Phase II Multi-institutional Clinical Trial Result of Concurrent Cetuximab and Nivolumab in Recurrent and/or Metastatic Head and Neck Squamous Cell Carcinoma

In the United States, approximately 67,000 cases of head and neck squamous cell carcinoma (HNSCC) are diagnosed per year (1). The major risk factors of HNSCC are tobacco use, heavy alcohol use, and human papillomavirus (HPV) infection (2–6). The incidence of HPV-related HNSCC, particularly in the oropharynx, is rapidly increasing (7, 8). While the prognosis of newly diagnosed, HPV-related HNSCC is favorable, with 3-year overall survival (OS) greater than 90%, non–HPV-related HNSCC has poor prognosis, with 3-year OS less than 50% (6, 9–11). Once patients develop recurrent and/or metastatic (R/M) HNSCC, they are mostly incurable.

The current standard of care for the management of previously untreated and incurable R/M HNSCC is a programmed cell death 1 (PD-1) inhibitor, pembrolizumab, for patients with a PD ligand 1 (PD-L1) combined positive score (CPS) of ≥1. A combination of pembrolizumab, cisplatin, and 5-fluorouracil (5-FU) followed by pembrolizumab maintenance therapy is recommended for patients with any PD-L1 CPS (12). Although these regimens are highly effective in selected patients, most patients ultimately have disease progression, with a median OS of 11.6 to 13.6 months and 1 year OS of 49% to 55% depending upon the PD-L1 expression. In addition, combination chemotherapy with pembrolizumab is relatively toxic compared with monotherapy. There have been attempts to combine two immunotherapy agents to improve efficacy. However, the combination of durvalumab (PD-L1 inhibitor) and tremelimumab [a cytotoxic T-lymphocyte–associated antigen 4 (CTLA-4) inhibitor] did not show an OS benefit in a phase III clinical trial of durvalumab with and without tremelimumab (13). An additional phase III trial comparing a combination of nivolumab (PD-1 inhibitor) and ipilimumab (CTLA-4 inhibitor) to the EXTREME regimen did not meet the primary endpoint goal of OS in the total and PD-L1 CPS ≥ 20 population.

Cetuximab is a human-mouse chimeric anti-EGFR monoclonal IgG1 antibody with FDA approval in the management of HNSCC. Among the EGFR inhibitors, cetuximab is unique in its ability to activate the antibody-dependent cellular cytotoxicity (ADCC) because it is an IgG1 antibody (14–16). As reviewed in detail by Ferris and colleagues (14), after cetuximab binds to EGFR on the tumor cells, CD16 on natural killer (NK) cells execute their killing routine on the cetuximab-bound tumor cell. Notably, the killing of tumor cells can result in the cross-priming of cytotoxic CD8⁺ T cells by activated dendritic cells. Moreover, activated NK cells also secrete IFNγ, other proinflammatory cytokines, and chemokines, which will mediate changes in the immune contexture that favor immune activation and the recruitment of other immune cells to the tumor microenvironment (TME). However, cetuximab-mediated immune stimulatory activity can also result in immunosuppressive signals through multiple negative feedback loops. For example, cetuximab-mediated immune response can also increase immunosuppressive regulatory T cell (Treg) infiltration and the recruitment of myeloid-derived suppressor cells (MDSC) which can dampen the immune stimulatory activity of cetuximab. Also, IFNγ from NK cells can lead to the expression of PD-L1. Given the data that higher expression of PD-L1 is associated with increased clinical benefits (12, 17), there is a strong rationale to combine PD-1/PD-L1 axis inhibitors with cetuximab as a means to synergy in their antitumor activities.

