Analysis of EGFR, KRAS, and PIK3CA gene mutation rates and clinical distribution in patients with different types of lung cancer

Background To analyze and evaluate EGFR, KRAS, and PIK3CA gene mutation rates and clinical distribution in patients with different types of lung cancer Method A total of 221 lung cancer patients treated in our hospital between January 2016 and June 2019 were enrolled. Tissue and whole blood samples were collected and analyzed to determine the mutation status of EGFR, KRAS, and PIK3CA genes. The gene exon mutation rates were determined. Relevant clinical data, such as age, gender, tumor sample type, treatment method, pathologic type, and lung cancer stage were recorded and statistically analyzed. Results The EGFR gene mutation rates in exons E18-E21 were 2.3%, 17.6%, 3.6%, and 20.4%, respectively. E18, E19, and E20 mutations were commonly detected in adenosquamous carcinoma, and E21 mutations were commonly detected in adenocarcinoma. Mutations in exons E18-E21 were frequently detected in patients with lung cancer stages IA, IB, IIA, or IIB, respectively. The KRAS gene mutation rate in lung cancer patients in exon E2 was higher in whole blood and tissue samples than other exon mutations, while the KRAS gene mutation rate in exons E2 and E3 was significantly higher in patients with lung cancer stages IIB and IA, respectively. PIK3CA gene mutations in exons E9 and E20 occurred in patients < 60 years of age. Exon E9-positive mutations were more common in men or patients with squamous cell carcinoma, while exon E20-positive mutations were more common in females. Conclusion The EGFR, KRAS, and PIK3CA gene exon mutation rates differ and were shown to be correlated with different clinical indicators, which have significance in clinical treatment.

individualized treatment has been proposed to achieve higher success rates than standardized treatment [4].
With the rapid advances in modern molecular biology technology, the treatment model for lung cancer has focused on targeting abnormal molecules in specific signaling pathways [5,6]. In the past 10 years, tyrosine kinase inhibitors (TKIs) for epidermal growth factor receptor (EGFR) have demonstrated remarkable clinical effects and paved the way for effective treatment of lung cancer [7][8][9]. Indeed, a recent phase III clinical trial (ADAURA [NCT02511106]) assessed the efficacy and safety of a 3rd-generation EGFR-TKI, osimertinib, which had superior efficacy when compared to EGFR-TKI (gefitinib/erlotinib) in treatment-naïve patients with EGFRmutated advanced NSCLC [10]. In addition, rapid and accurate determination of EGFR gene mutation status is important to correctly adjust the chemotherapy regimen and usage of drugs. In recent years, with the ongoing indepth studies of molecular biology and human genomics, it has been shown that EGFR-TKI treatment is effective in NSCLC patients with E19/21 EGFR mutations along with the metastases, and these patients had a longer progression-free survival (PFS) [11]. EGFR-TLI treatment of patients with KRAS mutations lacks efficacy, which indicates the different patterns of EGFR and KRAS gene mutations in lung cancer patients.
Phosphatidylinositol-3 kinase (PIK3CA) is a coding gene for the protein catalytic subunit of the phosphatidylinositol-3 kinase family (PI3Ks) [12]. It has been well-documented that activation of the PI3Ks pathway is involved in multiple human malignancies, while the effect of PIK3CA mutations on the prognosis of patients is controversial for different human cancers. It has been demonstrated that PIK3CA mutation status predicts the prognosis of breast cancer patients [13], while another study that PIK3CA mutation status is not associated with prognosis of colon cancer patients [14]. Few studies have systematically evaluated the relationship between the mutation status of EGFR, KRAS, and PIK3CA and lung cancer patients, especially among patients with different types of lung cancer.
In this study, we analyzed the mutation status of EGFR, KRAS, and PIK3CA in different types of lung cancer patients. These findings may provide theoretical insight for clinicians to make accurate and instant treatment plans for lung cancer patients.

