Invest New Drugs (2014) 32:1311–1315 DOI 10.1007/s10637-014-0146-x

SHORT REPORT

Epidermal growth factor receptor (EGFR) exon 20 mutations in non-small-cell lung cancer and resistance to EGFR-tyrosine kinase inhibitors Hyun Sun Woo & Hee Kyung Ahn & Ha Yeon Lee & Inkeun Park & Young Saing Kim & Junshik Hong & Sun Jin Sym & Jinny Park & Jae Hoon Lee & Dong Bok Shin & Eun Kyung Cho

Received: 23 May 2014 / Accepted: 28 July 2014 / Published online: 23 August 2014 # Springer Science+Business Media New York 2014

Summary Introduction In patients with non-small cell lung cancer (NSCLC), the predictive value of rare epidermal growth factor receptor (EGFR) exon 20 mutations in determining a patient’s response to EGFR tyrosine kinase inhibitor (TKI) treatment is unclear. Patients and Methods We reviewed data for NSCLC patients harboring EGFR exon 20 mutations from two hospitals in Korea. EGFR mutations were analyzed using directional sequencing. Results We identified eight patients carrying EGFR exon 20 mutations, seven of whom had insertional mutations. Three patients carried previously unreported insertional mutations. Among six patients who were treated with EGFR TKI, one showed stable disease and three showed primary resistance. Response evaluations were not performed for the other two patients because of their clinical deterioration. Conclusions EGFR exon 20 insertional mutations, including three that were previously unreported, were associated with the poor response of patients to TKI treatment.

Keywords EGFR . EGFR TKI . Exon 20 . Non-small cell lung carcinoma Hyun Sun Woo and Hee Kyung Ahn contributed equally to this work. H. S. Woo : H. K. Ahn : I. Park : Y. S. Kim : J. Hong : S. J. Sym : J. Park : J. H. Lee : D. B. Shin : E. K. Cho (*) Division of Hematology and Oncology, Department of Internal Medicine, Gachon University Gil Medical Center, 1198 Guwol-dong, Namdong-gu, Incheon 405-760, South Korea e-mail: [email protected] H. Y. Lee Department of Hematology and Oncology, KyungHee University Hospital at Gandong, Seoul, South Korea

Introduction Epidermal growth factor receptor (EGFR) somatic mutations in non-small cell lung cancer (NSCLC) drive oncogenesis [1] in 10–15 % of NSCLC patients in the West and up to 30 % of those in East Asia. EGFR tyrosine kinase inhibitors (TKIs) significantly improve the treatment responses and survival rates of patients with EGFR-mutant NSCLC compared to those of patients treated with platinum-doublet chemotherapy [2–6] and, thus, EGFR TKIs are a first-line treatment. The majority of the EGFR mutations are deletions in exon19 and the L858R point mutation in exon 21 [7, 8]. Other EGFR mutations in exon 18 or exon 20 are rarely found, and studies that report the clinical implications of these rare mutations are limited [8–11]. EGFR exon 20 mutations represent rare recurrent mutations in EGFR that account for 1–10 % of all EGFR mutations [8–11]. Of these mutations, insertional mutations are the most widely reported and studied. Mounting preclinical and clinical evidence shows that these insertional mutations are associated with resistance to EGFR TKI. As EGFR exon 20 insertional mutations are generally located after the C-helix of the EGFR tyrosine kinase domain, the functional and clinical implications of these mutations on drug binding have been studied [10, 12]. However, due to the rarity and wide variability of the mutation types and sites, few studies have examined the response of patients carrying these mutations to EGFR TKI. We examined the clinical characteristics of NSCLC patients carrying EGFR exon 20 mutations and their response to EGFR TKI treatment.

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Methods We identified and reviewed the clinical data of patients diagnosed with NSCLC from two hospitals in Korea between 2008 and 2013. EGFR mutation tests were not routine and were thus performed at the physician’s discretion prior to 2011. When the reimbursement policy of the National Insurance System was changed in 2011 with regard to EGFR TKI treatment, EGFR mutation analysis was ordered in nearly every patient with advanced lung adenocarcinoma. We collected the demographic information of the patients, as well as their clinical stage at diagnoses, histology and EGFR mutation analysis results, and treatment regimens and responses. This study was approved by the Institutional Review Board, and informed consent was obtained. Subjects who smoked less than 100 cigarettes in their life were categorized as non-smokers. Histology was analyzed according to the World Health Organization pathology classification criteria [13]. Assessment of the treatment response was performed by CT scan every 2–3 months, and the results were analyzed according to the Response Evaluation Criteria in Solid Tumors Criteria v.1.1 [14]. Tumor specimens in primary or metastatic lesions were obtained using needle biopsy. Tumor DNA was obtained from paraffin-embedded tissue and amplified using polymerase chain reaction. EGFR mutation analysis of exons 18–21 by directional sequencing was performed by ISU ABXIS Co. (Seoul, Korea), an independent commercial laboratory.

