Quantitative Differences in Levels of Immunohistochemical Biomarkers between Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix: Implications for Treatment Outcomes after Chemoradiotherapy

. Quantitative Differences in Levels of Immunohistochemical Biomarkers between Squamous Cell Carcinoma and Adenocarcinoma of the Uterine Cervix: Implications for Treatment Outcomes after Chemoradiotherapy. Journal of Women’s Health and Development 5 (2022): 271-281


Introduction
Uterine cervical cancer is both the fourth most common cause of cancer and the fourth most common cause of death from cancer in women worldwide [1]. Squamous cell carcinoma (SCC) has the highest incidence, but the incidence of adenocarcinoma (AC) has increased in recent decades, and constitutes approximately 10% to 20% of all cervical carcinomas [2][3][4]. Some studies indicated that cervical AC and SCC behave differently epidemiologically [3][4][5][6][7], and have different genomic expressions [8,9]. In addition, they have diverse prognostic factors and patterns of failure after similar treatments [5,7,[10][11][12][13]. As advances in molecular profiling have allowed for the identification of biomarkers of many biological characteristics in tumor cells, biomarkers in standard treatment are of interest for their potential role in the design of personalized therapeutic strategies targeting individual tumors, rather than therapy based on histological types alone. Given that chemoradiotherapy (CRT) has been the standard of care for patients with locally advanced cervical cancer worldwide, radioresistance or treatment failure is a clinically relevant problem. Patients with cervical AC primarily treated with radiotherapy have inferior outcomes compared with those with SCC [5,[12][13][14]. In cervical cancer, several biomarkers for radiotherapy-based treatment have been validated by patient survival or recurrence data [15,16]. These biomarkers fall into categories according to biological function including hypoxia, cell proliferation, cell adhesion, immunogenicity, and evasion of apoptosis [15]. To optimize the treatment outcomes for patients with advanced disease, there is a great need to understand the differences in the expression of the aforementioned biomarkers between cervical SCC and AC, particularly for CRT-based prognostic factors. Hence, this study was conducted to investigate the quantitative differences in immunohistochemical (IHC) biomarkers between the two pathologies. Thus, the impact of these IHC markers on CRT-based treatment beyond the histological types could be identified.

Study Population
For this study, incisional biopsy specimens from patients newly diagnosed with stage IB3 to III uterine cervical SCC and AC between July 2009 and December 2015 were analyzed. Because of missing tumor tissue in some specimens, this retrospective study included a cohort consisting of 76 SCC and 42 AC patients. According to institutional protocol, all patients had undergone F-18 fluorodeoxyglucose positron emission tomography/computed tomography (FDG-PET/ CT) for initial staging. In addition, all had received allocated external-beam radiotherapy and intracavitary brachytherapy. Concurrent chemotherapy consisted of weekly administration of 40 mg/m 2 cisplatin. The patients were staged in accordance with the International Federation of Gynecology and Obstetrics [17]. Accordingly, 14, 34, and 70 patients were classified as having stage IB3, II, and III disease, respectively. The median age of our patients was 55 years. Because FDG-PET/ CT has high sensitivity and specificity in detecting the nodal status in cervical cancer, PET/CT was used for the diagnosis of pelvic or paraaortic lymph node metastasis. We excluded patients who were diagnosed as having a histological type of adenosquamous carcinoma. This study was approved by the local institutional review board [CMUH-107-REC3-163]. Patient characteristics are listed in Table 1.

