Subjects aged 18 years or greater with a pathologically confirmed diagnosis of papilloma and with a clinical diagnosis of recurrent respiratory papillomatosis were eligible for both studies. Eligible subjects were required to have tracheal disease, or laryngeal disease with an anatomic Derkay score of 10 or greater, that required two or more clinically indicated interventions in the prior 12 months. Inclusion criteria included adequate organ function as measured by standard laboratory tests and an Eastern Cooperative Oncology Group performance status of 0 or 1. Exclusion criteria included evidence of infection with HIV, hepatis B or C, history of solid organ or bone marrow transplant, autoimmune disease or immunosuppression.

Both phase II studies, Avelumab for People with Recurrent Respiratory Papillomatosis (NCT 02859454) and M7824 for People with Recurrent Respiratory Papillomatosis (NCT03427411) were approved by the National Cancer Institute (NCI) Institutional Review Board, and informed consent was obtained from all subjects. Treatment consistent of intravenous avelumab (10 mg/kg) or intravenous bintrafusp alpha (1200 mg) administered every 2 weeks. On-treatment disease response was assessed by clinical endoscopic examination every 2 weeks for the duration of the study. Papilloma disease burden was quantified via calculation of an anatomic Derkay score,13 a validated objective disease measurement tool. At any point, if a subject developed clear disease progression, treatment was halted and surgical debridement of papilloma was performed. If subjects achieved a partial response after three doses, as defined by a reduction in anatomic Derkay score of 30% or greater, subjects received an additional three doses for a maximum of six doses. Adverse events were graded according to Common Terminology Criteria for Adverse Events V.4.03. Papilloma and normal mucosa biopsies for correlative studies were obtained before treatment and at the time of surgical debridement following treatment with either avelumab or bintrafusp alpha. Both avelumab and M7824 were provided by EMD Serono through a Cooperative Research and Drug Agreement with the NCI. Following completion of treatment with avelumab or bintrafusp alpha, all subjects underwent surgical debridement of papilloma with standard-of-care techniques and returned to their referring otolaryngologist to resume care. Instruction was given to each referring specialist to resume standard of care after the clinical trial, with decisions of when to proceed with a clinically indicate intervention after the trial to occur in a similar fashion as before the clinical trial. Documentation of clinically indicated interventions before and after each clinical trial was determined from medical records obtained from referring centers for each patient following consent.

TGF-b ELISA (R&D Systems, #DB100B) was performed on pretreatment and post-treatment peripheral blood serum per manufacturer recommendations.

Five micron sections from formalin-fixed paraffin-embedded papilloma blocks were deparaffinized and rehydrated using the Leica ST5020 Autostainer. Antigen retrieval was performed using a Leica Bond citrate-based solution (Leica AR9961). Single color automated staining was performed using anti-human CD4 (Abcam ab133616; 1:400 dilution) or CD8 (Abcam ab182729; 1:200) antibodies applied for 60 min. After washing, secondary antibody staining and colorimetric development was perfumed using the Leica BOND Polymer Refine Detection kit (#DS9800) per manufacturer recommendations. Coverslipping was performed with the Leica Automated Coverslipper (#CV5030). Individual subject pretreatment and post-treatment samples were always stained in the same batch with appropriate positive and negative controls. All conditions were optimized on human tonsil sections. Images of stained slides were acquired on a Vectra Polaris using brightfield illumination. Analysis of images was performed using QuPath. Percentage positivity of stained cells for entire FFPE sections was calculated using common detection thresholds for each subject’s paired pretreatment and post-treatment biopsies.

T lymphocyte cultures were initiated from fresh pretreatment and post-treatment papilloma fragments using interleukin-2 containing media. Autologous B lymphocytes were isolated from the Peripheral blood mononuclear cells (PBMC) using negative magnetic selection (StemCell) and expanded in the presence of irradiated (6000 rad) NIH3T3-CD40L feeder cells (1:1 ratio) and rhIL-4 (200 U/mL). Expanded papilloma T lymphocytes were assayed for reactivity against autologous B lymphocytes (1:2 ratio) electroporated with in vitro transcribed full-length mRNA from HPV 6 or 11 E2, E6 or E7 (mMESSAGE T7 ULTRA Transcription Kit, Thermo). T lymphocyte reactivity against these products or PMA/Ionomycin (positive control) was measured via ELISpot assay (R&D Systems). Spot counts were measured on an Immunospot ELISpot reader (Cellular Technology).

