Correlation between CXCR5 expression and prognosis in stage T1 non-small cell lung cancer

Background : Lung cancer is the leading cause of cancer-related death. Even if early detection has been applied and proved to be effective, the survival outcomes are still poor. Thus, to find out novel prognostic factors is of great significance to identify high-risk patients and guide individualized precise treatment in order to reduce recurrence and improve survival outcomes. Methods: Tissue samples and clinicopathologic data of 244 stage T1 NSCLC patients were collected. We investigated CXCR5 expression using immunochemical method and analyzed its correlation with pathologic subtypes and prognosis. Results: Elevated level of positive CXCR5 expression was found in tumor tissues ( p <0.0001) and patients with positive CXCR5 expression are more likely to have LVIs ( p =0.030) and recurrences ( p <0.043). Besides, CXCR5 expression is also correlated to histological type and differentiation. Lepidic predominant tumors have significantly lower expression of CXCR5 ( p <0.001). Survival analyses showed that patients with positive CXCR5 had a significantly lower DFS ( p =0.038) and patients with solid or micropapillary predominant NSCLC had a significantly worse prognosis. CXCR5 expression was proved to be an independent prognostic factor for DFS through multivariate analysis. Conclusions: CXCR5 expression has been proved to be an independent prognostic factor for stage T1 NSCLC patients. In addition, CXCR5 was also found to be relevant to cancer recurrence, and this indicated that CXCR5 may play an important role in lymph node and distant metastasis.


Background
Lung cancer is now the most prevalent malignant tumor and has become the leading cause of cancerrelated death both in China and worldwide. [1,2] Histologically, lung cancer is classified into non-small cell lung cancer (NSCLC), which accounts for approximately 85%, and small cell lung cancer (SCLC) accounts for the remaining 15%. [3] Despite advances in screening, diagnosis and multidisciplinary treatments made in recent years, the survival outcomes of lung cancer patients are still not satisfactory. Since early symptoms are not specific and conspicuous, most patients are diagnosed at advanced stages with poor prognoses. Fortunately, early detection with low-dose computed tomography (LDCT) has been reported to be effective and reduce lung cancer mortality up to 20% among high risk individuals. [4,5] However, the 5-year survival of all stages is only 18%. [6] Thus, apart from LDCT screening, following precise treatments also play a pivotal role in improving survival of lung cancer patients. Previous studies demonstrated that recurrence or metastasis occurs in about 20% of early stage NSCLC patients even though surgeries had been done, which indicated that other clinicopathological factors that would impact the prognosis of early NSCLC patients did exist other than TNM stage. Thus, to find out these factors is of great significance to identify high-risk patients and guide individualized precise treatment in order to reduce recurrence and improve survival outcomes.
Histologically speaking, NSCLC were classified into adenocarcinoma (AC), squamous cell carcinoma (SCC), large-cell carcinoma and etc. Among them, Adenocarcinoma is the most common histologic type. To address advances in oncology, molecular biology, pathology, radiology, and surgery of lung adenocarcinoma, an international multidisciplinary classification was sponsored by the International Association for the Study of Lung Cancer, American Thoracic Society, and European Respiratory Society (IASLC/ATS/ERS) in 2011. [7] This new classification has been shown to provide prognosesrelevant information for patients with lung adenocarcinoma after curative surgeries [8,9] and it has been incorporated into the 2015 World Health Organization classification of lung tumors for its reliability and practicability. [10] Chemokines are a superfamily of small (8-10kDa) proteins which can cause the directed migration of certain subsets of leukocytes and are induced by inflammatory cytokines, growth factors and pathogenic stimuli. [11][12][13] Chemokines are the major regulators of cell trafficking and adhesion in the body. [14] Based on the configuration of the first two conserved cysteine residues adjacent to the Nterminal, chemokines are classified into four subfamilies (C, CC, CXC and CXXXC families). [11] Chemokine receptors are a superfamily of seven transmembrane spanning proteins coupled to Gprotein-coupled receptors (GPCRs). Most of these receptors bind to more than one type of chemokine.
Emerging evidence revealed that elevated levels of CXCR5 activate downstream signals and contribute to tumorigenesis, cell growth, survival, and site-specific metastasis. [28][29][30][31][32][33] Previous study demonstrated that CXCR5 is overexpressed in NSCLC tissues and the interaction between CXCR5 and its ligand promote migration of NSCLC cells, which indicates that CXCR5 may play a role in the dissemination and/or metastasis of primary lung cancers. [34] To further investigate the correlation between CXCR5 expression and prognosis in stage T1 NSCLCs, we collected clinical and pathological data of stage T1 NSCLC patients who underwent curative lobectomies with systematic lymph node dissection, detected expression of CXCR5 in tumor tissues and corresponding non-neoplastic tissues using immunohistochemical method and then analyzed its correlation with prognosis, in order to evaluate the potential of CXCR5 to be a prognostic biomarker or a therapeutic target. In addition, we analyzed the survival outcomes of stage T1 NSCLC patients based on their pathological subtypes, and validated their relationship with CXCR5 expression.

