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Detection of SHOX2 DNA Methylation by Methylation-Specific PCR in Non-Small Cell Lung Cancer

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Detection of SHOX2 DNA Methylation by Methylation-Specific PCR in Non-Small Cell Lung Cancer 6077 Original Article Detection of SHOX2 DNA methylation by methylation-specific PCR in non-small cell lung cancer Hongxiang Feng1#, Weipeng Shao2#, Lanfang Du3#, Xin Qing4, Zhenrong Zhang1, Chaoyang Liang1, Deruo Liu1 1Department of Thoracic Surgery, China-Japan Friendship Hospital, Beijing, China; 2Department of Thoracic Surgery, Peking University China- Japan Friendship School of Clinical Medicine and China-Japan Friendship Hospital, Beijing, China; 3Department of Emergency, Peking University Third Hospital, Beijing, China; 4Department of Pathology, Harbor-UCLA Medical Center, Torrance, CA, USA Contributions: (I) Conception and design: W Shao, H Feng, L Du, D Liu; (II) Administrative support: D Liu, X Qing; (III) Provision of study materials or patients: All authors; (IV) Collection and assembly of data: W Shao, H Feng, L Du; (V) Data analysis and interpretation: W Shao, H Feng, L Du; (VI) Manuscript writing: All authors; (VII) Final approval of manuscript: All authors. #These authors contributed equally to this work. Correspondence to: Deruo Liu. Department of Thoracic Surgery, China-Japan Friendship Hospital, No. 2, Yinghua, East Rd, Beijing, China. Email: [email protected]. Background: Lung cancer is the leading cause of cancer-related death worldwide. Short stature homeobox 2 (SHOX2) methylation detected by real-time polymerase chain reaction (PCR) has recently been demonstrated to be a potential biomarker in the diagnosis of lung cancer. However, more cost-effective methods are still needed to help cancer detection in the early stage of lung cancer. The aim of this study was to examine the methylation status of the SHOX2 gene and to investigate its diagnostic value in non-small cell lung cancer (NSCLC) patients. Methods: A total of 89 Chinese NSCLC patients and 9 non-tumor patients was enrolled in this study. The methylation status of SHOX2 gene in NSCLC tumor tissues/corresponding non-neoplastic lung tissues and lung tissues from non-tumor patients was examined by methylation-specific PCR (MSP). Results: We found that SHOX2 methylation was significantly associated with NSCLC (P=0.003). We also analyzed the correlation of SHOX2 methylation with clinicopathological variables including sex, age, tumor pathologic classification, tumor differentiation degree, TNM stage, T stage, and nodal status, and found no significant correlation between them. Conclusions: These results suggested that SHOX2 gene methylation was closely associated with lung carcinogenesis. Thus, SHOX2 methylation could be used as a potential marker to help NSCLC detection. MSP might be used as a cost-effective method alternative to real-time PCR in detection of SHOX2 methylation in the early diagnosis of NSCLC. Keywords: Stature homeobox 2 methylation (SHOX2 methylation); non-small cell lung cancer (NSCLC); diagnosis; methylation-specific PCR (MSP) Submitted Feb 06, 2020. Accepted for publication Aug 21, 2020. doi: 10.21037/tcr-20-887 View this article at: http://dx.doi.org/10.21037/tcr-20-887 Introduction cancer incidence and mortality in 2012, respectively (2). Over one million people die from lung cancer globally Lung cancer is the leading cause of cancer-related deaths every year, among which approximately 0.6 million are worldwide (1). In China, lung cancer is the most prevalent from China. Worse yet, the incidence of lung cancer is still cancer type and accounted for 25.2% and 29.5% of the total increasing in China due to the high prevalence of cigarette © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(10):6070-6077 | http://dx.doi.org/10.21037/tcr-20-887 Translational Cancer Research, Vol 9, No 10 October 2020 6071 use and other factors (3). Compared with real-time PCR-based methods, MSP is Non-small cell lung cancer (NSCLC) accounts for much more cost-effective. It is also fast, sensitive, and approximately 85% of all lung cancers and carries a specific, and has been widely used for DNA methylation dismal 5-year survival rate of 15% (3). Nevertheless, analyses (15,16). Therefore, we chose to investigate the the International Early Lung Cancer Action Program diagnostic value of SHOX2 methylation detection by MSP Investigators reported that, among 302 participants in lung cancer as this method may reduce costs and thus with clinical stage I lung cancer who underwent surgical may facilitate the use of SHOX2 methylation in large-scale resection within 1 month after diagnosis, the survival rate lung cancer screening, especially in developing countries was 92% (4). These results suggest that timely detection where cost is a limiting factor. Specifically, we examined of lung cancer and appropriate treatment may prevent or SHOX2 methylation by the MSP method in tumor tissue interrupt cancer progression and improve overall survival and corresponding non-tumor lung tissue from a cohort rate. However, more than 75% of lung cancer patients are of Chinese NSCLC patients, and further