The response rate of cetuximab as a monotherapy in R/M HNSCC is 13% and has shown improved OS in combination with radiation or cisplatin and 5-FU (18–20). To determine more effective and less toxic treatments, we evaluated a combination of cetuximab and nivolumab in two R/M HNSCC cohorts: cohort A for patients with prior exposure to any systemic therapy and persistent or platinum-refractory R/M HNSCC and cohort B for patients without prior exposure to any systemic therapy for R/M HNSCC. The early results from cohort A have been published and showed that the combination was very well tolerated (21). In the current manuscript, we report on the updated survival data of cohort A with a longer follow-up, and the clinical results of cohort B.

In addition, we report on the biomarker analyses of p16 IHC staining and PD-L1 CPS in the combined cohort A and B patients. While positive p16 IHC is an established surrogate biomarker of HPV infection in oropharyngeal squamous cell carcinoma (SCC) and a robust prognostic biomarker of favorable outcomes in HNSCC, its predictive role in patients treated with PD-1 inhibitors or immunotherapy-containing regimen is not clearly established (12, 22–24). We also evaluated the role of PD-L1 CPS as a biomarker of response in patients treated with the combination. Furthermore, we quantified circulating tumor-tissue–modified human papillomavirus (TTMV) DNA and determined the HPV strains (25, 26) in patients with p16-positive tumors. While TTMV DNA has been evaluated in patients with newly diagnosed HNSCC treated with radiation or concurrent chemoradiotherapy as a tool for response assessment and recurrence surveillance, our study is the first to evaluate this biomarker in R/M HNSCC treated with the combination of cetuximab and nivolumab.

The phase II study was conducted at the Moffitt Cancer Center (Tampa, FL), The Ohio State University (Columbus, OH), and Emory University (Atlanta, GA; NCT03370276), and written consents were obtained. Institutional Review Board (IRB) approval was obtained in accordance with the Department of Health and Human Services Federal Policy for the Protection of Human Subjects (U.S. Common Rule) at each participating institution. The study was initiated after the IRB approvals. Patients were eligible for enrollment if they met the following criteria: histologically or cytologically confirmed SCC of oral cavity, oropharynx, paranasal sinuses, nasal cavity, hypopharynx, or larynx; p16-positive SCC of unknown primary in a cervical lymph node; or incurable R/M HNSCC by a local therapy (surgery or radiotherapy with or without chemotherapy). Cohort A enrolled patients with prior exposure to any systemic therapy for R/M HNSCC including cetuximab or PD-1 inhibitors and persistent or platinum-refractory disease; Fig. 1; ref. 21). Cohort B enrolled the patients with no prior exposure to any systemic therapy for R/M HNSCC. The detailed eligibility criteria are provided in the protocol (Supplemental File 1).

Patients were treated with lead-in cetuximab 500 mg/m² alone (day 14 before cycle 1 only) followed by nivolumab 240 mg i.v. + cetuximab 500 mg/m² every 2 weeks for 24 cycles or discontinuation (Supplementary Fig. S1). We chose to have a cetuximab lead-in to exclude patients with cetuximab-related infusion reaction. Patients having a cetuximab-related infusion reaction or who did not receive cycle 1 day 1 for any reason were deemed nonevaluable and replaced. The toxicities with possible, probable, and definite attribution were included in treatment-related adverse events (TRAE) and immune-related adverse events (IRAE) analyses. Nivolumab dose reduction was not allowed. Cetuximab dose reduction was allowed: dose 1 (250 mg/m² every 2 weeks) or dose 2 (125 mg/m² every 2 weeks). In cases where one agent was withheld, both were delayed and resumed concurrently.

For response assessment, CT or MRI imaging studies were obtained every 6 weeks for cycles 1 to 4, every two cycles during cycles 5 to 6, and then every three cycles during cycles 7 to 24 while on study drugs. The same types of scans were used for repeat measurements. Response was assessed by the RECIST 1.1 by the investigators.

