Non - small cell lung cancer (NSCLC) is the largest subgroup of lung cancers, responsible for about 80% of all lung cancers. Targeted cancer therapy has opened a new window into treatment of non - small cell lung cancer. This therapy is very effective for patients who have EGFR (Epidermal growth factor receptor) activating mutations, primarily located in the tyrosine kinase domain in the form of a base - pair deletion at exon 19 (LREA deletions, accounting for about 54%) or a point mutation at exon 21 (L858R, accounting for about 43%). EGFR activating mutations occur in about 20% of NSCLC patients.
JOURNAL OF MEDICAL RESEARCH TARGETING TKI RESISTANCE IN NON - SMALL CELL LUNG CANCER PATIENTS CAUSED BY SECONDARY EGFR T790M MUTATION Tran Quoc Dat, Tran Huy Thinh, Le Hoan, Tran Van Khanh, Ta Thanh Van Hanoi Medical University Non - small cell lung cancer (NSCLC) is the largest subgroup of lung cancers, responsible for about 80% of all lung cancers Targeted cancer therapy has opened a new window into treatment of non - small cell lung cancer This therapy is very effective for patients who have EGFR (Epidermal growth factor receptor) activating mutations, primarily located in the tyrosine kinase domain in the form of a base - pair deletion at exon 19 (LREA deletions, accounting for about 54%) or a point mutation at exon 21 (L858R, accounting for about 43%) EGFR activating mutations occur in about 20% of NSCLC patients However, acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) in NSCLC patients prevails after 10 - 20 months of treatment, in relation to several genetic alterations of the tumors The most frequent genetic alteration is the secondary mutation T790M in exon 20 of the EGFR gene In this study, we detected secondary T790M resistance mutations in 11 EGFR - mutant patients who had poor response to targeted therapy We also report two typical clinical cases with TKI acquired resistance, using a standard sequencing - based method followed by the Scorpions ARMS (Scorpions - Amplification Refractory Mutation System) method, which has enhanced analytical sensitivity The T790M mutation analysis of specimens from 21 patients detected a total of 10 mutants (47.6%), including mutants that were detected by both sequencing and the Scorpions ARMS method and mutants that were only detected by the Scorpions ARMS method Keywords: Non - small cell lung cancer, TKI - acquired resistance, Secondary T790M mutation I INTRODUCTION However, despite proven benefits in an appropriately selected population, the impact of tra- Lung cancer is the leading cause of cancer ditional chemotherapy on progression - free mortality worldwide In the United States, lung survival (PFS) and overall survival (OS) cancer accounted for an estimated 159,480 remains small Currently, the - year survival deaths in 2013, which is more than breast, rate for a patient with stage IIB/IV NSCLC is, colon, prostate and pancreatic cancer deaths at best, around 30%, with a median survival combined [Cancer Fact & Figure 2013] time of no more than 12 months [2; 3] Conventional treatment for advanced NSCLC Recently, targeted cancer therapy has opened has typically consisted of chemotherapy [1] a new window into treatment the of NSCLC This therapy is very effective for patients that Corresponding author: Tran Huy Thinh, Dept of Biochemistry, Center for Gene and Protein Research, Hanoi Medical University E-mail: tranhuythinh@hmu.edu.vn Received: 02 November 2016 Accepted: 10 December 2016 JMR 105 E1 (7) - 2016 have epidermal growth factor receptor (EGFR) activating mutations, the majority of which are located in the tyrosine kinase domains in the form of a base-pair deletion at exon 19 (LREA deletions, accounting for about 54% of EGFR 11 JOURNAL OF MEDICAL RESEARCH activating mutations) or a point mutation at II MATERIALS AND METHODS exon 21 (L858R, accounting for about 43% of EGFR activating mutations) EGFR activating Materials mutations occur in about 20% of NSCLC Twenty - one re - biopsied formalin - fixed patients, with significantly increased proportions paraffin embedded (FFPE) tissue samples in those patients with adenocarcinoma on were obtained from NSCLC patients with histology, female patients, patients of Asian acquired resistance to EGFR - targeted ethnicity, and patients who were never - therapy The patients came from Huu Nghi smokers [4; 5] Even though sensitizing Hospital, National Bach Mai Hospital, or the mutations in the EGFR gene are associated National Cancer Hospital All three hospitals with higher response rates to EGFR - TKI are therapies and prolonged progression - free diagnosed survival, resistant mutations eventually prevail stage IIIB - IV, based on clinical features and via cellular survival pressure [6] pathology results These patients had all been Many studies have postulated various located previously in with Vietnam NSCLC, biopsied