Analysis of the frequency of oncogenic driver mutations and correlation with clinicopathological characteristics in patients with lung adenocarcinoma from Northeastern Switzerland

Background Molecular testing of lung adenocarcinoma for oncogenic driver mutations has become standard in pathology practice. The aim of the study was to analyze the EGFR, KRAS, ALK, RET, ROS1, BRAF, ERBB2, MET and PIK3CA mutational status in a representative cohort of Swiss patients with lung adenocarcinoma and to correlate the mutational status with clinicopathological patient characteristics. Methods All patients who underwent molecular testing of newly diagnosed lung adenocarcinoma during a 4-year period (2014–2018) were included. Molecular analyses were performed with Sanger sequencing (n = 158) and next generation sequencing (n = 311). ALK, ROS1 and RET fusion gene analyses were also performed with fluorescence in situ hybridization and immunohistochemistry/immunocytochemistry. Demographic and clinical data were obtained from the medical records. Results Of 469 patients with informative EGFR mutation analyses, 90 (19.2%) had EGFR mutations. KRAS mutations were present in 33.9% of the patients, while 6.0% of patients showed ALK rearrangement. BRAF, ERBB2, MET and PIK3CA mutations and ROS1 and RET rearrangements were found in 2.6%, 1.9%, 1.9%, 1.5%, 1.7% and 0.8% of the patients, respectively. EGFR mutation was significantly associated with female gender and never smoking status. ALK translocations were more frequent in never smokers, while KRAS mutations were more commonly found in ever smokers. The association between KRAS mutational status and female gender was statistically significant only on multivariate analysis after adjusting for smoking. Conclusion The EGFR mutation rate in the current study is among the higher previously reported mutation rates, while the frequencies of KRAS, BRAF, ERBB2 and PIK3CA mutations and ALK, ROS1 and RET rearrangements are similar to the results of previous reports. EGFR and KRAS mutations were significantly associated with gender and smoking. ALK rearrangements showed a significant association with smoking status alone. Electronic supplementary material The online version of this article (10.1186/s13000-019-0789-1) contains supplementary material, which is available to authorized users.


Background
Lung cancer is the leading cause of cancer-related mortality worldwide [1]. Non-small cell lung cancer (NSCLC) is the most common histological subtype of lung cancer, accounting for approximately 80-85% of lung cancer cases [2,3]. Molecular testing for epidermal growth factor receptor gene (EGFR) mutations and ALK receptor tyrosine kinase (ALK) translocations has become the evidencebased standard of care for the management of advanced NSCLC. In the past, pivotal clinical trials have demonstrated clinical benefit from targeting EGFR mutations and ALK translocations, and currently a number of effective EGFR and ALK inhibitors are available for targeted therapy of NSCLC harboring the relevant aberrations [4]. More recently, new molecular profiling technologies have permitted the identification of other potential oncogenic drivers including mutations in the KRAS proto-oncogene (KRAS), B-Raf proto-oncogene (BRAF), erb-b2 receptor tyrosine kinase 2 gene (ERBB2), MET proto-oncogene (MET) and phosphatidylinositol-3 kinase catalytic subunit alpha gene (PIK3CA) as well as ROS proto-oncogene 1 (ROS1) and ret proto-oncogene (RET) rearrangements [4]. While a number of studies have already evaluated the frequencies of these genetic alterations in NSCLC patients from different countries, information on the prevalence of oncogenic driver mutations in the Swiss population are scarce and limited to population based epidemiological data derived from cancer registries and molecular test results based exclusively on Sanger sequencing rather than next generation sequencing (NGS) [5,6].
In Switzerland lung cancer is the most common cause of cancer-related death among men (approximately 2000 deaths per year) and the second most common cause of cancer-related death among women (approximately 1100 deaths per year) [7]. Adenocarcinoma is the predominant histological subtype with distinct molecular features, and incidence rates of lung adenocarcinoma are increasing among both sexes [8,9]. The aim of the study was to analyze the frequencies of ALK, RET and ROS1 gene rearrangements and EGFR, KRAS, BRAF, ERBB2, MET and PIK3CA mutations in a representative cohort of Swiss patients with lung adenocarcinoma using NGS as testing method in the majority of cases and to correlate the molecular findings with clinicopathological patient characteristics.

