ADCY7 mRNA Is a Novel Biomarker in HPV Infection and Cervical High-Grade Squamous Lesions or Higher

The effect of cervical cancer immunotherapy is limited. Combination therapy will be a new direction for cervical cancer. Thus, it is essential to discover a novel and available predictive biomarker to stratify patients who may benefit from immunotherapy for cervical cancer. In this study, 563 participants were enrolled. Adenylate cyclase 7 (ADCY7) mRNA was detected by real-time quantitative PCR (qPCR) with cervical cytology specimens. The relationship between ADCY7 and cervical intraepithelial neoplasia in grade 2 and higher (CIN2+) was analyzed, and the optimal cut-off values of the relative expression of ADCY7 mRNA to predict CIN2+ were calculated. In addition, the clinical significance of ADCY7 in cervical cancer was determined by the Kaplan–Meier Cox regression based on the TCGA database. The mean ADCY7 mRNA expression increased significantly with cervical lesion development, especially compared with CIN2+ (p < 0.05). Moreover, the expression of ADCY7 increased significantly in high-risk human papillomavirus (HR-HPV) infection but not in HPV-A5/6 species. The area under the receiver operating characteristic curve (AUC) of ADCY7 was 0.897, and an optimal cut-off was 0.435. Furthermore, ADCY7 had the highest OR (OR= 8.589; 95% CI (2.281–22.339)) for detecting CIN 2+, followed by HPV genotyping, TCT, and age (OR = 4.487, OR = 2.071, and OR = 1.345; 95% CI (1.156–10.518), (0.370–8.137), and (0.171–4.694), respectively). Moreover, this study indicated that higher ADCY7 levels could be a suitable predictor for poor prognosis in cervical cancer due to immune cell infiltration. A new auxiliary predictor of CIN2+ in cervical cytology specimens is ADCY7 ≥ 0.435. Furthermore, it may be a promising prognosis predictor and potential immunotherapy target for the combined treatment of cervical cancer and possibly further block HR-HPV persistent infection.


Introduction
Cervical cancer is the fourth most common type of cancer in women [1]. According to the World Health Organization's (WHO) Global Cancer Data Statistics Report, there are 604,127 emerging cases and 341,831 deaths annually [1]. China has 109,741 new cases and 59,060 deaths annually, which is an increasing trend [1]. Morbidity and mortality are higher in developing countries than in developed countries [2]. If no action is taken, the incidence of cervical cancer worldwide is expected to increase by at least 25 percent by 2030, to more in tumor immune regulation. However, there are few studies about ADCY7 expression status and its biological function in cervical cancer.
This present study aimed to evaluate the predictive value of ADCY7 expression in cervical cancer. We investigated whether the expression of ADCY7 could be a valuable biomarker to predict CIN2+ with cervical cytological specimens collected non-invasively. Additionally, this study demonstrated the prognosis of ADCY7. The results provide novel insights into the active role of ADCY7 in HPV infection and cervical cancer, thereby highlighting a potential mechanistic basis whereby ADCY7 influences immune cell interactions with tumors.

Patients and Study Design
We included 563 participants from the Fujian Maternity and Child Health Hospital College of Clinical Medicine for Obstetrics and Gynecology and Pediatrics, Fujian Medical University from December 2018 to May 2022. The populations had to reach the following criteria: (1) age above 20 years; (2) sexually active; (3) no history of cervical cancer, CIN, or HIV infection; and (4) did not undergo a hysterectomy or cervical surgery. The Hospital Ethics Committee approved the research of Fujian Provincial Maternity and Children's Health Hospital, an affiliated hospital of Fujian Medical University (2022KYLLR03050), and all individuals participating in this study signed written informed consents.

Specimen Collection and Management
Exfoliated cervical cells were collected from cervical canals using a cytobrush. The samples were collected in vials containing preservation solutions for HPV DNA testing or in bottles of ThinPrep ® PreservCyt ® solution (Hologic, Waltham, MA, USA) for cytology examination. The storage conditions of the samples are detailed in our previous study [28,29].

Liquid-Based Cytology, HPV Genotyping Test
Cytological samples were blindly examined, independent of the results from the other assays, by two experienced cytopathologists. The results were reported according to the Bethesda 2001 system [30]. If the diagnosis was inconsistent, the cervical specimens were re-analyzed, and a consensus diagnosis was reached. The PCR-RDB HPV genotyping kit (Yaneng Limited Corporation, Shenzhen, China) can discover 18 HR-HPV types and 5 LR-HPV types. All procedures were conducted according to the manufacturer's instructions [31].