Archived formalin-fixed, paraffin-embedded tumors were collected to assess p16 and PD-L1 expression. The p16 staining was performed using p16 mouse mAb predilute, CINtec, clone E6H4 (Roche Tissue Diagnostics) as previously described and scored following the guidelines for p16 interpretation endorsed by the College of American Pathology. The PD-L1 staining was performed using 22C3 pharmDx assay (Agilent Technologies), and CPS was determined as previously described (12). Whole blood was collected at five time points: (i) pretreatment, (ii) after cetuximab lead-in or 2 weeks after Cycle 1 Day 1, (iii) Cycle 4 Day 1, (iv) end of treatment, and (v) end of 2-year follow-up or at the time of disease progression, whichever was earlier, and the plasma was separated. TTMV DNA was quantified from cell-free DNA isolated from plasma samples (Naveris, Inc.), as previously described (25, 26). The lower limit of detection is <5 copies/mL.

For survival analyses, progression-free survival (PFS) time was defined as the time between the date of study enrollment and the date of progressive disease or death, whichever happened first, or otherwise censored at the last date known alive. OS time was defined as the time between the date of study enrollment and the date of death or censored at the last date known alive. The primary objective of the phase II study cohort A was to determine the 1-year OS rate of concurrent cetuximab and nivolumab in patients who had any prior treatments and persistent or platinum-refractory R/M HNSCC. The null hypothesis was that the 1-year OS rate with the combination would be no higher than 36% based on the historical data from pembrolizumab and nivolumab monotherapy (24, 27). Forty-five evaluable patients were enrolled to have 90% power to reject the null hypothesis and conclude the true 1-year OS rate was higher than 36% if the true rate was 36% or higher at α = 0.05, using the one-sample log-rank test assuming an exponential distribution.

The primary objective of the phase II study cohort B was to determine the 1-year OS rate of concurrent cetuximab and nivolumab in patients who had not had any prior treatments for their R/M HNSCC. The null hypothesis was that the 1-year OS rate with the combination would be no higher than 46% based on the historical data from pembrolizumab and nivolumab monotherapy (24, 27, 28). Forty-three evaluable patients were enrolled, to achieve 90% power to reject the null hypothesis and conclude the true 1-year OS rate was higher than 46% if the true rate was 46% or higher at α = 0.05, using the one-sample log-rank test assuming an exponential distribution. To be considered evaluable, patients must have completed the lead-in period and have received the Cycle 1 Day 1 doses of cetuximab and nivolumab. The exploratory objectives were to identify potential biomarkers related to response to concurrent cetuximab and nivolumab in patients with R/M HNSCC.

For the combined analyses of cohorts A and B, the clinical features of interest were summarized using descriptive statistics, including median and interquartile range for continuous variables, and proportions and frequencies for categorical variables. In general, Kruskal–Wallis tests for continuous variables and χ² tests for categorical variables were conducted to compare the differences among the multiple groups. For 2 × 2 tables, the Barnard unconditional test was applied. The Kaplan–Meier method was used for the PFS and OS analyses, and log-rank tests were adopted to compare survival differences between groups. The univariate Cox proportional hazards model was conducted to evaluate the association of OS with the sequential treatment and individual clinical features. All statistical analyses were performed using SAS (version 9.4, SAS Institute Inc.) or the R 3.6.0 software (https://www.R-project.org).

Data were generated by the authors and included in the article.

A total of 116 patients were screened, and 95 patients were enrolled between December 2017 and September 2020 to the phase II clinical trial assessing the combination of cetuximab and nivolumab (Fig. 1). Eighty-eight of 95 patients were evaluable for OS; the other 7 patients did not receive at least one dose of combination cetuximab and nivolumab after lead-in cetuximab (Fig. 1). The median follow-up time was 15.9 months [95% confidence interval (CI), 12.2–18.8]. The patient characteristics are detailed in Table 1.