General information
A total of 221 lung cancer patients (114 males [51.6%] and 107 females [48.4%]) who were treated in our hospital from January 2016 to June 2019 were enrolled in the current study. The following specimens were collected: 194 tissue samples (87.7%), 22 whole blood samples (10.0%), and 5 whole blood + tissue samples (2.3%). One hundred twelve patients (50.7%) were ≤ 60 years of age, 109 (49.3%) were > 60 years of age, and the average age was 59.62 ± 9.82 years. The clinical characteristics of the patients are presented in Table 1. All paraffin-embedded tissue sections and cytologic smears were examined and diagnosed by senior pathologists. The tumor areas were delineated, and the tumor cell number (> 200) and percentage (> 20%) were evaluated. Ten milliliters of peripheral venous blood samples was collected in blood collection tubes dedicated to protect free DNA for normal temperature transportation and preservation.

Reagents
Formaldehyde-fixed, paraffin-embedded tissue DNA extraction kits were purchased from Qiagen Company (Darmstadt, Germany). Tissue DNA extraction kits, plasma circulating DNA extraction kits, human EGFR gene mutation detection kits (ARMS method [15]), and human EGFR, KRAS, and PIK3CA gene mutation detection kits (super-ARMS method [16]) were purchased from AmoyDx (Xiamen, China). An ABI fluorescence quantitative PCR instrument (Foster city, CA, USA) was used.

Detection of EGFR, KRAS, and PIK3CA gene mutations
Sample processing and DNA extraction were performed according to the manufacturer's protocol. The DNA extraction protocol from paraffin-tissue samples was as follows: refrigerate the circled wax block and place the block on a slicer, cut the samples into 10 pieces (4-μm-thick slices), remove the non-cancerous tissue, and transfer the wax into a 2-ml centrifuge tube. Extract 50 μl of DNA according to the formaldehyde-fixed, paraffin-embedded tissue DNA extraction kit protocol. The DNA extraction from peripheral blood sample protocol was as follows: collect the samples and centrifuge the samples immediately at 2000×g for 10 min, aspirate the supernatant and transfer the sample to a new centrifuge tube, centrifuge the samples at 8000×g for 10 min, and aspirate the supernatant and transfer to a new centrifuge tube. PBS was added to the tube if the volume was < 4 ml, and 100 μl of DNA was extracted according to the plasma circulating DNA extraction kit protocol. The EGFR (E18, E19, E20, and E21), KRAS (E2, E3, and E4), and PIK3CA (E9 and E20) gene mutations in lung cancer samples were detected using liquid chip technology. The main research steps were as follows: the gene fragments containing common alleles in the exons of EGFR, KRAS, and PIK3CA genes were obtained using multiplex PCR; the reaction products were hydrolyzed by an exonuclease and alkaline phosphatase (EXO-SAP); the allele-specific primer extension (ASPE) was used for PCR product processing; the tag sequence on the amplified product and anti-tag sequence on polystyrene microspheres were specifically hybridized; and the hybridized product was analyzed using a Luminex 200 system (Austin, TX, USA) to obtain the median fluorescence value (MFI).

Statistical analysis
The SPSS 19.0 software was used for statistical analyses. The correlation between EGFR, KRAS, and PIK3CA gene mutations and clinicopathologic features of patients was analyzed using unpaired χ 2 tests or Fisher exact probability analysis [17]. The difference was statistically significant (p< 0.05).

Detection and analysis of EGFR gene mutations
As shown in Table 2, mutation of the EGFR gene in exon E18 was significantly related to sample type, ). In addition, stage IA lung cancer patients (13.64%) and patients who underwent surgical resection (5.97%) were diagnosed with a higher E18 mutation rate; however, there was no significant difference between the E19 and E20 mutation status and clinical indicators (Tables 3 and 4). The E19-positive mutation rate was higher in adenosquamous carcinoma (25.49%) and stage IB patients (28.57%), while the E20-positive mutation rate was higher in adenosquamous carcinoma (14.29%) and stage IIIB stage patients (20.00%). As shown in Table 5, mutation of the EGFR gene in exon E21 was significantly associated with gender, lung cancer stage, and treatment method. Specifically, the E21-positive mutation rate in females (29.91%) was significantly higher than males (11.40%), and the E21 mutation rate in patients with stage IB lung cancer (33.33%) was significantly higher than other stages. Like the E18 mutation rate, the patients who underwent surgical resection (37.31%) had a higher E18 mutation rate than patients who were treated with chemotherapy (10.81%) or surgical resection combined with chemotherapy (13.64%). Even though there was no significant difference between pathologic type and the E21-positive mutation rate, the results showed that the patients with adenocarcinoma had the highest E21 mutation rate (24.52%).

