Friday 12th August 2022

Expression of B7-H3 in patients with cervical cancer

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Introduction

Cervical cancer is the third most common cancer in women worldwide. Following the implementation of cervical cancer screening and development of effective preventative vaccines, the overall incidence of cervical cancer has declined during the past decade;1 however, the relative and absolute incidences of adenocarcinoma are still rising.2 Most patients with early-stage disease have favorable prognoses; nonetheless, those with advanced or recurrent disease have poor outcomes.

In recent years, immunotherapy targeting the immune checkpoint programmed cell-death 1 (PD-1) and its ligand programmed cell-death ligand 1 (PD-L1) has emerged as a novel modality in oncology and has been shown to be effective against a variety of tumor types. The United States Food and Drug Administration has approved the PD-1 inhibitor pembrolizumab for patients with recurrent or metastatic cervical cancer whose tumors express PD-L1 with a combined positive score (CPS) of ≥1. However, the objective response rate for pembrolizumab was found to be 14.6% in a Phase II study.3 Therefore, efforts continue to identify new targets and develop novel immunotherapeutic strategies for patients with this disease.

B7 homolog 3 (B7-H3), also known as CD276, belongs to the B7 superfamily that also includes PD-L1 and is an immune checkpoint protein that exhibits essential roles in T cells. Notably, B7-H3 has been reported to have both costimulatory and coinhibitory abilities in different tumor microenvironments.4 Chapoval et al demonstrated that B7-H3 increases the proliferation of CD4+ and CD8+ T cell populations and upregulates IFN-γ production.5 In contrast, Suh et al showed that B7-H3 is a negative regulator that inhibits T cell proliferation.6 B7-H3 protein is present at low levels in the liver, colon, and prostate but is rare in normal tissues overall; nevertheless, it is expressed in various types of malignant tissues including lung cancer,7 colorectal cancer,8 prostatic cancer,9 renal cell carcinoma,10 breast cancer,11 endometrial cancer,12 ovarian cancer,13 gestational trophoblastic neoplasia,14 and cervical cancer.15–18 B7-H3 is expressed in tumor cells (TCs) and stromal cells including tumor-associated vascular endothelial cells, fibroblasts, and immune cells.7,19,20 Generally, B7-H3 is associated with tumor aggressiveness, unfavorable clinicopathological features, and poor outcomes. It has been shown to promote invasion, migration, and angiogenesis in lung, colorectal, ovarian, and prostate cancers.21–24 These results suggest that B7-H3 may be able to induce cancer progression and metastasis beyond its ability to modulate tumor immunity.4

Although B7-H3 expression in cervical cancer has been reported in previous studies, including ours,12–15,18 its association with PD-L1 remains unclear. Furthermore, the numbers of patients included in previous studies were small, particularly those with adenocarcinoma. Therefore, the associations between B7-H3 and histological subtypes as well as with survival outcomes remain unknown. As such, the aims of this study were to explore B7-H3 expression within cervical cancer tissues and to assess the relationship between this expression and PD-L1, clinicopathological features, and patient outcomes.

Materials and Methods

Study Cohort and Tissue Microarray (TMA) Construction

This retrospective study included 552 radical hysterectomy specimens from patients diagnosed with either squamous cell carcinoma of the cervix (N = 406) between January 2012 and December 2015 or usual type endocervical adenocarcinoma (N = 146) between January 2008 and December 2017, at Qilu Hospital of Shandong University (Jinan, China). All patients in this study were diagnosed before 2018; therefore, we used the 2009 International Federation of Gynecology and Obstetrics (FIGO) staging system for carcinoma of the cervix. Considering the excellent odds of survival in patients with stage IA, we included only patients with IB1 and IIA1 disease, for whom the standard treatment was radical hysterectomy with pelvic lymph node dissection and/or para-aortic lymph node sampling or dissection. The following patients were excluded from the study: those with non-usual types of endocervical adenocarcinoma (adenosquamous, mucinous gastric/intestinal, adenoma malignum, serous, clear cell, or endometrioid), those with neuroendocrine carcinoma, those with insufficient data, those who died within 1 month after surgery, and those who received neoadjuvant therapy before surgery. Clinicopathologic data (collected from medical records) included age at diagnosis, tumor size, tumor histology, differentiation grade, depth of invasion, parametrial involvement, lymphovascular space invasion, number of positive lymph nodes, postoperative adjuvant treatment (chemotherapy, chemoradiotherapy, or radiotherapy), date of recurrence or last follow-up, and patient status on the last follow-up date. Representative areas within the tumor tissue were marked on hematoxylin and eosin-stained slides and sampled for the TMA blocks. TMAs with a single 2 mm core per case were constructed using a tissue arrayer (Mini Core, Mitogen, Hertford, UK). This study conformed to the ethical standards set forth in the Declaration of Helsinki and in national and international guidelines and was approved by the Institutional Review Board of Qilu Hospital of Shandong University (approval No. KYLL-2017-560). All investigators signed patient data confidentiality statements, and patient data were only accessible on confidential computers at Qilu Hospital of Shandong University. All patients, upon admission to the hospital, signed consent forms allowing review of their medical records, the use of their tissue samples, and possible publication of associated reports. None of the patients can be identified from their clinical information or images in this retrospective study, and informed consent to determine PD-L1 and B7-H3 expression was not required by the Institutional Review Board of Qilu Hospital of Shandong University.

Immunohistochemistry and Assessment

Immunohistochemistry was performed using our laboratory protocol as described previously.14,17,26 Briefly, 4 μm TMA serial sections were deparaffinized and subjected to heat-induced epitope retrieval with 10 mM sodium citrate (pH 6.0) at 95°C for 20 min. The endogenous peroxidase activity was quenched using a 0.3% hydrogen peroxide solution. TMA sections were incubated with primary antibodies against PD-L1 (dilution 1:200, clone E1L3N, Cell Signaling Technology, Danvers, USA) and B7-H3 (dilution 1:200, clone D9M2L, Cell Signaling Technology, Danvers, USA). Human placental tissues treated with primary antibodies were used as positive controls, while the same tissues without primary antibodies comprised negative controls. All the slides were stained using an automatic immunohistochemistry staining instrument (BOND-III; Leica Biosystems, Wetzlar, Germany) according to the manufacturer’s instructions. PD-L1 was evaluated using the CPS, which was calculated as the sum of the number of PD-L1-stained cells (TCs, lymphocytes, and macrophages) divided by the total number of viable tumor cells, with the quotient multiplied by 100. A CPS of ≥1 was considered indicative of positive PD-L1 expression. Samples were considered positive for B7-H3 when ≥5% of the TCs expressed this protein at any intensity; the 5% cut-off was chosen based on a previous publication.6 Staining of B7-H3 in stromal fibroblasts, endothelial cells, and immune cells was also recorded.

Statistical Analysis

The χ2 test was used to determine the association between categorical variables. Relapse-free survival (RFS) was defined as the interval between the date of surgery and that of the detection of the first local, regional, and/or distant relapse. Overall survival (OS) was defined as the time between the date of surgery and that of death from any cause, censoring, or the last follow-up. Survival curves were plotted using the Kaplan–Meier method and compared using the Log rank test. To identify prognostic predictors, univariate and multivariate survival analyses were performed using the Cox proportional hazards regression model, and hazard ratios (HRs) with 95% confidence intervals (CIs) for recurrence and death were calculated. All statistical analyses were conducted using the Statistical Package for the Social Sciences software (version 20.0; IBM Corp., Armonk, NY, USA). A 2-sided P-value <0.05 was considered statistically…

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