Exploratory analysis of the cervix tumoral HPV antigen-specific T-cell repertoire during chemoradiation and after brachytherapy.

BACKGROUND: Chemoradiation (CRT) may modulate the immune milieu as an in-situ vaccine. Rapid dose delivery of brachytherapy has unclear impact on T-cell repertoires. HPV-associated cancers express viral oncoproteins E6/E7, which enable tracking antigen/tumor-specific immunity during CRT. METHODS: Thirteen cervical cancer patients on a multi-institutional prospective protocol from 1/2020-1/2023 underwent standard-of-care CRT with pulsed-dose-rate brachytherapy boost (2 fractions). Cervix swabs at various timepoints underwent multiplex DNA deep sequencing of the TCR-ß/CDR3 region with immunoSEQ. Separately, HPV-responsive T-cell clones were also expanded ex vivo. Statistical analysis was via Mann-Whitney-U. RESULTS: TCR productive clonality, templates, frequency, or rearrangements increased post-brachytherapy in 8 patients. Seven patients had E6/E7-responsive evolution over CRT with increased productive templates (ranges: 1.2-50.2 fold-increase from baseline), frequency (1.2-1.7), rearrangements (1.2-40.2), and clonality (1.2-15.4). Five patients had HPV-responsive clonal expansion post-brachytherapy, without changes in HPV non-responsive clones. Epitope mapping revealed VDJ rearrangements targeting cervical cancer-associated antigens in 5 patients. The only two patients with disease recurrence lacked response in all metrics. A lack of global TCR remodeling correlated with worse recurrence-free survival, p = 0.04. CONCLUSION: CRT and brachytherapy alters the cervical cancer microenvironment to facilitate the expansion of specific T-cell populations, which may contribute to treatment efficacy.

Tumor-resident Lactobacillus iners confer chemoradiation resistance through lactate-induced metabolic rewiring.

Tumor microbiota can produce active metabolites that affect cancer and immune cell signaling, metabolism, and proliferation. Here, we explore tumor and gut microbiome features that affect chemoradiation response in patients with cervical cancer using a combined approach of deep microbiome sequencing, targeted bacterial culture, and in vitro assays. We identify that an obligate L-lactate-producing lactic acid bacterium found in tumors, Lactobacillus iners, is associated with decreased survival in patients, induces chemotherapy and radiation resistance in cervical cancer cells, and leads to metabolic rewiring, or alterations in multiple metabolic pathways, in tumors. Genomically similar L-lactate-producing lactic acid bacteria commensal to other body sites are also significantly associated with survival in colorectal, lung, head and neck, and skin cancers. Our findings demonstrate that lactic acid bacteria in the tumor microenvironment can alter tumor metabolism and lactate signaling pathways, causing therapeutic resistance. Lactic acid bacteria could be promising therapeutic targets across cancer types.

Cervicovaginal Microbiota Profiles in Precancerous Lesions and Cervical Cancer among Ethiopian Women.

Although high-risk human papillomavirus infection is a well-established risk factor for cervical cancer, other co-factors within the local microenvironment may play an important role in the development of cervical cancer. The current study aimed to characterize the cervicovaginal microbiota in women with premalignant dysplasia or invasive cervical cancer compared with that of healthy women. The study comprised 120 Ethiopian women (60 cervical cancer patients who had not received any treatment, 25 patients with premalignant dysplasia, and 35 healthy women). Cervicovaginal specimens were collected using either an Isohelix DNA buccal swab or an Evalyn brush, and ribosomal RNA sequencing was used to characterize the cervicovaginal microbiota. Shannon and Simpson diversity indices were used to evaluate alpha diversity. Beta diversity was examined using principal coordinate analysis of weighted UniFrac distances. Alpha diversity was significantly higher in patients with cervical cancer than in patients with dysplasia and in healthy women (p < 0.01). Beta diversity was also significantly different in cervical cancer patients compared with the other groups (weighted UniFrac Bray-Curtis, p < 0.01). Microbiota composition differed between the dysplasia and cervical cancer groups. Lactobacillus iners was particularly enriched in patients with cancer, and a high relative abundance of Lactobacillus species was identified in the dysplasia and healthy groups, whereas Porphyromonas, Prevotella, Bacteroides, and Anaerococcus species predominated in the cervical cancer group. In summary, we identified differences in cervicovaginal microbiota diversity, composition, and relative abundance between women with cervical cancer, women with dysplasia, and healthy women. Additional studies need to be carried out in Ethiopia and other regions to control for variation in sample collection.

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.

Serial Genotyping of the Human Papillomavirus in Cervical Cancer: An Insight Into Virome Dynamics During Chemoradiation Therapy.

PURPOSE: Human papillomavirus (HPV) is the primary driver of cervical cancer. Although studies in other malignancies correlated peripheral blood DNA clearance with favorable outcomes, research on the prognostic value of HPV clearance in gynecologic cancers using intratumoral HPV is scarce. We aimed to quantify the intratumoral HPV virome in patients undergoing chemoradiation therapy (CRT) and associate this with clinical characteristics and outcomes. METHODS AND MATERIALS: This prospective study enrolled 79 patients with stage IB-IVB cervical cancer undergoing definitive CRT. Cervical tumor swabs collected at baseline and week 5 (end of intensity modulated radiation therapy) were sent for shotgun metagenome sequencing and processed via VirMAP, a viral genome sequencing and identification tool for all known HPV types. The data were categorized into HPV groups (16, 18, high risk [HR], and low risk [LR]). We used independent t tests and Wilcoxon signed-rank to compare continuous variables and χ2 and Fisher exact tests to compare categorical variables. Kaplan-Meier survival modeling was performed with log-rank testing. HPV genotyping was verified using quantitative polymerase chain reaction to validate VirMAP results using receiver operating characteristic curve and Cohen's kappa. RESULTS: At baseline, 42%, 12%, 25%, and 16% of patients were positive for HPV 16, HPV 18, HPV HR, and HPV LR, respectively, and 8% were HPV negative. HPV type was associated with insurance status and CRT response. Patients with HPV 16+ and other HPV HR+ tumors were significantly more likely to have a complete response to CRT versus patients with HPV 18 and HPV LR/HPV-negative tumors. Overall HPV viral loads predominantly decreased throughout CRT, except for HPV LR viral load. CONCLUSIONS: Rarer, less well-studied HPV types in cervical tumors are clinically significant. HPV 18 and HPV LR/negative tumors are associated with poor CRT response. This feasibility study provides a framework for a larger study of intratumoral HPV profiling to predict outcomes in patients with cervical cancer.