Circulating neutrophils and tumor-associated myeloid cells function as a powerful biomarker for response to chemoradiation in locally advanced cervical cancer

Purpose: The immune system’s role in mediating the cytotoxic effects of chemoradiotherapy remains not completely understood. The integration of immunotherapies into treatment will require insight into features and timing of the immune microenvironment associated with treatment response. Here, we investigated the role of circulating neutrophils and tumor-associated myeloid cells (TSAMs) as potential agents and biomarkers for disease-related outcomes in locally advanced cervical cancer (LACC). Material and Methods: Hematologic parameters for two LACC patient cohorts, a retrospective clinical and a prospective translational cohort, were obtained at baseline, weekly during chemoradiotherapy for the retrospective cohort, biweekly during chemoradiotherapy for the prospective cohort, and at the first follow-up visit for both cohorts (mean 14.7 weeks, range 8.1–25.1 weeks for the prospective cohort and 5.3 weeks with a range of 2.7–9.0 weeks for the retrospective cohort). In both cohorts, baseline as well as mean and lowest on-treatment values for platelets, hemoglobin, absolute neutrophil count (ANC), and absolute lymphocyte count (ALC) were analyzed for correlations with disease-related outcomes. In the prospective cohort, circulating myeloid cells were isolated from peripheral blood mononuclear cells (PBMCs), and TSAMs were isolated from tumor tissue via a novel serial cytobrush sampling assay. The samples were analyzed by flow cytometry. Results: In both cohorts, the only hematologic parameter significantly associated with survival was elevated on-treatment mean ANC (mANC), which was associated with lower local failure-free and overall survival rates in the retrospective and prospective cohorts, respectively. mANC was not associated with a difference in distant metastases. CD11b+CD11c- TSAMs, which act as a surrogate marker for intratumoral neutrophils, steadily decreased during the course of chemoRT and nadier’d at week 5 of treatment. Conversely, circulating myeloid cells identified from PBMCs steadily increased through week 5 of treatment. Regression analysis confirmed an inverse relationship between circulating myeloid cells and TSAMs at this time point. Conclusions: These findings identify on-treatment mean neutrophil count as a predictor of disease-related outcomes, suggest that neutrophils contribute to chemoradiation treatment resistance, and demonstrate the importance of techniques to measure intratumoral immune activity

Top 50 gene alterations over time for 70 patients with paired normals.

Heat map displaying the top 50 genes ranked by occurrence for 70 patients (216 samples) grouped by timepoint collection during chemoradiation.</p

Computational pipeline for whole exome sequencing data.

Workflow depicting preprocessing, variant calling and data analysis tools and parameters implemented to analyze WES data acquired from tumor DNA collected by cervical swab.</p

Overall gene alterations from swab acquired tumor samples (patients 2–30) is similar to landscape of TCGA cervical squamous cell carcinoma dataset.

(A) Ninety-four percent (1339/1430) of altered genes in baseline samples (defined as substitutions, insertions or deletions in gene) were also identified in the TCGA dataset, suggesting accurate identification of mutated genes related to cervical cancer. (B) The distribution of the top 30 most altered genes in study samples 2–30 and in TCGA(C) was also similar. (TIF)</p

Quality characteristics for samples by timepoint.

Quality characteristics for samples by timepoint.</p