Detection and analysis of KRAS gene mutations
As shown in Table 6, mutation of the KRAS gene in exon E2 was significantly related to sample type. In contrast to mutation of the EGFR gene in exon E18, the E2-positive KRAS mutation rate in the whole blood + tissue sample (20.00%) was significantly higher than the whole blood (0%) or tissue sample (7.22%). There was no significant difference between the E2 mutation rate and other clinical indicators. Patients with adenosquamous carcinoma (14.29%) and patients with stage IIB lung cancer (11.11%) had a higher E2 mutation rate in the KRAS gene. Similar to mutation of the EGFR gene in exons E20 and E21, there was no significant difference between the KRAS gene mutation rate in exon E3 and clinical indicators ( Table 7). The E3-positive mutation rate was more common in adenocarcinoma patients (0.65%) and  (Table 8).

Detection and analysis of PIK3CA gene mutations
As shown in Table 9, mutation of the PIK3CA gene in exon E9 was significantly related to the sample and pathologic types. The positive E9 mutation rates in the whole blood + tissue sample (20.00%) and patients with squamous cell carcinoma (13.33%) were the highest. In addition, E9-positive mutations were more common in stage IIB lung cancer patients (11.11%) and patients treated with chemotherapy (5.41%). The mutation status of the PIK3CA gene in exon E20 was significantly associated with sample type (Table 10). The rate of positive E20 mutations in the whole blood sample (4.55%) was significantly higher than the other two sample types; however, there were no positive PIK3CA gene mutations detected in exon E20 among patients with different pathologic types or lung cancer stages (Table 10).