Immuno histochemistry
As reported in our previous study [18], IHC biomarkers, namely endogenous hypoxic (Glut1, CAIX, and HIF-1α), angiogenesis or metastasis (VEGF), cell proliferation [EGFR, c-Myc, insulin-like growth factor 1 receptor IGF-1R)], cell to cell adhesion (E-cadherin, Vimentin, calretinin), evasion to apoptosis [B-cell lymphoma 2 (Bcl-2), Bax, myeloid cell leukemia 1 (Mcl-1)], and immunogenic or inflammatory biomarkers [programmed cell death protein ligand 1 (PD-L1),} tumor necrosis factor-α (TNF-α), galectin-9, and chemokine ligand 5 (CCL5)] were analyzed using tissue microarrays from incisional biopsy specimens before treatment. Each tumor was represented by one tissue core on a tissue microarray. Paraffin sections 4-µm-thick were deparaffinized and microwaved according to standard procedures before being processed for IHC staining. The staining slides were scored by 2 pathologists blinded to the clinical outcome. Except for PD-L1, IHC results of the aforementioned biomarkers were scored by a semiquantitative approach used to assign an H-score to tumor samples [19]. The H-score takes into consideration the staining intensity in conjunction with the percentage of cells staining positively. Staining intensity was graded as 0, 1, 2, and 3 corresponding to negative, mild, moderate, and strong, respectively. The percentage of positive tumor cells was estimated by the observers. The total number of neoplastic cells in the field and the number of neoplastic cells stained at each intensity were counted. The following formula was applied: H-score = [% of cells stained at intensity category 1 (neoplastic cells with mild staining) x 1] + [% of cells stained at intensity category 2 (neoplastic cells with moderate staining) x 2] + [% of cells stained at intensity category 3 (neoplastic cells with strong staining) x 3]. Accordingly, the H-scores, ranging from 0 to 300, were calculated with 300 equal to 100% of tumor cells stained strongly (3 +). Tumor PD-L1 biomarker was evaluated through IHC staining using the DAKO clone 22C3 pharmDx kit (DAKO, Carpinteria, CA,USA). PD-L1 expression was scored according to the combined positive score (CPS), which is the number of PD-L1 stained cells (tumor cells, lymphocytes, macrophages) at any intensity divided by the total number of viable tumor cells, multiplied by 100 [20].

Treatment
The treatment was described previously [18,21]. All patients were treated with intensity-modulated radiotherapy. The total dose applied to the pelvis was 45 Gy, administered in 25 fractions over a 5-week period. Following pelvic irradiation, the bilateral parametrium was boosted from 50.4 to 54 Gy. After adequate tumor regression, high-doserate intracavitary brachytherapy was performed once or twice a week using an Ir-192 remote after loading technique concurrently with pelvic irradiation or parametrial boosting. Before January 2013, the standard prescribed dose for each session of brachytherapy was 6.0 Gy to Point A, with 5 sessions. After January 2013, patients were treated with threedimensional image-based brachytherapy according to the recommendations of the Groupe Européen de Curiethérapie and the guidelines specified by the European Society for Radiotherapy and Oncology [22]. The details of the cumulative dose are summarized in Table 1. Chemotherapy consisted of weekly 40 mg/m 2 -doses of cisplatin, administered intravenously.

Follow-up
After completion of radiotherapy, patients were regularly followed up every 2 months for the first year, and every 3 to 4 months thereafter. Besides a routine pelvic examination, the serum levels of tumor markers, namely carcinoembryonic antigens, were examined during each follow-up. A radiographic examination was performed every 6 months. Patients exhibiting symptoms of central-pelvic recurrence underwent a salvage hysterectomy or pelvic exenteration, if feasible. Patients with distant metastasis were treated with systemic chemotherapy.

Statistical Analysis
The quantitative differences in the calculated H-scores of the biomarkers between SCC and AC were examined using the Mann-Whitney U test. To examine correlations between the IHC biomarkers and lymph node status or  treatment outcome, receiver operating characteristic (ROC) curves were constructed to evaluate the optimal predictive performance among the various IHC and clinical parameters, such as maximum tumor dimension and pretreatment hemoglobulin [23]. In addition, binary logistic regression analysis was performed to determine the independent factors among all IHC biomarkers for predicting the lymph node status. The outcome endpoints were disease-free survival (DFS), distant metastasis-free survival (DMFS), and pelvic relapse-free survival (PRFS), all of which were calculated using the Kaplan-Meier method. The log-rank test and Cox regression analysis were performed to examine the effects of explanatory variables on these endpoints. The stage, age, histology, lymph node status, maximum tumor dimension, baseline hemoglobulin, and predictable IHC markers were included for analysis. Two-tailed tests were used, and P < .05 was considered statistically significant. All calculations were performed using SPSS, Version 13.0 for Windows (SPSS Inc., Chicago, IL, USA).