to Patients were adenocarcinoma identify EGFR possible resistance mechanisms, including: a sensitizing mutations (11/21 patients had secondary mutation gene LREA deletion mutations in EGFR exon 19, (a T790M mutation in exon 20, a L747S and 10/21 patients had L858R point mutations mutation, a D761Y mutation in exon 19, or a in EGFR exon 21) All patients had been T854A mutation in exon 21), amplification of treated with EGFR - TkI, with their responses signaling EGFR evaluated using the WHO/RECIST Criteria, inhibition (MET and HER2), mutations in other and rebiopsied to identify the EGFR - T790M genes that may substitute as oncogenic resistance mutation Jackman’s Evaluation drivers (PIK3CA and B - RAF), epithelial - to- Criteria was used to define more precisely the mesenchymal transition (EMT), and conver- patients’ acquired resistance to EGFR - TkI sion to small - cell lung cancer [7; 8] Among (Table 1) [9] molecules in the that EGFR bypass them, the T790M point mutation in EGFR exon Methods 20 is believed to be the main source of resistance, accounting for over half of the resistance seen to gefitinib and erlotinib The aim of this study was to identify secondary EGFR - T790M mutations in re - biopsied tissue samples from Vietnamese NSCLC patients with acquired resistance to TKis Responsible molecular alterations and mechanisms used to overcome TKI resistance were contemporarily assessed We also report two clinical cases of DNA was extracted from biopsied FFPE samples using a Qiamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the protocol for tissue samples in the manufacturer's instructions The DNA obtained was eluted in 50 µL of sterile bidistilled buffer, and the concentration and purity of the extracted DNA were assessed by spectrophotometry The extracted DNA was stored at −20°C until use women with NSCLC whose histories showed a Sequencing method: The EGFR gene was typical series of mutations and how their therapy amplified using specific primer pairs for exon was tailored based on molecular evidence 19, exon 20 and exon 21 PCR products were 12 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH directly sequenced using an Advant 3100 0.01% All reactions were carried out in 25 µL automated sequencer (Applied Biosystems volumes with µL of template DNA, 10 µL of Inc., Foster City, California, USA) Sequences primer mix and 0.1 µL of Taq polymerase were aligned and inspected using a reference Real - time PCR was carried out under the sequence from GeneBank (NM_005228) following conditions: initial denaturation at 95° Allele C for 10 minutes, 50 cycles of 95°C for 30 Refractory Mutation System) method: We seconds and a final 60 seconds at 62°C with used an EGFR Scorpion TM Kit (EGFR RGQ fluorescence reading (set to PCR Kit, Qiagen, Germany), which combines (6 - carboxy fluorescein) that allows optical two technologies - ARMS and Scorpion - to excitation at 480 nm and measurement at 520 detect mutations in real - time PCR reactions, nm) at the end of each cycle Scorpion ARMS (Scorpion FAM dye with an analytical sensitivity of approximately Table Jackman’s Evaluation Criteria to define acquired resistance to EGFR - TKi in NSCLC Previous treatment with a single - agent EGFR TKI (eg, gefitinib or erlotinib) Either or both of the following: + A tumor that harbors an EGFR mutation known to be associated with drug sensitivity or objective clinical benefit from treatment with an EGFR TKI; + Systemic progression of disease (Response Evaluation Criteria in Solid Tumors [RECIST] or WHO) while on continuous treatment with gefitinib or erlotinib within the last 30 days No intervening systemic therapy between cessation of gefitinib or erlotinib and initiation of new therapy Research Ethics: This research was approved by the ethics committee of the Hanoi Medical University, decree No.161/HMUIRB, signed on February 15th, 2014 III RESULTS Our results showed that T790M mutation analysis of the specimens from the 21 patients Twenty - one patients with NSCLC with in our study detected 10 mutants (47.6%), acquired resistance to EGFR - targeted ther- including mutants that were detected by both apy were enrolled in this study Rates of sequencing and the Scorpions ARMS method detection of the T790M mutation in exon 20 of and mutants that were only detected by the the EGFR gene using the sequencing method Scorpions ARMS method because of the and the Scorpion ARMS method are shown in limited cancer tissues obtained after re - table biopsy JMR 105 E1 (7) - 2016 13 JOURNAL OF MEDICAL RESEARCH Table Patient subtype pathology and EGFR mutation status before and after treated with TKis Code Subtype pathology Stage Adenocarcinoma IIIB Adenocarcinoma Mutation identified before treated with EGFR- TKis Mutation identified after treated with EGFR- TKis Sequencing method Scorpions ARMS method LREA LREA LREA, T790M IV LREA LREA