Lung cancer is one of the main leading causes of cancer death, and the most common cancer after prostate and breast cancers, in respect to prevalence [1]. The 5-year overall survival is 15.0% in patients with non-small cell lung cancer (NSCLC) [1]. Overall survival rates increase with surgical, chemotherapy and radiotherapy treatments in early stages of the cancer [2]. Recently, immunotherapy and molecular targeted therapy have been used to treat cancer patients. Erlotinib and gefitinib, which are the inhibitors of epidermal growth factor receptor (EGFR) tyrosine kinases are molecular targeted agents. These agents have been used to inhibit certain EGFR tyrosine kinase mutations. Both of them can pass through the phospholipid membrane into the cell, and these agents compete with adenosine triphosphate (ATP) to bind the ATP binding pocket of EGFR. The EGFR, a transmembrane glycoprotein of 170 kDa consisting 1186 amino acids, is localized in the 7 q12 chromosome region. It is known that the EGFR receptor has mutations in the tyrosine kinase region of 18-21 exons in NSCLC cells and correlate therapy response. These EGFR mutations are more common in Asian, Caucasian and non smoking women [4]. K-ras, a downstream protein, becomes constantly active as a result of EGFR auto-phosphorylation. The Kirsten ras sarcoma (
In our study, we investigated the frequency of EGFR and KRAS mutations and effects of anti-EGFR tyrosine kinase inhibitors treatment in Turkish NSCLC patients. This is the first large-scale study that investigates EGFR and KRAS mutations in 300 patients with NSCLC in Turkey.
Clinicopathological characteristics of the patients.
Characteristics | Patients ( |
---|---|
Age: | |
≤62 years | 148 (49.3) |
≥62 years | 152 (50.7) |
Gender: | |
males | 238 (50.7) |
females | 62 (20.7) |
Smoking habit: | |
smoker | 233 (74.3) |
never smoked | 77 (25.7) |
Histological type: | |
adenocarcinoma | 228 (76.0) |
squamous | 62 (20.7) |
other | 10 (3.3) |
Stage: | |
early (I; II; IIIA) | 70 (23.3) |
advanced (IIIB; IV) | 230 (76.7) |
After validation of the PCR products on the gel, 40 μL of the products were bound to streptavidin Sepharose HP (GE Healthcare, Waukesha, WI, USA), purified, washed, and denatured using a 0.2 mol/L sodium hydroxide solution, and washed again. Subsequently, 0.3 μmol/L pyrose-quencing primers were annealed to the purified single-stranded PCR products, and the pyrosequencing was performed on a PyroMark ID system (Qiagen GmbH), according to the manufacturer’s instructions and analyzed in the AQ mode of the PyroMark (Qiagen GmbH) software.
Detailed clinicopathological characteristics of patients with EGFR and KRAS mutations.
Characteristics | Patients | EGFR Mutations (32.33%) | KRAS Mutations (25.0%) | EGFR and KRAS Mutations (6.6%) | |||
---|---|---|---|---|---|---|---|
( | [+] ( | [–] ( | [+] ( | [–] ( | [+] ( | [–] ( | |
Age: | |||||||
≤62 years | 148 | 50 (32.9%) | 102 (67.1%) | 32 (21.1%) | 120 (78.9%) | 7 (4.6%) | 145 (95.4%) |
≥62 years | 152 | 47 (31.8%) | 101 (68.2%) | 43 (29.1%) | 105 (70.9%) | 13 (8.8%) | 135 (91.2%) |
Gender: | |||||||
males | 238 | 67 (28.2%) | 171 (71.8%) | 63 (26.5%) | 174 (73.5%) | 16 (6.7%) | 222 (93.3%) |
females | 62 | 30 (48.3%)a | 32 (51.7%) | 12 (19.4%) | 50 (80.6%) | 4 (6.5%) | 58 (93.5%) |
Smoking habit: | |||||||
smoker | 233 | 59 (26.5%) | 164 (73.5%) | 58 (26.0%) | 165 (74.0%) | 11 (4.9%) | 212 (88.4%) |
never smoked | 77 | 38 (49.4%)a | 39 (50.6%) | 17 (22.0%) | 60 (78.0%) | 9 (11.6) | 68 (88.4%) |
Histological type: | |||||||
adenocarcinoma | 228 | 77 (33.7%)a | 151 (66.3%) | 55 (24.1%) | 173 (75.9%) | 16 (7.0%) | 212 (93.0%) |
squamous | 62 | 13 (21.0%) | 49 (79.0%) | 18 (29.0%) | 44 (71.0%) | 2 (3.2%) | 60 (96.8%) |
other | 10 | 7 (70.0%) | 3 (30.0%) | 2 (20.0%) | 8 (80.0%) | 2 (20.0%) | 8 (80.0%) |
Stage: | |||||||
early (I; II; IIIA) | 70 | 18 (25.7%) | 52 (74.3%) | 16 (22.8%) | 54 (77.2%) | 2 (2.8%) | 68 (97.2%) |
advanced (IIIB; IV) | 230 | 79 (34.3%) | 151 (65.7%) | 59 (25.6%) | 171 (74.4%) | 18 (7.8%) | 212 (92.2%) |
a Found to be statistically significant.