Quantitative Differences in Immunohistochemical Biomarkers between the Two Histological Types
All aforementioned IHC biomarkers were retrieved. The quantitative differences in the IHC biomarkers are illustrated in Table 2 and Figure 1. When using the H-scores of the SCC IHC markers as a reference, increased expression of E-cadherin, calretinin, CAIX, and c-Myc and decreased levels of VEGF, TNF-α, galectin-9, CCL5, Bax, EGFR, and IGF-1R were found in AC tumors. The mean PD-L1 CPS score was higher in AC than SCC, but no statistical significance existed. In summary, cervical SCC exhibited strong expression of the inflammatory and angiogenesis markers, whereas the levels of cell to cell adhesion markers were higher in cervical AC. In addition, the Bax levels, an apoptotic activator, were higher in the SCC than in the AC specimens.

Predictive Ability for Lymph Nodes Metastases
Based on the baseline PET/CT, 60 and 15 patients were identified as having pelvic and paraaortic lymph node metastases, respectively. As shown in Supplemental Table 1

Predictive Ability for Treatment Outcomes
In total, 76 patients were alive and 42 patients had died of cancer progression after a median follow-up of 50 months (range, 7-122). Seventy-two patients had no evidence of cancer progression. Thirteen of the 46 patients with tumor progression had pelvic recurrence, 19 had distant metastasis, and 14 had both. None of the 27 patients with pelvic recurrence experienced sole relapse in the lymph nodes. In summary, 27 patients had local residual or recurrent tumors at primary sites, whereas 33 patients experienced distant metastasis. The 4-year DFS, PRFS, and DMFS for SCC and AC patients were 60% and 57% (P = 0.97), 80% and 67% (P = 0.06), 67% and 70% (P = 0.92), respectively. Table 3 lists the biomarkers and the AUC if the predictive value was greater than 0.6 or less than 0.4 for any endpoint. The H-scores of 4 IHC markers were associated with the presence of local residual or recurrent tumors, including c-Myc (AUC: 0.65, P = 0.018), Bax (AUC: 0.30, P = 0.002), E-cadherin (AUC: 0.64, P = 0.027), and calreticulin (AUC: 0.63, P = 0.047). ROC analysis disclosed that none of the other IHC biomarkers, including the hypoxia, cell adhesion, or immunogenicity biomarkers, appeared to be prognostic for distant metastasis or cancer progression. Logistic regression analysis revealed that the existence of pelvic lymph node disease was the sole factor that predicted distant metastasis (P = 0.016, OR = 2.91, 95% CI = 1.22-6.93), and the maximum tumor diameter was the only paramater that predicted cancer progression (P = 0.037, OR = 1.49, 95% CI = 1.03-2.17).

Prognostic Factors for DFS, PRFS, and DMFS
To test the prognostic values of the IHC markers, tumors were dichotomized using the median cut-offs of the 4 predictable IHC markers mentioned above. By combining with the clinical parameters, Cox regression analysis was performed. As summarized in Table 4, the results indicated that an E-cadherin H-score > 50% percentile [P = 0.006, hazard ratio (HR) = 2.35, 95% CI = 1.27-4.35] and stage III disease (P= 0.019, HR = 2.15, CI = 1.13-4.07) were two prognostic factors for an inferior DFS. The 4-year DFS of patients with low and high E-cadherin H-scores was 67% and 50% (P = 0.029). Cox regression analysis disclosed that high E-cadherin and low Bax H-scores were the two predictors of poor PRFS (P = 0.002, HR = 2.72, CI = 1. 17-6.35 and P = 0.001, HR = 0.31, CI = 0.12-0.76, respectively). As depicted in Figure 2, the 4-year PRFS of patients with tumors with high and low E-cadherin values was 53% and 84% (P = 0.007), and the 4-year PRFS of patients with high and low expression of Bax was 88% and 63% (P = 0.003) , respectively. In SCC patients, the impact of E-cadherin and Bax remained statistically significant (Supplemental Figure 1). None of IHC biomarkers were prognostic for DMFS. The major determinant for a low DMFS was pelvic lymph node disease (P = 0.01, HR = 2.66, CI = 1. 26-5.59). In multivariate analysis, histology, age, maximum tumor size, and pretreatment hemoglobulin were not identified as independent prognostic factors for the aforementioned endpoints.