Discussion
The collective evidence has demonstrated that targeting abnormal genes in specific signaling pathways is a significant and effective strategy to eliminate cancers [18,19]. In this study, we found that mutations of the EGFR gene in exons E18, E19, and E20 in lung cancer patients were common in adenosquamous carcinoma, while E21 mutations were common in adenocarcinoma. The E18 and E21 mutation rates were associated with lung cancer stage, gender, sample type, and treatment modality. Similarly, the KRAS gene mutation rate in exons E2 and E4 were also correlated with the sample type and treatment approach. In addition, the PIK3CA gene mutation status in exons E9 and E20 was associated with sample type, as well as lung cancer type. Taken together, these findings provide theoretical insight for clinicians to make accurate and timely treatment plans for lung cancer patients. EGFR is a transmembrane tyrosine kinase receptor that plays an important role in the growth, proliferation, and differentiation of cells under physiologic and pathologic conditions. It has been reported that   . In addition, a recent 3rd-generation EGFR-TKI, osimertinib, had a statistically significant and clinically meaningful improvement in disease-free survival in patients with common sensitive EGFR-mutated NSCLC after complete tumor resection and adjuvant chemotherapy [10,24]. Therefore, detection of EGFR mutation status in NSCLC patients before treatment is of great importance in the clinical setting. In this study, the positive EGFR mutation rates in exons E18-E21 were 2.3%, 17.6%, 3.6%, and 20.4% respectively.
Mutations in exons E19 and E21 represented the vast majority (87%) of all observed EGFR gene mutations in lung cancer patients, which is consistent with another study [25]. Interestingly, the mutation rates in exons E18-E21 were significantly different in patients with stages IA, IB, IIA, or IIB, respectively. Nie et al. [26] reported that stage I lung adenocarcinoma patients had the highest incidence of mutations when compared with other pathologic stages [26], which was consistent with our finding. In addition, even though there was no statistical difference between the E19-E21 mutation status and patient gender or age, females and patients ≤ 60 years of age had a higher mutation rate than males, females, and patients > 60 years of age, which was in agreement with the dominant population of EGFR mutations in females [24] and young patients [27]. KRAS mutations are key oncogenic regulators in the development of many human malignant cancers, including lung cancer. KRAS mutations regulate signal transduction networks, which are necessary for cell survival, proliferation, differentiation, and  [28,29]. Mutated KRAS leads to phosphorylation and activation of p21. Approximately 29% of NSCLC patients have KRAS gene mutations, among whom those with adenocarcinoma and a history of smoking are the most commonly diagnosed [30]. Collective evidence has demonstrated that the KRAS gene mutation status serves as a biomarker to predict the prognosis of NSCLC patients after chemotherapy [31]; however, KRAS and EGFR mutations are mutually exclusive, and NSCLC patients with KRAS mutations have a low response rate to EGFR-TKIs [32]. At present, several KRAS-targeting drugs, such as heat shock protein 90 (Hsp90) inhibitors, have been used in the treatment of NSCLC patients with KRAS-positive mutations with significant efficacy [33]. Therefore, except for detection of the EGFR gene, confirmation of KRAS gene mutation status is also important in the clinical setting. In this study, it was shown that the positive KRAS gene mutation rate in exon E2 was highest in whole blood + tissue samples, patients with stage IIB, and patients with adenosquamous carcinoma; however, patients with adenocarcinoma or stage IA lung cancer were significantly more frequent than other lung cancer types or stages. Notably, no E4-positive mutations were detected in this study, which might reflect the limited sample size.
PI3Ks play a vital role in many biological processes and can activate serine/threonine kinase Akt and the downstream mTOR pathway to regulate cell survival, proliferation, and the cell cycle. Somatic mutations in the PI3K-Akt-mTOR pathway are often found in cancer tumorigenesis and can be used as a target for treatment of cancer patients [34]. PI3Ks consist of catalytic (P110) and regulatory subunits (p85), while catalytic subunits contain 3 gene codes (PIK3CA, PIK3CB, and PIK3CD). Generally, PIK3CA is the most common mutation location in cancer patients [35]. The most common mutation sites of PIK3CA are located in the spiral domain, including E542K and E545K in exon 9, and in the kinase domain, including H1047R in exon 20 [36]. In this study, we found that PIK3CA-E9 and the E20-positive mutation rates were higher in patients ≤ 60 years of age. E9-positive mutations were more common in males, while E20-positive mutations were more common in females. In addition, the positive PIK3CA-E9 mutation rate was highest in whole blood + tissue samples and patients with squamous cell carcinoma, while the E20 mutation was highest in whole blood samples.
The current study showed that the positive mutation rates in EGFR, KRAS, and PIK3CA gene exon loci were different which had a correlation with different clinical indicators. These findings may provide significance for the direction and predictability of clinical lung cancer treatment. With respect to the diagnosis and treatment of lung cancer, further understanding of these biomarkers can be used to assess the risk of tumor occurrence, early diagnosis, formulation of treatment plan, and evaluation of patient prognosis. In recent years, because the importance of biomarkers has been gradually revealed by scientists, more and more new biomarkers have been investigated and applied in the clinical setting. Therefore, better illustrating the significance of each biomarker in the diagnosis, treatment, and prognosis of lung cancer could potentially reduce the mistakes made in the early diagnosis of cancer patients and improve the efficacy of personalized treatment plans for cancer patients.
There were some limitations in this study. First, this was a single-center study with a small size of patients with lung cancer. Second, there were few patients with KRAS gene mutations in exons E3 (n=2) and E4 (n=0) or with PIK3CA gene mutations in exon E20 (n=1), which may result in inaccurate conclusions for these cohorts. Therefore, further investigations with a large sample size, as well as a comparison of samples in multiple centers, are needed to confirm the current findings. Third, the comparison among the different mutations and the relationship with clinical characteristics was not performed. Finally, the mechanism underlying these gene mutations and the correlation with clinical indicators, especially patient staging, are not fully elucidated, which requires further in vitro and in vivo studies.