EGFR mutation heterogeneity and mixed response to EGFR tyrosine kinase inhibitors of non small cell lung cancer: a clue to overcoming resistance

Personalised medicine in non-small cell lung cancer (NSCLC) is a reality in our days and tumor genomic landscape is based on a single tumor biopsy results. Several studies have demonstrated that the presence of an EGFR activating mutation is predictive of benefit from reversible and irreversible EGFR tyrosine kinase inhibitor (TKI) in non-small cell lung cancers, with significant advantage compared to chemotherapy in progression free survival and response rate (RR) in first line. Among those EGFR mutant patients the tumor RR to first-line EGFR TKI is in the range of 58-84%, indicating that there are additional factors mediating the sensitivity of tumors to EGFR TKI (1-8). This phenomenon may be explained by heterogeneity in EGFR mutation status within an individual tumor. On the contrary to this theory, because the driver mutation is acquired in an early step of progression, subsequent clonal expansion distributes the mutation through the tumor. However, Gerlinger et al. (9) map out the remarkable intratumoral heterogeneity within a single renal cell cancer respect to somatic mutations in driver and passenger genes, which may foster tumor adaption and therapeutic failure via Darwinian selection. Intratumor heterogeneity may have important consequences for personalized-medicine approaches that commonly rely on single tumor-biopsy samples to portray tumor mutational landscape. This heterogeneity has been investigated regarding EGFR mutation in NSCLC.

Chen et al. (10) studied discordance in EGFR mutation status using direct DNA sequencing in paired samples of lung adenocarcinoma and regional lymph nodes or distant metastases in 180 Asian patients. In case of discordance between the primary tumor and the metastasis, results were confirmed using the high-resolution melting method (HRM). The overall discordance rate was 13.9%. Heterogeneity was significantly higher in patients with multiple pulmonary nodules (24.4%) than in patients with distant metastasis (14.3%), lymph nodes metastases (10.2%) or metachronous primary tumors (9.1%). Additionally, the discordance also was higher between paired samples from metachronous tumors (15.7%) than samples from synchronous tumors (7.5%). These results are in contrast to a study by Yatabe et al. (11) who did not find EGFR mutation heterogeneity by reverse transcriptase polymerase chain reaction among 77 EGFR mutant patients with paired primary and metastatic site samples or among 54 primary and recurrent tumor pairs. The authors also performed a transactional analysis of 50 lung adenocarcinomas carrying EGFR mutation. Three parts of each individual tumor were selected and examined for their EGFR mutation status and all three parts demonstrated identical mutations. Also, five tumors were dissected into more than 100 pieces and examined for EGFR status and again no EGFR mutation heterogeneity was found. The authors concluded that heterogeneous distribution of EGFR mutations is extremely rare and that pseudoheterogeneity is observed as a result of the use of less sensitive methods of detection. Other studies using heteroduplex analysis or Scorpion Amplification Refractory Mutation System (ARMS) method have reported EGFR mutation heterogeneity in the range of 16.8% to 27%, respectively (12). Tomonaga et al. (13) described intratumor heterogeneity of EGFR mutation by PCR in nine out of 38 patients with resected mixed-type lung adenocarcinoma and it was significantly associated with smoking history.

Recently, 45 tumors of patients with EGFR mutant stage IIIA-IV NSCLC with palliative surgery in which EGFR mutations were determined using Denaturing High Performance Liquid Chromotography and ARMS revealed 30% of intratumoral EGFR mutational heterogeneity, accompanying with low EGFR copy number. The prognosis of the patients was also related to the EGFR mutation heterogeneous status (14). These findings suggest that patients with advanced lung cancer harbor EGFR mutational heterogeneity and this heterogeneity might have clinical consequences in the efficacy of EGFR-TKI, and it could be a mechanism of resistance to EGFR TKI. Taniguchi et al. (15) demonstrated that those patients harboring heterogeneous tumors had a statistically significant decreased survival compared with those patients harboring mutation-positive tumors cells only after gefitinib treatment.