We previously reported the 1-year OS as 44% with a median follow-up for survival analysis of 21.0 months (21). Here, we report the updated survival analyses with a median follow-up of 32.1 months. The median OS was 11.4 months (90% CI, 9.4–14.4 months), and the estimated OS was 50% at 12 months (90% CI, 43–57; Fig. 2A). A 90% CI was used, as the lower bound is the minimum observed rate to reject the null hypothesis at a one-sided α = 0.05 with a true 1-year OS of 0.36; thus, we rejected the null hypothesis for the primary endpoint of the study. We also evaluated the OS and PFS using the Kaplan–Meier method. One-year OS was 46%, and 1-year PFS was 19% (Supplementary Fig. S2A and S2B). Median OS was 11.5 months, and median PFS was 3.4 months. The overall response rate (RR) was 22%, with 2 patients having a complete response (CR) and 9 patients having a partial response (PR) in this heavily previously treated cohort. Among the responders, 1 of 2 patients with CR and 3 of 9 patients with PR had prior exposure to a PD-1 inhibitor (21).

For the patients enrolled in cohort B, the median OS was 20.2 months (90% CI, 15.7–24.7 months). The estimated OS was 66% at 12 months (90% CI, 59–71; Fig. 2B). A 90% CI was used, as the lower bound is the minimum observed rate to reject the null hypothesis at a one-sided α = 0.05 with a true 1-year OS of 0.46; thus, we rejected the null hypothesis for the primary endpoint of the study. Using the Kaplan–Meier method, the 1-year OS was 61%, and 1-year PFS was 43% (Supplementary Fig. S2C and S2D). The median OS was not reached at the time of analyses, and the median PFS was 6.15 months. The PFS and OS evaluation based on the p16 status (Supplementary Fig. S2E and S2F) and PD-L1 CPS (Supplementary Fig. S2G and S2H) did not show a statistically significant difference.

Of the 43 patients in cohort B, 42 (98%) patients were evaluated for response assessment while 1 (2%) patient did not have any response assessment imaging studies. The overall RR was 37% (16/43), including CRs or PRs (Fig. 2C and D). Complete toxicity data of cohort B are detailed in Table 2 and Supplementary Table S1. The only grade 3 or higher TRAEs, occurring in ≥2 patients, were hypophosphatemia (2/43; 5%), skin- and nail-related (6/43; 14%), and hypomagnesemia (2/43; 5%). Hypomagnesemia was the only grade 4 TRAE (2%). The only grade 3 IRAE occurring in ≥2 patients was fatigue (2/43; 5%). No grade 4 IRAEs were observed.

We evaluated data from both cohorts A and B in the secondary biomarker analyses including p16 (N = 88) and PD-L1 IHC (N = 79). The patient characteristics based on these biomarkers are detailed in Supplementary Table S2 and Supplementary Table S3. The p16 IHC were positive in 40 (45%) and negative in 48 (55%). The PD-L1 CPS were ≥20 in 39 (49%), 1 to 19 in 26 (33%), and <1 in 14 (18%) of 79 patients, as there were insufficient numbers of tumor cells in 9 patients. The detailed overall RR assessments based on these biomarkers are described in Table 3. The RR was statistically signifcantly higher in p16-negative patients compared with p16-positive patients (P = 0.02, unconditional test). The RR was also higher in PD-L1 CPS ≥ 20 and 1 to 19 compared with CPS < 1, with statistical significance [P = 0.03, Cochran–Mantel–Haenszel (CMH) test]. There were no statistically significant differences in the RR among patients who had prior cetuximab or immunotherapy exposure or platinum-resistant HNSCC (defined as relapse within 6 months of platinum-containing curative therapy). The PFS and OS plots based on the p16 IHC status did not show a statistically significant difference (Fig. 3A and B). The PFS based on the PD-L1 CPS status also did not show a statistically significant difference (Fig. 3C). However, patients with PD-L1 CPS 1 to 19 had the longest median OS of 19.9 months, compared with CPS < 1 with 8.9 months, and compared with CPS ≥ 20 with 10.7 months (log-rank P = 0.04; Fig. 3D). No statistically significant differences were found when evaluating PFS and OS using both p16 status and PD-L1 CPS (Supplementary Fig. S3A and S3B).