The Wilcoxon matched-pairs signed-rank test was used to test for significant differences between pretreatment and post-treatment clinical and correlative outcomes. The Holm-Sidak method was used to compare multiple means over multiple timepoint to test for significant difference between Derkay scores. A p value of <0.05 was considered statistically significant.

Nine subjects with RRP were treated with the dual PD-L1/TGF-b inhibitor bintrafusp alpha (online supplemental table 1). Based on frequent clinical endoscopic examination, subjects experienced initial disease regression after the first treatment, then papilloma regrowth (figure 1A). In five of nine subjects, the rate of papilloma growth from 2 to 4 or 6 weeks after treatment was greater than the rate of papilloma growth in the 4 weeks prior to starting treatment (online supplemental figure 1). A previously reported clinical trial treated a similar patient population with the PD-L1 mAb avelumab following the same treatment schedule.12 Comparing clinical endoscopy results from these two separate clinical trials revealed that treatment with dual PD-L1/TGF-b inhibition in the currently reported trial controlled the growth or papillomas to a lesser degree compared with treatment with PD-L1 blockade alone in the previously completed trial (figure 1B,C). Peripheral TGF-b levels were significantly reduced following treatment with PD-L1/TGF-b inhibition but not PD-L1 blockade alone (online supplemental figure 2). Based on a follow-up period of 1 year after completion of the trial, four of eight (50%) evaluable subjects treated with dual PD-L1/TGF-b inhibition required more frequent surgical intervention compared with the 1 year prior to the study (figure 1D). In comparison, 11 of 12 (92%) subjects treated with PD-L1 blockade alone experienced durable responses with increased intersurgery interval after completion of treatment in the previously completed trial. These data indicated that the addition of TGF-b neutralization to PD-L1 blockade abrogated the clinical benefit observed with PD-L1 blockade alone and was associated with reduced papilloma growth control in adult subjects with recurrent respiratory papillomatosis.

Treatment with bintrafusp alpha was well tolerated. No subject experienced grade three or four adverse events. One subject experienced grade 2 diarrhea that quickly resolved spontaneously, and one subject experienced grade 2 dry eyes that spontaneously resolved after 6 months. Five of nine (56%) subjects treated with bintrafusp alpha developed grade 1 bleeding from the upper aerodigestive tract mucosa that resolved spontaneously within 3 months of completing treatment. Two of nine subjects (22%) developed a keratoacanthoma that resolved within 6 months of completing treatment.

Given the discordant clinical outcomes observed with the addition of TGF-b neutralization to PD-L1 blockade in these two similar clinical studies, correlative analysis of pretreatment and post-treatment papillomas treated with dual PD-L1/TGF-b inhibition was performed. As a comparison, post hoc analysis of papillomas treated with PD-L1 blockade alone in a previous clinical trial was performed concurrently. To determine if canonical TGF-b signaling was intact within papilloma cells and possibly altered by TGF-b neutralization, changes in expression or phosphorylation of key signaling components was assessed in samples from subjects treated with dual PD-L1/TGF-b inhibition or PD-L1 blockade alone via multiplex immunofluorescence (figure 2A). Clear differences in histologic architecture, expression of TGF-b signaling components and proliferative state were evident between the basal and suprabasal layers within papillomas. Basal and suprabasal layers within each papilloma were separately annotated and digitally scored for immunofluorescence positivity and intensity (online supplemental figure 3). The basal layers of post-treatment papillomas from subjects treated with dual PD-L1/TGF-b inhibition consistently demonstrated increased expression of the proliferation marker Ki67, decreased p21 cell cycle checkpoint and decreased pSMAD3 compared with pretreatment (figure 2B). One subject with slightly decreased basal layer Ki67 also demonstrated slightly increased basal layer p21. Increased post-treatment basal Ki67 positively correlated with increased papilloma disease burden (Derkay score) in subjects treated with PD-L1 blockade with or without TGF-b neutralization (online supplemental figure 4). Basal Ki67 decreased in 10 of 12 (83%) subjects treated with PD-L1 blockade alone. No consistent changes in basal p21 or pSMAD3 were measured in these samples (figure 2C). Furthermore, no consistent changes in basal pSMAD2 or suprabasal Ki67, p21, pSMAD3 or pSMAD2 were observed in papillomas from subjects treated with or without TGF-b neutralization (online supplemental figure 5). These data indicated that TGF-b signaling is intact and may be restraining proliferation within basal layer papilloma cells and that TGF-b neutralization correlates with decreased cell cycle checkpoint expression and increased papilloma cell proliferation.

