioritized to maintain cure rates, but also to decrease treatment-related side effects.
Treatment for advanced head and neck cancer includes radiation and platinum-based chemotherapy, which are associated with dose-related adverse side effects including acute toxicities like mucositis and loss of taste, as well as long-term problems of dysphagia, osteoradionecrosis, xerostomia, muscle fibrosis, and lymphedema. These side effects can lead to downstream infections, difficulty eating, and increased hospitalizations, which decrease a patient's quality of life. Limiting these side effects is especially important in HPV(+) patients, who present at a younger age. Despite their improved cure rates, 20-30% of HPV(+) tumors will recur and upon recurrence, effective treatment options are limited.
Given toxicities associated with aggressive therapy, recent studies have focused to improve treatment of HPV(+) OPSCC. The most common strategies include limiting platinum-based chemotherapies and decreasing radiation fields or dosage. At best, these strategies can minimize therapeutic side effects, but at the risk of decreased cure rates. Our group has taken a different approach with two major points of focus. The first has been to identify vulnerabilities of HPV-associated OPSCC that may lead to more targeted toward HPV(+) tumors treatments. The second has been to identify reliable biomarkers that predict which patients can benefit from and safely undergo treatment de-escalation. We hope that these research directions will not only decrease side effects while maintaining efficacy, but also provide treatment for those with recurrent or resistant HPV(+) OPSCC.
Recently, we became interested in 5-azacytidine (5-azaC), a DNA-demethylating agent, following the discovery that HPV(+) tumors have a hypermethylated genome [3]. Previous literature has shown that demethylation decreased expression of the major HPV oncogenes E6 and E7, which have been linked to hypermethylation and invasiveness in cervical cancer [5,6]. In our preclinical studies and in a window clinical trial at the Yale Cancer Center, 5-azaC markedly downregulated expression of all HPV genes. 5-azaC treatment of cultured cells inhibited growth and caused cell death that was dependent on stabilization of p53 (Figure 1) [7]. Increased p53 protein levels following 5-azaC correlated with decreased expression of the HPV E6, providing a mechanism for this effect. We also examined the ability of 5-azaC to inhibit tumor metastasis. Using cell culture, xenograft mouse model, and samples from the window clinical trial, we found that 5-azaC decreased expression of matrix metalloproteinases (MMPs) 1 and 10 that degrade the extracellular matrix to allow cancer cells to

Figure 1: Schematic of selective multimodal 5-aza toxicity in HPV+ HNSCC
spread and have been shown to be critical for carcinomas invasion. Consistent with downregulation of MMPs, we found that xenografted HPV(+) OPSCC had reduced metastatic potential after 5-azaC treatment [7].
The ability of 5-azaC to activate interferon (IFN) response and induce a local production of T cells attracting cytokines in some HPV(+) head and neck cancer cells was another important finding of our study, suggesting a therapeutic potential of a combination of 5-azaC with anti PDL1/PD1 immunotherapy. Together, our data proposes the potential of 5-azaC in the treatment of HPV(+) OPSCC and prevention of metastasis ( Figure 1) and warrants investigation in a larger clinical trial.
One challenge of therapeutic de-escalation is to determine the appropriate subset of patients that will not suffer worse prognosis as a result. To address this challenge, we sought biomarkers that have utility in selecting HPV(+) OPSCC patients with improved prognosis who may be candidates for de-escalated therapy. Our analysis of The Cancer Genome Atlas (TCGA) head and neck cancer database revealed nearly mutually exclusive inactivating mutations in TRAF3 and CYLD genes in 28% of HPV(+) specimens [8]. TRAF3 and CYLD serve functions in activating immune response and inhibiting NF-kB, which is known to be activated in many cancers. These gene defects were also found to occur in 25% of a separate cohort of 23 HPV(+) OPSCC patients treated at Yale (data not shown), confirming that a subset of HPV(+) OPSCCs with defective TRAF3/CYLD may rely on overactive NF-kB and impaired innate immunity. Remarkably, analysis of the TCGA cohort revealed improved survival in HPV(+) patients with TRAF3/CYLD mutations compared with wild-type TRAF3/CYLD, while survival of HPV(+) patients without these mutations was similar to that of HPV(-) negative patients. These results suggest that constitutive activation of NF-kB defines a subgroup of HPV(+) patients with improved survival for whom de-escalated therapy may be safe and effective.
Development of less toxic treatment options and identification of a patient population that may be best for testing these therapies are key to improving patient morbidity and mortality in HPV(+) OPSCC. As more clinicians and researchers recognize this distinct subgroup of tumors and more is discovered about HPV-driven carcinogenesis, the better equipped we will be to prevent and treat HPV(+) head and neck cancer patients.