Expression Profiles of CYP1A1 and CYP1B1 Enzymes in Non-Small Cell Lung Cancer of Turkish Patients

Tumors originating in the lung parenchyma or bronchi are termed lung cancer or bronchogenic carcinoma [1]. Lung neoplasms are the foremost contributors to both cancer incidence and mortality worldwide. In 2022, lung cancer emerged as the predominant malignancy globally, with approximately 2.5 million new cases, accounting for 12.4 % of the total cancer incidence. Furthermore, it exhibited the highest fatality rate among cancer types, causing about 1.8 million deaths, representing 18.7 % of all cancer-related mortalities [2].

Advancements in diagnostic techniques have significantly enhanced the precision of pathological and genetic classifications of lung tumors, facilitating the development of more effective therapeutic interventions. This progress is largely attributable to the integration of immunohistochemistry and molecular testing into classification protocols. The 2021 WHO Classification of Thoracic Tumors encompasses a comprehensive range of categories, including papillomas, adenomas, precursor glandular lesions, lung adenocarcinoma in situ, lung adenocarcinomas (AC), invasive nonmucinous lung adenocarcinoma, squamous precursor lesions, lung squamous cell carcinomas (SCC), large cell carcinomas (LCC), adenosquamous carcinomas, sarcomatoid carcinomas, neuroendocrine tumors, salivary gland-type tumors, neuroendocrine carcinomas (including small cell carcinoma (SCLC) and large cell neuroendocrine carcinoma (LCNEC)), tumors of ectopic tissues (melanoma and meningioma), mesenchymal tumors specific to the lung, and PEComatous tumors [3]. AC, SCC, and LCC are subtypes of non-small-cell lung carcinoma (NSCLC), which represents 85 % of all lung cancer cases [4].

Lung adenocarcinomas are pathologically distinguished by the formation of neoplastic glands, the presence of pneumocyte markers such as thyroid transcription factor 1 (TTF-1) with or without napsin expression, and intracytoplasmic mucin. Squamous cell pathology is identified by the presence of keratin and/or intercellular desmosomes [5].

CYP1A1 and CYP1B1 are enzymes belonging to the cytochrome P450 superfamily, playing significant roles in the metabolism of various carcinogens. CYP1A1 is primarily involved in the biotransformation of polycyclic aromatic hydrocarbons (PAHs) and other carcinogens. Its expression is notably higher in lung tissues exposed to environmental carcinogens and has been identified as a key player in lung carcinogenesis. CYP1A1 is markedly induced by PAHs, leading to increased DNA adduct formation, which is a critical step in the initiation of cancer. Elevated levels of CYP1A1 expression have been associated with a higher risk of lung cancer. In lung cancer tissues, CYP1A1 has been detected in a substantial proportion of lung adenocarcinomas, suggesting its potential role as a biomarker for lung cancer [6, 7]. CYP1B1 has been identified as a potential tumor marker, often overexpressed in various tumor tissues, including lung cancers, while being absent in normal tissues. This differential expression underscores its potential utility in cancer diagnostics and as a therapeutic target [6, 8].

Although CYP1A1 and CYP1B1 are known to be involved in the metabolism of carcinogens, their exact roles in lung cancer warrant further investigation. Particularly, studying the variability of CYP1A1 and CYP1B1 expression in lung tissues from different individuals and correlating this with clinical outcomes could help identify potential biomarkers for lung cancer prognosis. Hence, this study addresses the CYP1A1 and CYP1B1 expression profiles in two distinct types of NSCLC tumor samples: lung adenocarcinoma (AC) and lung squamous cell carcinoma (SCC), and explores the possible association with demographic and clinical features among Turkish patients.

Material and Methods

This study performed a retrospective analysis of clinical data from patients with lung adenocarcinomas and lung squamous cell carcinomas treated at the Dr. Lütfi Kırdar Education and Research Hospital's Pathology Clinic between 2017 and 2019. Archival sampling was carried out among patients who met the study's inclusion and exclusion criteria, resulting in the recruitment of 40 subjects (20 lung AC and 20 lung SCC) aged 46 to 83 years (mean 67.20 ± 8.5 years), with a gender distribution of 11 females and 29 males, representing the broader population. Each patient's cancer stage was determined at the time of surgery using the TNM staging method by the American Joint Committee on Cancer. Of the 40 surgically removed lung tumors, 13 were stage 1A, 7 were stage 1B, 8 were stage 2A, 9 were stage 2B, and 3 were stage 3A, with an average tumor diameter of 3.67 ± 0.38 cm. Histopathological analysis of the tumors and adjacent healthy tissues was conducted using immunohistochemical (IHC) staining to profile CYP1A1 and CYP1B1 enzymes.

Inclusion and exclusion criteria

Patients with NSCLC tumor samples, including lung adenocarcinomas and lung squamous cell carcinomas, were considered for the investigation if both tumor and adjacent healthy tissue samples were available. Additionally, patients should not have received any prior treatment for NSCLC, such as chemotherapy, radiation therapy, or surgery. Therefore, patients meeting these criteria, with sufficient clinical and pathological data listed in Table 1, were included in the study.

To ensure validity, exclusion criteria were applied to exclude patients with small cell lung cancer (SCLC), other histological types of lung cancer, or a history of other malignancies. Moreover, patients with severe comorbidities that might affect the interpretation of the study results, such as severe cardiovascular disease, liver or kidney failure, or autoimmune disorders, were excluded from the sample group.

Data collection

Demographic and clinical information was meticulously collected using a detailed checklist that included parameters such as age, gender, diagnosis, tumor grade, localization, presence of vascular and neural invasion, bronchial and pleural involvement, in situ status, metastasis, tumor size, lymph node involvement, stage, neoadjuvant therapy, and survival status. This comprehensive data collection enabled a thorough retrospective analysis of each subject's background. Tumor and adjacent healthy tissue samples were obtained from surgical sites following standardized protocols by skilled surgeons and preserved in paraffin for subsequent analysis. Immunohistochemical (IHC) methods were employed to evaluate the expression levels of CYP1A1 and CYP1B1 proteins.

Histopathological analysis of tissue is crucial for providing detailed insights into tumor characteristics, essential for accurate diagnosis, classification, and prognosis. This process begins with the procurement of tissue during surgical resection, followed by immersion in 10 % buffered formalin for preservation. Thin sections, 4 µm thick, are prepared by embedding the tissue in paraffin wax using a microtome. These sections are then mounted on glass slides and stained with hematoxylin and eosin to analyze tissue morphology. Additionally, immunohistochemical (IHC) staining is performed to detect the expression of CYP1A1 and CYP1B1 proteins. This comprehensive analysis aids in making informed therapeutic decisions and predicting prognosis based on the tumor's molecular profile.

For immunohistochemical staining, the formalin-fixed, paraffin-embedded tissues sections, after deparaffinization, were incubated for 10 minutes at room temperature in a 3 % hydrogen peroxide (v/v) in methanol solution to neutralize the natural peroxidase activity. After that, the parts were given a five-minute rinse with distilled water. Using 0.01 M citrate buffer (pH 6.0), antigen retrieval was carried out for three minutes using a home pressure cooker. To avoid nonspecific background staining, the sections were treated for 10 minutes at room temperature with super block (SHP125) from ScyTek Laboratories, USA. The sec-

Table 1/Òàблицà 1

Demographic and clinical data of patients Дåмîгðàфичåñêиå и êлиничåñêиå дàнныå пàциåнтîв

Notes: created by the authors.