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There has been significant interest in combining anti-PD-1/PD-L1 agents with other clinically active anticancer agents. Gefitinib, a first-generation inhibitor of the epidermal growth factor receptor (EGFR) tyrosine kinase, is approved for non-small cell lung cancer (NSCLC) patients with sensitizing EGFR mutations. Durvalumab has demonstrated clinical activity in NSCLC and is approved for Stage III, unresectable NSCLC that has not progressed following platinum-based chemotherapy and radiotherapy. A phase I trial (NCT02088112) combining gefitinib and durvalumab was initiated to establish the safety profile of this combination in tyrosine-kinase inhibitor (TKI)-naive patients with NSCLC containing EGFR-positive sensitizing mutations. As part of this trial, paired tumor biopsies and multiple blood samples were collected for biomarker evaluation. Peripheral blood samples were analyzed for gene expression, cytokine production, immunophenotyping, and circulating DNA (ctDNA). Paired tumor biopsies were evaluated for expression of multiple proteins by immunohistochemistry, including phosphorylated EGFR (pEGFR), PD-L1, CD73, and CD8. We saw ≥50% inhibition of pEGFR in 8 of 17 paired biopsies but no consistent directional changes with PD-L1. We observed known durvalumab-mediated effects such as increases in tumor CD8+ cells (3.7 fold, p=0.011), proliferating CD8+ cells in circulation (2.4 fold, p=0.074), and increased interferon gamma (10.3 fold, p=0.01). A cohort of patients (n=10) was treated with a 28-day gefitinib run-in prior to addition of durvalumab. Sample collection during this period allowed us to evaluate the effects of gefitinib alone on immunologic readouts. While treatment with gefitinib alone resulted in ≥50% reduction of pEGFR in 5 of 8 paired tumor biopsies, its effect on expression of immune-related genes, interferon-related cytokines, and immune subpopulations was minimal. Interestingly, we saw an association between higher baseline tumor PD-L1, pEGFR or CD73 expression and a numerical trend for longer progression-free survival. Finally, analysis of ctDNA showed robust decreases in mutant EGFR could be observed as early as Day 5 of gefitinib treatment, either alone or in combination with durvalumab. Altogether, these results demonstrate that each agent had its expected downstream biologic effects and there was no antagonism between the two agents. We did not observe any novel biologic effects nor any striking synergy for known readouts. New hypotheses (i.e., high PD-L1/pEGFR/CD73) for patient selection biomarkers were identified, although more follow-up is required to determine whether they reflect combination or single-agent treatment. These studies highlight the utility of collecting biomarker samples to confirm target engagement, understand downstream effects, identify patient selection biomarkers, and explore ctDNA biomarkers. Citation Format: Tammie C. Yeh, Helen K. Angell, Vicky Rowlands, Emma V. Jones, Vidalba Rocher Ros, Alison Pritchard, Martine P. Roudier, Nathan Standifer, Mike Kuziora, Philip Martin, Kirill Peskov, Rosie Taylor, J. Carl Barrett, Ben Sidders. Exploratory biomarker analyses of tumor and peripheral blood samples from the phase I durvalumab plus gefitinib trial in EGFR-mutated NSCLC [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2019 Nov 17-20; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2020;8(3 Suppl):Abstract nr B55.

In this study, we examine the T cell receptor (TCR) repertoire in a cohort of Head and Neck Squamous Cell Carcinoma (HNSCC) patients (n=50) at time of diagnosis. The TCR repertoire is indicative of the ability of the body to mount an immune response, with increased productive clonality within the tumor environment suggesting accumulation of tumor antigen specific T cells. Recent studies demonstrate that the intratumoral TCR repertoire is prognostic of response to anti-PD-1 (Tumeh et al., 2014) and anti-CTLA-4 (Page et al., 2016) therapies. Here we examine diversity and clonality of intratumoral T cell β repertoires in HNSCC patients independently and in conjunction with other biomarkers including tumor mutational burden/neoantigen load, expression of inhibitory checkpoints, and characterization of immune cell populations, as well as clinical metadata including HPV status, smoking history, and survival outcomes. The TCRβ repertoire was analyzed from FFPE primary tumor samples through targeted next generation DNA sequencing of the TCRβ loci and examination of the CDR3 sequences to determine clonality of the TCRβ repertoire (1 – Pielou’s evenness). Somatic genomic alterations, tumor mutational burden, and HLA typing were conducted through whole exome sequencing. Inhibitory checkpoint expression and characterization of immune cell populations were conducted through immunohistochemistry, RNA expression, or both methods. Preliminary results indicate productive clonality of the TCR repertoire in the range of 0.03-0.16. In examining HPV+ and HPV- patients, where we hypothesize the presentation of foreign peptides of viral etiology will produce an increase in T cell populations recognizing those neoantigens, we see no difference in either T cell infiltration or productive clonality. However, we observe that PD-L1 status is significantly correlated (p < 0.05) to productive clonality of the tumor infiltrating T cells, with a higher average productive clonality in PD-L1+ patients. Ongoing studies will characterize the landscape of somatic genomic alterations including tumor mutational burden, and gene expression profiles of the tumor microenvironment. Citation Format: Lara McGrath, Elizabeth Maloney, Maria Secrier, Helen Angell, Emma Jones, Brian Dougherty, Daniel Stetson, Patricia McCoon, Andrew Pierce, Craig Barker, Carl Barrett. Intratumoral T cell receptor β repertoire as a biomarker in head and neck squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2017 Oct 1-4; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2018;6(9 Suppl):Abstract nr B83.