Abstract
Background/Aim: The neonatal Fc receptor (FcRn) is a major histocompatibility class I-like molecule responsible for the transfer of passive humoral immunity from a mother to her newborn. Recent research revealed that FcRn is involved in antigen-presentation, humoral immunity and antitumor immunity of various types of cancer, such as lung, colon and breast. Lung cancer is the leading cause of cancer-related death and non-small cell lung cancer (NSCLC) accounts for 85% of all lung cancer. NSCLC is a highly heterogeneous disease and this affects the prognosis. Therefore, many studies have tried to identify factors that are associated with prognosis. The lungs are a major organ expressing FcRn. We aimed to evaluate FcRn expression in surgical specimens of NSCLC and determine its correlation with patient prognosis. Materials and Methods: We analyzed 140 NSCLC surgical specimens for FcRn expression using immunohistochemistry and correlated positivity with clinicopathology and survival of these patients. A chi-squared test and Kaplan–Meier analysis with log-rank tests were performed for statistical evaluation. Results: The FcRn-positive group had a significantly higher disease-free survival and a tendency towards increased disease-specific survival in patients with tumor-node-metastasis stage I NSCLC. Conclusion: Our study supports the hypothesis that FcRn down-regulation is associated with NSCLC progression.
Lung cancer is the leading cause of cancer-related deaths worldwide in both men and women. Approximately one-quarter of all cancer-related deaths are due to lung cancer. More than half of all patients with lung cancer are diagnosed with metastatic disease, and the 5-year survival rate is 8% (1).
Non-small-cell lung carcinoma (NSCLC) accounts for 85% of all lung cancer cases. The most common risk factor for NSCLC is tobacco smoke inhalation. Other causes of lung cancer include alcohol use, and exposure to secondhand smoke, radon, asbestos, arsenic, nickel and ionizing radiation. Long-term and cumulative exposure to air pollution has also been identified as a risk factor for lung cancer (2, 3), with fine particulate matter (with a diameter of 2.5 μmm or less) inducing motility and proliferation of NSCLC cells (4).
Biomarkers that can indicate disease prognosis and patient survival are necessary for the effective management of lung cancer (5, 6). Currently, most patients with stage I NSCLC do not receive adjuvant systemic treatment after local therapy as numerous studies have reported that chemotherapy is associated with toxic effects (7-10). Recently, advances in molecular markers of lung cancer have progressed significantly, and molecular markers have been used in targeted therapies, leading to a decline in mortality rates. Tests for epidermal growth factor receptor mutations and anaplastic lymphoma kinase gene rearrangements are becoming the standard for personalized therapies in patients with advanced NSCLC. Treatments targeted at programmed death ligand 1 (PD-L1) and its receptor (PD1) have improved survival in patients with NSCLC [reviewed in (11)]. In the near future, molecular prognostication could become the standard means of pathological diagnosis for patients with early-stage NSCLC, as is the case for breast and colon cancer. A successful practice in this field is the incorporation of molecular markers into the histological classification system of lung cancer (12).
Neonatal Fc receptor (FcRn) has attracted interest in the scientific community as it plays an important role in antitumor immunity. It is expressed by antigen-presenting cells, implicated in humoral immunity, and is involved in the activation of tumor-reactive T-cells and maturation of natural killer cells (13-15).
FcRn is expressed by a variety of tissues, including the intestines, mammary glands, placenta, lungs, spleen, kidneys, brain, and liver (16-19). The lung is a major organ that expresses FcRn (8). Most lung cancers are diagnosed at an advanced stage, and early detection is important for lung cancer treatment (20). Low-dose computed tomography has been adopted for screening high-risk persons and will increase the diagnostic rate of early-stage lung cancer (21). The discovery of new biomarkers of early-stage lung cancer is warranted because the accurate prediction of tumor behavior can help improve survival. Only a limited number of studies have been conducted on FcRn expression and its prognostic value in cancer (13, 14, 17, 18).
In this study, we evaluated FcRn expression in surgical specimens of early-stage NSCLC and its correlation with patient prognosis.
Materials and Methods
Patients and clinicopathological data. We obtained clinicopathological data of patients with NSCLC at Gyeongsang National University Hospital, Jinju, Republic of Korea, between January 2002 and December 2009 by reviewing their electronic clinical charts. In this study, 148 patients who underwent surgical resection for NSCLC were enrolled. Of the 148 surgical samples, those with metastatic carcinoma and those with no residual tumor in their paraffin blocks were excluded. Finally, samples from 140 patients were included.