For the exploratory analysis of TTMV DNA, 105 plasma samples were available from 37 of 40 (93%) p16-positive patients from cohorts A and B, although 1 patient did not have a pretreatment plasma sample and was thus excluded. All extracted DNA samples passed the quality assurance analysis. The patient characteristics based on these biomarkers are detailed in Supplementary Table S4. Two patients had undetectable TTMV DNA at all timepoints, one with a larynx SCC and the other with an oropharyngeal SCC. Two patients had undetectable TTMV DNA in the pretreatment samples, but at least one subsequent sample had detectable levels. The median TTMV DNA was 1,230 copies/mL (range 0–2,850 copies/mL). Among the patients with detectable TTMV DNA at any timepoint (N = 34), HPV strains could be determined in 33 (32 with HPV 16 and 1 with HPV 18) patients. One patient had indeterminant HPV 16/18/31/33/35, but the TTMV DNA levels were very low (pre-treatment, 3 copies/mL; end of treatment, 5 copies/mL) or undetectable (after lead-in cetuximab, 0 copies/mL; before Cycle 4 Day 1, 0 copies/mL).

Detailed RR assessments based on the TTMV DNA levels are described in Fig. 2E and F. When 35 patients with pretreatment TTMV DNA were evaluated, the RR was higher in patients below the median 1,230 copies/mL compared with those above the median, with a statistical significance (P = 0.01, unconditional test; Table 3). Patients with circulating TTMV DNA levels less than the median at baseline had longer median PFS and median OS times (7.9 months and 19.9 months, respectively) compared with patients with TTMV DNA levels higher than the median (3.1 months and 8.6 months, respectively). These differences in PFS and OS were statistically significant (log-rank P = 0.02 and P = 0.05, respectively; Fig. 3E and F). In addition, as an exploratory analysis, we evaluated PFS and OS using both TTMV DNA levels and PD-L1 CPS (Supplementary Fig. S3C and S3D). There was no statistically significant difference in PFS. However, OS was significanly better in groups: TTMV lower than median and PD-L1 CPS 1 to 19/CPS ≥ 20 and TTMV higher than median with PD-L1 CPS 1 to 19 (log-rank P = 0.016).

In our study, the 1-year OS was 50% in patients with prior exposure to systemic therapy and persistent or platinum-refractory R/M HNSCC and 66% in patients with no prior exposure to systemic therapy for R/M HNSCC treated with combination cetuximab and nivolumab. The safety profile was favorable. Our data suggests that p16-negative patients have comparable OS with p16-positive patients, when previous studies showed that p16-positive patients have overall better prognosis than p16-negative patients with both newly diagnosed and R/M HNSCC (6, 29, 30). The response rate was statistically higher in p16-negative patients than in p16-positive patients.

In a study by Ferris and colleagues which studied a similar population with cohort A, the ORR was 13.3%, and the 1-year OS given nivolumab monotherapy was 36% with median OS of 7.5 months (24). In cohort A of our study which included patients who failed prior PD-1 inhibitor therapies, the ORR was 22%, and the 1-year OS was 50% with median OS of 11.4 months. In the KeyNote 048 trial which studied the first-line R/M HNSCC, the ORR was 17%, and the 1-year OS given pembrolizumab monotherapy was 49% with median OS of 11.5 months in total population with any PD-L1 CPS (12). In the same study for patients treated with combination of pembrolizumab and chemotherapy, the ORR was 36%, and the 1-year OS was 53% with median OS of 13 months in total population with any PD-L1 CPS (12). In cohort B of our study, the ORR was 37%, and the 1-year OS was 61% using the Kaplan–Meier method as in the KeyNote 048. The median OS was not reached at the time of analyses. Even in the selected patients with PD-L1 CPS ≥ 1 in the KeyNote 048 trial, the 1-year PFS was 20%, and the 1-year OS was 51% with pembrolizumab monotherapy (12). In our study, the 1-year PFS was 45% and the 1-year OS was 58% in patients with PD-L1 CPS ≥ 1. Our study supports a strong rationale for a larger randomized study.

