Patients age 18 years or older with a pathologically confirmed diagnosis of squamous papilloma were eligible for the trial. For laryngeal disease, eligibility required an anatomic Derkay score [15] of 10 or greater, and two or more surgical interventions to control papillomas in the prior 12 months. For pulmonary disease, eligibility required measurable lung lesions by RECIST1.1. Inclusion criteria included adequate organ function as assessed by standard laboratory tests and Eastern Cooperative Oncology Group performance status of 0 or 1. Exclusion criteria included infection with HIV, hepatitis B, or hepatitis C; a history of solid organ transplant or autoimmune disease; immunosuppression; and prior treatment with an immune checkpoint inhibitor.

This study was designed as a phase II clinical trial. The protocol was approved by the National Cancer Institute (NCI) Institution Review Board and the National Institutes of Health Clinical Center, and informed consent was obtained from all patients. Treatment consisted of avelumab (10 mg/kg) iv every 2 weeks for three doses (one course). Patients who demonstrated a partial response (PR) after the first course were treated with a second course. Hence, patients could receive a total of up to six doses of avelumab over 12 weeks. Patients who did not demonstrate a PR after one course did not receive a second course. Following completion of treatment, all patients underwent surgical debridement of papillomas with standard-of-care techniques, and they returned to their referring otolaryngologist to resume standard-of-care treatment for their RRP. Avelumab was provided by EMD Serono through a Cooperative Research and Development Agreement with the NCI.

Clinical assessments including office-based endoscopy via flexible laryngoscopy and completion of the Voice Handicap Index-10 (VHI-10) questionnaire [16] were performed at the time of screening and prior to each dose of the study drug. Chest computed tomography (CT) evaluation was obtained on patients with known or suspected lung disease. Complete response was defined as complete resolution of all laryngeal papillomas visible on office-based endoscopy and all pulmonary papillomas evident by CT scan. PR for laryngeal disease was defined as a decrease in anatomic Derkay score of ≥50%. After four patients were treated, the definition was changed to ≥30% to lower the threshold for a second course of treatment as the treatment was well tolerated and appeared to have activity. Responses are reported based on the protocol criteria at the time of treatment. For pulmonary disease, response was defined using RECIST 1.1 criteria. All patients were examined under anesthesia prior to the first and second doses of avelumab to verify patency of the airway and to obtain biopsies. During the baseline and 2-week exams under anesthesia, < 1 mm biopsies were obtained under direct visualization from locations that would not change the Derkay score, such as tracheal or large supraglottic lesions. Normal mucosa was sampled from the posterior arytenoid or post-cricoid regions in all patients at all time points. Clinical laboratory tests were obtained weekly on patients throughout the duration of treatment. Determination of response was based upon changes in anatomic Derkay score obtained via office-based endoscopy and/or chest CT (when indicated) at 6 or 12 weeks.

The primary outcome measure was rate of complete response. Secondary outcome measures included the rate of PR, change in inter-surgery interval, change in VHI-10, rate of adverse events, and change in immune correlatives.

T cell cultures were initiated from papillomas and adjacent but normal appearing laryngeal mucosa fragments using interleukin-2 containing media [17]. Expanded T-cells from papilloma and normal mucosa were tested for reactivity against autologous feeder cells (dendritic cells or B cells) transfected with mRNA from individual HPV genes. T-cell reactivity against these products or PMA/Ionomycin (positive control) was assessed via a standard ELISPOT assay. HPV 6 or 11 copy number per cell was determined using real-time quantitative PCR primers specific for HPV L1 (type specific) and Ribonuclease P. For immunohistochemistry, sectioned FFPE tissue samples were stained with haematoxylin and eosin, anti-CD8, and anti-PD-L1 at the UCLA Anatomic Pathology Immunohistochemistry and Histology Laboratory. Antibodies used included CD8 clone C8/144B (Dako, 1:100, low pH retrieval) and PD-L1 (Spring Biosciences, Sp142, 1:200, high pH retrieval). IHC was performed on a Leica Bond III autostainer. Percentage cellularity (% positive cells/all nucleated cells) was calculated using the Halo platform (Indica Labs). For gene expression analysis, RNA was extracted from frozen tissues and NanoString PanCancer IO 360 Gene Expression Panel™ analysis (NanoString Technologies, Seattle, WA) was performed per the manufacturer’s recommendations. Expression of individual HPV genes was determined by Droplet Digital™ PCR (ddPCR™) analysis (Bio-Rad, Hercules, CA) using HPV type and gene specific primers. Bioinformatic analysis was performed by NanoString.

The Wilcoxon matched-pairs test was used to test for a significant difference in the surgery-free interval. A p value of < 0.05 was considered statistically significant.