Patients
A total of 475 consecutive patients who underwent molecular testing of newly diagnosed lung adenocarcinoma at the Institute of Pathology and Molecular Pathology, University Hospital Zurich (Zurich, Switzerland), between January 2014 and January 2018, were included in the study, independent of tumor stage. Molecular analyses were performed at the University Hospital Zurich according to National Comprehensive Cancer Network (NCCN) and Swiss Society of Pathology (SSPath) guidelines. Inclusion criteria were histologically and/or cytologically confirmed lung adenocarcinoma, chemotherapy, targeted therapy and radiotherapy naïve, and tissue blocks/cell blocks with adequate tumor cellularity. Exclusion criteria were non-adenocarcinoma histology, previous chemotherapy, targeted therapy or radiotherapy, and insufficient tumor material. Of the initial study population, 469 patients had adequate tumor material for molecular testing, while 6 patients had insufficient tumor samples and were not further evaluated. The results of molecular analysis were recorded for each patient and correlated with demographic and tumor related data such as gender, age, smoking status, clinical stage, and TNM stage (as defined by the Union for International Cancer Control (UICC) TNM classification of malignant tumors, 8th edition [10]). Smoking status was defined as never smokers (< 100 lifetime cigarettes), ex-smokers (≥100 lifetime cigarettes and currently not smoking) and current smokers (≥100 lifetime cigarettes and currently smoking). The cutoff date for data collection was 15 May 2018. The study was approved by the Cantonal Ethics Committee of Zurich (StV-No. 2009/14-0029).

Molecular analysis
Nucleic acids (DNA and RNA) were isolated from formalin-fixed paraffin-embedded (FFPE) tissue blocks or FFPE cell blocks using the Maxwell

Statistical analysis
Descriptive statistics were employed to describe the patient characteristics of the study cohort. The results are presented as frequencies and percentages for categorical variables and as mean ± standard deviation, median and range for continuous variables. Associations between mutation status and clinicopathological characteristics were tested using univariate and multivariate analyses. Univariate analysis was performed by chi-square test or Fisher exact test for categorical variables and by t test or nonparametric Mann-Whitney test for continuous variables. Multivariate analysis was performed by logistic regression. P-values < 0.05 were considered statistically significant. All statistical analyses were performed using SPSS Statistics software (version 24.0, IBM, Ehningen, Germany).  Table 2). Doublet EGFR mutations were found in 5 (5/90, 5.6%) tumors, including 2 tumors with L858R and non-L858R missense mutations, 2 tumors with two non-L858R missense mutations and 1 tumor with non-L858R missense mutation and T854A primary resistant mutation (Additional file 1: Table S1). The highest prevalence of EGFR mutations was observed in never smokers (69/115, 60.0%) and was considerably lower in ex-smokers (10/160, 6.3%) and current smokers    (Tables 8 and 9). By contrast, ALK rearrangement was significantly more common in never smokers than in ever smokers (12/84, 14.3% vs 16/292, 5.5%, p = 0.007) ( Table 8). The association between ALK rearrangement and smoking status remained statistically significant on multivariate analysis after adjusting for gender (p = 0.008, beta 1.081, OR 2.948, CI 95% 1.323-6.567) ( Table 9). Among the patients tested for ALK rearrangement, females were more likely to be never smokers than males (