Our efficacy data are consistent with published results by Sacco and colleagues based on the combination of pembrolizumab 200 mg i.v. every 3 weeks and cetuximab initial loading dose 400 mg/m² i.v. followed by 250 mg/m² i.v. weekly in each 21-day cycle in patients with R/M HNSCC who had no prior exposure to immunotherapy or EGFR inhibition (31). The primary endpoint of that study was overall RR (ORR) at 6 months. The total sample size was 33 patients with a follow-up time of 7.3 months, and ORR at 6 months was 45%. The median PFS was 6.5 months, and the median OS was 18.4 months. In our study, the 6-month ORR was 23% in cohort A and 36% in cohort B. However, we recognized that their study also included platinum-resistant or platinum-ineligible R/M HNSCC. In our study, this population was in cohort A. Therefore, we combined the 11 patients who failed platinum and enrolled as the first-line therapy from cohort A and 43 patients in cohort B. Among the 54 patients, 52 were evaluable for response assessment. The 6-month ORR was 37%, the median PFS was 7.8 months, and the median OS was 14.5 months. The differences in the ORR and survival between these studies could be from differences in the clinical characteristics of the study population and the different dosing schedule of cetuximab.

In terms of the toxicities, the combination regimen was very well tolerated. In the first reported study of cetuximab monotherapy in R/M HNSCC by Vermorken and colleagues (20), characteristic cetuximab-related toxicities were described in detail including rash (49% for all grades and 1% for grade 3–4) and acne (26% for all grades with no grade 3–4) while hypomagnesemia was not reported. In a more recent study by Adkins and colleagues (32), 53% had all-grade rash, and 11.7% had all-grade hypomagnesemia while none were grade 3 to 4, in patients treated with cetuximab and placebo. In our study, 84% had rash of all grades including 9% grade 3, and 47% had hypomagnesemia of all grade including 2% grade 3. Furthermore, in Sacco and colleagues (31), the most common grade 3 to 4 TRAE was oral mucositis (3/33 patients, 9%). We observed 3 of 45 (7%) grade 1 and 1 of 45 (2%) grade 2 oral mucositis in cohort A (21) and 2 of 43 (5%) grade 1 and 2 of 43 (5%) grade 2 in cohort B (Supplementary Table S1). We did not observe any grade 3 to 4 oral mucositis in both cohorts. However, in Sacco and colleagues, the grade 3 skin- and nail-related toxicity including acneiform rash was only 2/33 (6%). Our study had a numerically higher rate of grade 3 skin- and nail-related toxicities with 4 of 45 (9%) in cohort A and 6 of 43 (14%) in cohort B. IRAEs included PD-1 inhibitor–related grade 3 maculo-papular rash in 1 of 45 (2%) in cohort A and in 1 of 43 (2%) in cohort B while Sacco and colleagues did not report IRAE separately. The difference could be from a higher dose of cetuximab 500 mg/m² given every 2 weeks in our study instead of cetuximab 250 mg/m² given every week in all previous studies as above. In addition, a significant portion of our patients had prior exposure to cetuximab before enrolling on our study. Compared with the Sacco and colleagues study, our study had a much longer follow-up potentially allowing cumulative toxicities of cetuximab.

Cetuximab has been extensively studied in both newly diagnosed and R/M HNSCC (10, 18, 19, 33–37). In patients with newly diagnosed HNSCC, cetuximab added to concurrent cisplatin and radiation did not show significant survival benefit. Cetuximab was also evaluated for treatment deintensification compared to cisplatin given concurrently with radiation in patients with p16-positive oropharynx cancer but was inferior to cisplatin, without a significantly favorable safety profile compared with cisplatin. However, cetuximab may be more effective in p16-negative R/M HNSCC. When ficlatuzumab and cetuximab combination therapy was evaluated as a part of randomized phase II trial in pan-refractory, advanced HNSCC, the ORR was 19%, and only p16-negative patients had a response suggesting that cetuximab may have a preferential efficacy in HPV-negative patients (38). While our study is unable to answer whether there is a clear synergy between cetuximab and PD-1 inhibitors, considering that it is a single-arm study, it supported a preferential activity of this regimen in p16-negative HNSCC.