Patients
From January 2011 to January 2012, information of patients with stage T1 NSCLC who underwent curative lobectomy with systematic lymph node dissection at Department of Thoracic Surgery,

Tumor Specimens
Tumor and its surrounding tissues were fixed in 10% (v/v) formalin right after resection. After embedded in paraffin, the specimens were prepared in serial sections of 5 μm and reserved for hematoxylin-eosin (HE) staining. Then the sections would be examined by professional pathologists to determine the pathological type, subtype (if AC) and the existence of lymphovascular invasion (LVI) of the tumor.

Immunohistochemistry
We performed immunohistochemistry (IHC) for CXCR5 on 244 NSCLC samples and their corresponding non-neoplastic samples. The slides were de-paraffinized in xylene (I, II and III) for 10 min each, then rehydrated through ethanol of different gradient (100%, 85% and 75%) for 10 min each and washed in deionized water and phosphate-buffered saline (PBS). Antigen retrieval was implemented by incubating with 0.01 M sodium citrate buffer (pH=6.0) in boiling water for 20 min and then cool down to room temperature and the activity of endogenous peroxidase was blocked by incubating with 3% hydrogen peroxide (H 2 O 2 ) for 10 min. PBS washing was applied after each step for 4 times. After incubation in a non-specific stain blocking agent, slides were incubated for 2 hours at 37°C with primary antibody: anti-human CXCR5 antibody (GR297692-6; Abcam, Cambridge, UK; 1:100). This was followed by PV-9000 Polymer Detection System (ZhongShanJinQiao, Beijing, China) which contains PV 9000 Kit Polymer Helper and PV 9000 Kit polyperoxidase-anti-mouse/rabbit IgG, incubating for 20 min at 37℃ and then washed by PBS for 3 times. After incubation, slides were stained with a 3, 3'-diaminobenzidine (DAB) (ZhongShanJinQiao, Beijing, China) as a chromogen.
Subsequently, slides were washed with water and ammonia, dehydrated in 75%, 85%, and absolute alcohol for 5 min each, and sealed with resin for following evaluation.
To analyze the immunohistochemical staining of CXCR5, slides were evaluated by 2 professional pathologists who were blinded to each other. The intensity of staining was evaluated and graded from 0 to 3, where 0 for no staining (negative), 1 for light yellow (weak), 2 for yellow (moderate) and 3 for brown (strong). The numbers of positively stained cells were scored as follows: 0, ≤25%; 1, 25%-50%; 2, 51%-100%. The two values obtained were multiplied to calculate a final score (maximum value, 6).
Samples were classified into negative (score 2) or positive (score ≥ 2) for further analysis.

Statistical Analyses
The clinicopathologic characteristics were compared between the groups using Student's t tests for continuous variables with normal distributions, Mann-Whitney U tests for continuous variables with abnormal distributions, and chi-square tests for categorical variables. OS was defined as the period between the date of surgery and death due to any cause or the last follow-up. DFS was defined as the period after successful treatment during which there were no signs or symptoms of the disease that was treated. The cumulative OS and DFS rates were estimated by the Kaplan-Meier method, and the differences were compared between groups by the log-rank test. To identify prognostic factors, univariate and multivariate analyses were performed using the Cox proportional hazards regression model. Baseline variables that were considered clinically relevant or that showed a univariate relationship with the outcome were entered into multivariate Cox proportional hazards regression model. Variables for inclusion were carefully chosen, given the number of events available, to ensure parsimony of the final model.
All statistical analyses were performed using IBM SPSS software version 24.0 (IBM Corp., Armonk, NY, USA) and GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA). The results were considered statistically significant when p value was less than 0.05.

Patient clinicopathological characteristics
A total of 244 qualified patients who were diagnosed with stage T1 NSCLC were enrolled in this study, and the clinicopathological characteristics are shown in Table 1 Figure 1 showed the expression of CXCR5 in tumor and non-neoplastic tissues. Immunohistochemical analyses revealed that CXCR5 mainly expresses on the membrane of tumor cells. Positive CXCR5 expression was found in 155 (63.5%) tumor tissues and 15 (6.1%) non-neoplastic tissues. As it showed in Figure 2, the positive rate of CXCR5 expression in tumor tissues is significantly higher than that in non-neoplastic tissues (p<0.0001).

Correlation between CXCR5 expression and clinicopathological characteristics
We then evaluated the correlation between CXCR5 expression and clinicopathological characteristics and the results were listed in Table 2. Patients were classified into two groups depending on the CXCR5 expression in tumor tissues. From the table, we can see that CXCR5 expression was positive in 155 patients and negative in 89 patients. Sex, age, tumor size, tumor location and N stage were similar between the groups, while histological type (p<0.001), tumor differentiation (p<0.001) and LVI (p=0.030) differs significantly. In addition, recurrence was significantly higher in CXCR5 positive group (p=0.043), while survival was similar between the groups. Further analyses were conducted and showed in Figure 2. We found that positive rate of CXCR5 is significantly higher in patients with LVI than those without LVI (71.9% vs. 58.1%, p<0.05) and in AC than SCC (69.8% vs. 40.0%, p<0.001). Moreover, positive CXCR5 expression was significantly higher in moderate and poor differentiated tumors when comparing to well differentiated ones (Moderate vs. Well = 71.0% vs. 32.4%, p<0.0001, Poor vs. Well = 65.8% vs. 32.4%, p<0.01). Nonetheless, no significant differences were found in the positive rate of CXCR5 in tumor location and N stage.