Discussion
This study presents for the first time data on the EGFR, KRAS, ALK, ROS1, RET, BRAF, ERBB2, MET and PIK3CA mutation frequencies in a representative Swiss cohort of patients with stage I-IV lung adenocarcinoma using NGS as testing method in the majority of patients. Molecular testing was performed in all patients at the time of initial diagnosis during a 4-year period at a primary referral center for lung diseases in Northeastern Switzerland. We also comprehensively studied types of mutations and associations of mutational status with demographic and clinicopathological patient characteristics. The reported EGFR mutation rate in patients with lung adenocarcinoma varies widely between different populations worldwide, ranging from 10 to 20% in European and North American cohorts [5,6,[13][14][15][16][17][18][19][20][21][22][23]] to more than 50% in Asian populations [24,25]. The wide range of reported EGFR mutation rates among European cohorts might be explained by differences between the published studies with respect to patient selection criteria and methods used for molecular analysis. In a French study by Vallee et al. [19], one of the largest single center studies in Europe, EGFR mutations were detected in 13.5% of patients with NSCLC and in 14.7% of patients with lung adenocarcinomas. The authors used allele-specific PCR for evaluation of L858R point mutation and DNA fragment analysis to detect exon 19 deletions. Because other EGFR mutations were not evaluated, the true prevalence of EGFR mutations in this study remains unknown. The INSIGHT study, a large multicenter study comprising 1785 NSCLC patients (including 1393 patients with lung adenocarcinoma), showed an EGFR mutation frequency of 13.8% in NSCLC patients and of 15.4% in patients with lung adenocarcinoma [14]. The study analyzed tumor samples from 14 cancer centers in six Central European countries, each with different patient inclusion criteria and testing methods, which makes comparison with other studies more difficult. In addition, mutation testing was not performed at a fixed time point, which could induce bias as mutations may arise during the disease course [23]. Our study results show a prevalence of EGFR mutations that is similar to that reported by Moiseyenko et al. [20] (19.8%) in a Russian cohort and by Hlinkova et al. [22] (20%) in a Slovakian cohort, but lower than the EGFR mutation rates reported in two previous studies from Switzerland [5,6]. Ess et al. [5] retrospectively analyzed population based data on the  [18][19][20][21] for EGFR mutation analysis and FISH with a break-apart probe for ALK rearrangement testing, EGFR mutations (exclusively exon 19 deletions and exon 21 L858R mutations!) were detected in 11% of patients with advanced non-squamous NSCLC and in 13% of patients with lung adenocarcinoma, while 12% of patients with non-squamous NSCLC and 10% of patients with lung adenocarcinoma harbored ALK rearrangements. Other oncogenic driver mutations or associations between EGFR mutation/ALK rearrangement status and clinicopathological characteristics of patients with lung adenocarcinoma were not evaluated. More recently, Schwegler et al. [6] prospectively analyzed population based epidemiological data on overall survival of patients with mutated stage IV lung adenocarcinoma, mostly residents in rural areas of Central Switzerland, from 2010 to 2014. EGFR mutations were detected with Sanger sequencing in 14% of the patients, while KRAS, ERBB2, BRAF and MET mutations and ALK and RET translocations were found in 20%, 2%, 1%, 0.5%, 6% and 0.5%, respectively [6]. In contrast to our study, the types of mutations were not analyzed, and mutational status was not correlated with demographic or clinicopathological features. Possible reasons for the reported lower EGFR mutation rates compared with that of our study may be different modes of patient selection (selection from the molecular database of University Hospital Zurich vs selection from cancer registries), different patient selection criteria (patients with stage I-IV lung adenocarcinoma vs patients with stage IV or relapsed non-squamous NSCLC [5] and patients with stage IV lung adenocarcinoma [6]) and different methods used for mutational analysis (NGS and Sanger sequencing vs Sanger sequencing alone). In contrast to the studies by Ess et al. [5] and Schwegler et al. [6], the majority of patients in our study underwent   [2,26]. In addition, in the study by Schwegler et al. [6] patients with stage I-III lung adenocarcinoma were excluded from the analysis, and 20% of stage IV lung cancer patients were not tested for oncogenic driver mutations, while in the study by Ess et al. [5] 38% of patients did not receive molecular analysis. Although we did not assess mutation testing rates at our institution, it can be assumed that the molecular testing rates in the period from 2014 to 2018 were higher than those of previous years and that patients treated at an institution active in clinical research are more regularly tested for predictive biomarkers than patients treated at an institution not participating in clinical research [5]. In accordance with published literature [13][14][15][16]23], we found a significant association of EGFR mutation status with female gender and never smoking status. When we restricted the analysis to female never smokers, we achieved a high EGFR mutation rate of 65.7% (46/70), a finding consistent with previous reports [13,24,25].