It is intriguing to see the longest median OS of 19.9 months in the patients with PD-L1 CPS 1 to 19 compared with the CPS < 1 or CPS ≥ 20. We considered an imbalance in the clinical characteristics among these three groups but did not find any significant characteristics (Supplementary Table S3). We also examined a potential imbalance in patients who presented with distant metastatic disease at diagnosis and received no prior systemic therapy. There were only 4 patients in this category and received the cetuximab and nivolumab combination as the first-line therapy. Two patients had PD-L1 CPS ≥ 20, 1 patient had CPS 1 to 19, and 1 patient did not have any sufficient tumor to determine the score. The potential biological mechanisms mediating these differences are unknown. CPS represents the ratio between the number of PD-L1–positive cells (tumor, lymphocytes, and macrophages) in relation to the total number of viable tumor cells multiplied by 100. Thus, a simple model can be proposed that can explain the longer PFS and OS in patients with higher CPS. However, this model fails to explain the CPS ≥ 20 group, as their OS is shorter than in the CPS 1 top 19 group, as one would expect that a higher CPS would correlate with a longer OS (12). One possible explanation would be that the tumors in the CPS 1 to 19 group are the ones benefiting from inhibition of the EGFR pathway as the result of subsequent TME remodeling mediated by cetuximab-mediated NK activation, tumor ADCC, antigen cross-presentation, and T-cell activation (14, 39). In contrast, cetuximab-induced changes of the immune contexture within the TME in the CPS ≥ 20 group may be characterized by the immune dampening suppressive cells, such as Tregs and/or MDSC (40). The median OS of pembrolizumab monotherapy and pembrolizumab and chemotherapy combination in the first line R/M HNSCC with any PD-L1 CPS were only 11.5 months and 13 months, respectively (12). The median OS of 19.9 months in the CPS 1 to 19 group is very promising with a caveat that it is a small subset analysis.

Our data regarding the circulating TTMV DNA levels obtained from cell-free DNA extracted from the plasma are also intriguing. Assessing the response to immunotherapies based on the RECIST is known to be challenging with the concern of pseudo-progression (41). The longer PFS and OS in patients with lower median TTMV DNA levels at baseline compared with higher median are difficult to explain. While it would make an intuitive sense that the higher levels simply reflect higher tumor burdens in our patients with R/M HNSCC, the current data in newly diagnosed patients by Chera and colleagues suggest that the tumor burden alone does not fully explain the circulating TTMV DNA levels at baseline and the episomal versus integrated HPV genome in the tumors may influence the circulating TTMV DNA levels at baseline (25). However, the potential benefit of this biomarker may be greater in response assessment and surveillance by evaluating the changes over time as an adaptive biomarker demonstrated by patients with no evidence of disease having undetectable TTMV DNA after the definitive treatments when the TTMV DNA was detectable at baseline (26). While our data is preliminary, it warrants further evaluation of the circulating TTMV DNA as a potential surveillance tool. In addition, it may be useful to determine HPV strains in patients without archived tumors for HPV genotyping rather than conducting an invasive biopsy, as there are rapidly increasing developments in HPV strain–specific novel therapeutics such as an HPV16 vaccine.

In summary, our data suggest that the combination of cetuximab and nivolumab is a promising regimen, with significant antitumor activity and acceptable toxicity profile in patients with and without prior treatment for incurable R/M HNSCC. Our correlative studies also suggest that p16-negative patients have similar OS and a higher response rate compared with p16-positive patients given the combination.