LREA Adenocarcinoma IV L858R L858R L858R Adenocarcinoma IV LREA LREA LREA, T790M Adenocarcinoma IIIB L858R L858R, T790M L858R, T790M Adenocarcinoma IV LREA LREA LREA Adenocarcinoma IV L858R L858R L858R, T790M Adenocarcinoma IV L858R L858R L858R Adenocarcinoma IIIB LREA LREA, T790M LREA, T790M 10 Adenocarcinoma IIIB LREA LREA LREA 11 Adenocarcinoma IV L858R L858R L858R 12 Adenocarcinoma IIIB LREA LREA LREA 13 Adenocarcinoma IV L858R L858R L858R 14 Adenocarcinoma IIIB LREA LREA LREA, T790M 15 Adenocarcinoma IIIB LREA LREA LREA, T790M 16 Adenocarcinoma IV L858R L858R L858R 17 Adenocarcinoma IIIB L858R L858R L858R 18 Adenocarcinoma IV L858R L858R, T790M L858R, T790M 19 Adenocarcinoma IV LREA LREA LREA 20 Adenocarcinoma IIIB L858R L858R L858R, T790M 21 Adenocarcinoma IV LREA LREA, T790M LREA, T790M Type of mutation: LREA (E746_A750del): in - frame deletion mutation in exon 19; L858R (c.2573T > G): point mutation in exon 21; T790M (c.2369C > T): point mutation in exon 20 Reference sequence: (NM_005228) 14 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH Figure Results of the direct sequencing method and the Scorpion Amplified Refractory Mutation System (ARMS) method in patient no.05 before (A) and after (B) treated with EGFR-Tkis Before treatment with EGFR - TKis, a sensitizing mutation (L858R exon 21) was detected in this patient’s tumor After treatment with EGFR - TKis, not only was the L858R sensitizing mutation seen, but an EGFR - T790M secondary mutation was also detected in the re - biopsied tissue sample These mutations were seen using both the sequencing method and the Scorpion ARMS method This mutation is associated with resistance to EGFR - TKis Figure Results of the direct sequencing method and the Scorpion Amplified Refractory Mutation System (ARMS) method in patient no.01 before (A) and after (B) treated with EGFR-TKis JMR 105 E1 (7) - 2016 15 JOURNAL OF MEDICAL RESEARCH Before treatment with EGFR - TKis, a deletion mutation in EGFR exon 19 was detected in the patient’s biopsied tissue This activating mutation is predicted to be a sensitizing mutation to EGFR-TKI 12 months after treatment with the EGFR TKI, when tissue was re - biopsied, sequencing revealed a suspicious signal in amino acid 790 in exon 20 This result was verified using the Scorpion ARMS method and the results showed two mutations in the re - biopsied tissue: the deletion mutation in exon 19 and a T790M secondary mutation Patient no.05 is one of the most typical clinical cases of a NSCLC patient with acquired resistance to TKIs caused by a secondary EGFR - T790M mutation At the time of diagnosis, the patient had symptoms of persistent sinus congestion, cough, and mild progressive dyspnea Computed tomography (CT) of the chest revealed a large left upper lobe pulmonary mass with bilateral pulmonary metastases and right hilar, prevascular, pretracheal, and subcarinal lymphadenopathy CEA concentration was found to be 27.7 ng/ mL, and no distant metastatic lesions were The patient was started on oral Gefitinib for first - line treatment (250mg daily) After months, the patient had a complete response The symptoms of persistent sinus congestion, cough and dyspnea decreased However, after 13 month of gefitinib treatment, the patient developed acquired resistance to TKIs A lesion appeared in the left lower lobe, along with left pleural effusion and a mediastinal lymph node metastasis near the right lung Re - biopsy of the second lesion located in left lower lobe revealed detected CT - guided biopsy of the primary adenocarcinoma The re - biopsied tissue was mass differentiated found to have a T790M mutation in exon 20, non - small - cell carcinoma, consistent with an which is associated with resistance to first- adenocarcinoma Point mutation L858R in generation EGFR ‑ TKIs The re - biopsied EGFR exon 21 was detected in the biopsy tissue was also found to have an activating tissue from this patient This EGFR activating mutation mutation is believed to be a sensitizing mutations were detected by both the sequen- mutation to EGFR tyrosine kinase inhibitors cing method and the Scorpion ARMS method A 16 revealed a poorly (L858R exon 21) These two B JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH C Figure Chest computed tomography (CT) scan of patient no.05 (A) before treatment with EGFR - TKis, (B) months after starting gefitinib, the primary tumor decreased in size (C) 13 months after treatment with gefitinib, disease progression was seen A B C Figure Chest computed tomography (CT) scan and bone scintigraphy of patient no.01 (A) before treatment with EGFR - TKis, (B) months after starting gefitinib, the tumor decreased in size (C) 15 months after treatment with erlotinib, disease progression was seen Patient no.01 was a 38 year old woman the left middle lobe that was connected to the with no notable occupational exposure, no pleura Left