Histology
The colposcopy and needle biopsy in women who were HPV-16/18 positive, with or without abnormal cytology (a grade higher than atypical squamous cells of undetermined significance (ASC-US)), were performed according to the guidelines. Women with a punch biopsy diagnosis of more than high-grade squamous intraepithelial lesions (HSIL) underwent conization by the cold knife or loop electrosurgical excision procedure cone biopsy (LEEP). Specimens were fixed in 10% formalin and routinely processed for paraffin embedding. Then, according to standard methods, 4 µm thick tissue sections were cut and stained with hematoxylin and eosin.

RT-PCR and Analysis of ADCY7
RT-qPCR was carried out according to the protocol. ADCY7 PCR was used with primer pairs 5 -GAT GTA CGT CGA GTG TCT CCT-3 and 5 -CTT TGT CCA TGC GTC GAA CA-3 . GAPDH was used as an experimental control for sample quality and adequacy during the PCR process. GAPDH PCR was applied with primer pairs 5 -GGT GTG AAC CAT GAG AAG TAT GA-3 and 5 -GAG TCC TTC CAC GAT ACC AAA G-3 . Relative levels of ADCY7 mRNA were quantified by qPCR and calculated by the 2−∆∆CT method.

Prognosis Analysis
We analyzed the OS, DSS, and PFI according to different characteristics to individualize the prognosis prediction in cervical cancer patients. All clinicopathological data were acquired from TCGA-CESC datasets, including 306 cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) sample tumor tissues and 13 normal tissues [31].

TIMER Database Analysis
The Tumor Immune Estimation Resource (TIMER2.0) (https://timer.cistrome.org, accessed on 20 January 2023) is a database used for the analysis of tumor-infiltrating immune cells and various gene expression levels in different types of cancer [32]. We assessed the relationship between ADCY7 and TILs via gene modules. In addition, the relationship between ADCY7 mRNA expression and gene markers of TILs has been investigated through gene correlation research in the exploration module [33]. The statistical significance of the estimation and correlation of Spearman was analyzed by the correlation module.

Statistical Analysis
ADCY7 expression was calculated in unpaired samples using the Wilcoxon ranksum test, while paired samples were analyzed using the Wilcoxon signed-rank test. Cox regression analyses and Kaplan-Meier analyses were performed to assess the prognostic factors. We conducted an ROC analysis using the pROC package to determine whether ADCY7 could accurately distinguish cervical intraepithelial neoplasia grade two or more (CIN2+) from healthy specimens. Furthermore, we compared the impact of ADCY7 levels on the incidence of CIN2+ using a multivariate Cox analysis. Different risks were estimated using odds ratios (ORs) and 95% confidence intervals (CIs). In all statistical analyses, p-values below 0.05 were considered statistically significant.

Clinical Characteristics and Associations between Cervical Lesions and Clinicopathological Factors
The analysis of the 563 cervical tissue samples indicated that 143 (25.400%) were standard, 116 (20.604%) were classified as CIN1, 202 (35.879%) were categorized as CIN2/3, and 102 (18.117%) were diagnosed as cancer. As shown in Table 1, age, cytology, and HPV genotyping infection were significantly associated with cervical lesions. This demonstrates that abnormal cytology and older age promote the development of CIN2+. Furthermore, with the development of cervical lesions, ADCY7 mRNA levels increased, especially compared with CIN2+. This suggests that ADCY7 mRNA levels could predict the development of CIN2+ lesions (p < 0.001). The relationship between ADCY7 expression and cervical lesions and cytology is summarized in Figure 1A,B. We discovered a significant difference in means of ADCY7 expression in four groups of cervical lesions (p < 0.001, ANOVA test) ( Figure 1A). Moreover, ADCY7 expression increased significantly in the ASCUS group and higher ASCUS group. Importantly, we found the expression of ADCY7 among different HPV genotyping. This revealed that the expression of ADCY7 increased significantly in HR-HPV positive, HPV A7 species, HPV A9 species, HPV-16/18 positive, HPV-16 positive, and HPV-18 positive samples. However, no significant difference between HPV A5/6 species positive groups was found.
ADCY7 expression is a valuable predictor for the detection of ≥CIN2.

Association between Immune Infiltration and ADCY7 mRNA Levels in Cervical Cancer
Immune infiltration is a crucial factor related to tumor progression. In cervical canc

Association between Immune Infiltration and ADCY7 mRNA Levels in Cervical Cancer
Immune infiltration is a crucial factor related to tumor progression. In cervical cancer, ADCY7 mRNA levels are associated with immune cell infiltration levels using TIMER platforms. Figure 5 shows a strong correlation between ADCY7 mRNA levels and TIL

