Small cell lung cancer (SCLC) is an aggressive cancer with high recurrence rate after conventional chemotherapeutic treatment. SCLC presents unique biological characteristics, high genetic mutation load, dysregulation of tumor suppressor genes and oncogenes, and upregulated level of receptor tyrosine kinases, growth factors, and cellular markers. Currently, the analyses of genomic aberrations and searching new molecular-targeted drugs for SCLC are underway. However, only a few studies have investigated the molecular map of SCLC, and to date, none of the molecular-targeted drugs have exhibited clinical activity against SCLC. Reportedly, epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) has been successfully being used in SCLC in the event of seldom EGFR mutation seldom occurs (1,2). Although the EGFR mutations have been previously identified in SCLCs, those in the other EGFR signaling pathway genes, such as Kirsten rat sarcoma viral oncogene homolog (KRAS), human EGFR 2 (HER2), and v-raf murine sarcoma viral oncogene homolog B1 (BRAF) are yet unknown. Thus, additional studies are needed to determine the molecular characteristics of SCLC and seek other new effective targeted therapies.
The phosphoinositide 3-kinase (PI3K)/AKT pathways are vital for the proliferation, energy metabolism, cellular architecture advantages, and survival of malignant cells. Intriguingly, the suppression of the PI3K-AKT-mammalian target of rapamycin (mTOR) pathway through the lipid phosphatase activity activates phosphatase and tensin (PTEN) homolog gene that regulates a plethora of cellular processes. Moreover, the PI3K/PTEN pathway functions downstream of the receptor tyrosine kinases, including EGFR. Currently, two studies have used the targeted gene panels to identify the two common genetic aberrations in patients with SCLC: phosphatidylinositol-3-kinase catalytic subunit alpha (PI3KCA; 15%), and MET (4.4%) (3,4). Another analysis of 98 cancer patients using next-generation sequencing demonstrated that the most common aberrations occurred on the rapamycin-insensitive companion of mTOR (RICTOR; 10%), PI3KCA (6%) EGFR (5%), PTEN (5%), and KRAS (5%) (5). The identification of molecular aberrations of SCLC might facilitate the targeted therapies for the SCLC treatment.
The present study aimed to investigate the genomic aberrations in patients with SCLC in a Chinese cohort. The mutations in SCLC primarily encompass the EGFR/Ras/Raf pathway and PIK3CA/PTEN pathway genes and provide a novel potential target for SCLC. Furthermore, the identification of genomic aberrations linked to SCLC would facilitate the recognition of potential targets. A total of 5 genes (EGFR E18, EGFR E19, EGFR E20, EGFR E21, KRAS E2, BRAF E15, PTEN E5, PTEN E6, PTEN E8, PIK3CA E9, PIK3CA E20) were detected using direct sequencing in SCLC tumor samples.
A total of 30 SCLC patients treated at the Zhejiang Cancer Hospital between November 2012 and November 2016 were recruited for this study. The pathological diagnosis of SCLC in each case was based on the standard criteria defined by WHO classification. The median age of the study group was 59 (range, 35–79) years. The cohort consisting of 28 (93.3%) males and 2 (6.7%) females was further categorized into 4 non-smokers, 1 light smoker, 8 moderate smokers, and 17 heavy smokers. The stages involved were as follows: 20 were extended stage and 10 were limited stage. The study was approved by the Medical Ethics Review Committee of Zhejiang Cancer Hospital (Hangzhou, China). The patient samples of SCLC were consecutively collected, and all participants provided written informed consent.
