Immunotherapy in HPV-Related Oropharyngeal Cancers

Opinion statement Human papillomavirus (HPV)–related oropharyngeal squamous cell carcinoma (OPSCC) incidence has been increasing in recent decades. Treatment of the locally advanced HPV-related OPSCC includes a multidisciplinary approach. Immunotherapy with immune checkpoint inhibitors is used in the treatment of patients with recurrent/metastatic head and neck squamous cell carcinomas (HNSCC), including HPV-related OPSCC patients. There is increasing knowledge of the role of HPV in the tumor immune microenvironment. Therefore, HPV status of OPSCC plays an essential role in the design of immunotherapy clinical trials in both curative intent and metastatic settings. Moreover, HPV has become a potential therapeutic target, with vaccines and adoptive T-cell therapies being developed against HPV for the treatment of OPSCC. Several novel studies are designed to target HPV in combination with immune checkpoint inhibitors. Thus, HPV-related OPSCC remains a unique subgroup in the immunotherapy era.


Introduction
The incidence of HPV-related OPSCC has been increasing in recent decades [1]. HPV is a prognostic factor in head and neck cancers, and patients with HPV-positive OPSCC have better overall survival [1,2]. Immune checkpoint inhibitor (ICI) treatment with pembrolizumab and nivolumab has recently been approved in recurrent and metastatic head and neck squamous cell carcinoma (R/M HNSCC) [3][4][5].
The clinical trials leading to the approval of these agents included both HPV-related and unrelated HNSCC patients, and immunotherapy is used regardless of HPV status. However, HPV changes the immune microenvironment by modulating cytotoxic T cells, NK cells, and regulatory T cells [6]. Therefore, there are novel studies explicitly designed for HPVr e l a t e d O P S C C w i t h I C I s . M o r e o v e r , E 6 / E 7 oncoproteins have become a potential target for vaccine development [7]. Here we discuss the role of ICI treatment in HPV-related OPSCC and new treatment approaches.

Response to anti-PD1 immune checkpoint inhibitors
Conventional cytotoxic chemotherapy drugs, targeted therapy, and checkpoint inhibitors have all been shown to have activity in R/M HNSCC [3][4][5]8]. The PD-1 inhibitor pembrolizumab has activity alone or in combination with chemotherapy in patients with advanced HNSCC. Both pembrolizumab and PD-1 inhibitor nivolumab have been shown to benefit patients whose disease progressed on or after platinum-based regimens.

