Tadalafil Enhances Immune Signatures in Response to Neoadjuvant Nivolumab in Resectable Head and Neck Squamous Cell Carcinoma

Abstract

Purpose: We hypothesize that the addition of the phosphodiesterase-5 inhibitor tadalafil to the PD-1 inhibitor nivolumab, is safe and will augment immune-mediated antitumor responses in previously untreated squamous cell carcinoma of the head and neck (HNSCC).

Patients and methods: We conducted a two-arm multi-institutional neoadjuvant randomized trial in any-stage resectable HNSCC (NCT03238365). Patients were stratified at randomization by human papillomavirus (HPV) status. Patients in both arms received nivolumab 240 mg intravenously on days 1 and 15 followed by surgery on day 28. Those in the combination therapy arm also received tadalafil 10 mg orally once daily for 4 weeks. Imaging, blood, and tumor were obtained pretreatment and posttreatment for correlative analysis.

Results: Neoadjuvant therapy was well-tolerated with no grade 3 to 5 adverse events and no surgical delays. Twenty-five of 46 (54%) evaluable patients had a pathologic treatment response of ≥20%, including three (7%) patients with a complete pathologic response. Regardless of HPV status, tumor proliferation rate was a negative predictor of response. A strong pretreatment T-cell signature in the HPV-negative cohort was a predictor of response. Tadalafil altered the immune microenvironment, as evidenced by transcriptome data identifying enriched B- and natural killer cell gene sets in the tumor and augmented effector T cells in the periphery.

Conclusions: Preoperative nivolumab ± tadalafil is safe in HNSCC and results in more than 50% of the patients having a pathologic treatment response of at least 20% after 4 weeks of treatment. Pretreatment specimens identified HPV status-dependent signatures that predicted response to immunotherapy while posttreatment specimens showed augmentation of the immune microenvironment with the addition of tadalafil.

Figure 1.

Trial schema and overall response. A, Trial schema. B, Example of radiographic and pathologic representation of treatment response. A 6-cm pretreatment lymph node decreased to 3.4 cm posttreatment and on final pathology had 95% pTR with only 5% viable tumor in the lymph node (yellow arrow). C, Waterfall plot of overall pTR by treatment group and HPV status at time of surgery. Threshold of ≥20% was considered a “responder” in the correlative analysis. Analysis of tumor tissue used pTR at the primary site only. Analysis of PBMCs used overall pTR including primary and lymph nodes. OC, oral cavity; HP, hypopharynx; NC, nasal cavity.

Figure 2.

HPV⁻ Rs have increased pretreatment T-cell abundance. A, Pretreatment biopsy CD8⁺ T-cell RNA signature did not demonstrate significance between Rs and NRs. Subgroup analysis with respect to HPV status reveals the HPV⁻ cohort having a significant difference in overall T cells, Tregs, CD8, and CD4 that is not identified in the HPV⁺ cohort. Pretreatment CD8, FoxP3, and CD163 IHC confirm a significant difference for HPV⁻but not positive tumors pointing to an immune desert as a significant correlate of nonresponse. B, Quantitation by RNA-seq of PD-L1 and semiquantification by PD-L1 IHC staining demonstrating no predictive significance for response (left graph, y-axis shows log₂ expression). In pre- to posttreatment samples PD-L1 staining remained stable in the NRs and trended upward in the Rs (right graph). C, High tumor proliferation rate signatures in the pretreatment samples are associated with nonresponse. The tumor proliferation signature consists of cell cycle– and tumor progression–associated genes.

Figure 3.

Flow cytometric analysis of peripheral blood cell subsets shows that Rs have different pretreatment peripheral compartments that support cytotoxic T cells. Scatter graphs depict representative subjects with numbers indicating the percentage of cells within a given gate. Graphs show NRs and Rs baseline pretreatment values compared with posttreatment nivolumab versus nivolumab + tadalafil. Rs demonstrated a greater CD8:CD4 ratio in the periphery with the tadalafil cohort significantly augmenting this ratio after treatment regardless of response. In pretreatment samples, B lymphocytes are increased. Peripheral PD-L1 levels and CD163 macrophages are significantly decreased in Rs compared with the NRs, suggesting the peripheral compartment plays role in treatment response. Statistical significance was assessed using ANOVA (*P < 0.05, **P < 0.001, ***P < 0.0005). Nivo, nivolumab; Tad, tadalafil.

Figure 4.

HPV⁺ tumors demonstrate the greatest overall immune-related transcriptomic change with the addition of tadalafil. A, Heatmap showing expression of a set of 292 immune signature genes comparing pre- and posttreatment separated out by HPV status and treatment cohort regardless of response. Individual genes (demarcated by rows) are subdivided based on immune cell association (vertical legend). Each column represents a cohort prior to treatment or following treatment. B, Gene signatures demonstrate dynamic changes caused by tadalafil in the B-cell population, with upward trend in the T-cell population and no effect on CAFs. *P < 0.05, ** P < 0.01. Nivo, nivolumab; Tad, tadalafil.

Figure 5.

Unbiased GSEA of bulk RNA sequences, indicating enrichment of B- and NK-cell gene sets in Rs to nivolumab and tadalafil. A, Enrichment plots and heat maps. Top, left: Result from a canonical pathway collection (C2 cp) query showing enrichment of CD22-mediated B-cell antigen receptor regulation in HPV⁻ and HPV⁺ Rs, posttreatment. Top, right: Cell-type signature (C8) query revealing enrichment of B cells. Heatmaps show the 12-most upregulated genes, posttreatment. Bottom, left: Cell-type signature collection (C8) query illustrating enrichment of an NK-cell gene set in HPV⁻ and HPV⁺ Rs. Bottom, right: Enrichment of a second NK-cell gene set, with portions of heatmaps. B, Scatter plots of normalized transcript counts from bulk RNA sequence data. Top: Expression of B-cell genes, selected from heat maps in panel A. Enrichment, posttreatment, manifested in the 20% to 100% pTR group that received nivolumab + tadalafil (left; one-way ANOVA; P < 0.0001), but not nivolumab-alone (right). Bottom: Similarly, a subset of five NK-cell genes were found to be enriched by tadalafil in the 20% to 100% pTR group, posttreatment (one-way ANOVA; P = 0.0003). C, Venn diagram of the GSEA leading edges and table of the 66 genes of an NK-cell signature unique to the tadalafil group. NS, not significant.

Figure 6.

Flow cytometric analysis of exosomes in pre- and posttreatment plasma and supernatants from short-term ex vivo tumor cell cultures identified increase exosomes in NRs. A, Gating strategies for single events (left) and exosomes (right). B, The frequency of circulating exosomes in pretreatment plasma (n = 41; left) and overnight biopsy cultures (n = 23; right) as they relate to clinical response. Statistical significance was assessed using ANOVA (*P = 0.03). C, Bar charts show expression of immune checkpoint receptors on exosomes present in posttreatment tumor culture supernatant as they relate to clinical response (n = 23). Statistical significance was assessed using Student t test (*P < 0.05). Exo, exomes; MFI, mean fluorescence intensity; FSC-A, Forward scatter area; FSC-H, forward scatter height; DiO, 3,3′-dioctadecyloxacarbocyanine; TCS, tumor culture supernatant.