Background Pre-operative immunotherapy results in pathologic tumor responses (pTR) for some patients with head and neck squamous cell carcinoma (HNSCC), but response mechanisms remain poorly defined.^1,2 We evaluated T cell profiles and clonal dynamics associated with pTR in a phase II trial of two doses of neoadjuvant pembrolizumab.

Methods 29 patients with stage III/IV HPV-unrelated HNSCC were enrolled in a multicenter phase 2 clinical trial of anti-PD-1 antibody pembrolizumab (2 doses, Q3 weeks) as neoadjuvant immunotherapy over 5 weeks prior to surgery. pTR to PD-1 blockade was assessed based on histologic reduction of tumor cell-fraction, as previously published, with responders defined as pTR of >10%.¹ We profiled tumor-infiltrating lymphocytes (TILs) from 14 tumor biopsies from 4 Responders (Rs) and 4 Non-Responders (NRs), collected either before or after PD-1 blockade through single-cell RNA (scRNA-seq) and T-cell receptor sequencing (scTCR-seq). Quality and quantity of TILs were assessed with multiplex immunofluorescence. Data were validated via comparison with a similar cohort of 36 HNSCC patients receiving single-dose neoadjuvant pembrolizumab.¹

Results pTR was detected in surgical specimens from 15 patients (53%), with two-year overall survival (OS) and progression-free survival (PFS) rates of 92.2% (95% CI: 72.1-97.9) and 92.3% (95% CI: 72.6-98.0%), respectively. Single-cell analysis of CD8+ TILs identified 12 transcriptionally-defined clusters. The microenvironment of Rs compared to NRs showed higher pre-treatment frequencies of exhausted TILs (T_Ex-TILs) (p<0.0001, figure 1a), which were more clonally expanded and expressed a previously-defined gene signature associated with tumor-specificity.³ R T_Ex-TILs were dominated by T_TE-CTX, a subpopulation with characteristics of cytotoxicity and high expression of ZNF683, suggesting a tissue-resident memory (TRM) program.^4,5 Multiplex immunofluorescence of pre-treatment biopsies confirmed that Rs were more highly infiltrated with CD3+ TILs with a TRM phenotype, identified through CD103 co-expression (figure 1b).[4 6 pTR following PD-1 blockade was associated with contraction of highly cytotoxic T_TE-CTX clones and likely unleashing of their antitumor activity (figure 1c). Within this timeframe, immunotherapy response was predominantly attributable to activity of pre-existing CD8+ TIL clones, while phenotypic revival of persisting clones and clonal replacement were modest. For NRs, the baseline microenvironment exhibited a relative absence of ZNF683+CTX+ TILs with post-therapy accumulation of extremely exhausted clones lacking evidence of post-therapy reinvigoration.

Conclusions A larger pre-treatment proportion of T_Ex-TILs retaining cytotoxic potential and a TRM signature are associated with pTR in HNSCC. Expanded T_TE-CTX clones were diminished in number after immunotherapy treatment, consistent with release of their anti-tumor activity and subsequent contraction due to antigen clearance.

Acknowledgements This study was supported by the Alvin J. Siteman Cancer Center at Washington University School of Medicine and Barnes-Jewish Hospital in St. Louis, Missouri and the Clinical Trials Core. The Siteman Cancer Center is supported in part by the NCI Cancer Center. RU is funded by the National Cancer Institute, the National Institute for Dental and Craniofacial Research (NIH/NCI/NIDCR U01DE029188 and NIH/NIDCR R01DE027736) and a V Foundation Translational Research Award. Single-cell data acquisition was supported by Robert A. and Renee E. Belfer Foundation and Expect Miracles Foundation. Clinical trial support was through a Merck Investigator Studies Program award to R. Uppaluri/D.R. Adkins.

Trial Registration clinicaltrials.gov unique identifier: NCT02296684

References

1. Uppaluri R, et al. Neoadjuvant and adjuvant pembrolizumab in resectable locally advanced, human papillomavirus–unrelated head and neck cancer: a multicenter, phase II Trial. Clin. Cancer Res. 2020; 26:5140–5152.

2. Wise-Draper TM, et al. Phase II clinical trial of neoadjuvant and adjuvant pembrolizumab in resectable local-regionally advanced head and neck squamous cell carcinoma. Clin Cancer Res. 2022;28:1345–1352.

3. Oliveira G, et al. Phenotype, specificity and avidity of antitumour CD8+ T cells in melanoma. Nature 2021;596, 119–125.

4. Mackay LK, et al. Hobit and Blimp1 instruct a universal transcriptional program of tissue residency in lymphocytes. Science (80-. ). 2016; 352:459–463.

5. Parga-Vidal L, et al. Hobit identifies tissue-resident memory T cell precursors that are regulated by Eomes. Sci. Immunol 2021;6.

6. Duhen T, et al. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun. 2018;9.

Ethics Approval This study was approved by the Institutional Review Boards of Dana-Farber/Harvard Cancer Center (DFCI#16-385), Washington University (#201412118) and Memorial Sloan-Kettering Cancer Center (MSKCC) (#18-379).

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Abstract 503 Figure 1

Dynamics of exhausted CD8 T cells expressing markers of cytotoxicity and a tissue-resident memory program in HNSCC tumors. a, Frequencies of principal phenotypes among CD8+ TILs collected from Responders (R, circles, n=3) or Non-Responders (NR, diamonds, n=4) at pre-treatment timepoints (Pre). Box plots – median percentage of TILs with phenotypes corresponding to CD8+ non-exhausted memory cell states (TNExM, blue), exhausted states (TEx, red), or unclassified clusters (Other, grey). Whiskers: min-max values; horizontal bars: medians. Boxes: 25th-75th percentiles. P values: significant comparisons (two-tailed Welch's t-test). b, Multiplexed Immunofluorescence of tumor biopsies collected prior to treatment from 3 Rs (left) and 3NRs (right) patients. The representative images demonstrate the pre-existing high levels of tissue resident memory-like (CD103+) and exhausted (PD1) TILs (CD3+) in Rs within the tumor bed, marked by expression of cytokeratin (Cytok). c, Bidimensional plot quantifying the expression ZNF683 expression (x axis) and cytotoxicity genes (summarized in a score [3], y axis) in CD8+ TILs with TEx-TCR clonotypes. Cells are colored according to the size of the TCR clonotype family they belong to. The percentage of cells in each quadrant is calculated based on thresholds representing the average values of variables (vertical and horizontal lines), as measured in the entire dataset of CD8+ TILs.