DNA extraction and PCR amplification (direct sequencing)
The specimens from fine-needle biopsy or surgical operations were fixed using formalin and embedded in paraffin. Genomic DNA was isolated from 10 g-paraffin sections using DEXPAT® D9091 Kit (TaKaRa, Dalian, China) according to the manufacturer’s instructions. The target DNA fragment containing mutation loci, including EGFR exon 18, EGFR exon 20, EGFR exon21, KRAS exon2, BRAF exon15, PTEN exon5, PTEN exon6, PTEN exon8, PIK3CA exon9, PIK3CA exon20, was amplified using PCR. The amplification and sequencing primers were designed (Table 1) and synthesized by Invitrogen (Shanghai Invitrogen Biotechnology Co., Ltd, Shanghai, China). Primers with AF/AR post-fixed were the amplification primers, and those with SF post-fixed were the sequencing primers. The 25-µL PCR reaction system comprised of 12.5 µL of PrimeStar® Premix (TaKaRa), 2.5 µL of DNA sample, 1.5 µL of 10 µmol/mL AF and AR primers, and 7 µL of ddH2O. The PCR reaction was as follows: an initial denaturation step at 94 °C for 1 min, followed by 35 cycles of denaturation at 94 °C for 10 s, annealing at 58 °C for 5 s, elongation at 72 °C for 15 s, and final extension at 72 °C for 5 min; the products were held at 4 °C. The products were analyzed and identified by 2% agarose gel electrophoresis.
PCR product purification
The PCR products were purified in a reaction system consisting of 25 µL PCR products, 2 µL exonuclease I, and 5 µL alkaline phosphatase (ThermoFisher Scientific Inc., Waltham, USA). The reaction mixture was incubated at 37 °C for 30 min, inactivated at 85 °C for 15 min, and stored under cold conditions until further usage.
The purified PCR products were sequenced using BigDye® Terminator V3.1 Cycle Sequencing Kit (ABI, Foster City, USA). The 20 µL reaction system consisted of 6 µL of 10 mol/mL sequencing primer, 8 µL of 2.5× BigDye, 4 µL of ddH2O, and 2 µL of the purified PCR product. The sequencing reaction conditions were as follows: an initial pre-denaturation at 96 °C for 60 s, followed by 25 cycles of denaturation at 96 °C for 10 s, annealing at 50 °C for 5 s, and elongation at 60 °C for 4 min.
The sequencing was conducted on an ABI 3130XL genetic analyzer (ABI) after the purification of the sequencing reaction products. The sequencing data were collected using the 3130XL software and analyzed by Sequencing Analysis software v5.4.
The statistical significance of the mean values was determined by SPSS 22.0. A P value <0.05 indicated a statistically significant difference.
Genomic aberrations of SCLC
We detected 10 genomic aberrations in 30 cases (33.3%): an EGFR mutation (n=6, E19, E21), a KRAS mutation (n=1, E2), PIK3CA mutations (n=1, E20), a PTEN mutation (n=2, E5, E8) (Tables 2,3). In addition, 3 cases of synonymous mutation included 2 cases of EGFR E20, 1 case of E18, and 1 case of PIK3CA E9.
Comparison of patient characteristics classified by genomic aberrations
The patient characteristics are classified according to the status of the genomic aberrations (Table 4). No significant differences were detected in age, sex, disease extent, smoking history, and carcinoembryonic antigen (CEA) and squamous cell lung carcinoma (SCC) level between patients with and without genomic aberrations (P>0.05).
Furthermore, no significant differences were detected in age, sex, disease extent, smoking history, and CEA and SCC level in patients with and without EGFR mutation (P>0.05, Table 5).
Cases of common mutations
EGFR E19 c.2235–2249 del 15: EGFR codes 746 for glutamate to 750 for alanine loss
This variation, localized in the EGFR protein kinase domain, is a common variant of EGFR exon 19 (6). The E746_A750 Del mutation was reported to maintain the activity of EGFR, promote the MAPK and AKT phosphorylation downstream, and promote the tumor growth in transplanted mice (7,8) (Figure 1A,B,C).
EGFR E19 c.2239–2256 del 18 (p.L747_S752del)
Cases of rare mutations
PTEN E5 461delT
PTEN p.F154fs*5, PTEN 154th codon, wherein phenylalanine becomes serine, and according to the new open reading frame, 5th codon is the termination codon. The prediction analysis of this mutation led to amide-mediated mRNA degradation, resulting in the loss of protein expression. The mutation was detected in gliomas, according to the Cosmic database (Figure 2A).