Results A total of 139 patients carrying EGFR mutations were identified. Among them, 123 patients carried a deletion in exon 19 or the L858R point mutation. Mutations in exon 20 comprised 6 % of all EGFR mutations in these patients. Six patients carried the G719X mutation in exon 18, while the other two patients had mutations in exon 21 (one with N700Y and one with H835L). Eight patients carrying exon 20 mutations were included in the final analysis, five of whom were female. All of the patients had adenocarcinoma and four were nonsmokers. Six patients had distant metastases at diagnosis, whereas the other two had stage III cancers. One patient had the missense mutation R803Q, whereas the remaining seven patients had insertional mutations located after the C-helix of the tyrosine kinase domain. Six different insertional mutations were found in seven patients, and three of these mutations were previously unreported. Insertional mutations A767_V769 dup ASV (n=2), S768_D770 dup SVD (n=1), and N771_H773 dup NPH (n=1) were reported in previous studies. N771 del ins SY, N771_P772 in. G, and N771_P772 in. TQPNP were identified in three patients, and were not reported before.

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Six patients were treated with EGFR TKI (one with erlotinib and five with gefitinib) as first- or second-line chemotherapy. Only one patient with N771 del ins SY showed stable disease, for whom EGFR TKI treatment was continued for 6 months. In three patients, the disease was primarily refractory to EGFR TKI, and the response was not evaluated in the other two patients because of clinical deterioration. Treatment duration was less than 1 month in four patients (Table 1).

Discussion Mutations in exon 20, especially insertional mutations, are the most frequent of the rare EGFR mutations, accounting for 1– 17 % of all EGFR mutations [8–12, 15]. Reports of EGFR TKI treatment in patients with exon 20 mutations are limited, with less than 60 published cases [8–12, 15]. Insertional mutations are the most studied mutations occurring in exon 20. Exon 20 insertional mutations are highly heterogeneous in length and insertion position. To date, approximately 40 different exon 20 insertional mutations have been reported among 160 patients in nine studies [8–11, 15–19]. The most common mutations were A763_Y764 in. FQEA, A767_769 dup ASV, S768_D770 dup SVD, N771_H773 dup NPH, H773_V774 in. H, and H773_V774 in. PH. Three mutations (N771_P772 in. G, N771_P772 in. TQPNP, and N771 del ins SY) in the present study were not reported previously, in line with the high variability of insertional mutations in exon 20. The most common insertion site is the N-lobe of the tyrosine kinase domain, which is located after the C-helix (A767– C775), and is thought to have clinical implications in catalytic activity and drug binding [10, 12]. The computational model presented by Arcila et al.[10] further divided the predicted functional impact of exon 20 insertional mutation by the insertional site. They reported that insertional mutation in A764–770 seemed to have less effect on the drug-binding pocket. Conversely, the insertional mutations in A773–775 lead to primary resistance to EGFR TKI due to direct effects to the drug-binding pocket [10]. However, the most current available data show that primary resistance to EGFR TKI occurs in patients with insertional mutation after A767 (Table 2), with only two responding patients (one with S768_D770dupSVD [15] and one with N771 del insKPP [8]). Among the 12 patients with insertional mutations in this region who were reported in a small case series [12], only one patient with S768_D770dupSVD had partial response (PR) for 24 months [15]. However, three other patients with the same mutation showed no response to EGFR TKI treatment [15]. Four larger studies [8–11] in Asian and Western populations noted similar primary EGFR TKI resistance in patients with EGFR exon 20 distal insertion mutations (Table 2). French group [8] performed the largest study of EGFR rare

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Table 1 Clinicopathologic features and response to EGFR TKI in eight patients with exon 20 mutation Case