Tumor stage was determined following the guidelines of the eighth edition of the American Joint Committee on Cancer Tumor Node Metastasis (TNM) staging system (22). Histological type determination and tumor differentiation were performed using the fourth edition of the World Health Organization classification system (23). Cancer recurrence was diagnosed via surgical biopsy or radiological examination. Disease-free survival (DFS) was defined as the length of time between the date of surgery and the date of detection of cancer recurrence. Disease-specific survival (DSS) was defined as the length of time between the date of surgery and the date of death from NSCLC. Smoking history was classified as non-smoking or smoking, including current smokers and ex-smokers.
The hematoxylin and eosin-stained tumor slides were reviewed by two pathologists. The study was approved by the Institutional Review Board of Gyeongsang National University Hospital (2020-04-006) and the need for informed consent was waived.
Tissue microarray. Specimens were surgically obtained and fixed overnight in buffered neutral formalin (20%). The samples were embedded in paraffin blocks. Two 3-mm tissue cores derived from each paraffin block were collected and transplanted into new recipient tissue microarray (TMA) blocks. The two cores were selected from the center and periphery of the tumor.
Immunohistochemical analysis. Immunohistochemistry was performed on 4 μm-thick sections from the TMA blocks. The tissues were stained with a monoclonal antibody to FcRn (dilution, 1:50; sc-271745; Santa Cruz Biotechnology Inc., Dallas, TX, USA) using an automated immunostainer (Benchmark Ultra, Ventana Medical Systems Inc., Tucson, AZ, USA).
FcRn expression. FcRn expression was evaluated in each TMA sample by immunohistochemically staining tumor cells. The staining result was defined as positive when the cytoplasm of the tumor cells was stained with a signal stronger than that of the stroma or as negative (not stained). To confirm reproducibility, two pathologists scored all samples in a blinded manner.
Statistical analysis. The correlation between FcRn expression and clinicopathological parameters was analyzed using chi-squared test and Fisher’s exact test. Survival probability was analyzed using the Kaplan–Meier method with the log-rank test for DFS and DSS. Results were considered statistically significant when the p-value was less than 0.05. SPSS version 21.0 (IBM Corp., Armonk, NY, USA) was used for the analysis.
Results
Clinicopathological patient data. Clinicopathological data of the patients are summarized in Table I. The median age of the patients was 65 years (range=31-77 years). Histological types of the tumors included 96 (64.9%) squamous cell carcinomas, 37 (25%) adenocarcinomas, eight (5.4%) large-cell neuroendocrine carcinomas, and seven (4.7%) other types, including pleomorphic and mucoepidermoid carcinomas. The most prevalent histological feature of squamous cell carcinoma was moderate differentiation in 58 patients (39.2%) and that of adenocarcinoma was an acinar pattern in 15 (10.1%) patients. Of all the recruited patients, 130 (87.8%) underwent lobectomy, and the remaining 18 (12.8%) underwent bi-lobectomy, sleeve lobectomy, or pneumonectomy. Regarding the TNM stage, most tumors were classified as stage I or II (74.3%).
Clinicopathological data of patients (n=148).
Correlation of FcRn expression with clinicopathological data. Out of the 140 patient samples, FcRn was positive in 68 and negative in 72 samples. Specimens with positive FcRn expression exhibited strong and diffuse cytoplasmic staining patterns in tumor and inflammatory cells. Stained cells were evenly distributed within single cores (Figure 1).
Tumor cells showing neonatal Fc receptor expression, with strong and diffuse cytoplasmic staining pattern.
The correlations between expression of FcRn and the clinicopathological data are shown in Table II. Considering all 140 patients assessed for FcRn expression, FcRn positivity was significantly correlated with sex (p<0.001) and tumor histological type (p<0.001) but not with age, smoking history, surgical method, tumor stage, lymph node metastasis, distant metastasis, or TNM stage. FcRn positivity was more frequent in men than in women and in patients with adenocarcinoma than in those with squamous cell carcinoma.
Correlation of neonatal Fc receptor (FcRn) expression with clinicopathological data in patients with non-small-cell lung cancer.
Out of all the patients, 58 had TNM stage I disease. In these patients, FcRn positivity was also significantly correlated with sex (p<0.001), smoking (p=0.026) and histological type (p<0.001). Among patients with stage I disease, all females (12, 100%) and most patients with adenocarcinoma (n=19, 95%) exhibited positivity for FcRn.
FcRn expression and survival analysis. We analyzed patient prognosis according to tumor stage. Survival probability was analyzed using the Kaplan–Meier method. In the group with TNM stage I NSCLC, the FcRn-positive group exhibited significantly increased DFS and DSS (p=0.005 and p=0.027, respectively) (Figure 2). However, there was no statistically significant correlation in DFS and DSS (p=0.273 and p=0.732, respectively) in the FcRn-positive group with TMN stage II and above.