Correlation between CXCR5 expression and pathological subtypes of lung adenocarcinoma
CXCR5 expression and other variables were compared among different pathological subtypes ( Table   3). It revealed in the table that CXCR5 expression (p<0.001) was significantly different among the groups, besides, the differences in N stage (p<0.001), recurrence (p<0.001) and survival (p<0.001) were also significant. In subgroup analyses (Figure 3), difference in CXCR5 expression (p=0.0001) was found to be between lepidic predominant (LP) group and the other four groups, and differences in N stage (p<0.0001), recurrence (p<0.0001) and survival (p<0.0001) were between solid predominant (SP), micropapillary predominant (MPP) and the other three groups.

Survival analyses based on CXCR5 expression
Survival outcomes based CXCR5 expression was compared between CXCR5 positive group and CXCR5 negative group using Kaplan-Meier method ( Figure 4). Patients in CXCR5 positive group tended to have a lower 5-year OS than them in CXCR5 negative group, but the difference was not significant (CXCR5 positive vs. CXCR5 negative, 78.7% vs. 86.5%, p=0.220). On the contrary, the difference in 5-year DFS was significant between the groups (CXCR5 positive vs. CXCR5 negative, 65.2% vs. 79.8%, p=0.038).

Survival analyses based on pathological subtypes of lung adenocarcinoma
Survival analyses were completed based on pathological subtypes of lung adenocarcinoma ( Figure 5). p=0.434). Besides, DFS between LP group and AP group was also significantly different (p=0.048).

Prognostic factors for survival of 244 NSCLC patients
Univariate and multivariate analyses were conducted using the Cox proportional hazards regression model (

Discussion
Lung cancer is now the leading cause of cancer-related death [1] and even for early stage patients, recurrence occurs in about 20% of them after surgery. Therefore, it is significant to find out the prognostic factors to guide individualized precise treatment to improve the survival outcomes. To our knowledge, this is the first study that analyzes the correlation with CXCR5 and prognosis.
This study examined the level of CXCR5 expression in tissues using immunohistochemical methods, analyzed the correlation among CXCR5 expression, pathological subtypes, patients' prognosis and other clinicopathological characteristics and investigated the prognostic factors in NSCLC patients. In this study, we demonstrated that CXCR5 expression is significantly higher in tumor tissues. Patients with positive CXCR5 expression are more likely to have LVIs and recurrences. We also found that patients with ACs had a significantly higher level of CXCR5 expression than those with SCCs (Patients with other types of NSCLC were not included in the calculation because of the small sample size) and the positive rate of CXCR5 expression in patients with moderate or poor differentiation was significantly higher than patients with well differentiation, which indicated that CXCR5 expression is correlated to histological type and differentiation of NSCLC. As for pathological subtypes, we found that CXCR5 expression, N stage, recurrence and survival all differed significantly among pathological subtypes. Further analyses showed that positive CXCR5 expression in LP tumors was significantly lower than the other four groups, which may be part of the reason for the pretty good survival outcomes. In addition, SP and MPP group were found to have a more advanced N stage, higher recurrence and worse survival, and these results are consistent with previous studies. [8,9,66] Survival analyses showed that patients with positive CXCR5 expression had a significantly lower DFS while OS was similar, which supported our results that CXCR5 expression is related to cancer recurrence. These results indicate that CXCR5 may be involved in recurrence and metastasis of Whether these signaling pathways are involved in the tumorigenesis and progression of lung cancer remains to be validated.
In following researches, we would validate the expression of CXCL13, ligand of CXCR5, in tumor and metastatic tissue and peripheral blood samples, investigate how CXCL13/CXCR5 impact cell function and the exact signaling pathway behind.

Conclusions
In conclusion, the present study demonstrated that positive rate of CXCR5 is significantly higher in tumor tissues and positive CXCR5 expression was an independent prognostic factor for stage T1 NSCLC patients. CXCR5 was also proved to be relevant to cancer recurrence, and this indicated that

Declarations Declarations
The authors declare no conflicts of interest.

Ethics approval and consent to participate
This study was approved by the Ethics Committee of Cancer Hospital, Chinese Academy of Medical Sciences (NCC2014ST-07).

Consent for publication
All patients' consent forms were signed and collected.

Availability of data and materials
The datasets generated during and/or analyzed during the current study are available.

Competing interests
The authors declare that they have no competing interests.

Funding
This study was supported by Beijing Municipal Science & Technology Commission (Capital specialty clinical research project, Grant number: Z141107002514047).

Authors' contributions
Zhao Yue analyzed and interpreted the data, drafted and modified this manuscript, Ding Ningning