|Ahead of print
Extended-spectrum of KRAS and NRAS mutations in lung cancer tissue specimens obtained with bronchoscopy
Muserref Basdemirci1, Adil Zamani2, Ayse G Zamani3, Siddika Findik4, Mahmut S Yildirim3
1 Department of Medical Genetics, Konya Training and Research Hospital, Konya, Turkey
2 Department of Pulmonary Medicine, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey
3 Department of Medical Genetics, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey
4 Department of Pathology, Meram Medical Faculty, Necmettin Erbakan University, Konya, Turkey
|Date of Submission||28-Aug-2019|
|Date of Decision||06-Sep-2019|
|Date of Acceptance||10-Jun-2020|
|Date of Web Publication||21-Jun-2021|
Department of Medical Genetics, Konya Training and Research Hospital, Konya
Source of Support: None, Conflict of Interest: None
Background: Mutations in the RAS genes, HRAS, KRAS, and NRAS, are the most common modifications in many types of human tumors and are found in approximately 30% of all human cancers. These mutations are usually found in codons 12, 13, or 61.
Methods: The aim of this study is to evaluate mutations in codons 59, 117, and 146 of KRAS and NRAS genes in addition to codons 12,13, and 61 of KRAS gene in lung cancer tissue specimens obtained with bronchoscopy. KRAS and NRAS mutation analyses with pyrosequencing were performed on DNA isolated from formalin-fixed paraffin-embedded (FFPE) tissue samples of 64 patients histopathologically diagnosed as lung cancer after bronchoscopic biopsy.
Results: In all, 20 patients (31.2%) had mutations in KRAS gene (8/27 squamous cell carcinoma, 8/11 adenocarcinoma, 3/16 small cell carcinoma, and 1/1 pleomorphic carcinoma). The most common mutation in codon 12 was in c.35G>T (G12V). When the mutation rate of adenocarcinoma (72.7%) and squamous cell carcinoma (22.9%) patients was compared with each other, a statistically significant difference was observed (P = 0.008). There were no mutations in codons 59, 117, or 146 of KRAS and NRAS genes in patients with lung cancer.
Conclusion: In this study, we firstly examined mutations in codons 59, 117, and 146 of KRAS and NRAS genes in addition to codons 12, 13, and 61 of KRAS gene in Turkish lung cancer patients both in non-small cell lung cancer and small cell lung cancer. Although no mutation was detected in codons 59, 117, and 146 of KRAS and NRAS genes, the frequency of KRAS gene mutation was higher than the rate of mutation in both Asian and Western countries, and multicenter studies including more cases should be performed to further explore our results.
Keywords: Bronchoscopy, KRAS, lung cancer, mutation, NRAS
The absence of mutations in codons 59, 117 and 146 of KRAS and NRAS genes suggests that mutations in these codons perhaps don't contribute to pathogenesis of lung cancer in Turkey.
|How to cite this URL:|
Basdemirci M, Zamani A, Zamani AG, Findik S, Yildirim MS. Extended-spectrum of KRAS and NRAS mutations in lung cancer tissue specimens obtained with bronchoscopy. Indian J Cancer [Epub ahead of print] [cited 2021 Oct 28]. Available from: https://www.indianjcancer.com/preprintarticle.asp?id=318901
| » Introductıon|| |
Non-small cell lung cancer (NSCLC), which is the most common histological type of lung cancer, has an increasing incidence and the highest cancer-related mortality rate worldwide in recent years. Results from previous studies have shown that somatic mutations in genes such as EGFR, KRAS, BRAF, NRAS, PIK3CA, Her-2, and TP53 are associated with the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors, metastasis, or total survival. Therefore, molecular analyses of mutations in these genes are widely used in the management of individualized NSCLC patients' treatment.,
The incidence of oncogene mutations in NSCLC patients varies from society to society. In Western countries, the oncogene most commonly mutated in patients with NSCLC (and highly associated with smoking) is Kirsten rat sarcoma viral oncogene homolog (KRAS). In humans, there are four RAS isoforms encoded by three genes (KRAS, HRAS, and NRAS). RAS proteins are small GTPases which function as molecular switches that control signaling pathways for cell proliferation and regulation of cell survival. Irregular RAS signals are associated with oncogenesis and a class of developmental disorders called RASopathies. KRAS is the most commonly mutated RAS isoform (69% of RAS-mutant patients) found in approximately 20–25% of lung adenocarcinomas (ADC) in western countries and in 10–15% of cases in Asia. NRAS mutations with potential susceptibility to MEK inhibitors occur in lower rates in lung cancers (adenocarcinoma: 0.9%, squamous cell carcinoma: 1.2%)., However, to our knowledge, there is no extensive study of NRAS mutations.