It is not well understood if systemic therapy may influence the expression of different biomarkers such as EGFR mutation in the tumor. In the Chen et al. (10) study, those patients that had received systemic therapy had a higher EGFR mutation discordance than those without exposure to any systemic therapy, suggesting potential mutagenic effects of chemotherapy. In a cohort of 264 advanced NSCLC patients, chemotherapy significantly decreased frequency of EGFR mutations from 34.5% in the prechemotherapy plasma samples to 23.1% in the postchemotherapy plasma samples (PEGFR mutation changes after chemotherapy were from mutant state to wild type (16). Notwithstanding these results, Rosell et al. (17) demonstrated no statistically significant differences in RR, PFS and OS in EGFR mutant patients receiving EGFR TKI in either first- or second-line setting. Also, data from the SATURN trial showed a compelling PFS HR of 0.10 in patients positive for EGFR mutation who received erlotinib as a maintenance treatment after standard chemotherapy (18). Chen et al. (10) also reported in a multivariable analysis that heterogeneity was significantly higher in patients with EGFR TKI exposure. The EGFR mutation heterogeneity accounted 8.9% of TKI-resistant cases. It is difficult to estimate whether discordance biomarker expression between pre and post treatment samples is due to a change in a biomarker status or simply a reflection of the pre-existing tumor genetic heterogeneity that can influence tumor phenotype after EGFR TKI treatment. Taniguchi et al. (15) tested EGFR mutation in multiples areas in 21 resected tumors, and six of them had both EGFR-mutated and wild type NSCLC cells. This fact could explain why patients with multiple pulmonary nodules had a higher heterogeneity in EGFR mutation status in the Chen et al. study (10).

EGFR mutant heterogeneity could explain mixed responses to EGFR TKI, suggesting that EGFR TKIs should be continued beyond progression in combination with other therapies such as chemotherapy to act in all cell clones that are part of the tumor in those patients. The IMPRESS study (NCT01544179) is currently evaluating the role of gefitinib combined with chemotherapy in patients with EGFR mutations that have progressed to gefitinib.

Tailored treatment in advanced NSCLC is going to improve in the next years based on new research on druggable biomarkers. Treatment of patients with advanced NSCLC and a positive biomarker requires that all tumor clones are eradicated. The question is if a single biopsy might represent the mutation status of the entire tumor, and the answer would probably be no. Then, it gets increasingly important to elucidate the relevance and degree of heterogeneous distribution of the targeted biomarker regarding the metastasis localisation, previous systemic antineoplastic treatments and interval between primary tumor and metastasis (synchronous or metachronous). Furthermore, and based on these results, to perform a rebiopsy when the treatment fails to offer an individually tailored treatment would be crucial to determine the status of the druggable biomarker. Since not all patients are suitable for a rebiopsy of all tumor lesions, new techniques such as liquid biopsies might help us to distinguish those patients who could have higher EGFR mutation heterogeneity (19,20). These findings would also help us to design new strategies for patients with lung cancer harboring heterogeneous EGFR mutations.

Acknowledgements

Disclosure: The authors declare no conflict of interest.