(A) Types of EGFR mutations in Turkish NSCLC patients.
Mutation | Exon | Nucleotide Number | Amino Acid Changes | Mutation |
---|---|---|---|---|
Point mutation | 18 | 2155 (G>T) | G719C | 1 (0.8%) |
Point mutation | 18 | 2134 (G>A) | G719S | 3 (2.5%) |
Point mutation | 18 | 2155 (G>A) | G719A | 9 (7.6%) |
Deletion | 19 | 235-2249del (GGA ATT AAG AGA AGC) | delE746-A750 | 39 (33.4%) |
Del/Ins | 19 | 2235-2252del (GGA ATT AAG AGA AGC insCA) | L747-6751insP | 10 (8.5%) |
Del/Ins | 19 | 2235-2252del (GGA ATT AAG AGA AGC insAAT) | delE746-T751insI | 5 (4.2%) |
Del/Ins | 19 | 2235-2258del (GGA ATT AAG AGA AGC insAAT CCA) | delE746-A750insIP | 1 (0.8%) |
Del/Ins | 19 | 2235-2249del (GGA ATT AAG AGA AGC insAGA) | delL747-P753insS | 3 (2.5%) |
Del/Ins | 19 | 2235-2258del (GGA ATT AAG AGA AGC insAAT) | delE746-A750insI | 3 (2.5%) |
Del/Ins | 19 | 2235-2258del (GGA ATT AAG AGA AGC insCCA) | delA746-A750insP | 1 (0.8%) |
Del/Ins | 19 | 2235-2258del (GGA AAT AAG AGA AGC insCAA) | delL747-T751insQ | 1 (0.8%) |
Point mutation | 20 | 2369 (C>T) | T790M | 3 (2.5%) |
Point mutation | 21 | 2573 (T>G) | L858R | 10 (8.5%) |
Point mutation | 21 | 2582 (T>A) | L861Q | 29 (24.6%) |
Del/Ins: deletions and insertions.
We detected both EGFR-KRAS mutations in 20 patients, four females and 16 male patients. Histopathologically, two of them were squamous, 16 were adenocarcinoma and two were in other types. Two were in early stage, 18 were in advanced stages, 13 of them were older than 62. Eleven of them are smokers, while nine are not. Detailed information is given in Table 2.
(A) The progression-free survival rate was statistically significantly higher in patients carrying EGFR mutations receiving EGFR-TKI therapy in comparison to patients having no such treatment. (B) The overall survival rate was statistically significantly higher in patients carrying EGFR mutations receiving EGFR-TKI therapy in comparison to patients having no such treatment.
Twenty patients who have EGFR-KRAS mutations and were not treated with erlotinib, had the lowest survival rate (34 ± 16 weeks) (
(A) The overall survival rate was statistically significantly higher in patients not carrying the KRAS mutations receiving EGFR-TKI therapy in comparison to patients having no such treatment. (B) The overall survival rate was statistically significantly higher in patients carrying the KRAS mutations receiving EGFR-TKI therapy in comparison to patients having no such treatment.
