Original Article

Oncogenic Mutation Frequencies in Lung Cancer Patients

10.4274/hamidiyemedj.galenos.2020.74429

  • Ömer Ayten
  • Tayfun Çalışkan
  • Kadir Canoğlu
  • Neslihan Kaya Terzi
  • Levent Emirzeoğlu
  • Oğuzhan Okutan

Received Date: 03.09.2020 Accepted Date: 17.09.2020 Hamidiye Med J 2020;1(1):17-21

Background:

The data on mutation frequencies in patients with lung cancer are limited in Turkey. We aimed to determine the frequencies of EGFR, ALK, ROS1, and BRAF gene mutations in patients with lung cancer in this study.

Materials and Methods:

Data of 329 patients with lung cancer, who underwent molecular examination at the İstanbul Sultan 2. Abdülhamid Han Training and Research Hospital, Clinic of Medical Pathology Laboratory, were analyzed retrospectively.

Results:

A total of 329 patients with lung cancer, of whom 257 were male and 71 were female, were included in the study. The average age of women was 65.7±11.2 years and the average age of men was 64.6±10.3 years. Thirty nine patients (11.8%) were non-smokers. The prevalence of smoking was 97.2% (n=251) in male patients and 54.9% (n=39) in female patients. Two hundred sixty one (79.3%) of the patients had adenocarcinoma, 50 (15.2%) squamous cell lung cancer, 13 (3.9%) non-small cell lung cancer, 4 (1.3%) small cell lung cancer, 1 patient (0.3%) was diagnosed with large cell lung cancer. Gene mutation was detected in 52 patients (15.8%). EGFR mutation was detected in 32 patients (9.7%), ALK in 17 patients (5.5%), ROS1 in 2 patients (0.6%), and BRAF mutation in 1 patient (0.3%). The frequency of mutation was 30.7% (n=12) in non-smoking patients, and 18.2% (n=53) in smokers. The most common genetic alteration was deletions in the EGFR gene in exon 19.

Conclusion:

In our study, we found the frequencies of EGFR and ALK mutations similar to the studies conducted around the world but BRAF and ROS1 mutations frequencies were lower compared to studies conducted around the world. In addition, we found that all mutation frequencies were lower than in studies conducted in our country. We thought this was related to the low number of cases in the studies and more selective patient selection.

Keywords: Lung cancer, oncogenic mutations, EGFR, ALK, ROS1, BRAF

Introduction

Lung cancer is still the leading cause of cancer deaths worldwide (1). There are 1.8 million new cases with lung cancer and 1.6 million people die due to lung cancer per year (2). Non-small cell lung cancers (NSCLC) are 80% of lung cancers (3). Since the majority of cases are diagnosed at an advanced stage, the mortality is high. The standard chemotherapy regimens applied so far have not had a dramatic effect on the prognosis (5-year survival 15%) (4). In recent years, a better understanding of the pathogenic genomic changes of NSCLC and determination of molecular tests and biomarkers used to identify patients with these genomic changes have enabled the use of molecular target therapies and immunotherapy in advanced stage patients with NSCLC, especially for adenocarcinoma. Pharmacological treatments guided by epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), proto-oncogene receptor tyrosine kinase 1 (ROS1) and V-Raf murine sarcoma viral oncogene homolog B (BRAF) driver mutations have opened a new era in the treatment of advanced lung cancer. Although many mutations have been identified in lung cancer, the mutation status is still unknown in more than 50% of patients with lung cancer and therapeutic targets can be determined only in 20% of the patients (5).

The detection of EGFR, ALK, ROS1 and BRAF in patients with advanced stage or metastatic adenocarcinoma and NSCLC, whose histologically clear distinction cannot be made, and in patients with squamous cell carcinoma and no history of smoking is currently recommended (6). Therefore, adequate biopsies are needed to perform molecular examination along with histopathological diagnosis.

The data on mutation frequencies in patients with lung cancer are limited in Turkey. We aimed to determine the frequencies of EGFR, ALK, ROS1, BRAF gene mutations in patients with lung cancer in this study.


Material and Methods

A total of 329 patients with lung cancer were included in the study. The samples were performed molecular examination at the İstanbul Sultan 2. Abdülhamid Han Training and Research Hospital, Clinic of Medical Pathology Laboratory between 2017 and 2019. Ethics committee approval of the study was obtained (number: 57 and date: 18/03/2020). Preoperative approvals were obtained from all patients. Age, gender, smoking history and histopathological diagnoses of the patients were recorded.