Platinum refractory disease
In a phase I study, KEYNOTE-012, patients with R/M HPV-positive HNSCC demonstrated a higher ORR with pembrolizumab alone as first-line or later treatment when compared directly with the HPV-negative cohort (25% versus 14%) [9]. Later data from an expansion cohort of KEYNOTE-012 demonstrated an even more pronounced ORR benefit (32% versus 14%), which also translated into an improvement in progression-free survival (PFS) (37% versus 20%) and overall survival (OS) (70% versus 56%) in those patients with HPVpositive disease [10]. However, this finding was not proven in large randomized phase III studies.
One of the first of these such trials was CheckMate-141, which was a randomized, open-label phase III trial in which 361 patients with R/M HNSCC whose disease had progressed within 6 months after platinumbased chemotherapy were assigned in a 2:1 ratio to receive nivolumab every 2 weeks or standard, single-agent systemic therapy of the physicians' choice (methotrexate, docetaxel, or the anti-EGFR-monoclonal antibody cetuximab) [3]. Nivolumab was found to improve OS when compared to standard chemotherapy. In 178 of the patients on the trial, p16 status was reported as a marker for HPV-positive head and neck cancers. Median survival was longer with immunotherapy when compared to chemotherapy, regardless of p16 status. Exploratory subgroup analysis suggested that the effect of anti-PD-1 therapy may be more pronounced in HPV-positive tumors, with a median OS of 9.1 versus 4.4 months, compared to 7.5 versus 5.8 months in those with HPV-negative tumors. This study led to the approval of nivolumab as a treatment option in R/M HNSCC after platinum therapy. The 2-year results of this trial also showed that nivolumab demonstrated a prolonged OS benefit with a consistent safety profile [11]. This OS benefit was again detected regardless of PD-L1 expression and HPV/p16 status. Nivolumab demonstrated~40% reduction in the risk of death compared with investigators' choice standard single-agent chemotherapy in both the HPV-positive subgroup (HR=0.60 [95% CI 0.37-0.97]) and HPV-negative subgroup (HR=0.59 [95% CI 0.38-0.92]). Treatment with nivolumab improved the overall response rate (ORR) as compared with standard chemotherapy in patients with HPV-positive tumors, while the ORR was similar across all treatment arms in patients with HPVnegative tumors. Subgroup analysis was done according to both tumor PD-L1 expression and HPV status. The greatest OS benefit with nivolumab was noted in the PD-L1 expressors with HPV-positive tumors (HR=0.39 [95% CI 0.18-0.81]).
Pembrolizumab is another anti-PD-1 immune checkpoint inhibitor used in platinum-refractory HNSCC. KEYNOTE-040 was a randomized, open-label phase III study that enrolled patients with HNSCC that progressed during or after platinum-based systemic treatment for recurrent or metastatic disease [4•]. Patients whose disease recurred or progressed within 3-to-6 months of chemotherapy were randomly assigned in a 1:1 fashion to receive pembrolizumab monotherapy or investigators' choice of standard, single-agent chemotherapy (methotrexate, docetaxel, or cetuximab). Median OS in the intent-to-treat population given pembrolizumab was 8.4 months (95% CI 6.4-9.4), and in those given standard of care chemotherapy was 6.9 months (95% CI 5.9-8.0; HR=0.80 [95% CI=0.65-0.98]). There was no difference in response to immunotherapy based on p16 status in this study. There were 119 patients in the study with a positive p16 status in the oropharynx and 376 patients with a negative p16 status. There was no notable difference in response to pembrolizumab in these two groups, with HR of 0.97 (95% CI 0.63-1.49) in the HPVpositive subgroup and HR of 0.77 (95% CI 0.61-0.97) in the HPV negative subgroup.
First-line treatment  was an open-label phase III trial where 882 patients with R/M HNSCC, which was deemed to be incurable by surgery and with no prior systemic therapy, were randomly assigned to receive single-agent pembrolizumab versus pembrolizumab plus platinum chemotherapy and fluorouracil combination versus cetuximab plus platinum chemotherapy and fluorouracil combination [5•]. PD-L1 status was assessed using the combined positive score (CPS), which stains lymphocytes, macrophages, and other cells in addition to tumor cells and reports these cells in relation to the total tumor cells in order to capture tumor and immune cells in a single score [12]. KEYNOTE-048 enrolled platinum-eligible patients to evaluate the effectiveness of chemoimmunotherapy or immunotherapy when compared to standard-of-care chemotherapy and cetuximab combination. Patients were stratified by PD-L1 expression, p16 status, and performance status. KEYNOTE-048 established pembrolizumab with or without chemotherapy as a first-line treatment option for patients with R/M HNSCC. This study demonstrated that the addition of pembrolizumab to platinum chemotherapy and fluorouracil improves OS when compared with cetuximab plus platinum chemotherapy and fluorouracil. OS was improved with single-agent pembrolizumab as compared to cetuximab plus platinum chemotherapy and fluorouracil in patients whose tumors had high PD-L1 expression (CPS ≥1). A subgroup analysis demonstrated that single-agent pembrolizumab had the biggest impact in those patients whose tumors had a CPS ≥20.