The overall survival rate patients treated with EGFR-TKI without progression was also found to be significantly higher. Patients who were treated with EGFR-TKI: 288 ± 11 and those who were not: 119 ± 11 weeks (
Non-small cell lung cancer has different subtypes, mainly adenocarcinoma, squamous and other subtypes. Of these, adenocarcinoma is seen more often in patients with NSCLC [10, 11, 12, 13]. Previous studies that showed correlation can be found in literature regarding to the subtypes of NSCLC in the Turkish population [9,14]. In this study, we found 76.0% adenocarcinoma, 20.7% squamous, 3.3% other subtypes in 300 patients. The distribution of gender has been reported in the literature as: in East Asian communities 54.0-65.0% males, 35.0-46.0% females [10], in England 55.0% males, 45.0% females [1], in America 58.0% males 42.0% females [1]. Two previous studies performed in the Turkish population, found 85.0% males and 15.0% females by Unal
We detected EGFR mutations in 33.0% of all 300 patients and 84.0% of EGFR mutation-carrying patients had adenocarcinoma. Previous studies also showed similar results, 66.0 and 81.0% EGFR mutation rates, respectively [9,14]. In our study, we found EGFR mutations in 48.0% female patients and in 28.0% male patients. This result is similar to previous reports that female patients who had NSCLC have higher EGFR mutations [10, 11, 12, 13]. Regarding smoking habits, patients who do not smoke have more EGFR mutations [9, 10, 11, 12, 13, 14, 15]. In our study, we detected EGFR mutations in 49.0% of non smoking patients and in 28.0% of the smoking patients (Table 2). In the literature, EGFR mutations are seen more often in non smokers and female Asian patients [10]. Mutation rate also increases with advanced stages of NSCLC cancer (IIIB-IV) [10, 11, 12, 13]. In our study, similar results were obtained in 18.6% early stage and 81.4% advanced stage in NSCLC patients with EGFR mutations.
The KRAS mutation rates in NSCLC patients were reported as 2.3-9.4% in East Asian populations, 11.0-29.0% in North America, 12.0-31.0% in Europe, 25.0% in Mexico, 12.0% in Columbia and 12.0% in Peru [10, 11, 12]. In our research, we found 20.8% patients had KRAS mutations (Table 2). We detected 65.0% KRAS mutations in codons 12 and 13 and 35.0% in codon 64 [Table 3(B)].
(B) Types of KRAS mutations in Turkish NSCLC patients.
Mutation Types | Codon | Nucleotide Number | Amino Acid Changes | Mutations |
---|---|---|---|---|
Point mutation | 12 | 34 (G>A) | G12S | 10 (12.7%) |
Point mutation | 12 | 34 (G>T) | G12C | 8 (10.1%) |
Point mutation | 12 | 35 (G>A) | G12D | 6 (6.7%) |
Point mutation | 12 | 35 (G>T) | G12V | 20 (25.2%) |
Point mutation | 12 | 35 (G>A) | G12A | 1 (1.3%) |
Point mutation | 13 | 37 (G>A) | G13D | 1 (1.3%) |
Point mutation | 61 | 182 (A>G) | Q61R | 18 (22.8%) |
Point mutation | 61 | 183 (A>C) | Q61H | 15 (19.0%) |
We also found that 6.6% of the patients carry both EGFR and KRAS mutations (Table 2). In the literature, this rate varied between 1.0 and 15.0% [10, 11, 12]. This is the first study in the Turkish population showing KRAS mutations and erlotinib therapy in NSCLC patients.
Fifty-nine of 97 patients with EGFR mutations were treated with EGFR-TKI agent (erlotinib). The average overall survival rates are 146 ± 22 weeks in EGFR-TKI treated patients and 84 ± 12 weeks in untreated patients. Similar results were published in the literature that erlotinib therapy increases overall survival [16] [Figure 1(B)].
The effect of EGFR-TKI treatment on a patient’s survival without progression was found to be statistically significant. Erlotinib-treated patients’ survival rate was 288 ± 11 weeks and 119 ± 11 weeks in untreated patients. This result is in accordance with the literature [16]. EGFR-TKI treatment provided longer survival rates in patients carrying only EGFR mutations compared to the patients with both mutations [Figure 2(B)]. We report that the overall survival rates are 34 ± 16 weeks in patients with KRAS-EGFR mutations and 98 ± 16 weeks in EGFR mutations without KRAS mutations in erlotinib-treated patients. Thus, the longest overall survival rates were observed in patients with EGFR but without KRAS mutations, and the lowest overall survival rates were found in patient with EGFR and KRAS mutations in EGFR-TKI-treated patients. Therefore, this indicated that KRAS is a prognostic factor for NSCLC.
The mutations on the