Zhiming Jiang  ( Zhejiang Key Laboratory of Diagnosis & Treatment Technology, China. )
Chenghui Li  ( Department of Thoracic Medical Oncology, Zhejiang Cancer Hospital, China. )
Hongyang Lu  ( Zhejiang Key Laboratory of Diagnosis & Treatment Technology, China. )

Objective: To analyse clinical and molecular features in patients with surgically resected patients with lung cancer harbouring anaplastic lymphoma kinase fusion.

Methods: The retrospective study was conducted at Zhejiang Cancer Hospital, Hangzhou, China, and comprised data from November 2013 to August 2015 of lung cancer patients. Anaplastic lymphoma kinase, epidermal growth factor receptor, kirsten rat sarcoma viral oncogene, v-raf murine sarcoma viral oncogene homolog, REarranged during Transfection  proto-oncogene, c-ros oncogene 1 receptor kinase, V-Erb-B2 avian erythroblastic leukaemia viral oncogene homolog 2 and mesenchymal epithelial transition factor were noted using next generation sequencing. Clinicopathological parameters were also investigated. All patients were followed up till August 10, 2017. Data was analysed using SPSS 22.

Results: Of the 19 patients, 15(79%) were non-smokers. Anaplastic lymphoma kinase rearrangements occurred in the acinar predominant in 6(31.6%), solid predominant 6(31.6%) and mucinous predominant 4(21%) adenocarcinomas. There was 1(5.2%) patient with epidermal growth factor receptor 21 G863D mutation. The 3-year disease-free survival rate in 5(26.3%) cases of anaplastic lymphoma kinase variant 1 was 5(100%), while in the 14(73.7%) cases of non-variant 1 group it was 9(64.3%) (p=0.257).

Conclusion: Anaplastic lymphoma kinase rearrangements did not tend to be accompanied with other driver genes.  Difference between variant 1 and non-variant 1 patients was uncertain and needs to be further investigated.

Keywords: Lung cancer, ALK genotype, Next generation sequencing, Disease-free survival, Molecular characteristic. (JPMA 71: 531; 2021)

DOI: https://doi.org/10.47391/JPMA.1295

 

Introduction

 

Echinoderm microtubule-associated protein-like4 (EML4)-anaplastic lymphoma kinase (ALK) has been identified as one of driver genes in non-small-cell lung cancer (NSCLC) in preclinical experiments where inhibition of EML4-ALK was found to be leading to apoptosis of tumour cells expressing corresponding fusion protein.¹ EML4-ALK fusion transcript is found in approximately 4-5% of NSCLCs.¹ In most cases, ALK rearrangements are considered to be mutually exclusive with epidermal growth factor receptor (EGFR) and Kirsten rat sarcoma viral oncogene homolog (KRAS) mutations.² ALK inhibitors have shown to be more effective for lung cancer patients with ALK rearrangements compared to chemotherapeutic drugs. In comparison with standard firstline chemotherapy -- pemetrexed combined with platinum -- crizotinib is considered a superior choice for treating ALK-positive NSCLC patients.³ The benefits of crizotinib include symptom improvement and better life quality.^4,5 Furthermore, patients with brain metastases treated with crizotinib had a significantly higher intracranial disease control rate compared to those treated with chemotherapy.⁶

Crizotinib has shown to be more efficient for patients with ALK variant 1 versus non-variant 1; median progression-free survival (PFS) of patients with ALK variant 1 treated with crizotinibi iwas significantly longer compared to those with ALK non-variant 1 (11.0 months vs 4.2 months, respectively; p< 0.05), which suggests that the efficacy of ALK inhibitors might be sensitive to the structurally diverse ALK kinase inhibitors, such as crizotinib and TAE684, varies in patients with dissimilar ALK fusion genes and EML4-ALK variants, and the differential sensitivity is related to the difference in protein of EML4-ALK expression cells.⁷ The data could partly explain the phenomenon that ALK-positive tumours exhibit diverse responses to ALK inhibitors in clinical practices. Consequently, targeted therapy for patients with lung cancer harbouring ALK positivity should be further selected according to precise ALK genotype.⁸ Moreover, it appears necessary to clear ALK genotype in lung cancer harbouring ALK fusion protein. It still remains unclear whether there is any difference among patients with resected lung cancer and different ALK fusion genes; especially with reference to disease free survival (DFS), as well as which patients are easier to relapse; those with ALK variant 1 or non-variant 1.