Discussion
In cervical cancer, several biomarkers for radiotherapybased treatment have been validated by patient survival and recurrence data [15,24]. In view of radioresistance, these biomarkers fall into categories according to biological function including hypoxia, cell proliferation, cell adhesion, immunogenicity, and evasion of apoptosis [15]. There are few IHC comparison studies available for clinical practice which distinguish the differences in CRT-based biomarkers    between SCC and AC. This study explored the quantitative differences in a wide range of IHC biomarkers between the two histological types, as well as their roles in determining CRT-based outcomes in patients with locally advanced disease. Herein, we disclosed that cervical SCC exhibited prominent expression of the inflammatory and angiogenesis markers. In contrast, expression of cell to cell adhesion biomarkers were distinct in cervical AC. Additionally, our results first found that high E-cadherin and low Bax H-scores in tumors were the two determinants for inferior local control. Because expression of high E-cadherin and low Bax H-scores were more common in AC than in SCC, it would be interesting to clarify if the profile might contribute to the inferior radiosensitivity reported in cervical AC. Before initiating a novel predictive model for cervical cancer, however, validation studies are required to confirm the findings. E-cadherin is critical to the maintenance of the epithelial phenotype and provides a structural link between adjacent cellular cytoskeletons, which is important for tissue architecture. Loss of E-cadherin is regarded as a common feature of epithelial-mesenchymal transition (EMT) and is associated with a majority of epithelial cancers [25]. However, many invasive carcinomas infiltrate surrounding tissues as multicellular clusters in which tumor cells remain connected to neighboring tumor cells, which is known as collective invasion [26]. Additionally, recent data demonstrated that a partial EMT resulting in a hybrid epithelial/mesenchymal phenotype with retention of E-cadherin is essential for cancer cell dissemination, and E-cadherin or E-cadherinbased adherens junctions are required for collective invasion and tumor migration [27]. Furthermore, a molecular study disclosed that pretreatment with anti-E-cadherin antibody significantly decreased stromal cell-induced radiation resistance in human prostate cancer [28]. The investigators suggested that cell adhesion molecules such as E-cadherin in cancer cells induce cell survival signals and mediate resistance to cancer treatments such as radiation. The expression of E-cadherin is regulated by genetic and epigenetic mechanisms related to cancer, and its function is modulated by mechanical forces at the junctions, and by multiple signaling pathways [29]. Therefore, integrated molecular studies are required to clarify the biological mechanism by which higher E-cadherin expression in tumors is related to poor radiosensitivity. Resistance to apoptosis plays an important role in tumors that are refractory to ionizing radiation. Apoptosis regulator Bax, also known as Bcl-2-like protein 4, forms a heterodimer with Bcl-2, and functions as an apoptotic activator. This protein is reported to interact with, and increase the opening of, the mitochondrial voltage-dependent anion channel, which leads to the loss of membrane potential and the release of cytochrome c. In a study investigating a human breast cancer cell line [30], the degree of enhancement of radiosensitivity was dependent on the expression level of Bax. In addition, a superior radiotherapy-based response has been reported in patients with higher Bax expression cervical cancers [31,32]. On the other hand, Bax is a p53 primary-response gene, presumably involved in a p53-regulated pathway for induction of apoptosis [33]. In an IHC study of cervical carcinomas [34], a total of 66% of the tumors expressed the mutated p53 protein. The overall survival was better for patients expressing the mutated p53 protein in the nucleus. Hence, we have been working to clarify the relation between p53 gene and Bax expression levels in our patients, and their impact on final outcomes. It will be interesting to investigate whether radiation combined with drugs that activate Bax can have a synergistic effect in anticancer treatments by inducing apoptosis in lower Bax expression tumors.
Our study has several limitations. First, this was a retrospective study in a single institution. External validation studies using an independent data set are necessary to confirm these findings. Particularly, future studies should enroll patients prospectively and employ a standardized IHC protocol. Second, the precise molecular pathway that E-cadherin and Bax confers to poor CRT-based local control could not be clarified through association molecular studies, animal experiments, or clinical trials. Finally, the association between DNA sequencing and the protein product should be investigated to understand the comprehensive molecular mechanism of radioresistance and distant metastasis in these patients. Nevertheless, the strengths of this study include the uniform treatment strategies, and wide-ranging analyses of IHC biomarkers. Our findings provide a hint that future studies can clarify the mechanisms related to failure of CRT. In addition, this study initiated a pilot step to enable the tailoring of CRT to the specific biological characteristics of patients with cervical cancers instead of histological types. Our findings disclosed that certain IHC information from cervical tumors might supplement well-known clinical prognostic factors in predicting CRT-based treatment outcomes.