Combinations with CTLA-4 inhibitors
The EAGLE study was a randomized, open-label phase III clinical trial for patients with R/M HNSCC [13•]. Patients were randomly assigned to receive in a 1:1 ratio the anti-PD-L1 monoclonal antibody durvalumab, durvalumab plus the anti-CTLA-4 monoclonal antibody tremelimumab, or standard of care chemotherapy (single-agent methotrexate, a taxane, cetuximab, or a fluoropyrimidine). This study demonstrated that there were no statistically significant differences in OS for durvalumab or durvalumab plus tremelimumab versus standard of care. The number of patients with HPV+ oropharyngeal squamous cell carcinoma (OPSCC) was small in this trial: 30 patients received durvalumab, 30 patients received durvalumab plus tremelimumab, and 31 patients received standard of care. However, there was no difference in response to treatment in those with HPV-positive tumors in subgroup analysis as well. The negative results of this trial with ICI combination and ICI alone may be in part related to the unexpectedly high survival rate in the standard of care control arm.
CheckMate 651 study was another randomized phase 3 clinical trial assessing efficacy of anti-PD-1 inhibitor and anti-CTLA-4 inhibitor in R/M HNSCC. This study enrolled patients with no prior systemic treatment and randomized patients to cetuximab plus chemotherapy or nivolumab plus anti-CTLA-4 antibody ipilimumab. Patients were stratified by PD-L1 and p16 expression status. There was no statistically significant improvement in OS with nivolumab plus ipilimumab [14•].
These phase 3 studies have shown no OS benefit of addition of anti-CTLA-4 inhibitor for R/M HNSCC including HPV-related OPSCC. There are several ongoing studies on the role of the other novel checkpoint inhibitors including TIGIT, TIM-3, and LAG3 and also costimulatory agents such as OX40, 4-1BB, and CD40 in treatment of HPV-related OPSCC patients [15].

Anti-PD1 immune checkpoint inhibitor and radiation therapy combinations
The most common presentation for HNSCC is locally advanced disease requiring multidisciplinary treatment, including surgery, chemotherapy, and radiation therapy. Preclinical studies suggested the potential role of immune checkpoint inhibitors as a radiosensitizer [16,17]. Chemoradiation and immunotherapy was shown to be safe in early-phase trials in HNSCC [18,19].
Both studies enrolled locally advanced head and neck squamous cell carcinoma (LAHNSCC) patients, including select HPV-positive OPSCC patients with advanced disease (including T4, N2c, or N3). These studies did not show any OS benefit of the addition of an anti-PD1/PD-L1 inhibitor to concurrent chemoradiation.
Cetuximab and radiation therapy have been considered standard-of-care treatments for cisplatin-ineligible LAHNSCC patients. The GORTEC 2015-01 (PembroRad) study was a phase II randomized study that assessed the efficacy of pembrolizumab with radiation therapy to cetuximab with radiation therapy [22•]. The pembrolizumab arm did not improve 2-year PFS or OS. This study enrolled 60% OPSCC patients, of whom 46% were p16+. In a similar patient population, NRG-HN004 was a phase II/III trial to compare durvalumab with radiation to cetuximab for patients with LAHNSCC not eligible for cisplatin [23•]. 47% of the patients had p16+ OPCSCC or unknown primary. Phase II part of this study also failed to show improvement of PFS on durvalumab with radiation arm, and they decided not to move to phase III.
The GORTEC REACH study was a phase III study designed to assess the efficacy of avelumab plus cetuximab with concurrent radiation to cisplatin and radiation or cetuximab and radiation for cisplatin-unfit patients. This study enrolled patients with LAHNSCC including p16+ OPSCC. In the cisplatin-unfit cohort, primary end point for PFS was not met for avelumab and cetuximab. In cisplatin-fit cohort, futility boundary was crossed for standard of care cisplatin arm.
Trials using concurrent immunotherapy and radiation therapy with or without chemotherapy have not shown activity for LAHNSCC. Therefore, the effective sequence of radiation and immunotherapy remains unanswered. An institutional phase II study randomized LAHNSCC to receive concurrent chemoradiation with pembrolizumab or chemoradiation followed by sequential pembrolizumab [24]. Two-year PFS was longer in the sequential pembrolizumab arm.
Immunotherapy and radiation therapy studies for HPV-related OPSCC EA3161 is an ongoing study enrolling only patients with HPV-associated disease. This is a phase III trial of maintenance nivolumab versus observation in patients with locally advanced, intermediate-risk HPV-positive OPSCC. Patients are receiving nivolumab versus placebo following definitive concurrent chemoradiotherapy with cisplatin to determine whether this can improve PFS and OS in this population. For enrollment, patients must have HPV-related OPSCC with ≥ 10 pack-year smoking history, stage T1-2N2-N3 or T3-4N0-3 disease or G 10 pack-year smoking history, stage T4N0-N3 or T1-3N2-3 disease (locally advanced, intermediate risk features). This study will give a better insight into the role of sequential immunotherapy for HPV-related OPSCC.
Immunotherapy has also been incorporated into the treatment deescalation trials for HPV-related OPSCC. NRG-HN005 is a clinical trial that is focused specifically on patients with HPV-positive OPSCC. This phase II/ III study looks into how well reduced intensity treatment may work in those with HPV-positive OPSCC. The primary endpoint is non-inferiority in terms of PFS of concurrent reduced-dose RT with cisplatin or concurrent reduceddose RT with nivolumab compared with the current standard of care, which is standard-dose RT with cisplatin. In addition, the phase III portion aims to demonstrate the co-primary endpoints of non-inferiority of PFS as well as the superiority of quality of life for these patients undergoing these curativeintent therapies.
There are several other clinical trials for LAHNSCC that is not specifically designed for HPV-related OPSCC but include HPV-positive tumors ( Table 1).