Oncogenic drivers in NSCLC are considered mutually exclusive. Nevertheless, some studies have revealed that EGFR mutations and ALK rearrangements can  concomitantly occur.^9-11 Both EGFR inhibitors and ALK inhibitors have durable effect on patients harbouring both gene alterations  in the second line once the tumour has progressed.^9,10 EGFR mutations and ALK rearrangements may coexist in the same tumour cells.¹⁰ Li et al. have reported no EGFR/KRAS mutation in seven EML4-ALK-positive patients.¹² Furthermore, Shaozhang et al. found no EGFR mutations in their patients; while KRAS mutation was observed only in one patient.¹³ Moreover, a concurrent EGFR or KRAS mutation was detected in one among 200 cases of ALK-positive patients.¹¹

The current study was planned to investigate clinical and molecular characteristics and prognostic value in postoperative patients with lung adenocarcinoma harbouring ALK fusion protein variant 1 or non-variant 1.

 

Materials and Methods

 

The retrospective study was conducted at Zhejiang Cancer Hospital, Hangzhou, China, and comprised data from November 2013 to August 2015 of lung cancer patients. After approval from the institutional ethics review committee, ALK-positive specimens were collected that were were diagnosed by immunohistochemistry (IHC) using a highly sensitive anti-ALK (D5F3) rabbit monoclonal primary antibody (Ventana Medical Systems Inc, Roche, Inc., Tuscon, AZ). They were analysed using OptiView Amplification Kit and an OptiView DAB IHC Detection Kit (Ventana Medical Systems Inc, Roche, Inc., Tuscon, AZ). All specimens were obtained from resected lung cancer tumours. Pathological diagnosis was based on the standard criteria defined by the World Health Organisation (WHO).¹⁴ The breakdown of the stages was defined by the eighth edition of the Tumour, Node, Metastasis (TNM) classification for lung cancer.¹⁵

All stage IA patients with lung adenocarcinoma received no adjuvant chemotherapy; 1 stage IB patient received four cycles of pemetrexed plus cisplatin as adjuvant chemotherapy; and all stage IIA, IIB and IIIA patients received four cycles of adjuvant chemotherapy with gemzar combined with cispaltin or pemetrexed combined with cisplatin, and among them a patient with stage IIIA underwent adjuvant thoracic radiotherapy. Clinicopathological features including gender, age, stages, pathological types, smoking history and adjuvant treatment were noted.

Next-generation sequencing (NGS) was applied to detect ALK, EGFR, KRAS, v-raf murine sarcoma viral oncogene homolog (BRAF), Rearranged during Transfection proto-oncogene (RET), c-ros oncogene 1 receptor kinase (ROS1), V-Erb-B2 avian erythroblastic leukaemia viral oncogene homolog 2 (CerbB-2) and mesenchymal epithelial transition factor (MET).

For NGS, tissue deoxyribonucleic acid (DNA) was extracted using QIAamp DNA formalin-fixed, paraffin-embedded (FFPE) tissue kit (Qiagen) according to the manufacturer’s instructions. DNA concentration was measured using Qubit dsDNA assay.