Immunosuppression mediated by TGF-b represents a mechanism of resistance to effective immune checkpoint blockade immunotherapy in patients with malignancy.4 5 To determine if TGF-b neutralization enhanced anti-HPV immunity within papillomas, samples from subjects treated with dual PD-L1/TGF-b inhibition or PD-L1 blockade alone were assessed for changes in T lymphocyte infiltration and specificity for HPV6 or 11. Determined by T lymphocyte IHC (online supplemental figure 6), papillomas from patients treated with dual PD-L1/TGF-b inhibition did not consistently increase CD8 or CD4 T lymphocyte infiltration into papillomas (figure 3A). No subjects harbored HPV specific T lymphocytes pretreatment, and treatment with bintrafusp alpha induced HPV immunity in one of eight (13%) evaluable subjects. In comparison, papillomas from subjects treated with PD-L1 blockade alone revealed significantly increased papilloma accumulation of CD8 T lymphocytes and a trend toward increased accumulation of CD4 T lymphocytes (figure 3B). Assessment of specificity of cultured papilloma infiltrating lymphocytes for HPV type specific E2, E6 or E7 revealed that no subjects harbored HPV specific T lymphocytes pretreatment, and treatment with avelumab induced HPV immunity in 3 of 11 (27%) evaluable subjects. These data indicated that dual PD-L1/TGF-b inhibition did not enhance HPV specific immunity beyond that observed with PD-L1 blockade alone within the papillomas of subjects with recurrent respiratory papillomatosis.

Dual PD-L1/TGF-b inhibition with bintrafusp alpha provides clinical benefit for patients with relapsed malignancy.26 Genomic alterations in TGF-b superfamily genes are frequent events in cancers,27 suggesting that genetic or epigenetic alterations in malignant cells may abrogate the antiproliferative effects of TGF-b signaling observed in non-malignant epithelial cells. We hypothesized that the rate of genomic alterations in key TGF-b superfamily genes27 would be less frequent in normal mucosa and papillomas from subjects with recurrent respiratory papillomatosis compared with head and neck squamous cell carcinoma (HNSCC). First, total somatic SNVs and small insertions and deletions (INDELs) were quantified in pretreatment papillomas (n=21) and laryngeal normal mucosa (n=8) using peripheral leukocytes as a germline reference and compared with HPV-positive HNSCC (n=74) and HPV-negative HNSCC (n=314) from TCGA.15 Both HNSCC cohorts harbored a greater number of mutations compared with mucosa or papilloma (figure 4A). Considering SNVs or INDELS, greater than 25% of all HNSCC samples harbored mutations in one or more TGF-b superfamily genes27 (online supplemental table 2) compared with infrequent mutations in these genes within normal mucosa or papilloma (figure 4B). Study of copy number alterations revealed a greater number of TGF-b superfamily genes either amplified or deleted in carcinomas compared with papillomas or mucosa (figure 4C). These data revealed that genetic alterations in TGF-b superfamily genes are infrequent events in normal mucosa or papilloma consistent with intact antiproliferative TGF-b signaling. Given the high frequency, genetic alterations in TGF-b superfamily genes may be advantageous for carcinoma cells, possibly through loss of TGF-b antiproliferative signaling.