The aim of this study is to evaluate mutations in 59, 117, and 146 codons of KRAS and NRAS genes in addition to 12, 13, and 61 codons of KRAS gene in bronchoscopic materials of patients with lung cancer.
| » Methods|| |
Consecutive patients who were suspected to have lung cancer because of signs, symptoms, and imaging findings on chest radiography, thoracic computed tomography, and/or fluorodeoxyglucose positron-emission tomography underwent fiberoptic bronchoscopy. Fiberoptic bronchoscopy using an Olympus BF-1T60 bronchoscope (Olympus Corp., Tokyo, Japan) was performed in 64 patients with endoscopically visible tumoral lesions between January 2014 and June 2016. Exclusion criteria for this study were as follows: patients under the age of 18 years, unfit to undergo an endoscopy, and unwillingness to provide written informed consent. Biopsy specimens were obtained with disposable oval-cup forceps (jaw outside diameter 1.8 mm). All bronchoscopies were performed by a single bronchoscopist. The study was approved by the medical ethics committee of the university. All patients that participated in the study signed informed consent release forms.
Demographic data collection
Patients' demographic characteristics, smoking status, and histopathological subtypes were extracted from electronic medical records (Hospital Information Management System).
A total of 64 formalin-fixed paraffin-embedded (FFPE) bronchoscopic tissue samples of patients, which were histopathologically diagnosed as lung cancer, were obtained from the pathology laboratory. Of the 64 samples, 48 were NSCLCs (35 squamous cell carcinomas, 11 adenocarcinomas, 1 large cell carcinoma, and 1 pleomorphic carcinoma) and 16 were SCLCs.
DNA isolation from FFPE tissue
For each specimen, a total of three sections and one corresponding hematoxylin and eosin-stained section were taken. For a maximum measure of tumor material, tumor-rich areas on the hematoxylin and eosin-stained slide were marked by a pathologist. Genomic DNA was isolated from the selected areas by using the QIAamp DNA FFPE Tissue Kit (Qiagen, Germany) according to the manufacturer's protocol. The concentration and quality of extracted DNA samples were measured by NanoDrop 2000c spectrophotometer (Thermo Scientific, USA). All isolated DNA samples were maintained at 4°C before use.
The mutational analysis of codon 59, 117, 146 of the KRAS and NRAS genes in addition to codon 12, 13, 61 of the KRAS gene was performed by pyrosequencing. The Therascreen KRAS Pyro Kit (Qiagen, Germany) and Therascreen RAS Extension Pyro Kit (Qiagen, Germany) were used to query the mutation status of codon 12, 13, and 61 of KRAS gene and codon 59, 117 and 146 of KRAS and NRAS genes, respectively. Target regions for mutation analysis of KRAS and NRAS genes were amplified by polymerase chain reaction (PCR) (Sensoquest, Thermocycler 48, Germany). PCR was conducted in a total volume of 25 μL, using the following reagents:
12.5 μL 2x PCR master mix, 2.5 μL 10x CoralLoad, 1 μL each PCR primer, 4 μL H2O (supplied) and 5 μL genomic DNA. Thermal cycle program for amplification was carried out as follows: the initial activation step at 95°C for 15 minutes was followed by 42 cycles of denaturation at 95°C for 20 seconds, annealing at 53°C for 30 seconds, and extension at 72°C for 20 seconds, with a final extension step of 5 minutes at 72°C. All PCR products were immobilized using immobilization mix consisting of PyroMark Binding Buffer, Streptavidin Sepharose High-Performance beads (GE Healthcare), and high-purity water. The immobilized amplicons were eluted at PyroMark Q24 Vacuum Workstation (Qiagen) using the following reagents: Ethanol (70%), PyroMark Denaturation Solution, PyroMark Wash Buffer, and high-purity water. Then, each pyrosequencing sequence primer was annealed to the purified single-stranded PCR products on the wells of PyroMark Q24 plate and pyrosequencing was performed on a PyroMark Q24 system (Qiagen) with PyroMark Gold Q24 Reagents (Qiagen) following the manufacturer's instructions. Results were analyzed using PyroMark Q24 software. The targeted sequences for KRAS (codons 12, 13, 59, 61, 117, and 146) and NRAS (codons 59, 117, and 146) are listed below.