For NGS library preparation, DNA shearing was performed using Covaris M220, followed by end-repair, phosphorylation and adaptor ligation. Fragments 200-400 bp in size were selected by bead (Agencourt Ampure XP Kit, Beckmann-Kurt, California, USA) followed by hybridisation with capture probes baits, hybrid selection with magnetic beads and polymerase chain reaction (PCR) amplification. A bioanalyser high-sensitivity DNA assay was then performed to assess the quality and size of the fragments and the indexed samples were sequenced on Nextseq500 sequencer (Illumina, Inc., California, US) with pair-end reads.

Genetic profiles of all tissue samples were assessed by performing capture-based targeted deep sequencing using the 8-gene panel (Burning Rock Biotech Ltd.), covering 76kb of human genome. DNA quality and size were assessed by high-sensitivity DNA assay using a bioanalyser. All the indexed samples were sequenced on a NextSeq 500 (Illumina, Inc., USA) with pair-end reads.

Sequence data was mapped to the human genome (hg19) using BWA aligner 0.7.10. Local alignment optimisation, variant calling and annotation were performed using GATK 3.2, MuTect, and VarScan. Plasma sample was compared against its own white blood cells (WBCs) to identify somatic variants. Variants were filtered using the VarScan fpfilter pipeline, with loci with depth <100 being filtered out. At least 2 and 5 supporting reads were used for  insertion or deletion of bases in the genome of organisms (INDELs) in plasma and tissue samples, respectively, while 8 supporting reads were used for  single nucleotide variants (SNVs) in both plasma and tissue samples. According to the Exac, 1000 genomes, Single Nucleotide Polymorphism Database (dbSNP), Exome Sequencing Project (ESP) 6500SI-V2 databases, variants with population frequency over 0.1% were grouped as SNP and excluded from further

analysis.^16-19 The remaining variants were annotated with ANNOVAR and SnpEff v3.6. DNA translocation analysis was performed using both Tophat 2 and Factera 1.4.3.

All patients were followed up till August 10, 2017. Survival time was calculated from the date of pathological diagnosis.

Data was analysed using SPSS 22. Overall data was screened and 3-year DFS was analysed using Fisher’s test. Log-rank test was used to estimate and compare DFS. P<0.05 was considered statistically significant.

 

Results

 

Of the 19 patients, 10(52.6%) were males and 9(47.4) were females. The overall media age was 56 years (interquartile range [IQR]: 33-66 years), and 15(79%) were non-smokers (Table 1).

ALK variant 1 was found in 5(26.3%) cases, variant 2 in 1(5.3%) case, variant 3 in 6(31.6%) cases, variant 5 in 2(10.5%) cases, and other variants in 5(26.3%) cases (Figure 1).

ALK rearrangements occurred in the acinar predominant in 6(31.6%), solid predominant 6(31.6%) and mucinous predominant 4(21%) adenocarcinomas. The amplification of CerbB-2 and MET, and negative fluorescence in situ hybridization (FISH) detection were observed in 1(5.3%) patient (Figure 2).

In 1(5.3%) patient, EGFR-21 G863D mutation was observed (Figure 3).

There were no mutations or amplifications of EGFR, KRAS, BRAF, RET, ROS1, CerbB-2 and MET in other patients (Table 2).

There were no differences in terms of gender, tumour stage, pathological type and smoking history between patients with ALK variant 1 and non-variant 1 (p>0.05). No relapse was observed in the patients with ALK variant 1, while it was observed in 4(28.6%) patients with non-ALK variant 1; 3(75%) of them relapsed due to brain metastasis. The 3-year DFS rate in the ALK variant 1 group was 5(26.3%), while it was 14(73.7%) in non-variant 1 group it was 9(64.3%) (p=0.257) (Table 3; Figure 4).