Age/sex

Histology

Smoking

TKI

TKI line

Response

EGFR mutation

TKI treatment period, months

1 2 3 4 5 6 7 8

72/M 34/F 69/F 56/F 66/F 57/M 46/F 66/M

AD AD AD AD AD AD AD AD

Yes Yes Never Never Never Yes Never Yes

Erlotinib Gefitinib Gefitinib Gefitinib Gefitinib Gefitinib None None

2nd 1st 1st 2nd 2nd 1st

PD PD SD N/A N/A PD

N771_P772insG A767_V769dupASV N771 del insSY A767_V769dupASV N771_H773dupNPH S768_D770dupSVD N771_P772insTQPNP R803Q

2.1 0.9 5.9 0.3 1.0 1.0

AD adenocarcinoma, TKI tyrosine kinase inhibitor, PD progressive disease, SD stable disease, N/A non applicable

the previously reported primary resistance observed in patients with EGFR exon 20 distal insertion mutations, as four of these patients had no response to treatment. The response was not evaluated in the other two patients because of clinical deterioration within 1 month of beginning EGFR TKI treatment.

mutations, and showed that the majority of the patients with EGFR exon 20 insertions had primary resistance. Nineteen patients were treated with EGFR-TKI; only one patient had PR, and six patients had stable disease. In our study, six patients carrying exon 20 distal insertion mutations were treated with EGFR TKI. Our study confirms Table 2 Clinical response to EGFR TKIs in patients with specific types of EGFR exon 20 insertion

Reference

This study

Beau-Faller [8]

Arcila [10]

Wu [15]*

Oxnard [11]**

Number of patients with exon 20 insertions who were treated with EGFR TKI Number of responses

6

19

5

6

8

1SD

2PR

2PR***

1PR

3SD

3PD

5SD

2PD

5PD

2PD

2NE Type of insertional mutation in exon 20 A763 Y764 in. FQEA M766 V769 in. WPA A767 S768 in. SVR A767 V769 dup ASV 1PD

12PD

1NE

1PR 1SD 1PD 2SD

1PR***

1NE 1PD

1PD 1SD

S768 D770dupSVD

*Original data includes cases with complex mutations, however were excluded in this table **Specific type of mutation were not reported ***Treated with EGFR TKI combined with chemotherapy

N771 del insKPP N771 del insSY N771_P772insG N771_H773dupNPH P772 C775dupPHVC P772 H773insDNP H773_V774dupH H773_V774dupHV

1PD 1PR

2PD 1PD 2PD

V769 D770delinsGI D770 N771insSVD D770 N771insD D770 N771insGT N771 dupN PD, progressive disease; SD, stable disease; PR, partial response; NE, not evaluated

1NE

3NE

3PD

1PD 1PD 1PD 1PR 1SD 1PD 1NE 1PD 1PD 2SD, 1PD 1PR*** 1PD

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Proximal insertions (E762–Y764) were predicted to significantly affect drug binding in a computational model, as the disturbance of the C-helix of EGFR kinase domain may reduce the protein’s drug-binding affinity [10]. Unexpectedly, these mutations were associated with the effectiveness of EGFR TKI treatment. Among five patients reported in four studies [8, 10, 20, 21], three had stable disease and one patient showed PR. The drug response in patients with point mutations in EGFR exon 20 is not known, except for T790M, which is associated with EGFR TKI resistance. In two studies [8, 15], ten patients with EGFR exon 20 point mutations were treated with EGFR TKI. Two patients with single point mutations (V774M and K806E) showed primary resistance. Eight patients had complex mutations involving point mutations in exon 20. Among six patients with concomitant sensitive mutations (G719A, L858R, or L861Q), four had stable disease. Two patients had a complex mutation of H773L+V774M; one had stable disease. The literature suggests that patients with proximal insertional mutations, point mutations, or complex mutations have different drug sensitivities. Through this study, we have contributed six rare cases to the literature in which EGFR TKI treatment was used in patients carrying EGFR exon 20 insertional mutations, and we have noted the poor response of these patients to the treatment. Three of the identified insertion locations in this study were not previously reported, confirming the high variability of EGFR insertional mutations in exon 20. The uniformly poor response of these patients to EGFR TKI treatment suggests that we should consider cytotoxic chemotherapy first in these patients. The development of strategies to overcome and treat primary drug resistance in patients carrying such mutations is warranted.

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4.

5.

6.

7.

8. Conflicts of interest There are no conflicts of interest.

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