For EGFR gene exon 18, 19, 20, 21 and BRAF gene exon 15 mutation analyses, the area containing the most tumor cells in the Hematoxylin & Eosin (H&E) stained section was determined. Tumor areas determined from 10 unstained histological tissue sections of 10 µm thicknesses were manually macro dissected with a scalpel. After standard deparaffinization and hydration, DNA was isolated with the QIAamp FFPE Tissue kit. DNA concentration of the samples was measured spectrophotometrically (nanodrop). The 18th, 19th, 20th, 21st exons of the EGFR gene and the 15th exons of the BRAF gene were amplified by polymerase chain reaction (PCR) in the thermal cycler using the HotStar Taq DNA Polymerase kit and appropriate primers. The sample was run in pairs with positive, negative and noDNA template controls. Density and contamination control of PCR products was done by gel electrophoresis process. PCR products were purified with the QIAquick PCR Purification kit. Two-way (forward and reverse) DNA sequence analysis was performed on the purified amplicons using the Big Dye Terminator v3.1 Cycle Sequencing kit with the Sanger sequence technique. After ethanol precipitation, PCR products were run on the ABI-3730 (48 capillary) automatic sequencing device. The resulting electropherograms were visually evaluated using SeqScape Software 3.0 by comparing them with the reference sequences NM_005228.3 (EGFR gene) and NM_004333.4 (BRAF gene), together with their positive and negative controls.

In order to investigate the ALK and ROS1 genes by FISH technique, the area containing the most tumor cells in the H&E stained section was determined. After the macrodissection of the 2 µm thick section taken from the formal-fixed paraffin embedded block, it was prepared by standard prehybridization steps. ‘‘ZytoLight SPEC ROS1 Dual Color Break Apart Probe, Z-2144-200, Zytovision’’ (proximal part of the breakpoint region of the ROS1 gene (6q22.1): green spectrum, distal part: orange spectrum) and reconstruction in ALK gene for evaluation of rearrangement in the ROS1 gene; using ‘‘ZytoLight SPEC ALK Dual Color Break Apart Probe, Z-2124-200, Zytovision’’ (proximal part of the breakpoint region of the ALK gene (2p23.1-p23.2): green spectrum, distal part: orange spectrum) for the evaluation of regulation, standard FISH process was applied. One hundred tumor cells were evaluated using Leica DM 2.500 fluorescence microscope and Argenit Akas imaging system.


Statistical Analysis

No statistical analysis was needed.


Results

A total of 329 patients with lung cancer, of whom 257 were male and 71 were female, were included in the study. The average age of the patients was 64.9±10 years. The mean age of women was 65.7±11.2 years and the mean age of men was 64.6±10.3 years. 39 patients (11.8%) were non-smokers. The prevalences of smoking of male and female patients were 97.2% (n=251) and 54.9% (n=39), respectively. Two hundred sixty one (79.3%) of the patients were diagnosed as adenocarcinoma, 50 (15.2%) of the patients were diagnosed as squamous cell lung cancer (SCC), 13 (3.9%) of the patients were diagnosed as NSCLC, 4 (1.3%) of the patients were diagnosed as small cell lung cancer (SCLC) and 1 patient (0.3%) was diagnosed as large cell lung cancer (Table 1). Gene mutation was detected in 52 patients (15.8%). EGFR mutation was detected in 32 patients (9.7%), ALK was positive in 17 patients (5.5%), ROS1 was positive in 2 patients (0.6%), and BRAF mutation was positive in 1 patient (0.3%). Thirty of the patients, whose biopsies were positive for EGFR, were diagnosed as adenocarcinoma, 1 patient was diagnosed as SCLC (transformed from adenocarcinoma) and 1 patient was diagnosed as SCC. One of the patients with positive ALK mutation was diagnosed as SCC and 16 were diagnosed as adenocarcinoma. All of the patients with positive ROS1 and BRAF mutations were diagnosed as adenocarcinoma (Table 2). The frequency of mutation was 30.7% (n=12) in non-smoking patients, and 18.2% (n=53) in smokers. EGFR (25.6%) was positive in 10 and ALK mutation was positive in 2 of the non-smoking patients (5.1%). The most common genetic alteration was deletions in the EGFR gene in exon 19 (Table 3).