Neoadjuvant anti-PD1 immune checkpoint inhibitor
There are several key studies on the potential benefits of neoadjuvant ICI in patients with HNSCC. Currently, more data is available on those with HPVnegative cancers, and they suggest pathologic response may predict response to treatment [22,23]. However, some trials also included those with HPV-positive disease.
One such study is CheckMate-358, a phase I/II study in HPV-associated cancers, which assessed neoadjuvant nivolumab in patients with previously untreated, resectable HPV-positive, and HPV-negative HNSCC [25•]. These patients received nivolumab on days 1 and 15, with surgery planned by day 29. This study found that neoadjuvant nivolumab was safe and induced pathologic responses in 23.5% of HPV-positive tumors and 5.9% of HPVnegative tumors. This trial sparked interest in potential combination neoadjuvant treatment regimens as well as adjuvant ICI for those with high-risk tumors.
Another phase II study enrolled resectable OPSCC patients and randomized to receive durvalumab or durvalumab plus tremelimumab [26•]. Twenty-four of 28 patients were p16-positive. The study found it was safe to administer neoadjuvant durvalumab; however, durvalumab plus tremelimumab did not increase CD8+TIL density more than durvalumab alone which was the primary endpoint of the study.
We still need a better understanding of the role of neoadjuvant immunotherapy in HPV-related OPSCC. Keynote 689 is a phase 3 randomized study of neoadjuvant and adjuvant pembrolizumab in patients with resectable HNSCC. This study enrolls HPV-related OPSCC patients with stage III disease (T4, N0-2).