pleural effusion was also seen No family history of lung cancer, and no history of affected lymph nodes were detected How- smoking or exposure to second - hand smoke ever, the tumor had already spread to the bone The patient visited the National Cancer Hospi- and metastases were detected in the spine (fig tal for evaluation of a chronic cough The initial 4) The patient was diagnosed with lung adeno- PET-CT scan showed a 3,5 x cm tumor in carcinoma by transbronchial lung biopsy and JMR 105 E1 (7) - 2016 17 JOURNAL OF MEDICAL RESEARCH an activating EGFR mutation (exon 19 dele- tumor cells’ acquired resistance to TKIs: i) the tion) the T790M mutation can change the kinase sequencing method and the Scorpion ARMS domain’s interactive region and block the method The patient was started on Erlotinib binding of the EGFR TKI, so the transduction followed firstline pathway can still be activated and cancer cells treatment, and her response to treatment was can continue with proliferation, migration, evaluated invasion and angiogenesis; ii) changing amino was detected by Progression using both Bisphotphonat using - the free for RECIST survival Criteria (PFS) was acid 790 from tyrosine to methionine physically measured from the first day of target treatment impedes access to the ATP binding site by TKI to the day of disease progression After drugs such as Erlotinib and Gefitinib [12] In this months, the patient had a complete response study, both the sequencing method and the and stable disease At the time of the Scorpion ARMS method was used to look for 15 monthS follow-up after treatment with er- mutations However, the detection rate of the lotinib was begun, the patient began to ex- T790M mutation was lower than that found in perience severe thoracic pain, shortness of previous studies, possibly due to sample size breath, and analgesia refractory to medication limitations A PET - CT scan revealed new metastatic suggest lesions in the contralateral lung and spine (fig should be used to detect secondary T790M - 4C) A new biopsy was taken Besides the EGFR mutations previously identified activating mutation in sequencing method The direct sequencing exon 19 of the EGFR gene, an additional method can be used initially as a screening secondary T790M point mutation in exon 20 was method to detect EGFR mutations now seen, conferring resistance to erlotinib In this case, the secondary T790M mutation was confirmed only by the Scorpion ARMS method [11; that 13] the Overall, our results scorpion ARMS method instead of the direct V CONCLUSION In this study, secondary EGFR- with an analytical sensitivity of approximately T790M mutation analysis of specimens from 0.01% This was because of the limited cancer 21 Vietnamese NSCLC patients detected 10 tissues obtained after re - biopsy mutants (47.6%), including mutants that were detected by both sequencing and the IV DISCUSSION Scorpions ARMS method and mutants that Many previous studies have found that a were only detected by the Scorpions ARMS gene method because of the limited cancer tissues (T790M mutation) and amplification of the obtained after re - biopsy Further studies MET proto - oncogene could be the causes of should be conducted better elucidate rates of resistance mechanisms in small cell lung EGFR cancer transformation Moreover, the T790M Vietnam secondary mutation in the EGFR point mutation in exon 20 of the EGFR gene is the main mutation believed to cause mutations in NSCLC patients in Acknowledgements resistance [10; 11] There are two mechanisms We would like to express our sincere which have been proposed to explain the thanks to the patients and their families for 18 JMR 105 E1 (7) - 2016 JOURNAL OF MEDICAL RESEARCH their voluntary involvement in this study This secondary mutations J Clin Oncol, 26, 1182 - work was supported by Research Grant 1184 KC.04.16/11-15 from the Ministry of Science and Technology in Vietnam REFERENCE Coudert B, Ciuleanu T, Park K et al (2012) Survival benefit with erlotinib mainte- Bean J., Brennan C., Shih JY et al (2007) MET amplification occurs with or without T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or erlotinib Proc Natl Acad Sci USA, 104, 20932 - 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2016 resistance to gefitinib: The 19 ... and conver- patients acquired resistance to EGFR - TkI sion to small - cell lung cancer [7; 8] Among (Table 1) [9] molecules in the that EGFR bypass them, the T790M point mutation in EGFR exon... rates of resistance mechanisms in small cell lung EGFR cancer transformation Moreover, the T790M Vietnam secondary mutation in the EGFR point mutation in exon 20 of the EGFR gene is the main mutation. .. ME small- cell lung cancer (NSCLC) according to (2012) Screening for germline EGFR T790M response to first-line chemotherapy Ann On- mutations through lung cancer genotyping J col, 23(2), 388 -