P16INK4A expression might be associated with a favorable prognosis for cervical adenocarcinoma via dysregulation of the RB pathway

Previous studies have largely failed to clarify the relationship between p16INK4A status and cervical adenocarcinoma prognosis. The current study aimed to examine the clinical and pathological significance of p16INK4A expression in several cervical adenocarcinoma subtypes. Eighty-two samples collected from patients with cervical adenocarcinoma were formalin fixed and paraffin embedded. Next, p16INK4A levels were analyzed with immunohistochemistry. Additionally, the relationship between p16INK4A expression and clinicopathological factors as well as prognosis was evaluated. The expression of p16INK4A was mostly detected in all usual cervical adenocarcinoma subtypes. In the gastric type, only a few cases were positive for p16INK4A expression. Results of the Kaplan–Meier analysis indicated that the positive p16INK4A expression in tumor cells was significantly associated with favorable progression-free survival and overall survival in patients with cervical adenocarcinoma (p = 0.018 and p = 0.047, respectively, log-rank test). Our findings suggest that the status of p16INK4A expression may influence prognosis. Thus, p16INK4A expression could be used as a biomarker for improving the prognosis of patients with cervical adenocarcinoma.

Definition of p16 INK4A and CD8/PD-1/ PD-L1 positivity by IHC. The expression of p16 INK4A in both cytoplasm and nucleus was evaluated by staining for three tumor density categories as follows: 0 (undetectable), 1 + (low density), and 2 + (high density). Intensities of + 1 and + 2 were considered as strong staining. In a previous study, over 70% of cervical cancer cells with strong p16 staining of the nuclei and cytoplasm were regarded as p16 positive 13 . Therefore, in the current study, most of the tumor cells with strong p16 staining were considered as p16 positive. The population density of tumor infiltrating lymphocytes was stratified by CD8 staining into three categories as follows: 0 (undetectable), 1 + (low density, 0-30%), and 2 + (moderate-high density, ≥ 30%). Samples that were categorized as 2 + were considered positive. For PD-L1, tumors with ≥ 5% of stained tumor cells (membranous and cytoplasmic staining) were considered positive. For PD-1, tumors with ≥ 5% of tumorinfiltrating stained lymphocytes were considered positive. Statistical analysis. The correlation between p16 INK4A expression and clinicopathological characteristics and patient prognosis was analyzed by chi-square test. Furthermore, the correlation between p16 INK4A expression and other immune escape mechanism-related factors was analyzed using the chi-squared test. Progressionfree survival (PFS) and overall survival (OS) were analyzed using the Kaplan-Meier method with the log-rank test. The univariate and multivariate Cox proportional hazard regression analyses were followed by binomial logistic regression for ordered categorical variables. Statistical analyses were performed using IBM SPSS (IBM, Armonk, NY, USA), version 23. Statistical significance was set at p < 0.05. Consent to participate. After appropriate explanation, patients provided written informed consent for the procedure and for participation in the study. For patients who could not visit hospitals again, we had clearly announced that the opportunity to opt out is always available by taking measures such as carrying the information on the opportunity to opt out on the website, as well as making arrangements so that patients can opt out via the website at any time.

Results
Clinicopathological characteristics of the patients. Histological  www.nature.com/scientificreports/ There were several histological subtypes in cervical adenocarcinoma, including the usual type, gastric type, and others. The usual type was the most popular type (59.0%), followed by the gastric type (16%). The clinicopathological characteristics of the test patients are summarized in Table 2 and Supplementary Table S1. In the present study, we identified clinical stages according to the definition of the International Federation of Gynecology and Obstetrics (FIGO) 2014 as stages IA, IB1, B2, IIA, IIB, IIIB, and IVB. Treatments received by the test patients were as follows: 72 underwent radical hysterectomy and received adjuvant therapy, such as concurrent chemoradiotherapy (CCRT); 9 patients with advanced stage cancer received CCRT; and 1 patient received chemotherapy without surgery due to multiple distant metastases. Radiotherapy (whole pelvic irradiation) or chemotherapy (paclitaxel 175 mg/m 2 and carboplatin area under the curve = 5 mg/m 2 ) was administered postoperatively in patients with a high recurrence risk because of locally advanced stage, non-SCC type of histology, bulky tumor > 4 cm, deep infiltration depth of cervical tumor, grade 2 or 3, lymph node metastasis, or lympho-vascular space invasion.
The chemotherapy regimens adopted were paclitaxel plus carboplatin, paclitaxel plus cisplatin, docetaxel plus carboplatin, irrinotecan plus cisplatin, and gemcitabine. In the CCRT regimen, 5-6 courses of 40 mg/m 2 cisplatin were administered weekly.  Clinical features of cervical adenocarcinoma with p16INK4A expression. Kaplan-Meier analysis was performed to determine the potential correlation between p16 INK4A expression and prognosis. Cervical adenocarcinoma patients with p16 INK4A negativity presented significantly worse PFS and OS than those with p16 INK4A positivity (p = 0.018 and p = 0.047, respectively, log-rank test; Fig. 3a,b).