Discussion

Genomic changes and mutations in lung cancer vary in different populations. While the incidence of EGFR, ALK, ROS1 and BRAF mutations in patients with adenocarcinoma is approximately 30% in the Uninted States of America, this rate is 60% in Japan (7,8). Çalıskan et al. (9) detected at least one gene mutation in 37 (46.2%) of 80 patients with lung cancer in a Turkish population. Bilgin et al. (10) detected at least one gene mutation in 60 (22.9%) of 260 NSCLC patients and EGFR was positive in 38 (14.6%), ALK was positive in 20 (7.69%), and ROS1 was positive in 2 (0.76%) of them. In our study, we found at least one gene mutation in 15.8% (n=52) of the patients. High mutation detection rate in the studies of Çalıskan et al. (9) and Bilgin et al. (10) may be related to the low number of patients and the more specific patient selection.

Although the true incidence of EGFR mutation in lung cancers is unknown, the incidence varies between 0 and 13% (11,12). EGFR mutation is more common in female non-smoking patients with adenocarcinoma. In our study, the frequency of EGFR mutation was found to be 9.7%, and 93.7% (n=30) of the patients with positive EGFR mutation had adenocarcinoma. One patient was diagnosed as SCC, and the diagnosis of one patient was transformed from adenocarcinoma to small cell lung cancer. Similar to the literature, the frequency of EGFR mutations was higher in patients who never smoked (n=10, 25.6%).

The frequency of ALK rearrangement in NSCLC is between 4% and 5% (13). In our country, Bilgin et al. (10) reported that 20 (7.69%) of 260 NSCLC patients were positive for ALK mutation, Seymen and Gümüşlü (14) detected ALK rearrangement in 8 (16%) of 14 50 NSCLC patients. Aytekin (15) detected ALK rearrangement in 2 (5.1%) of 130 lung cancer patients, 36 of whom had adenocarcinoma. In our study, the frequency of ALK rearrangement was 5.5%, similar to the literature. The frequency of ALK rearrangement was higher in female patients (n=6, 8.4%).

The frequency of ROS1 rearrangement in NSCLCs varies between 1% and 2% (16). ROS1 rearrangement is observed more frequently in young and non-smoking patients. In a limited number of studies on the frequency of ROS1 rearrangement in Turkey, the frequency varies between 0% and 0.76% (10,15). In our study, in contrast to the literature, ROS1 rearrangement was detected in 2 male patients (0.6%) over 65 years of age with a history of smoking and they were diagnosed as adenocarcinoma.

BRAF mutations are observed in 1-3% of patients with NSCLC. It is observed more frequently in patients with a smoking history. Dogan et al. (17) detected BRAF mutation in 1 (2.38%) of 42 patients with advanced NSCLC in their study. Çalıskan et al. (9) detected BRAF mutation in 1 patient (1.25%) of 80 NSCLC patients. In our study, BRAF mutation was found in 1 male patient (0.3%) with an active smoking and diagnosis of adenocarcinoma.


Conclusion

In our study, we found the frequencies of EGFR and ALK mutations similar to the studies conducted around the world but the frequencies of BRAF and ROS1 mutations were lower compared to studies conducted around the world. In addition, we found that all mutation frequencies were lower than in studies conducted in our country. We thought this was related to the low number of cases in the studies and more selective patient selection. Our study is one of the largest series of studies on mutation frequencies in lung cancers in Turkey. For this reason, it is of great importance to reveal mutation frequencies with more molecular studies in our country and to popularize the treatments for this.


Ethics

Ethics Committee Approval: Ethics committee approval of the study was obtained (number: 57 and date: 18/03/2020).

Informed Consent: Preoperative approvals were obtained from all patients.

Peer-review: Internally peer-reviewed.

Authorship Contributions

Concept: Ö.A., T.Ç., K.C., O.O., Design: Ö.A., T.Ç., L.E., O.O., Data Collection or Processing: Ö.A., T.Ç., K.C., N.K.T., L.E., Analysis or Interpretation: Ö.A., T.Ç., K.C., N.K.T., L.E., Literature Search: Ö.A., T.Ç., K.C., O.O., Writing: Ö.A., T.Ç.

Conflict of Interest: No conflict of interest was declared by the authors.

Financial Disclosure: The authors declared that this study received no financial support.

Images

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