Association between Immune Infiltration and ADCY7 mRNA Levels in Cervical Cancer
Immune infiltration is a crucial factor related to tumor progression. In cervical cancer, ADCY7 mRNA levels are associated with immune cell infiltration levels using TIMER platforms. Figure 5 shows a strong correlation between ADCY7 mRNA levels and TIL abundance. For instance, ADCY7 mRNA levels were strongly correlated with the infiltrating degree of CD4+ T cells (rho = 0.342), T cells gamma delta (rho = 0.323), macrophages (rho = 0.325), neutrophils (rho = 0.316), M1 macrophages (rho = 0.312), myeloid dendritic cells (rho = 0.293), mast cells (rho = 0.268), endothelial cell (rho = 0.242), cancer-association fibroblasts (rho = 0.236), and NK cells (rho = 0.212). All the p-values were below 0.001. These results demonstrate that ADCY7 mRNA levels play an essential role in the immune infiltration in cervical cancer. In addition, this research revealed that ADCY7 mRNA levels were significantly connected with immune inhibitors, as shown in Figure 6 and Table 3. These results demonstrate that ADCY7 mRNA levels play an essential role in the immune infiltration in cervical cancer. In addition, this research revealed that ADCY7 mRNA levels were significantly connected with immune inhibitors, as shown in Figure 6 and Table  3.