Twelve patients were treated (Table 1). The median age was 51 years (range 21 to 67 years). Four patients had juvenile onset RRP, and eight had adult onset RRP. The median number of years since diagnosis of RRP was 18 (range 2 to 45 years). Eligibility was based on laryngeal disease for eight patients, pulmonary disease for three patients, and both laryngeal and pulmonary disease for one patient. The median Derkay score of patients who qualified for the study based on laryngeal disease was 13 (range 10 to 26). Ten patients had required at least 20 surgeries to control their RRP lesions since initial diagnosis, and three patients had required greater than 100 surgeries. Most patients had received adjuvant local or systemic treatment with agents such as cidofovir or bevacizumab prior to enrollment. RRP was associated with HPV 11 in six patients and HPV 6 in six patients. There was a trend toward greater likelihood of experiencing a partial response to avelumab in patients with RRP associated with HPV 6 compared to HPV 11, although this failed to reach statistical significance in this small cohort (Additional file 1: Figure S1). There was no correlation between HPV subtype and the presence of pulmonary disease (Additional file 1: Figure S2).Tab1

Characterization of Patient and Prior Treatments

Treatment with avelumab was associated with a decrease in Derkay score in all patients (Fig. 1a&b). Six of nine patients with qualifying (i.e. Derkay score ≥ 10) laryngeal disease experienced a partial response. Patient 2 received only one dose of avelumab due to laboratory abnormalities but demonstrated a 46% reduction in Derkay score. Another patient demonstrated a 33% reduction in Derkay score but received systemic steroids after completion of course one for an issue unrelated to the protocol, making him ineligible for further treatment. Pulmonary disease did not respond to treatment in any of the four patients with pulmonary disease (Fig. 1c&d). One patient with both laryngeal and pulmonary disease demonstrated a PR in the larynx but no response in the lung. No patients achieved a complete response. Three of twelve patients had received polyvalent HPV vaccine after their RRP diagnosis but prior to enrollment in this protocol. There was no correlation between patients who received the HPV vaccine and patients who experienced a partial response to avelumab (Additional file 1: Figure S3).Fig1Fig. 1

Clinical response following initiation of avelumab in patients with recurrent respiratory papillomatosis. a, A spider plot of change in laryngeal disease burden for each patient, as measured by anatomic Derkay score, is shown. The dotted line shows the time that avelumab was initiated. The negative time points reflect screening assessments prior to starting treatment. Patients 5, 7, 8, 9 and 11 received 6 doses of avelumab over 12 weeks. Patients 1, 2, 3, 4, 6, 10 and 12 received three doses over 6 weeks. b, Representative endoscopic images of RRP lesions for patients 5, 6 and 9 at the timepoints indicated are displayed. c, A spider plot of the change in pulmonary disease burden for each patient, as measured by CT scan imaging per RECIST1.1 guidelines, is shown. d, Representative images from CT scans from patients 6, 10 and 11 are displayed. Red arrows point to index lesions

There were no grade 3 or 4 AEs (Table 2). The most common AE was grade 1 or grade 2 dry mouth, which developed in 5 of 12 patients. All patients reported ≥50% subjective recovery of dry mouth within 9 months of cessation of avelumab treatment, without systemic steroid administration. One patient experienced a flare of facial eczema/dry skin (pre-existing diagnosis) following her second dose of avelumab that was controlled with topical steroids and lasted for approximately 3 months after completion of treatment. One patient experienced a grade 2 infusion reaction that interrupted the first infusion. He was able to complete the infusion and did not experience infusion reactions with subsequent avelumab treatments. Patient 2 demonstrated asymptomatic grade 2 leukopenia and neutropenia on clinical laboratory assessment after one dose of avelumab, and the decision was made to discontinue treatment. Leukocyte and neutrophil counts normalized within weeks without the use of systemic steroids.Tab2

Summary of Adverse Events

To study if PD-L1 immune checkpoint blockade induced anti-viral T cell responses in patients with clinical responses, four patients (patients 5, 7, 8 and 9) were selected (based on PR and tissue availability) for immune correlative analysis of tissues. Patient 5 demonstrated HPV6 E2-specific responses at the 12-week time point in T-cell cultures from papilloma but not normal mucosa (Fig. 3a). This correlated with a reduction in papilloma HPV6 viral load. Patient 9 demonstrated HPV11 L1-specific responses at the 2-week time point in papilloma but not normal mucosa, but this response was not detected at 12 weeks. This also correlated with a reduction in HPV11 viral load. Patients 7 and 8 did not demonstrate HPV-specific T-cell responses or a reduction in viral load in papilloma tissues. HPV gene expression within papillomas revealed consistently high expression of early genes E2, E4 and E5γ, with inconsistent E6 and E7 expression and low expression of late genes L1 and L2 (Additional file 1: Figure S4). HPV gene expression was not detected in the normal mucosa of any sample (Fig. 3b).Fig3Fig. 3

HPV-specific T-cell responses and viral burden in patients treated with avelumab. a, HPV-specific responses in papilloma-infiltrating T-cells from patients 5 and 9 were measured by interferon gamma enzyme-linked immunospot assays. Antigen presenting cells were loaded with RNA encoding individual transcripts of HPV 6 (patient 5) or HPV 11 (Patient 9) genes. Patients 7 and 8 did not demonstrate HPV-specific responses in papilloma-infiltrating T-cells. b, HPV viral load for HPV 6 (patient 5) or HPV 11 (Patient 9) in papilloma and normal mucosa was determined by quantitative-polymerase chain reaction. c, The Tissue Inflammation Signature was calculated from NanoString IO 360 analysis for patients 5, 7, 8, and 9