 

Discussion

 

The accuracy of Ventana (D5F3) IHC assay for detecting ALK rearrangement is nearly consistent with FISH in NSCLC patients.²⁰ In our cohort study, all specimens were collected from patients with positive ALK fusion protein tumour diagnosed using IHC (Ventana) technique. Furthermore, all ALK-IHC-positive specimens were confirmed by NGS. This suggested that application of IHC to detect EML4-ALK rearrangement could be preferentially recommended, while NGS may be considered in undefined cases. Reverse transcription PCR (RT-PCR) should be considered an alternative or supplemental method to detect ALK fusion oncogene in NSCLC patients.²¹ Our fata regarding patient age and gender distribution is in line with Li et al.¹¹ Findings of Inamura et al.²² and Sasaki et al²³ regarding different ALK variants are similar to our findings, which are different from the findings of another srudy.⁷

A study revealed that the acinar pattern is related to lung adenocarcinomas harbouring ALK rearrangements in Asian populations,²² whereas in the Western patients, the signet-ring cell histology is more frequently reported.²⁴ The current study indicated that ALK rearrangement frequently occurred in the acinar predominant, solid predominant and mucinous predominant tumours. Additionally, the study supports the difference between pathological subtype of ALK-rearranged lung adenocarcinomas in Asian populations and in the Western patients. Moreover, most of our patients were non-smokers, which is consistent with earlier findings.²⁵

First-line profiling of using broad, hybrid capture-based NGS testing for lung adenocarcinomas is a more comprehensive and efficient strategy compared to non-NGS testing.²⁶ A more precise and clinically useful classification for lung adenocarcinoma at the molecular level may be brought about by targeted NGS.²⁷ Traditional capillary-based single-gene sequencing using first-generation technique, known as Sanger sequencing, has been replaced by NGS since it allows massive parallel sequencing with lower cost and higher output.²⁸ Metastatic breast cancer patients harbouring human epidermal growth factor receptor 2 (HER2) positivity can clinically benefit from trastuzumab combined with chemotherapy. HER2 3+/FISH-positive NSCLC patients could also benefit from trastuzumab.^29,30 A durable response to crizotinib in ALK-negative NSCLC patients with de novo MET amplification has also been observed, indicating that crizotinib is also a MET inhibitor.³¹ EGFR mutations and ALK translocations can coexist in part of NSCLCs, while coexistent prevalence of both increases in line with sensitivity of applied detection method for EGFR mutations. A study showed that EGFR and ALK alterations concomitantly occurred in 4.4% (4/91) of ALK-translocated NSCLCs, while detection ratio of concomitant EGFR mutations and ALK translocations increased to 8.8%, 12.1% and 15.4% when applying additional peptide nucleic acid real-time PCR, NGS and mutant-enriched NGS in 91 ALK-translocated NSCLCs, respectively.³² EGFR 21 (G863D) is very rare and it has even been reported in gastric cancer and thymoma.^33,34 In our study, amplification of CerbB-2 and MET was observed in only one patient, with very low and negative abundance for FISH detection. EGFR 21 (G863D) mutation was observed in another patient. Consequently, we assume that patients with ALK rearrangement rarely have other driver gene mutations or amplifications. This indicates that patients with advanced NSCLC harbouring ALK rearrangements may not need further detections for other driver genes alternations such as EGFR, MET, KRAS, ROS1, BRAF and RET, though parallel sequencing is still strongly recommended.

A study has suggested that overall survival for NSCLC patients with ALK positive is inferior compared to ALK-negative patients following surgical resection.³⁵ In addition, five-year risk of progression or recurrence is doubled for patients with ALK-positive tumours compared with ALK-negative tumours. In our cohort, there were no patients who relapsed in the ALK variant 1 group, while four patients relapsed due to brain metastasis in ALK non-variant 1 group. It can be deduced that patients with ALK non-variant 1 the relapse easily occurs, especially brain metastasis. Based on the limited sample size, there was no statistical significance in neither 3-year DFS rate nor DFS between the two groups, while improvement trends in 3-year DFS rate and DFS were observed in ALK variant 1 group. Further studies with larger sample size are necessary to verify these findings. A randomised phase II study has shown that adjuvant chemotherapy of pemetrexed plus carboplatin followed by gefitinib could improve DFS in postoperative stage IIIA-N2 NSCLC patients harbouring EGFR mutations.³⁶ Since ALK inhibitors have shown effectiveness in advanced NSCLC patients with ALK positivity, the application of ALK inhibitors in ALK-positive patients with surgically resected NSCLC is a potential therapeutic option to improve DFS and to lessen the risk of brain metastasis. Postoperative adjuvant crizotinib treatment for patients with lung adenocarcinoma patients harbouring ALK needs to be evaluated. Since patients with ALK variant 1 respond better to crizotinib compared to patients with non-variant 1, future studies should investigate whether ALK variant subtype could be used as a biomarker for population selection in ALK inhibitor adjuvant therapy for resected lung adenocarcinoma with ALK positivity. Studies with larger sample size are recommended to examine the role of ALK inhibitors in adjuvant treatments.