Discussion
This study demonstrated that cervical specimens with an over-expression of ADCY7 mRNA were significantly associated with CIN2+ lesions. Thus, the present study may provide information on a novel and valuable biomarker to predict the development of CIN2+ with non-invasive methods. Interestingly, this study demonstrated that a high expression of ADCY7 suggested HR-HPV infection. In this study, ADCY7 mRNA levels increased significantly with the development of the cervical lesion. Moreover, ADCY7 could be a valuable biomarker to predict CIN2+. The AUC for ADCY7 mRNA levels was 0.898. Moreover, an optimal cut-off for ADCY7 mRNA levels was 0.435. Additionally it was demonstrated that higher ADCY7 mRNA levels (≥0.435) had the highest OR (OR= 8.589; 95% CI (2.281-22.339)). Thus, this suggests that ADCY7 mRNA levels in cervical specimens can be a valuable biomarker to predict CIN2+ with non-invasive procedures. Our results may offer a helpful basis for the accurate cytological diagnosis of cervical pre-malignancy and cancer in future transitional research.
Furthermore, we also revealed that the expression of ADCY7 mRNA levels increased significantly in HR-HPV positive, HPV A7 species, HPV A9 species, HPV-16/18 positive, HPV-16 positive, and HPV-18 positive samples, but not in the HPV A5/6 species. We divided the high-risk HPV types into three groups: the HPVA5/6 species contains HPV 51, 56, and 66; the HPVA7 species contains probes for HPV 18, 39, 45, 59, and 68; and the HPVA9 species contains probes for HPV 16,31,33,35,52, and 58. It has been reported that persistent infection with high-risk human papillomavirus (HPV) is a high-risk factor for cervical precancerous lesions and cervical cancer [5,6], and HPV16 /18 accounts for most of the HPV-positive cases [34]. This study suggests that ADCY7 mRNA levels may be an auxiliary indicator for HR-HPV infection, except for HPV-A5/6 species.
Nearly 95% of cervical cancer patients have a high risk for HPV infection [35]. However, it is not expressed in healthy tissues, so it provides a specific therapeutic target for immunotherapy of cervical cancer. T-cell receptor-engineered T cell (TCR-T) therapy, a kind of immunotherapy method, uses tumor-specific antigens as the target to obtain TCR sequences that recognize tumor antigens and then introduces the TCR sequence into the patient's T cells through genetic engineering technology. Thus, obtained TCR-T cells can specifically recognize and kill specific tumors after in vitro expansion [36]. In addition, TCR cells also resist immune evasion by tumors [37]. A completed Phase I/II clinical trial investigating the use of E6 TCR-T cells in HPV-16-associated tumors showed that E6 TCR-T cell therapy could lead to the regression of HPV-associated epithelial cancer cells [38]. TCR-T therapy is effective in hematologic malignancies, but there is currently insufficient evidence to confirm the efficacy of TCR-T therapy in solid tumors and HPV-associated malignancies [39]. Several preclinical studies of HPV-associated tumors have shown that combining anti-PD-1 antibodies with therapeutic HPV16 E6/E7 vaccines significantly increases tumor clearance and more CD8+ T cells [40,41]. With a better understanding of cervical cancer antigen-specific T cells and immunosuppressive reversal mechanisms in the context of the tumor microenvironment (TME), it will be possible to discover more effective and safer immunotherapies for cervical cancer. Combination therapy with ICIs and targeting HPV-associated antigens will be promising.
ADCY7 encodes a membrane protein. It is involved in extracellular signaling into intracellular reactions [19]. It is reported that external stimuli bind to their receptors and then pass to G proteins, which can activate ADCY7. This promotes the conversion of adenosine triphosphate (ATP) into cyclic adenosine-phosphate (cAMP), which acts as a second messenger to transmit signals into the cell, which in turn undergoes a series of biochemical cascades [25,26]. In addition, the adenylate cyclase catalytic product cAMP upregulates the inhibitory molecule CTLA-4 on the surface of CD4+ T cells, which enables tumor cells to evade immune surveillance by T cells and continue to develop [27]. These findings confirm that ADCY7 is a critical molecule in tumor immune regulation.
Our study revealed that higher ADCY7 mRNA levels were significantly related to poor OS (HR = 2.43, 95% CI = 1.46-4.04, p = 0.001), DSS (HR = 2.29, 95% CI = 1.29-4.05, p = 0.005), and PFI. Similarly, Giorgio Bogani. et al. [42] pointed out that HPV persistence is the only risk factor for recurrence after treatment of cervical lesions. This study further demonstrated that ADCY7 mRNA levels are significantly associated with HPV infection. Thus, we have further demonstrated that the ADCY7 expression level can be used as a valuable biomarker for HPV infection and as a prognostic indicator. Moreover, this study analyzed the correlation of ADCY7 with immune cell infiltration. We found that ADCY7 mRNA levels were strongly correlated with the infiltrating degree of CD4+ T cells, T cells gamma delta, macrophages, neutrophils, M1 macrophages, myeloid dendritic cells, mast cells, endothelial cells, cancer-association fibroblasts, and NK cells. These results demonstrate that ADCY7 mRNA levels play an essential role in the immune infiltration of cervical cancer. It is well known now that CD8 T cells play a central role in mediating anti-tumor immunity. Their effector CTLs eliminate tumor cells by recognizing tumorassociated antigens presented on primary histocompatibility complex class I (MHCI) by their expressed T cell receptor (TCR). Studies have reported that the infiltration of T cells, especially CD4+ T cells and CD8+ T cells, into the tumor microenvironment (TME) demonstrate a good prognosis in cancers, such as breast, lung, melanoma, colorectal, brain, and cervical cancer [43][44][45][46]. Conversely, suppressive immune cells, such as Treg cells, indicate a poor prognosis. Higher percentages of Tregs were significantly associated with a poor clinical outcome for patients with cervical carcinoma [45,46]. Insufficient T cell priming likely contributes to cold tumors (no T cell infiltration in TME) and unresponsiveness to immune checkpoint blockade (ICB) therapy [47]. In addition, this research revealed that ADCY7 mRNA levels were significantly associated with immune inhibitors, such as PDCD1LG2, CTLA4, TIGIT, TNFRSF14, TNFRSF25, CD40, LAIR1, LGALS9, ADORA2A, ICOS, CD276, and TNFSF4. This evidence suggests that targeting ADCY7 may enhance the anti-tumor effect by increasing the infiltration of antitumor immune cells and promoting the expression of the immune checkpoint. Therefore, this study further demonstrates that ADCY7 can be used as a new target for cervical cancer treatment. Moreover, it provides a new research direction for the combined treatment of cervical cancer with immunotherapy.
If treatments for persistent HPV infection are developed, persistent HPV infection will become less frequent, significantly reducing the occurrence of high-grade intraepithelial neoplasia and cervical cancer in the cervix. Therefore, immunotherapy against HPV may have important clinical implications for preventing persistent HPV infection. This study indicates that targeting ADCY7 can serve as a new direction for immunotherapy for cervical cancer and further block HR-HPV persistent infections. This study fills the gaps in the primary and clinical research of the ADCY7 protein in the field of cervical cancer; provides theoretical support for the prevention, diagnosis, and treatment of cervical cancer; and seeks new targets for the treatment of cervical cancer. It provides valuable clinical data for ADCY7 in human biological function research, with good innovation and progress. However, there are some limitations, as follows: Firstly, we did not verify the relationship between ADCY7 expression levels and the prognosis of cervical cancer based on our clinical data in this study, which is worth further study. Secondly, the specimen size was not big enough. Therefore, increasing the sample size may be necessary in the future.

Conclusions
This study revealed that ADCY7 expression levels significantly increased in cervical cytology and is strongly related to CIN2+ lesions. ADCY7 expression levels can be a suitable biomarker to predict CIN2+. Moreover, higher ADCY7 expression levels predict a poor prognosis in cervical cancer due to the promotion of the expression of the immune checkpoint. In addition, ADCY7 mRNA levels may be an auxiliary indicator for HR-HPV infection. These results may provide a valuable research direction for the combined treatment of cervical cancer with immunotherapy and possibly further block HR-HPV persistent infection.

Institutional Review Board Statement:
The Hospital Ethics Committee of Fujian Provincial Maternity and Children's Hospital, an affiliated hospital of Fujian Medical University, approved the study (2022KYLLR03050) and complied with the Declaration of Helsinki.

Informed Consent Statement:
And all individuals participating in this study signed written informed consent.