- KRAS codon 12 and 13: GGTGGCGTAGG
- KRAS codon 59: ACAGCAGGT
- KRAS codon 61: AGGTCAAGAG
- KRAS codon 117: ATAAATGTG
- KRAS codon 146: ATCAGCAAAG
- NRAS codon 59: ACAGCTGGA
- NRAS codon 117: AGTGTGA
- NRAS codon 146: CAGCCAAGAAC.
Pyrosequencing reading for codon 61 of KRAS is a reverse component of the target sequence (CTCTTGACCT) because its sequencing primer is a reverse primer.
Interpretation of analysis results and detection of low-level mutations
Control DNA was included in every run for comparison and as a control for background levels. The measured frequency of the control sample should be smaller or equal to the limit of blank (LOB). All samples were examined in relation to the limit of detection (LOD) and interpreted as follows:
Mutation frequency <LOD: Wild type
Mutation frequency ≥LOD and ≤LOD + 3% units: Potential low-level mutation
Mutation frequency >LOD + 3% units: Mutation
LOB and LOD were determined according to the recommendations in the Clinical and Laboratory Standards Institute (CLSI) Guideline EP17-A “Protocol for determination of limits of detection and limits of quantitation; approved guideline”.
T-test and χ2 test were used to compare the data. A P value of less than 0.05 was considered statistically significant. Statistical analysis of all data obtained from this study was performed using SPSS version 20.0 (SPSS Inc., Chicago, IL, USA).
| » Results|| |
The average age of the patients was 62 years (range: 41–84 years). Most patients were smokers and men, only two patients were women. The patients' characteristics are shown in [Table 1].
Twenty (31.2%) of the 64 patients had mutations in the KRAS gene: 14 mutations in codon 12 (21.9%), 2 mutations in codon 13 (3.1%), 3 mutations in codon 61 (4.7%), and 1 tumor showed a double mutation (codon 12 and 61) (3%). No mutation in codons 59, 117, and 146 of KRAS or the NRAS gene was observed in patients. The most common mutation in codon 12 was c.35G>T (G12V). [Table 2] shows the results of the KRAS and NRAS genes' mutational analysis of all patients.
|Table 2: KRAS and NRAS mutational analysis of bronchoscopic tissue samples of 64 patients|
Click here to view
KRAS mutations were identified in 17 of 48 NSCLC and 3 of 16 SCLC. Among the 17 KRAS mutations in NSCLC, 8 were in squamous cell carcinomas, 8 were in adenocarcinomas, and 1 was in a pleomorphic carcinoma. Although the KRAS mutations were more prevalent in the NSCLC patients than in the SCLC patients (17/48 (35.4%) versus 3/16 (18.8%)), there were no statistically significant differences between the two groups (P = 0.213). However, the KRAS mutation in adenocarcinomas was significantly more frequent compared with squamous cell carcinomas (P = 0.008). There were also no statistically significant differences in the KRAS mutational frequencies between ≤60 and >60 year old patients (P = 0.945). Interestingly, although KRAS and NRAS mutations are rare in SCLC, one SCLC patient had two mutations in codon 61 of the KRAS gene (c.182A>T (Q61L) and c.183A>C (Q61H)).
| » Discussion|| |
Changes in the genome affecting the expression or function of genes that control cell growth and differentiation are considered the main cause of cancer. The first gene family found to be mutated in human tumors is the RAS oncogene family. Point mutations in specific codons of RAS genes lead to the continuous activation of the RAS proteins, ultimately triggering uncontrolled cell proliferation. RAS oncogenes are activated in approximately 30% of malignant tumors in humans. The most mutated gene is the KRAS gene (22%), while NRAS (8%) and HRAS (3%) genes are less affected according to the COSMIC dataset.
Most of KRAS mutations (80%) occur in codons 12 or 13, whereas very few mutations are observed at codon 61. By contrast, approximately 60% of NRAS mutations are occur in codon 61 and 35% in codon 12. Mutations in codon 12, 13, or 61 of KRAS are reported as 20–30%, in patients with lung cancers, especially in NSCLC.