 

Conclusion

 

Patients with ALK rearrangement rarely had other driver gene mutations or amplifications. NSCLC patients harbouring ALK variant 1 tended to have better prognosis compared to those with ALK non-variant 1.

 

Disclaimer: None.

Conflicts of interests: None.

Source of Funding: Zhejiang Province Medical Science Fund Project of China; Zhejiang Province Public Welfare And Technology Application Project of China; Zhejiang Provincial Natural Science Foundation of China; and 1022 Talent Training Program of Zhejiang Cancer Hospital.

Ethical conduct of research: This study was approved by the Medical Ethical Committee of Zhejiang Cancer Hospital and the ethics committee reference number is IRB-2016-86. The specimens were firstly obtained from Biological Sample Bank of Zhejiang Cancer Hospital which signed the written informed consent before surgery. Exempt written informed consent was also approved in this retrospective study by the Medical Ethics Committee of Zhejiang Cancer Hospital.

 

References

 

1.      Soda M, Choi YL, Enomoto M, Takada S, Yamashita Y, Ishikawa S, et al. Identification of the transforming EML4-ALK fusion gene in non-small-cell lung cancer. Nature 2007; 448: 561-6.

2.      Gainor JF, Varghese AM, Ou SH, Kabraji S, Awad MM, Katayama R, et al. ALK rearrangements are mutually exclusive with mutations in EGFR or KRAS: an analysis of 1,683 patients with non-small cell lung cancer. Clin Cancer Res 2013 1; 19: 4273-81.

3.      Solomon BJ, Mok T, Kim DW, Wu YL, Nakagawa K, Mekhail T, et al. First-line crizotinib versus chemotherapy in ALK-positive lung cancer. N Engl J Med 2014; 371: 2167-77. 

4.      Blackhall F, Kim DW, Besse B, Nokihara H, Han JY, Wilner KD, et al. Patient-reported outcomes and quality of life in PROFILE 1007: a randomized trial of crizotinib compared with chemotherapy in previously treated patients with ALK-positive advanced non-small-cell lung cancer. J Thorac Oncol 2014; 9:1625-33.

5.      Toyokawa G, Seto T, Takenoyama M, Ichinose Y. Insights into brain metastasis in patients with ALK+ lung cancer: is the brain truly a sanctuary? Cancer Metastasis Rev 2015; 34: 797-805.

6.      Solomon BJ, Cappuzzo F, Felip E, Blackhall FH, Costa DB, Kim DW, et al. Intracranial Efficacy of Crizotinib Versus Chemotherapy in Patients With Advanced ALK-Positive Non-Small-Cell Lung Cancer: Results From PROFILE 1014. J Clin Oncol 2016; 34: 2858-65.

7.      Yoshida T, Oya Y, Tanaka K, Shimizu J, Horio Y, Kuroda H, et al. Differential Crizotinib Response Duration Among ALK Fusion Variants in ALK-Positive Non-Small-Cell Lung Cancer. J Clin Oncol 2016; 34: 3383-9.