KRAS mutation is one of the most frequent mutations in NSCLC, at least in Caucasian populations, with reported frequencies reaching up to 30% of lung adenocarcinomas [13,23,27,28], while its prevalence in Asian populations is approximately 10% [29][30][31]. KRAS mutations are predominantly found in smokers [32], but they may occur in up to 15% of non-smokers [27]. To date, no effective anti-KRAS agent has been released, although a number of preclinical studies and clinical trials are currently underway, exploring novel therapeutic approaches to target KRAS mutated NSCLC [33][34][35][36]. The KRAS mutation rate in our study was slightly higher than was previously reported for Caucasian populations (which might be related to different smoking habits in this Swiss cohort), but was almost identical to the KRAS mutation rate reported by Brcic et al. [13] in a Croatian cohort. The presence of KRAS mutation in our study was significantly associated with a history of smoking on both univariate and multivariate analyses, while the association of KRAS mutation with gender was statistically significant only on multivariate analysis after adjusting for smoking. This finding adds to a mixed body of literature. Some studies have shown increased incidence of KRAS mutations among females [23], while others found equal frequencies in both men and women [13,37,38].
ALK rearrangements are detected in 3-7% of NSCLC [39][40][41][42][43][44]. They predominantly occur in non-smokers, lung adenocarcinomas and non-Asian vs Asian populations, while men and women seem to be equally affected [3]. The frequency of ALK rearrangement in our study is consistent with previous reports, as is the association with smoking status (higher frequency in never smokers). Interestingly, our study showed a higher frequency of ipsilateral mediastinal or subcarinal lymph node metastasis (N2) in   ALK-rearranged tumors compared with non-rearranged, EGFR mutated and KRAS mutated tumors, while no significant differences were found between ALK-rearranged and non-rearranged/EGFR mutated/KRAS mutated tumors with regard to N0, N1 and N3 stages. In addition, ALK rearranged lung adenocarcinomas were more frequently pT1 tumors compared with ALK-non rearranged lung cancer. In a previous study, evaluating surgically resected stage I-III NSCLCs, Paik et al. [45] found that ALK FISH-positive NSCLC cases showed lower tumor stage (pT1), but had more frequently lymph node metastases compared with ALK FISH-negative NSCLC cases. The authors suggested that ALK-rearranged lung cancer might have unique biological features with a tendency to early lymph node metastasis despite the small primary tumor size, which could explain higher incidences of ALK rearrangement in advanced NSCLC compared with surgically resectable lung cancer [45]. The frequency of BRAF mutations in the current study seems to be among the higher previously reported mutation rates [46][47][48], but is still lower than the mutation rate reported by Illei et al. [ [26,58,59], respectively). While our study with a limited sample size of RET and ROS1 rearranged lung cancer showed no significant differences between RET or ROS1 rearranged and non-rearranged tumors regarding clinicopathological characteristics, previous investigations have reported a higher incidence of RET and ROS1 rearrangements in younger age group and never smokers [60,61] as well as a significant association of RET rearranged NSCLC with small primary tumor size and lymph node involvement [60,62]. According to previous reports [63], ERBB2 mutations in NSCLC are more common in females, Asian cohorts and never-smokers. While our study showed no significant association of ERBB2 mutation with female gender, we could confirm the higher prevalence of ERBB2 mutations in never smoking patients. PIK3CA mutations are more commonly encountered in squamous NSCLC [58,59,64] and seem to confer inferior prognosis in lung adenocarcinoma [65]. Interestingly, PIK3CA mutations have been reported to occur in parallel with other oncogenic driver mutations [66,67], as was the case in 5 of 7 PIK3CA mutated tumors in the present study. Regarding the clinicopathological characteristics of MET exon 14 skipping mutation-positive tumors, three retrospective studies showed that MET exon 14 skipping positivity in NSCLC patients is significantly associated with advanced age [68][69][70]. In the current study, we found no significant difference in mean age between patients with and those without MET exon 14 mutated tumors. However, we acknowledge that the sample size of MET exon 14 mutated tumors was too small to draw meaningful conclusions.

Conclusion
Our study presents data on the frequency of oncogenic driver mutations in a Northeastern Swiss population with stage I-IV lung adenocarcinoma using NGS as testing method in the majority of cases. A number of studies already analyzed oncogenic driver mutation frequencies, notably EGFR, KRAS and ALK mutation rates, in different populations from European countries. However, based on the available data, the true prevalence of mutations in lung adenocarcinoma is often difficult to determine due to patient selection bias, different testing platforms used for analysis and the histological heterogeneity of tumors included in the studies. Although we cannot exclude some selection toward patients with higher likelihood of mutated tumors in the current study, a major selection bias is unlikely to have occurred because the epidemiological characteristics of our study population are similar to those of the INSIGHT study and other previous investigations. We found a relatively high EGFR mutation rate, while KRAS, BRAF, ERBB2, MET and PIK3CA mutation and ALK, RET and ROS1 rearrangement frequencies were

Additional file
Additional file 1: Table S1. Doublet EGFR mutations in 90 lung cancers. Table S2. Comparison of EGFR and KRAS mutated tumors. Table S3.
Comparison of EGFR mutated and ALK rearranged tumors. Table S4.