Expression and prongostic impact of galectin-7 in human lung cancer
Lung cancer is worldwide the leading cause of cancer-associated mortality.  Lung cancer is divided into small cell lung cancer (SCLC) and non-SCLC (NSCLC). The latter is further subdivided into adenocarcinoma (AC) and squamous cell lung carcinoma (SQCLC).  The treatment of lung cancer depends on the TNM classification and Union for international cancer control, UICC stage.  Surgery is the treatment of choice for patients with stage I or II lung cancer if there are no contraindications. The treatment claim is curative.  Patients with stage III NSCLC are rarely treated surgically. Most patients receive radiotherapy and/or chemotherapy.  In addition, lung cancer can be divided into many molecularly heterogeneous subgroups that are constantly evolving and potentially actionable. New strategies for molecular diagnostics and targeted therapies for lung cancer are being researched in order to detect and treat them.  The protein galectin-7 is in humans encoded by the LGALS7 gene.  Galectin-7 was originally considered a marker for the differentiation status of keratinocytes.  Apoptotic keratinocytes expressed a high amount of galectin-7.  In colon cancer cells, galectin-7 has been shown to act as an apoptosis regulator.  Furthermore, it was shown that galectin-7 makes tumor cells more susceptible to apoptotic stimuli.  On the other hand, the growth of neuroblastoma cells was reduced by extracellular binding of galectin-7 to cell surface receptors. There was no evidence of classical apoptosis.  In breast cancer cells, overexpression of galectin-7 led to a significant increase in lung metastases and osteolytic lesions. Galectin-7 is therefore being further investigated as a potential target for the specific detection and therapeutic inhibition of metastatic breast cancer.  In patients with breast cancer, galectin-7 was investigated as a prognostic marker and found to be associated with disease-free survival.  In addition to breast cancer, it has also been shown in lymphomas that overexpression of galectin-7 leads to increased metastasis.  In melanoma cells, the expression of galectin-7 was shown on primary tumors and lung metastases. The overexpression of galectin-7 increased the resistance of melanoma cells to apoptosis, but the overexpression had no effect on tumor growth.  In a mouse model of lung cancer with squamous cell carcinoma cells, galectin-7 was shown to be a key mediator of metastasis in conjunction with immunosuppression. Galectin-7 was identified as a metastasis-promoting factor.  In this study, the expression of galectin-7 was investigated in patients with non-small cell (adenocarcinoma (AC) and squamous cell carcinoma (SQCLC)) and SCLC in relation to clinicopathological characteristics and overall survival.   Tissue samples of lung cancer from patients who underwent surgery at the Department of Thoracic and Cardiovascular Surgery, University Medical Center Göttingen, Germany were analyzed for galectin-7 expression. All patients signed an informed consent. In accordance with the Declaration of Helsinki (October 2013 version) and institutional, state and federal guidelines and after approval by the ethics committee of the University Medical Center Göttingen (#1-2-08), the study was conducted. Inclusion criteria were age > 18 years and presence of histology of AC, SQCLC or SCLC. Exclusion criteria were a different histology, patients with inoperable tumor or with neoadjuvant treatment. 2.1. Immunohistochemical staining The 2-mm tissue sections were incubated in EnVision Flex Target Retrieval Solution at low PH (Dako/agilent, Santa Clara, CA), followed by incubation with the primary antibody against galectin-7 (Novus Bio, #NPB2-33995, 1:500) for 20 minutes at room temperature. Visualization was performed with a polymeric secondary antibody coupled to horseradish peroxidase (EnVision Flex+, Dako) and DAB (Dako). Prior to the light microscopy analysis of the tissue samples, counterstaining with Meyer’s hematoxylin was performed. The findings were classified according to the intensity of the staining into negative, weak expression and strong expression. 