analyzed the diagnosed at a locally advanced stage or with metastatic diagnostic value of SHOX2 methylation measured by MSP disease, partially due to the lack of an effective screening in NSCLC detection. We present the following article in program for early detection (5). Therefore, simple, safe, accordance with the STARD reporting checklist (available and cost-effective methods to aid in early detection of lung at http://dx.doi.org/10.21037/tcr-20-887). cancer are greatly needed. Tumor markers are molecules specifically expressed by tumor cells or produced by the host in response to Methods tumors. Epigenetic changes of certain genes, such as DNA Patients methylation, could occur at early stage of carcinogenesis, thus could be used as a type of tumor marker. DNA methylation The study was conducted in accordance with the based diagnostic devices has been used in screening of Declaration of Helsinki (as revised in 2013). The patients malignant diseases including colorectal cancer (6). SHOX2 underwent surgery after providing informed consent, and gene is located on human chromosome 3. SHOX2 gene is the requirement for the individual consent of patients widely expressed in tissue including skeleton, branchial whose records were evaluated was waived because they arches, heart tissue and central nervous system (7). SHOX2 were de-identified in the study. The study was approved methylation is closely related to tumor occurrence and by institutional ethics board of China-Japan Friendship progression. In colorectal cancer, SHOX2 methylation Hospital (No. 2018-13-K08). A total of 89 NSCLC patients could be used as a powerful biomarker for cancer screening and 9 non-tumor patients were included in this study. and post-therapeutic monitoring (8). SHOX2 gene Clinicopathological characteristics of the participating methylation status could also be used as potent prognostic patients with cancer or non-cancer are presented in Tables 1 parameter for predicting patient survival in lower-grade and 2, respectively. All tumors in these patients were glioma patients (9). Recently, Dietrich et al. reported completely resected (R0 category) by surgery. Additionally, that SHOX2 methylation is a reliable biomarker for the histologically normal lung tissue specimens obtained diagnosis of lung cancer (10-14). Based on this potential, at surgery from 9 patients with no evidence of cancer SHOX2 methylation has been developed as a commercially were used as a control group (including 8 patients with available assay, the Epi proLung BL Reflex Assay, for early pulmonary bulla and 1 patient with benign tumor). In lung cancer detection (13). the control group, 6 patients were men and 6 were under Specific DNA methylation can be measured by several 60 years old. polymerase chain reaction (PCR)-based methods, including methylation-specific PCR (MSP) and real-time quantitative Tissue acquisition and DNA extraction PCR. The aforementioned commercial SHOX2 methylation assay detects SHOX2 methylation through the Tissues for DNA analysis were obtained immediately after generation of bisulfate-converted template DNA followed lung resection before starting mediastinal lymphadenectomy by real-time PCR using TaqMan probes (13). While this and were frozen in liquid nitrogen. Tissues were analyzed is sensitive and quantitative, it is also relatively expensive. as tumors or uninvolved lung tissues taken from the farthest © Translational Cancer Research. All rights reserved. Transl Cancer Res 2020;9(10):6070-6077 | http://dx.doi.org/10.21037/tcr-20-887 6072 Feng et al. MSP and SHOX2 methylation Table 1 Clinicopathological characteristics of 89 non-small cell distance from tumors. Ten µm thick frozen sections were lung cancer patients taken from blocks of tumor tissues and stained with H&E Characteristics N (%) for histopathological evaluation. Sections were pooled Sex for analysis from areas of estimated 75% malignant cells. Genomic DNA was isolated from frozen tissue by standard Male 63 (70.8) methods of proteinase K digestion and phenol-chloroform Female 26 (29.2) extraction using Tiangen Cells and Tissue DNA Isolation Age* Kit (Tiangen Biotech Co., LTD, Beijing, China) according <60 42 (47.2) to the manufacturer’s instructions. ≥60 47 (52.8) Pathologic classification Detection of SHOX2 methylation using MSP Squamous cell carcinoma 34 (38.2) A total of 500 ng genomic DNA extracted from tissue Adenocarcinoma 47 (52.8) samples was modified by sodium bisulfite with DNA Methylation Detection Kit (BioChain, USA) according to Other 8 (9.0) the manufacturer’s instructions. 7 µL modified DNA was Differentiation degree used for PCR in a total reaction volume of 25 µL. PCR Undifferentiated/poorly 39 (43.8) program was set as 35 cycles of melting (95 for 30 s), annealing (58 for 30 s) and extension (72 for 30 s). Moderately/well 50 (56.2) ℃ The 25 µL reaction mixture contained 1× PCR Buffer ℃ ℃ TNM stage (Mg2+ Plus), 200 µM of each dNTP, 0.5 µM forward primer, I/II 52 (58.4) 0.5 µM reverse primer, and 0.5 unit of GoTaq Hot Start III/IV 37 (41.6) Polymerase (Promega, USA). PCR products were separated in 3% agarose gel supplemented with ethidium bromide.
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