Univariate analysis of prognostic factors in patients with cervical adenocarcinoma. The uni-
variate and multivariate Cox regression analyses of the prognostic factors in patients with cervical adenocarcinoma are shown in Tables 4 and 5. The univariate and multivariate logistic regression models were used for proportional hazards analysis of prognostic factors with a hazard ratio (HR) and 95% confidence interval. The results of univariate analysis indicated a significant correlation between PFS and p16 INK4A expression (HR: 0.376, p5%; CI 0.162-0.873, p = 0.023) ( Table 4). A significant correlation was also observed between PFS and FIGO stage, LVSI, tumor size, and metastasis of pelvic lymph node, paraaortic lymph node, or distance. The www.nature.com/scientificreports/ multivariate analysis revealed a significant correlation between PFS and LVSI or tumor size. We also performed a stratified multivariate analysis in early-stage cases; however, no significant correlation was observed between PFS and p16 expression (Supplementary Table S3). The univariate analysis revealed a significant correlation between OS and FIGO stage, LVSI, or tumor size, and the multivariate analysis demonstrated a significant correlation between LVSI and OS.

Discussion
This study demonstrated that strong p16 INK4A expression is related to a favorable prognosis of cervical adenocarcinoma. In contrast, the results of previous studies on the association between p16 INK4A expression and cervical adenocarcinoma have been unclear [13][14][15] . The 2018 International Endocervical Adenocarcinoma Criteria and Classification (IECC) distinctively explained the criteria pertaining to the pathological diagnosis of cervical adenocarcinoma, compared with the WHO criteria 16 . According to the IECC, diagnosis was defined using the HPV infection status, and it reflected patient prognosis. Furthermore, they substantiated their opinion by publishing clinical outcomes of patients with cervical adenocarcinomas associated or unassociated with HPV infection 17 . In the current study, we replicated their results. At the start of this study, we assumed that tumors with strong p16 INK4A expression predicted good prognosis because p16 INK4A is a surrogate marker of HPV infection. As viruses express antigens, lymphocytes expressing CD8 attack cancer cells 18 . According to our predictions, cervical adenocarcinoma patients with strong p16 INK4A expression showed the trend of favorable prognosis. However, a positive relationship between p16 INK4A expression and the expression of immune-check point-related molecules, such as CD8, PD-1, and PD-L1, was not verified (Supplementary Table S2). Previously, we reported the association between cervical adenocarcinoma and immune characteristics 19 . We demonstrated that a high PD-1 expression may be associated with a poor prognosis in patients with cervical adenocarcinoma. However, in that study, we did not analyze the relationship between the expression of p16 and immune-check point-related molecules. The lack of a significant positive relationship between p16 INK4A expression and immune-check point-related molecules can be attributed to the fact that cervical adenocarcinomas with HPV infection possibly do not continue to express p16 INK4A following malignant alteration. A previous study reported that some patients with cervical adenocarcinoma have reduced expression of p16 when the tumor is malignant 20 . In the current study, the positive rate of p16 INK4A expression was considerably higher in early-stage cervical adenocarcinoma than in advanced-stage cervical adenocarcinoma (Table 3).
Several studies have reported that p16 INK4A overexpression may be considered as a surrogate marker for high-risk HPV infection in the cervix [21][22][23] . As HPV infection induces tumor immune-environmental activity, immunotherapy has been recognized as an attractive treatment strategy for cervical carcinoma, as for other malignant tumors 24 .
Although carcinogenesis is associated with HPV infection, these tumors do not maintain steady p16 INK4A (HPV infection) expression during carcinogenesis and tumor growth. The expression of p16 INK4A changes under Figure 2. HE staining and immunohistochemistry of specimens obtained from patients with cervical adenocarcinoma. The expression of p16 INK4A was evaluated in three categories of tumor density via staining: 0 (undetectable); 1 + (low density); 2 + (high density). Cases that were 2 + were considered positive and those with 0 and + 1 were considered negative.    