The incidence of KRAS mutations in lung cancer is dependent upon tumor histopathological subtypes, and reported in 22–34% of adenocarcinomas, 0–25% of squamous cell carcinomas, 8–33% of large cell carcinomas, and 8–11% of adenosquamous cell carcinomas. In our study, KRAS gene mutations were detected in 20 of 64 patients (31.2%), a higher mutation rate than in similar studies in the literature. Seventeen of the detected mutations (35.4%) were in patients with NSCLC and 3 (18.8%) were in cases with SCLC. There was no statistically significant difference between these two groups. In one of the cases with SCLC, double mutation was detected in codon 61 of KRAS.
In Western countries, KRAS mutation rate is higher in patients with NSCLC, especially in those with ADC (30–50). In contrast, most studies from East Asian regions reported a much lower mutation rate in patients with NSCLC than the rate reported in Western countries. However, the mutation rate of KRAS gene in patients with ADC was reported as >10%in 2 studies, including one study from Japan (13%) and one study from Korea (31.2%).,
In most studies that analyzed KRAS mutation status in NSCLC, the patient groups consisted adenocarcinoma cases. However the number of studies with squamous cell cancer cases is limited. In a largest study from Japan by Noda et al., the KRAS mutation rate was 8% (33 of 410 patients) in NSCLC, 9.2% (21 of 217 patients) in ADC and 5.5% (8 of 145 patients) in SCC. Another of the largest, prospective study from Asian patients with SCC by Kenmatsu et al., KRAS mutation rate was 4%. In a study with SCC patients from Prague by Vachtenheim et al., KRAS mutation rate was reported as 8% (9 of 118). In our report, we have summarized the results of the KRAS mutations studies in patients with NSCLC from different countries, including our results [Table 3].,,,,,,,,,,,,,,,,,,,,,,
|Table 3: Comparison of the mutation rates in KRAS gene in patients with NSCLC from different countries|
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In the present study, the rate of KRAS mutation were 35.4% in NSCLC (17 of 48 patients), 72.7% in ADC (8 of 11 patients), and 22.9% in SCC (8 of 35 patients) as higher than the mutation rate in both East Asian and Western populations. KRAS mutation rates obtained in NSCLC cancer studies conducted in Turkey and the results of the present study are presented in [Table 4].,,,
|Table 4: Comparison of the mutation rates in KRAS gene in patients with NSCLC from different regions in Turkey|
Click here to view
High mutation rate in our study may be related to the low number of patients included in the study. In addition, the number of patients with adenocarcinoma who were included in the study was very low (17.2%). In many studies, only codon 12 of KRAS gene mutations were evaluated. In our study, we also looked for mutations in codon 13 or 61 of KRAS gene and detected mutations in these codons as well. These might partially explain higher mutation rate than other studies.
The number of studies evaluating KRAS gene mutations in SCLC was limited. Wakuda et al. detected KRAS gene mutation in only one of 60 Japanese patient with SCLC. In our study, we observed that KRAS mutations were detected more frequently (3 of 16 patients with SCLC) than in patients from Japan.
The reason of the changes in KRAS mutation rates between countries and regions are evaluated as ethnical and geographical differences.
All studies evaluating the frequency of KRAS gene mutations in lung cancer were focused mainly on codons 12, 13, or 61. In addition to these known KRAS mutations, a large-scale analysis which also includes codons 59, 117, and 146 of both KRAS and NRAS was performed in this study. Due to the design of the primers used for these regions, codons 58, 60, 118, 145, and 147 were also analyzed for mutations. No mutation was detected in these 8 codons of KRAS and NRAS. However, in studies with colorectal cancers, mutations were detected in these codons.,,, In fact, data have shown that these mutations play a role in response to chemotherapy. Based on our study, the absence of mutations in additional codons suggests that these mutations perhaps do not contribute to pathogenesis among patients in this region.
In conclusion, according to our literature review, this is the first study to evaluate extended spectrum of KRAS and NRAS mutations in lung cancer patients. KRAS mutation rate in codon 12, 13, or 61 reported herein is the highest among all reported series to date [Table 3]. In addition, there were no mutations in codons 59, 117 or 146 of KRAS and NRAS genes. In our opinion, multicenter studies should be conducted with a larger number of patients to further explore these findings.
Declaration of patient consent
The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient(s) has/have given his/her/their consent for his/her/their images and other clinical information to be reported in the journal. The patients understand that their names and initials will not be published and due efforts will be made to conceal their identity, but anonymity cannot be guaranteed.
Financial support and sponsorship
This work was supported by the Scientific Research Project of Necmettin Erbakan University (grant number 151518011).
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]