C.H. Chung reports personal fees and nonfinancial support from Bristol Myers Squibb, grants and nonfinancial support from Lilly Oncology, and personal fees from Merck during the conduct of the study; C.H. Chung also reports personal fees from CUE Biopharma, Sanofi-Aventis, Mirati, Brooklyn ImmunoTherapuetics, and Exelixis outside the submitted work. C.E. Steuer reports personal fees from Eli Lilly and Company, Sanofi-Aventis, Mirati, and Caris outside the submitted work. M. Johnson reports grants from James and Esther King Biomedical Research Grant, Eli Lilly and Company, and Naveris, Inc., as well as nonfinancial support from Bristol Myers Squibb during the conduct of the study. S. Kumar reports other support from Naveris, Inc. outside the submitted work; in addition, S. Kumar has a patent for US Patent 11,168,373 issued, licensed, and with royalties paid from Naveris, Inc. C. Kuperwasser reports other support from Naveris, Inc. outside the submitted work. P.J. Stephens reports other support from Naveris, Inc. during the conduct of the study, as well as other support from Naveris, Inc. outside the submitted work. D.M. Shin reports grants from Enzychem, Bristol Myers Squibb, and SNS Global Pharma outside the submitted work. N.F. Saba reports personal fees from Merck, BioNTech, GlaxoSmithKline, Eisai, AstraZeneca, WebMD, CUE, Kura, Aduro, and UpToDate outside the submitted work. M. Bonomi reports grants from Moffitt Cancer Center during the conduct of the study. No disclosures were reported by the other authors.

C.H. Chung: Conceptualization, resources, data curation, formal analysis, supervision, funding acquisition, validation, investigation, visualization, methodology, writing–original draft, project administration, writing–review and editing. J. Li: Formal analysis, methodology, writing–original draft, writing–review and editing. C.E. Steuer: Data curation, supervision, investigation, project administration, writing–review and editing. P. Bhateja: Data curation, investigation, writing–review and editing. M. Johnson: Data curation, formal analysis, writing–review and editing. J. Masannat: Data curation, project administration, writing–review and editing. M.I. Poole: Data curation, project administration, writing–review and editing. F. Song: Data curation, project administration, writing–review and editing. J.C. Hernandez-Prera: Data curation, investigation, writing–review and editing. H. Molina: Data curation, methodology, writing–review and editing. B.M. Wenig: Data curation, supervision, writing–review and editing. S. Kumar: Data curation, writing–review and editing. C. Kuperwasser: Data curation, writing–review and editing. P.J. Stephens: Data curation, writing–review and editing. J.M. Farinhas: Data curation, writing–review and editing. D.M. Shin: Data curation, writing–review and editing. J.A. Kish: Data curation, investigation, writing–review and editing. J. Muzaffar: Data curation, investigation, writing–review and editing. K. Kirtane: Data curation, investigation, writing–review and editing. J.W. Rocco: Conceptualization, data curation, supervision, methodology, writing–review and editing. M.J. Schell: Conceptualization, data curation, formal analysis, supervision, investigation, methodology, writing–review and editing. N.F. Saba: Conceptualization, data curation, supervision, funding acquisition, investigation, methodology, writing–review and editing. M. Bonomi: Conceptualization, data curation, supervision, investigation, methodology, project administration, writing–review and editing.

This work has been supported in part by the James and Esther King Biomedical Research Grant (grant no. 7JK02; C.H. Chung). Lilly Oncology provided the clinical trial funding and cetuximab. Bristol Myers Squibb provided nivolumab. It is also supported in part by Moffitt's Biostatistics and Bioinformatics and Tissue Core Facilities at the H. Lee Moffitt Cancer Center & Research Institute, an NCI-designated Comprehensive Cancer Center (P30-CA076292). The content is solely the responsibility of the authors and does not necessarily represent the official views of the Grant Sponsor or the H. Lee Moffitt Cancer Center & Research Institute. We appreciate the staffs at the Moffitt Tissue Core, Biostatistics Shared Resource, and Clinical Trials Office for their support in tissue collection and processing.

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