Kaplan–Meier survival analysis based on neonatal Fc receptor (FcRn) expression in patients with TNM stage I non-small-cell lung cancer. The group without FcRn expression had significantly lower disease-free survival (A) and a tendency towards reduced disease-specific survival (B).
Discussion
FcRn is a major histocompatibility class I-like molecule and is the receptor involved in the transfer of passive humoral immunity from mother to newborn (24). Contrary to its name, FcRn is expressed throughout life (19). It protects immunoglobulin (Ig) G and albumin from catabolism and mediates recycling and transcytosis of IgG across epithelial cells. This extends the half-life of IgG in the human body (17).
There have been several studies on FcRn as a prognostic factor. In a study by Jansen et al., down-regulation of FCGRT, the gene encoding the alpha chain of FcRn, was observed in progressive breast cancer and was correlated with poor patient prognosis (25). FCGRT down-regulation was also correlated with poor patient prognosis in hepatocellular carcinoma (26). In a study by Dalloneau et al., which is the only study of FcRn as a prognostic factor in NSCLC, FcRn down-regulation was correlated with poor prognosis (17).
In our study, Kaplan–Meier survival analysis was used to determine the overall survival of 140 patients with NSCLC by FcRn expression in tumor cells. FcRn negativity was associated with significantly lower DFS and reduced DSS in patients with TNM stage I disease (p=0.005 and p=0.027, respectively).
In a study by Dalloneau et al., FcRn expression was determined at both mRNA and protein levels in cancerous and non-cancerous tissue from 80 patients with NSCLC. Unlike our study, FcRn expression in patients with NSCLC was mainly attributed to resident and tumor-infiltrating immune cells and was at very low levels in tumor cells. They concluded that high FCGRT mRNA levels in cancerous and non-cancerous tissue are associated with a favorable prognosis. Like our study, they also analyzed prognostic value within the early-stage subgroup. A high FCGRT mRNA level was associated with favorable prognosis in early-stage (I/II) and metastasis-free patients with NSCLC. However, the prognostic value of FCGRT mRNA was studied in total tumor material and there was no precise description about which cell type was related to expression of FcRn (17).
Lung cancer progresses in a stepwise manner. Driver mutations and epigenetic alterations play important roles in tumorigenesis. Therefore, tumor heterogeneity and mutation burden are lower in patients with stage I NSCLC (27, 28). In the advanced stages, these factors affect FcRn. We found FcRn expression and its correlation with prognosis to be limited to patients with TNM stage I NSCLC. The limitation of our study is that the exact mechanism underlying this has not been elucidated. Further studies are required to determine the mechanism. In our study, FcRn expression by inflammatory cells was also observed, mainly by macrophages. We also evaluated FcRn expression by these inflammatory cells by calculating the percentage of inflammatory cells with FcRn expression. Inflammatory cells were scored by the proportion of tumor area that was occupied by FcRn-stained immune cells of any intensity. FcRn expression in ≥10% of the inflammatory cells were evaluated as positive. Out of the 140 patient samples, FcRn was positive in 50 (35.8%) and negative in 90 (64.2%) of the inflammatory cells. Kaplan–Meier survival analysis of the FcRn-positive inflammatory cell group showed no correlation with survival (DFS and DSS, p=0.170 and p=0.758, respectively).
To the best of our knowledge, this is the first study to precisely correlate FcRn expression in tumor cells and prognosis of NSCLC. Our findings support the hypothesis that FcRn down-regulation is correlated with NSCLC progression. Therefore, FcRn might be useful as a biomarker for determining prognosis in patients with NSCLC.
Conclusion
FcRn may be a potential early biomarker of prognosis in patients with NSCLC.
Acknowledgements
This research was supported by the Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2017R1C1B5014837).
Footnotes
Authors’ Contributions
MHK, DHS and MCS conceived and designed this study. MHK, GHK, JHL, JSL, DCK, JWY, JMN, HJA and DHS collected samples, performed pathological diagnosis, and analyzed the immunostained samples. MHK and DHS analyzed all the data. MHK and DHS wrote the first draft of the article. GHK, JHL, JSL, DCK, JWY, HJA, JMN, MCS and DHS critically reviewed and corrected the article. All Authors reviewed and approved the final version of the article.
Conflicts of Interest
The Authors declare that they have no competing interests.
- Received September 23, 2022.
- Revision received October 11, 2022.
- Accepted October 14, 2022.
- Copyright © 2022, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved
This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY-NC-ND) 4.0 international license (https://creativecommons.org/licenses/by-nc-nd/4.0).
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