References

1. Mitsudomi T, Morita S, Yatabe Y, et al. Gefitinib versus cisplatin plus docetaxel in patients with non-small-cell lung cancer harbouring mutations of the epidermal growth factor receptor (WJTOG3405): an open label, randomised phase 3 trial. Lancet Oncol 2010;11:121-8. [PubMed]
2. Zhou C, Wu YL, Chen G, et al. Erlotinib versus chemotherapy as first-line treatment for patients with advanced EGFR mutation-positive non-small-cell lung cancer (OPTIMAL, CTONG-0802): a multicentre, open-label, randomised, phase 3 study. Lancet Oncol 2011;12:735-42. [PubMed]
3. Rosell R, Carcereny E, Gervais R, et al. Erlotinib versus standard chemotherapy as first-line treatment for European patients with advanced EGFR mutation-positive non-small-cell lung cancer (EURTAC): a multicentre, open-label, randomised phase 3 trial. Lancet Oncol 2012;13:239-46. [PubMed]
4. Inoue A, Kobayashi K, Maemondo M, et al. Updated overall survival results from a randomized phase III trial comparing gefitinib with carboplatin-paclitaxel for chemo-naïve non-small cell lung cancer with sensitive EGFR gene mutations (NEJ002). Ann Oncol 2013;24:54-9. [PubMed]
5. Sequist LV, Yang JC, Yamamoto N, et al. Phase III Study of Afatinib or Cisplatin Plus Pemetrexed in Patients With Metastatic Lung Adenocarcinoma With EGFR Mutations. J Clin Oncol 2013;31:3327-34. [PubMed]
6. Wu YL, Zhou C, Hu CP, et al. LUX-Lung 6: A randomized, open-label, phase III study of afatinib versus gemcitabine/cisplatin as first line treatment for Asian patients with EGFR mutation-positive advanced adenocarcinoma of the lung. J Clin Oncol 2013;31:abstr 8016.
7. Mok TS, Wu YL, Thongprasert S, et al. Gefitinib or carboplatin-paclitaxel in pulmonary adenocarcinoma. N Engl J Med 2009;361:947-57. [PubMed]
8. Han JY, Park K, Kim SW, et al. First-SIGNAL: first-line single-agent iressa versus gemcitabine and cisplatin trial in never-smokers with adenocarcinoma of the lung. J Clin Oncol 2012;30:1122-8. [PubMed]
9. Gerlinger M, Rowan AJ, Horswell S, et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. N Engl J Med 2012;366:883-92. [PubMed]
10. Chen ZY, Zhong WZ, Zhang XC, et al. EGFR mutation heterogeneity and the mixed response to EGFR tyrosine kinase inhibitors of lung adenocarcinomas. Oncologist 2012;17:978-85. [PubMed]
11. Yatabe Y, Matsuo K, Mitsudomi T. Heterogeneous distribution of EGFR mutations is extremely rare in lung adenocarcinoma. J Clin Oncol 2011;29:2972-7. [PubMed]
12. Jakobsen JN, Sørensen JB. Intratumor heterogeneity and chemotherapy-induced changes in EGFR status in non-small cell lung cancer. Cancer Chemother Pharmacol 2012;69:289-99. [PubMed]
13. Tomonaga N, Nakamura Y, Yamaguchi H, et al. Analysis of intratumor heterogeneity of EGFR mutations in mixed type lung adenocarcinoma. Clin Lung Cancer 2013;14:521-6. [PubMed]
14. Bai H, Wang Z, Wang Y, et al. Detection and clinical significance of intratumoral EGFR mutational heterogeneity in Chinese patients with advanced non-small cell lung cancer. PLoS One 2013;8:e54170. [PubMed]
15. Taniguchi K, Okami J, Kodama K, et al. Intratumor heterogeneity of epidermal growth factor receptor mutations in lung cancer and its correlation to the response to gefitinib. Cancer Sci 2008;99:929-35. [PubMed]
16. Bai H, Wang Z, Chen K, et al. Influence of chemotherapy on EGFR mutation status among patients with non-small-cell lung cancer. J Clin Oncol 2012;30:3077-83. [PubMed]
17. Rosell R, Moran T, Queralt C, et al. Screening for epidermal growth factor receptor mutations in lung cancer. N Engl J Med 2009;361:958-67. [PubMed]
18. Cappuzzo F, Ciuleanu T, Stelmakh L, et al. Erlotinib as maintenance treatment in advanced non-small-cell lung cancer: a multicentre, randomised, placebo-controlled phase 3 study. Lancet Oncol 2010;11:521-9. [PubMed]
19. Murtaza M, Dawson SJ, Tsui DW, et al. Non-invasive analysis of acquired resistance to cancer therapy by sequencing of plasma DNA. Nature 2013;497:108-12. [PubMed]
20. Nakamura T, Sueoka-Aragane N, Iwanaga K, et al. A noninvasive system for monitoring resistance to epidermal growth factor receptor tyrosine kinase inhibitors with plasma DNA. J Thorac Oncol 2011;6:1639-48. [PubMed]