8.      Heuckmann JM, Balke-Want H, Malchers F, Peifer M, Sos ML, Koker M, et al. Differential protein stability and ALK inhibitor sensitivity of EML4-ALK fusion variants. Clin Cancer Res 2012; 18: 4682-90.

9.      Baldi L, Mengoli MC, Bisagni A. Banzi MC, Boni C, Rossi G. Concomitant EGFR mutation and ALK rearrangement in lung adenocarcinoma is more frequent than expected: report of a case and review of the literature with demonstration of genes alteration into the same tumor cells. Lung Cancer 2014; 86: 291-5.

10.    Chiari R, Duranti S, Ludovini V, Bellezza G, Pireddu A, Minotti V. Long-term response to gefitinib and crizotinib in lung adenocarcinoma harboring both epidermal Grosth factor receptor mutation and EML4-ALK fusion gene. J Clin Oncol 2014; 32: e30-2.

11.    Li T, Maus MK, Desai SJ, Beckett LA, Stephens C, Huang E, et al. Large-scale screening and molecular characterization of EML4-ALK fusion variants in archival non-small-cell lung cancer tumor specimens using quantitative reverse transcription polymerase chain reaction assays. J Thorac Oncol 2014; 9: 18-25.

12.    Li Y, Li Y, Yang T, Wei S, Wang J, Wang M, et al. Clinical significance of EML4-ALK fusion gene and association with EGFR and KRAS gene mutations in 208 Chinese patients with non-small cell lung cancer. PLoS One 2013; 8: e52093.

13.    Shaozhang Z, Xiaomei L, Aiping Z, Jianbo H, Xiangqun S, Qitao Y. Detection of EML4-ALK fusion genes in non-small cell lung cancer patients with clinical features associated with EGFR mutations. Genes Chromosomes Cancer 2012; 51: 925-32.

14.    Travis WD, Brambilla E, Burke AP, Marx A, Nicholson AG, et al. WHO Classification of tumours of the lung, pleura, thymus and heart. 4th ed. Lyon: International Agency for Research on Cancer 2015: 9-96.

15.    Detterbeck FC, Boffa DJ, Kim AW, Tanoue LT. The Eighth Edition Lung Cancer Stage Classification. Chest 2017; 151:193-203.

16.    Karczewski KJ, Weisburd B, Thomas B, Solomonson M, Ruderfer DM, Kavanagh D, et al. The ExAC browser: displaying reference data information from over 60 000 exomes. Nucleic Acids Res 2017; 45: D840-5.

17.    1000 Genomes Project Consortium, Auton A, Brooks LD, Durbin RM, Garrison EP, Kang HM, et al. A global reference for human genetic variation. Nature 2015; 526: 68-74.

18.    Kitts A, Sherry S. The Single Nucleotide Polymorphism Database (dbSNP) of Nucleotide Sequence Variation. In: McEntyre J, Ostell J, editors. The NCBI Handbook. USA: National Center for Biotechnology Information; 2002.

19.    Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP), Seattle, WA. [Online] [Cited 2019 May 20]. Available from: URL :http://evs.gs.washington.edu/EVS/)

20.    Ma H, Yao WX, Huang L, Jin SH, Liu DH, Liu Y, et al. Efficacy of D5F3 IHC for detecting ALK gene rearrangement in NSCLC patients: a systematic review and meta-analysis. Oncotarget 2016; 7: 70128-42.

21.    Wang Y, Zhang J, Gao G, Li X, Zhao C, He Y, et al. EML4-ALK Fusion Detected by RT-PCR Confers Similar Response to Crizotinib as Detected by FISH in Patients with Advanced Non-Small-Cell Lung Cancer. J Thorac Oncol 2015; 10: 1546-52.

22.    Inamura K, Takeuchi K, Togashi Y, Hatano S, Ninomiya H, Motoi N, et al. EML4-ALK lung cancers are characterized by rare other mutations, a TTF-1 cell lineage, an acinar histology, and young onset. Mod Pathol 2009; 22: 508-15.