Immunotherapy targeted against HPV Cancer vaccines
Based on the pathogenesis of the HPV virus, it is known that the development of HPV-related malignancies relies on the continual expression of the viral oncogenic proteins E6 and E7 [27]. Therefore, antigens derived from these proteins have become the target of many of the therapeutic HPV vaccines in development. The most frequently utilized strategies in HPV therapeutic cancer vaccine development have been peptide-based, vector-based (either viral or bacterial), and DNA-or RNA-based [6]. Given the remarkable T-cell responses in the pre-invasive malignancy setting, there is also a rationale to combine vaccines with ICI to further enhance T-cell responses [7]. Here we list the ongoing therapeutic vaccine trials for patients with HPV-positive OPSCC by vector used in the vaccine ( Table 2). The first is MEDI-0457, a VGX-3100 plasmid and INO-9012 plasmid vector DNA-based vaccine expressing HPV 16/18 E6 and E7 proteins and IL-12. A phase I/IIa trial studied the safety, tolerability, and immunogenicity of MEDI-0457 by electroporation (EP) in patients with HPV-positive HNSCC. These data demonstrated that MEDI-0457 generates HPV-specific peripheral humoral and cellular immune responses and affects the composition of immune cell infiltration into tumor tissue in this patient population [28•]. This vaccine was studied as part of a phase I/II trial of MEDI-0457 with durvalumab in recurrent and metastatic HPVpositive HNSCC [29]. Safety data was presented at the 2020 ESMO meeting; MEDI-0457 plus durvalumab was well-tolerated and showed clinical benefit. This study is active but not recruiting. Another trial is studying this vaccine in all R/M HPV+ tumors; preliminary results of this trial are pending. Another phase I/II dose escalation study (NCT03418480) is evaluating an HPV mRNA+ anti-CD40 vaccine (HARE-40) in patients with advanced HPV-positive cancer (head and neck, anogenital, penile, or cervical). This trial is actively recruiting. Another trial is studying BNT113, a vaccine with HPV 16 E6 and E7 antigens. This is an open-label, interventional two-arm phase II trial of BNT113 in combination with pembrolizumab versus pembrolizumab monotherapy as first-line treatment of unresectable recurrent or metastatic HPV-positive HNSCC.
There are also several viral and bacterial vector vaccine trials that are ongoing or recently completed. The first of these is ADXS11-001 with vector Listeria monocytogenes and antigen HPV 16 E7. A recent phase II trial studied ADXS11-001 prior to robotic surgery for HPV-positive OPSCC. Early results demonstrated safety and activity levels warranting further study. However, systemic Listeria infections led to the early termination of studies related to other listeria-based HPV E6 vaccines. Another trial is studying TG4001, a modified vaccinia (MVA) vector in a phase Ib/II single-arm trial of TG4001 plus avelumab in the recurrent and metastatic setting in HPV-positive OPSCC. This study is actively recruiting. A phase I/Ib dose-escalation trial of MG1-E6E7 (which has an adenovirus vector and Maraba virus boost) with atezolizumab in patients with HPV-positive cancers of the head and neck, and cervix remains active but is closed to accrual. There is also a phase I/II trial of PRGN-2009 (gorilla adenovirus vector) plus or minus anti-PD-1/TGF-β trap (M7824) in patients with recurrent or metastatic HPV-positive tumors, which is currently recruiting. A phase I/II dose multi-arm expansion study of TheraT® (LCMV and Pichinde virus vector) vaccine plus anti-PD-1 therapy in patients with HPV-positive OPSCC is ongoing and actively recruiting. Finally, another exciting ongoing study is a randomized phase I/II trial of TheraT® vectors expressing HPV 16 specific antigens in combination with neoadjuvant chemotherapy followed by transoral robotic surgery (TORS) or concurrent chemoradiotherapy for locoregional HPV-positive OPSCC. TheraT® is based on live-attenuated lymphocytic choriomeningitis virus (artLCMV) and works by delivering tumor-associated antigen-specific immunization with the release of IL-33 [30•]. IL-33 is an important driver of CD8+ cytotoxic effector T-cell function.

Peptide vaccines
The last major vaccine category to discuss is the peptide vaccines. Peptide vaccines deliver short or long peptides encoding HPV-oncoproteins; they contain a synthetic tumor-related epitope that is recognized by the immune system and generates an increase in immune activation, memory, and clearance of tumor cells. There are many peptide vaccine trials ongoing, with some of the results available. One such vaccine is ISA101, which has several completed and ongoing trials. A single-arm phase II trial of ISA101 plus nivolumab in recurrent or relapsed HPV-positive OPSCC demonstrated an ORR of 33% and median OS of 17.5 months, which shows promise when compared to PD-1 inhibition alone in a similar patient population [31••]. Further study of this vaccine in this group is warranted based on these results. Another phase II trial of ISA101 plus utomilumab (an IgG2 agonist monoclonal antibody that selectively binds human 4-1BB/CD137, resulting in NF-κB activation) in patients with incurable HPV-positive OPSCC is ongoing, with results expected soon. There are several other ongoing trials looking into ISA101, including a phase II study of ISA101 plus the anti-PD-1 monoclonal antibody cemiplimab in patients with HPVpositive OPSCC, a phase II study in which patients will be randomly assigned 1:1 to cemiplimab plus placebo or cemiplimab plus ISA101, and a phase II study of intensity-modulated radiotherapy (IMRT), pembrolizumab, cisplatin, and ISA101. A phase I study established the safest dose of Hespecta, an HPV 16 E6 protein conjugated to synthetic TLR2 ligand, in patients with HPV-positive malignancies [32•]. Results of this trial demonstrated that Hespecta was able to induce robust HPV 16-specific T-cell immunity in these patients, and that an increase in dose resulted in both increased mild adverse events and stronger Tcell immunity. A pilot study of P16_37-63 with cisplatin in patients with HPVpositive tumors was completed as well as a phase I/IIa trial of P16_37-63 plus vaccine adjuvant Montanide® ISA-51 VG in patients with advanced HPVpositive tumors; P16_37-63 was found to induce cellular and humoral immune responses and did not appear to cause any severe toxicities [33]. Another of these peptide vaccine trials is a phase Ib/II trial of DPX-E7 in patients with HPVpositive OPSCC, cervical, or anal cancer. This study is active but no longer recruiting. A phase I/II trial of PepCan, HPV 16 E6 peptides, is being studied versus placebo in patients with HPV-positive OPSCC. There are also studies ongoing with liposomal HPV 16 E6 and E7 peptides (PDS0101 vaccine); one is a phase II trial of pembrolizumab plus PDS0101 in recurrent and metastatic HPV-positive HNSCC (VERSATILE002) and a phase I/II trial of PDS0101 plus anti-PD-1/TGF-β trap (M7824) plus IL12 in recurrent and metastatic HPVpositive tumors.

