IMPORTANCE: The problem of recurrence in head and neck squamous cell carcinoma (HNSCC) remains challenging with persistently high rates and generally poor prognosis despite advances in detection and treatment. Recently approved immunotherapies highlight a potential key interaction between the tumor and host immune system in the progression of HNSCC; however a large number of patients will fail to achieve a sustained response to immunotherapy, bringing increased urgency to understanding the dynamic tumor-immune interaction in order to better stratify patients and identify alternative therapeutic targets. Historically, evaluation of the tumor-immune microenvironment has been constrained by inherent limitations in immunohistochemical (IHC) analysis and tissue availability; recent technological innovations have mitigated this issue.
OBJECTIVE: To comprehensively characterize the tumor-immune microenvironment in primary and recurrent HNSCC using a novel multiplex immunohistochemistry (mIHC) technology.
DESIGN, SETTING, AND PARTICIPANTS: A novel mIHC and computational image processing workflow was developed and used to evaluate surgical/biopsy specimens from matched primary and recurrent HNSCC tumors from 30 patients. A single formalin-fixed paraffin-embedded tissue section from each specimen was sequentially stained with a panel of 29 immune biomarkers. The stainings were converted to pseudo-fluorescent images, which were then combined to enable simultaneous evaluation of all 29 biomarkers within the single tissue section. Image cytometry analysis was applied to interpret chromogenic signals from the combined images to identify and quantify individual tumor, structural, and immune cells.
MAIN OUTCOMES AND MEASURES: Immune cell density and distribution within each tissue section were measured using visualization and quantification technology analogous to flow cytometry. Data from the matched primary and recurrent HNSCC tumors were then compared using the paired t-test.
RESULTS: 29-plex mIHC analysis of the matched primary and recurrent tumor tissues from 30 patients enabled quantitative analysis of 17 different cell lineages in a single surgical/biopsy tissue section. In recurrent, as compared to primary HNSCC tumors, the percentage of immunosuppressive TH2-polarized CD163+ tumor-associated macrophages (TAM) was significantly increased (p=0.023) while a decreased ratio of CD163-/CD163+ TAMs (p=0.032) was observed. In addition, there was a trend towards a decreased presence of CD8+ T cells in recurrent HNSCC tumors, associated with an increased percentage of PD1+Eomes+CD8+ T cells expressing exhaustion markers.
CONCLUSIONS AND RELEVANCE: This study establishes the validity of a novel 29-plex mIHC technology for analysis of single surgical/biopsy tissue sections and reveals marked differences in the tumor-immune microenvironment of recurrent vs. primary HNSCC. These data reveal a myeloid-type inflammatory status and decreased anti-tumor T cell phenotypes within recurrent HNSCC tumors, and provide important insights into potential mechanisms of tumor recurrence and therapeutic failure that will guide future development of biomarker and therapeutic targets in HNSCC.
FUNDING: The authors acknowledge support from the following sources: NIH/NCI, DOD BCRP Era of Hope Scholar Expansion Award, Susan B. Komen Foundation, Stand Up To Cancer-Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant to Lisa M. Coussens, PhD.