KRAS c.35G>A (p.G12D)
P. G12d, KRAS 12th code subglycine mutation to aspartic acid. KRAS 12D mutated GTP enzyme activity was weaker than wild-type KRAS, which enhanced the binding state of active KRAS-GTP and promoted the KRAS signaling pathway to cause cancer (10,11) (Figure 2B).
EGFR E19 c.2209_2219delinsCCACACTGGGA (p.K737_I740delinsPHWD)
The 713th–740th coding positions of EGFR are deleted and inserted with proline, histidine, tryptophan, and aspartic acid. This mutation is located in exon 19 of EGFR, and no such mutation has been reported (Figure 2C).
EGFR E21 c.2547_2548delinsTA (p.Q849_H850delinsHN)
Histidine and asparagine were inserted after the deletion of the coding sequence at positions 849-950 of EGFR (Figure 2D).
Cancer gene therapy has low toxicity than conventional chemotherapy, but no targeted drugs have been approved for the treatment of SCLC. The application of genomic technologies has begun to elucidate a large number of genetic abnormalities in SCLC. In a Japanese cohort, genomic aberrations were detected in 15% patients, including PIK3CA amplification (10%), PIK3CA mutations (5%), EGFR mutation (2%), and KRAS mutation (2%). However, in the Chinese population, the genomic aberrations in SCLC are specific. The present study demonstrated that 10 patients had genomic aberrations; EGFR mutation accounted for 60% of the patients, while PTEN mutations were found in 2 patients (20%).
The EGFR mutations found in the current study included E19 and E21, of which, E19 mutation occurred in 6 SCLC patients. Interestingly, EGFR mutations that commonly occurred in exon 19 were deletions, and those in exon 21 (L858R) were mutations. Also, in these 30 SCLC patients, rare EGFR-mutated exon 18 was found. The mutation rate of EGFR in our study was higher than in the Japanese patients, i.e., in 4% of 122 Japanese patients with SCLCs. This phenomenon might be associated with small sample size in the current research. Furthermore, patients with the p.E746_A750del variant in non-SCLC (NSCLC) are sensitive to EGFR TKI (6). Preclinical data and clinical trial results showed that exon insertion or deletion mutation 19 of EGFR was sensitive to EGFR TKI (12-14). Moreover, patients with NSCLC with L747_S752del mutation benefit from the EGFR TKI treatment (15,16).
The Ras oncogene controls several cellular functions including cell proliferation, apoptosis, migration, and differentiation, while the KRAS mutations are noted in >20% of all cancers. In this study, KRAS E2 mutation occurred in one patient (3.3% in all SCLCs), and this variation was mainly carcinogenic as identified via the PI3K-AKT, JNK, p38, and FAK signaling pathways (17,18).
PI3K might be activated by the receptor kinases and Ras, which in turn, activates AKT. However, the PI3K and EGFR signaling pathways closely interact, and PI3K signaling pathway is effectuated by additional activators and downstream targets (4). Herein, we found 1 patient with PIK3CA mutations (E20), and 2 patients harbored the PTEN mutation (E5, E8).
Furthermore, we compared the clinical characteristics between SCLC patients with and without genomic aberrations as well as between SCLC patients with and without EGFR mutation. However, no significant differences were detected in age, sex, disease extent, smoking history, and CEA and SCC level in either of the groups, which might be attributed to the small sample size as one of the underlying factors.
Nevertheless, the genomic aberrations of SCLC identified in this study offered mutational data to clinicians that might be applied for assigning patients to appropriate clinical study, especially the anti-EFGR treatment.
Funding: This work was supported by the Zhejiang Province Medical Science Project of China (2015ZHA006), the 1022 Talent Training Program of Zhejiang Cancer Hospital, and the Zhejiang Provincial National Science Foundation of China (Q18H290004).
Conflicts of Interest: The authors have no conflicts of interest to declare.
Ethical Statement: The study was approved by the Medical Ethics Review Committee of Zhejiang Cancer Hospital (Hangzhou, China). The number of ethics approval is -10-20. Written informed consent was obtained from the patient for publication of this manuscript and any accompanying images.
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