23.    Sasaki T, Rodig SJ, Chirieac LR, Jänne PA. The biology and treatment of EML4-ALK non-small cell lung cancer. Eur J Cancer 2010; 46:1773-80.

24.    Rodig SJ, Mino-Kenudson M, Dacic S, Yeap BY, Shaw A, Barletta JA, et al. Unique clinicopathologic features characterize ALK-rearranged lung adenocarcinoma in the western population. Clin Cancer Res 2009; 15: 5216-23.

25.    Shaw AT, Yeap BY, Mino-Kenudson M, Digumarthy SR, Costa DB, Heist RS, et al. Clinical features and outcome of patients with non-small-cell lung cancer who harbor EML4-ALK. J Clin Oncol 2009; 27: 4247-53.

26.    Drilon A, Wang L, Arcila ME, Balasubramanian S, Greenbowe JR, Ross JS, et al. Broad, Hybrid Capture-Based Next-Generation Sequencing Identifies Actionable Genomic Alterations in Lung Adenocarcinomas Otherwise Negative for Such Alterations by Other Genomic Testing Approaches. Clin Cancer Res 2015; 21: 3631-9.

27.    Han JY, Kim SH, Lee YS, Lee SY, Hwang JA, Kim JY, et al. Comparison of targeted next-generation sequencing with conventional sequencing for predicting the responsiveness to epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) therapy in never-smokers with lung adenocarcinoma. Lung Cancer 2014; 85:161-7.

28.    Wu K, Huang RS, House L, Cho WC. Next-generation sequencing for lung cancer. Future Oncol 2013; 9: 1323-36.

29.    Cappuzzo F, Bemis L, Varella-Garcia M. HER2 mutation and response to trastuzumab therapy in non-small-cell lung cancer. N Engl J Med 2006; 354: 2619-21.

30.    Gatzemeier U, Groth G, Butts C, Van Zandwijk N, Shepherd F, Ardizzoni A, et al. Randomized phase II trial of gemcitabine-cisplatin with or without trastuzumab in HER2-positive non-small-cell lung cancer. Ann Oncol 2004; 15: 19-27.

31.    Ou SH, Kwak EL, Siwak-Tapp C, Dy J, Bergethon K, Clark JW, et al. Activity of crizotinib (PF02341066), a dual mesenchymal-epithelial transition (MET) and anaplastic lymphoma kinase (ALK) inhibitor, in a non-small cell lung cancer patient with de novo MET amplification. J Thorac Oncol 2011; 6: 942-6.

32.    Won JK, Keam B, Koh J, Cho HJ, Jeon YK, Kim TM, et al. Concomitant ALK translocation and EGFR mutation in lung cancer: a comparison of direct sequencing and sensitive assays and the impact on responsiveness to tyrosine kinase inhibitor. Ann Oncol 2015; 26: 348-54.

33.    Liu Z, Liu L, Li M, Wang Z, Feng L, Zhang Q, et al. Epidermal growth factor receptor mutation in gastric cancer. Pathology 2011; 43: 234-8.

34.    Yoh K, Nishiwaki Y, Ishii G, Goto K, Kubota K, Ohmatsu H, et al. Mutational status of EGFR and KIT in thymoma and thymic carcinoma. Lung Cancer 2008; 62: 316-20.

35.    Yang P, Kulig K, Boland JM, Erickson-Johnson MR, Oliveira AM, Wampfler J, et al. Worse disease-free survival in never-smokers with ALK+ lung adenocarcinoma. J Thorac Oncol 2012; 7: 90-7.

36.    Li N, Ou W, Ye X, Sun HB, Zhang L, Fang Q, et al. Pemetrexed-carboplatin adjuvant chemotherapy with or without gefitinib in resected stage IIIA-N2 non-small cell lung cancer harbouring EGFR mutations: a randomized, phase II study. Ann Surg Oncol 2014; 21: 2091-6.