Afatinib and Pembrolizumab for Recurrent or Metastatic Head and Neck Squamous Cell Carcinoma (ALPHA Study): A Phase II Study with Biomarker Analysis

Hsiang-Fong Kao, Bin-Chi Liao, Yen-Lin Huang, Huai-Cheng Huang, Chun-Nan Chen, Tseng-Cheng Chen, Yuan-Jing Hong, Ching-Yi Chan, Jean-San Chia, Ruey-Long Hong, Hsiang-Fong Kao, Bin-Chi Liao, Yen-Lin Huang, Huai-Cheng Huang, Chun-Nan Chen, Tseng-Cheng Chen, Yuan-Jing Hong, Ching-Yi Chan, Jean-San Chia, Ruey-Long Hong

Abstract

Purpose: EGFR pathway inhibition may promote anti-programmed cell death protein 1 (PD-1) responses in preclinical models, but how EGFR inhibition affects tumor antigen presentation during anti-PD-1 monotherapy in humans remain unknown. We hypothesized that afatinib, an irreversible EGFR tyrosine kinase inhibitor, would improve outcomes in patients treated with pembrolizumab for recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) by promoting antigen presentation and immune activation in the tumor microenvironment.

Patients and methods: The ALPHA study (NCT03695510) was a single-arm, Phase II study with Simon's 2-stage design. Afatinib and pembrolizumab were administered to patients with platinum-refractory, recurrent, or metastatic HNSCC. The primary endpoint was the objective response rate (ORR). The study applied gene expression analysis using a NanoString PanCancer Immune Profiling Panel and next-generation sequencing using FoundationOne CDx.

Results: From January 2019 to March 2020, the study enrolled 29 eligible patients. Common treatment-related adverse events were skin rash (75.9%), diarrhea (58.6%), and paronychia (44.8%). Twelve patients (41.4%) had an objective partial response to treatment. The median progression-free survival was 4.1 months, and the median overall survival was 8.9 months. In a paired tissue analysis, afatinib-pembrolizumab were found to upregulate genes involved in antigen presentation, immune activation, and natural killer cell-mediated cytotoxicity. Unaltered methylthioadenosine phosphorylase and EGFR amplification may predict the clinical response to the therapy.

Conclusions: Afatinib may augment pembrolizumab therapy and improve the ORR in patients with HNSCC. Bioinformatics analysis suggested the enhancement of antigen presentation machinery in the tumor microenvironment.

Trial registration: ClinicalTrials.gov NCT03695510 NCT04839471.

©2022 The Authors; Published by the American Association for Cancer Research.

Figures

Figure 1.
Figure 1.
Clinical response to pembrolizumab–afatinib combined therapy in patients with HNSCC. A, Waterfall plot showing the best overall response (n = 29). B, Progression-free survival (n = 29). C, Overall survival (n = 29). D, The duration of response in responders (n = 12).
Figure 2.
Figure 2.
Paired tissue analysis. A, mRNA differential expression. Red dots represent genes with significant differences in mRNA expression (Benjamini–Yekutieli method, adjusted P < 0.05); B and C, Gene expression differences between paired pre- and post-treatment biopsy samples. *, P < 0.05; **, P < 0.01; ***, P < 0.001. D, Gene network analysis (by StringApp), showing the network of the upregulated genes in the tumor microenvironment. E, Gene set enrichment analysis of paired tissue mRNA samples. In the GO Biological Process analysis, only the top 10 downregulated gene sets are listed. F, Leading-edge analysis of three gene sets with immune cell regulatory functions.
Figure 3.
Figure 3.
Targeted gene mutation analysis. A, Genes were selected if three or more patients had mutations. TMB, tumor mutational burden in mutations/megabase. Survival analysis according to MTAP status. B, Progression-free survival. C, Overall survival. D, Comparing patients with altered MTAP versus unaltered MTAP by gene set enrichment analysis. In the present study and TCGA HNSCC analyses, tumors with altered MTAP had a more suppressed microenvironment. E, CIBERSORTx analysis: In this study, patients with MTAP loss or mutation had a low fraction of CD8+ T cells in the tumor microenvironment (Mann–Whitney U test, P = 0.0037, FDR q = 0.08).
Figure 4.
Figure 4.
Differences in mutation allele frequency (MAF) between pre-treatment and post-progression specimens. A, Best overall response: partial response. B, Best overall response: partial response. In the post-progression biopsy, a new MTAP loss and a new CDKN2A/B loss were detected. C, The patient had a good initial response to treatment. However, tumor regrowth occurred rapidly before the first imaging assessment. The best overall response of the patient was disease progression.

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