www.nature.com/scientificreports/ varying conditions, and there are currently no markers associated with the loss of p16 expression in some HPVpositive tumors before confirming malignant tumors. In addition, microenvironmental immunoactivity around HPV-infected tumors has not yet been conclusively proven, owing to which an association between the condition (stage, tumor size, and propensity of invasion) of the tumors themselves and the function of immune-related lymphocytes has not been demonstrated. Thus, further examination may be required to assess the changes in microenvironmental immunoactivity around tumors that have been already infected with HPV and have outgrown their previous conditions. Another possibility is that p16 INK4A also functions as a tumor suppressor gene, and the loss of p16 INK4A accentuates the phosphorylation of the retinoblastoma (RB) protein (pRB). This pathway may be more pathophysiologically important. P16 INK4A is a tumor suppressor gene, and it regulates the cell cycle by specifically inhibiting the cyclin D/CDK4/6 activity. P16 INK4A and pRB form a negative feedback loop and the inactivation or mutation of pRB leads to the overexpression of p16 INK4A , resulting in CDK4 and CDK6 dysregulation 25,26 .
Therefore, cell proliferation is suppressed and tumors that express p16 INK4A would have a good prognosis. Llucia et al. further indicated that p16 INK4A expression reflected not only the status of HPV infection but also dysregulation of the RB pathway, particularly in head and neck malignant tumors 27 .
Overall, the findings of previous studies and the current study indicate that p16 INK4A expression may be a favorable prognostic factor, associated with the pRB pathway, rather than with HPV infection, in cervical adenocarcinoma. Several studies have indicated that immunocyte invasion by HPV infection may contribute to the effectiveness of its treatment, whereas p16 INK4A expression reflects tumor suppressor properties, as well as its role in inhibiting CDK4 and maintaining pRB. Missaoui et al. reported that p16 INK4A expression primarily affects the RB protein-related pathway, rather than the HPV-independent pathway 28 . In the future, we aim to study mechanism(s) underlying the changes in p16 INK4A expression at the onset of HPV infection, during tumorigenesis and tumor growth. We will also investigate the changes in the expression of immune-related factors associated with tumor condition and p16 INK4A expression.
Another notable point is that the prognosis of gastric type was very poor. Mucinous gastric type was associated with a very poor prognosis; however, we could not unravel the mechanism underlying the correlation between poor prognosis and negative HPV infection in gastric-type tumors. The expression of p16 could be affected by various factors. We want to emphasize that p16-negative tumor subtypes such as gastric-type tumors are not www.nature.com/scientificreports/ associated with HPV infection and show a poor prognosis because of the inactivation of a tumor suppressor gene. Currently, we are conducting genetic analysis of gastric-type tumor using whole-exome sequencing. Our study had some limitations. Our series of cervical adenocarcinomas with confirmed p16 INK4A -negativity were frequently found in an advanced FIGO stage, showing a higher rate of lymph node metastasis. Therefore, the possibility of better responses of p16 INK4A -positive cases in advanced FIGO stage to chemotherapy and radiation therapy, as observed in head and neck cancer cases 27,29 , cannot be excluded. Availability of a higher number of cases may have allowed comparisons within the same FIGO stage, leading to more information regarding each p16 INK4A condition.
A further limitation was that only p16 INK4A expression was examined. Addition of PCR analyses or IHC of HPV may have aided in the clarification of the precise relationship between p16 INK4A expression and HPV infection status. However, some studies have reported that while p16 INK4A expression reflects HPV infection 30 , it may not necessarily reflect tumors with HPV infection 31 . Due to difficulties in correctly and easily establishing whether a tumor has HPV infection, only p16 INK4A expression was taken into consideration.
In summary, results of the current study indicated that p16 INK4A expression might be associated with favorable outcomes in patients with cervical adenocarcinoma. Thus, p16INK4A expression could serve as a biomarker for improving the prognosis of patients with cervical adenocarcinoma. Further research is needed to clarify that the expression of p16 INK4A may function as a tumor suppressor rather than an HPV infection suppressor, activating invasive immune system cells.

Data availability
Data of current study was available from corresponding author (K.N.).
Received: 4 February 2020; Accepted: 30 August 2021 www.nature.com/scientificreports/ Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.