2.2. Statistical analysis Data were collected using Microsoft Excel 2019 and statistical analysis was performed using GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla, CA, www.graphpad.com ). The correlation between clinicopathological features and protein expression of galectin-7 was analyzed using Chi-Square test and Student t test. The Kaplan–Meier estimator was used to investigate patient survival in relation to galectin-7 expression. Differences were calculated using the Mantel-Cox log-rank test. P -values < .05 were considered statistically significant.   The 2-mm tissue sections were incubated in EnVision Flex Target Retrieval Solution at low PH (Dako/agilent, Santa Clara, CA), followed by incubation with the primary antibody against galectin-7 (Novus Bio, #NPB2-33995, 1:500) for 20 minutes at room temperature. Visualization was performed with a polymeric secondary antibody coupled to horseradish peroxidase (EnVision Flex+, Dako) and DAB (Dako). Prior to the light microscopy analysis of the tissue samples, counterstaining with Meyer’s hematoxylin was performed. The findings were classified according to the intensity of the staining into negative, weak expression and strong expression.   Data were collected using Microsoft Excel 2019 and statistical analysis was performed using GraphPad Prism (version 7.00 for Windows, GraphPad Software, La Jolla, CA, www.graphpad.com ). The correlation between clinicopathological features and protein expression of galectin-7 was analyzed using Chi-Square test and Student t test. The Kaplan–Meier estimator was used to investigate patient survival in relation to galectin-7 expression. Differences were calculated using the Mantel-Cox log-rank test. P -values < .05 were considered statistically significant.   3.1. Patient characteristics A total of 308 patients (226 men, 82 women) were included in this study. The average age of the patients was 66 years (range 34–85 years), with two/thirds of the patients being older than 60 years. Histologically, non-SCLC (NSCLC) was the leading type. Squamous cell carcinoma (SQCLC) was detectable in 193 patients and adenocarcinoma in 108 patients. Only 7 patients had small cell lung carcinoma (SCLC). Lymph node metastasis could be ruled out in over 50% of patients. R0 resection status was achieved in around 90% of patients. Patients in clinical stage III (IIIa n = 66, IIIb n = 8) and stage IV (n = 6) received neoadjuvant treatment prior to surgery. Surgery of patients in stage IV was performed with palliative intention. Table and Supplementary Table 1, http://links.lww.com/MD/N665 show the detailed data on patient characteristics, clinical status and degree of tumor differentiation. 3.2. Impact of galectin-7 expression and survival In the immunohistochemical study, strong or weak expression of galectin-7 was most evident in 70 patients (36.27%) with SQCLC, followed by 6 patients (5.55%) with AC. Negative expression for galectin-7 was found in all patients with SCLC (100%), followed by 102 patients (94.44%) with AC and 123 patients (63.73%) with SQCLC (Fig. ). The correlation between galectin-7 expression and overall survival of patients with AC and SQCLC was demonstrated using the Kaplan–Meyer-Estimation. The median follow-up time was 24 months (range 6–128). No significant correlation was found in patients with SQCLC (Fig. ). 3.3. Protein expression of galectin-7 in lung cancer patients and correlation with clinicopathological characteristics In patients with AC, a significant correlation was found between the expression of galectin-7 and the age of the patients (Table ). In addition, a significant correlation between the expression of galectin-7 and the degree of differentiation was found in patients with SQCLC (Table ).   A total of 308 patients (226 men, 82 women) were included in this study. The average age of the patients was 66 years (range 34–85 years), with two/thirds of the patients being older than 60 years. Histologically, non-SCLC (NSCLC) was the leading type. Squamous cell carcinoma (SQCLC) was detectable in 193 patients and adenocarcinoma in 108 patients. Only 7 patients had small cell lung carcinoma (SCLC). Lymph node metastasis could be ruled out in over 50% of patients. R0 resection status was achieved in around 90% of patients. Patients in clinical stage III (IIIa n = 66, IIIb n = 8) and stage IV (n = 6) received neoadjuvant treatment prior to surgery. Surgery of patients in stage IV was performed with palliative intention. Table and Supplementary Table 1, http://links.lww.com/MD/N665 show the detailed data on patient characteristics, clinical status and degree of tumor differentiation.   