
==== Front
medRxiv
MEDRXIV
medRxiv
Cold Spring Harbor Laboratory

10.1101/2024.09.10.24313432
preprint
1
Article
Immune Cell Densities Predict Response to Immune Checkpoint-Blockade in Head and Neck Cancer
Ruiz Torres Daniel A. http://orcid.org/0000-0001-6215-3369

Bryan Michael E.
Hirayama Shun
Merkin Ross D. http://orcid.org/0000-0003-3418-2220

Evelyn Luciani
Roberts Thomas
Patel Manisha
Park Jong C.
Wirth Lori J. http://orcid.org/0000-0002-2629-2289

Sadow Peter M. http://orcid.org/0000-0003-1036-6367

Sade-Feldman Moshe
Stott Shannon L. http://orcid.org/0000-0002-0349-0522

Faden Daniel L. http://orcid.org/0000-0001-5284-7762

12 9 2024
2024.09.10.24313432https://creativecommons.org/licenses/by-nc-nd/4.0/ This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which allows reusers to copy and distribute the material in any medium or format in unadapted form only, for noncommercial purposes only, and only so long as attribution is given to the creator.
http://medrxiv.org/lookup/doi/10.1101/2024.09.10.24313432
nihpp-2024.09.10.24313432.pdf
Immune checkpoint blockade (ICB) is the standard of care for recurrent/metastatic head and neck squamous cell carcinoma (HNSCC), yet efficacy remains low. The current approach for predicting the likelihood of response to ICB is a single proportional biomarker (PD-L1) expressed in immune and tumor cells (Combined Positive Score, CPS) without differentiation by cell type, potentially explaining its limited predictive value. Tertiary Lymphoid Structures (TLS) have shown a stronger association with ICB response than PD-L1. However, their exact composition, size, and spatial biology in HNSCC remain understudied. A detailed understanding of TLS is required for future use as a clinically applicable predictive biomarker. Methods: Pre-ICB tumor tissue sections were obtained from 9 responders (complete response, partial response, or stable disease) and 11 non-responders (progressive disease) classified via RECISTv1.1. A custom multi-immunofluorescence (mIF) staining assay was designed, optimized, and applied to characterize tumor cells (pan-cytokeratin), T cells (CD4, CD8), B cells (CD19, CD20), myeloid cells (CD16, CD56, CD163), dendritic cells (LAMP3), fibroblasts (alpha-Smooth Muscle Actin), proliferative status (Ki67) and immunoregulatory molecules (PD1). Spatial metrics were compared among groups. Serial tissue sections were scored for TLS in both H&E and mIF slides. A machine learning model was employed to measure the effect of these metrics on achieving a response to ICB (SD, PR, or CR). Results: A higher density of B lymphocytes (CD20+) was found in responders compared to non-responders to ICB (p=0.022). A positive correlation was observed between mIF and pathologist identification of TLS (R2= 0.66, p-value= <0.0001). TLS trended toward being more prevalent in responders to ICB (p=0.0906). The presence of TLS within 100 um of the tumor was associated with improved overall (p=0.04) and progression-free survival (p=0.03). A multivariate machine learning model identified TLS density as a leading predictor of response to ICB with 80% accuracy. Conclusion: Immune cell densities and TLS spatial location within the tumor microenvironment play a critical role in the immune response to HNSCC and may potentially outperform CPS as a predictor of ICB response.
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