Adoptive T-cell therapy
Adoptive T-cell therapy is a type of immunotherapy in which T-cells are taken from the patient's own body (typically blood or tumor tissue), grown in large numbers, and adapted in the laboratory setting to make them better able to target the patient's own tumor cells, and then are given back to the patient [34]. The re-infusion of T-cells is typically preceded by lymphodepleting chemotherapy to help facilitate the engraftment of the newly adapted cells. The most common types of adoptive T-cell transfer are chimeric antigen receptor T-cell (CAR T-cell) therapy, tumor-infiltrating lymphocytes (TIL) therapy, and genetically modified T-cell receptors (TCRs). This has been an attractive strategy for HPV-related malignancies due to the fact that HPV viral antigens are tumor-specific and are ubiquitously shared among patients with these HPV-related malignancies.
A recent first-in-human phase I clinical trial of T-cells engineered with TCRtargeted HPV 16 E7 for metastatic HPV-related cancers was published, which showed objective clinical responses in 6 of 12 patients, including 4 of 8 patients with anti-PD-1 refractory disease [35••]. This led to the expansion of an ongoing phase II trial to assess the safety and efficacy of these E7 TCR cells. Another phase I/II study assessed the efficacy of TCR-directed therapy against HPV16 E6 [36•]. Among the 12 patients, no dose-limiting toxicity was observed, and two patients had an objective response. Another trial (KITE-439), a phase Ia/Ib study to evaluate the safety and efficacy of HPV 16 E7 T-cell Receptor Engineered T-cells (KITE-439) in human leukocyte antigen (HLA)-A*02:01+ patients with relapsed or refractory HPV-positive cancers was stopped; however, the participants who received an infusion of KITE-439 will complete the remainder of the 15-year follow-up assessments in a separate long-term follow-up study, KT-US-982-5968.

Conclusions
Taking into consideration all of the findings from the above trials and ongoing clinical trials, ICI has changed the landscape of treatment in HPV-related HNSCC. In general, HPV-positive HNSCC has a lower tumor mutational burden (TMB) than is typically seen in other cancers and generally portends a worse response to ICI; however, it appears that HPV viral antigens act as immunogens allowing HPV antigen-specific CD8+ T cell infiltration [37,38]. The results of these trials indicate that ICI has activity in HNSCC, though whether there is a difference in treatment effect in HPV-positive and HPV-negative tumors remains unclear. For previously untreated R/M HNSCC, treatment choice depends on PDL1 CPS. Immunotherapy with nivolumab or pembrolizumab can be considered for platinum-refractory patients regardless of their PDL1 and HPV status. Concomitant chemoradiation and immunotherapy combinations have not been proven to be effective in LAHNSCC. But the ideal chemoradiation and immunotherapy sequence remains unanswered. There are ongoing studies evaluating role of radiation therapy and immunotherapy for HPVrelated OPSCC. Along with the changes in the treatment landscape, HPVdirected therapies are emerging. These studies may provide more targeted and precise treatment approaches for HPV-related OPSCC patients.

Compliance with Ethical Standards
Conflict of Interest Logan Roof declares that she has no conflict of interest. Emrullah Yilmaz declares that he was on the advisory board of Astellas Pharma

Human and Animal Rights and Informed Consent
This article does not contain any studies with human or animal subjects performed by any of the authors.