In the immunohistochemical study, strong or weak expression of galectin-7 was most evident in 70 patients (36.27%) with SQCLC, followed by 6 patients (5.55%) with AC. Negative expression for galectin-7 was found in all patients with SCLC (100%), followed by 102 patients (94.44%) with AC and 123 patients (63.73%) with SQCLC (Fig. ). The correlation between galectin-7 expression and overall survival of patients with AC and SQCLC was demonstrated using the Kaplan–Meyer-Estimation. The median follow-up time was 24 months (range 6–128). No significant correlation was found in patients with SQCLC (Fig. ).   In patients with AC, a significant correlation was found between the expression of galectin-7 and the age of the patients (Table ). In addition, a significant correlation between the expression of galectin-7 and the degree of differentiation was found in patients with SQCLC (Table ).   Lung cancer continues to be the most common cause of death from cancer worldwide.  Patients are treated according to the TNM classification and the UICC stage of the cancer. Surgical resection is the treatment of choice in stages I and II of NSCLC. From stage II onwards, adjuvant therapy increases.  It has recently been shown that lung cancer can be divided into molecularly heterogeneous subgroups and that individualized therapy is important.  In this study, immunohistochemical detection of galectin-7 expression was most evident in SQCLC, followed by AC. Negative expression of galectin-7 was found in all patients with SCLC. Statistically significant correlations between the expression of galectin-7 and some clinicopathologic features of the patients were found in AC and SQCLC. Galectin-7 has been shown to be an important mediator in various tumor entities. In breast cancer, galectin-7 promotes metastatic potential and resistance to apoptosis.  Overexpression of galectin-7 has been found in squamous cell carcinoma of the esophagus.  In melanoma, galectin-7 was detected both in the primary focus and in the lung metastasis.  It has also been shown that galectin-7 promotes bone and lung metastasis of breast cancer.  It was found that galectin-7 is elevated in lung cancer patients with a squamous cell histology.  In squamous cell carcinoma, the expression of galectin-7 has been shown to be induced during tumorigenesis, particularly in the microenvironment of immunosuppression. During tumor progression, galectin-7 is released extracellularly. By deleting galectin-7, metastasis could be suppressed without affecting the growth of the primary tumor. Therefore, galectin-7 is considered a mediator of tumor metastasis and could be a potential target of cancer immunotherapy.  In our study, we were able to show that galectin-7 can serve as a prognostic biomarker, particularly in patients with non-SCLC, and may be a potential new drug target. The mechanistic role of galectin-7 in the tumorigenesis of non-SCLC and the importance of galectin-7 as a therapeutic target should be further investigated. Due to the design of a single center, the results of the current study are limited. A multicenter study with a larger patient cohort is therefore proposed.   The authors acknowledge support by the Open Access Publication Funds of Göttingen University. The authors thank Jennifer Appelhans for her technical support.   Conceptualization: Philipp Ströbel, Hanibal Bohnenberger. Data curation: Anna Tirilomi, Petros Tirilomis, Omar Elakad, Sha Yao, Marc Hinterthaner, Hanibal Bohnenberger, Alexander von Hammerstein-Equord. Formal analysis: Anna Tirilomi, Petros Tirilomis, Omar Elakad, Sha Yao, Theodor Tirilomis, Hanibal Bohnenberger. Funding acquisition: Sha Yao, Philipp Ströbel. Investigation: Omar Elakad, Marc Hinterthaner, Philipp Ströbel, Hanibal Bohnenberger. Methodology: Omar Elakad, Sha Yao, Marc Hinterthaner, Philipp Ströbel, Hanibal Bohnenberger. Project administration: Philipp Ströbel, Hanibal Bohnenberger, Alexander von Hammerstein-Equord. Resources: Philipp Ströbel. Software: Omar Elakad, Philipp Ströbel. Supervision: Bernhard C. Danner, Philipp Ströbel, Hanibal Bohnenberger, Alexander von Hammerstein-Equord. Validation: Anna Tirilomi, Theodor Tirilomis, Bernhard C. Danner, Philipp Ströbel, Hanibal Bohnenberger. Visualization: Theodor Tirilomis, Bernhard C. Danner, Philipp Ströbel, Hanibal Bohnenberger. Writing – original draft: Anna Tirilomi. Writing – review & editing: Alexander von Hammerstein-Equord.      