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.
Trang 1Corresponding author: Tran Huy Thinh, Dept of
Biochem-istry, Center for Gene and Protein Research, Hanoi
Medi-cal University
E-mail: tranhuythinh@hmu.edu.vn
Received: 02 November 2016
Accepted: 10 December 2016
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) activat-ing 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 rela-tion to several genetic alterarela-tions of the tumors The most frequent genetic alterarela-tion 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 4 mutants that were detected by both sequencing and the Scorpions ARMS method and 6 mutants that were only detected by the Scorpions ARMS method.
Keywords: Non - small cell lung cancer, TKI - acquired resistance, Secondary T790M mutation
I INTRODUCTION
Lung cancer is the leading cause of cancer
mortality worldwide In the United States, lung
cancer accounted for an estimated 159,480
deaths in 2013, which is more than breast,
colon, prostate and pancreatic cancer deaths
combined [Cancer Fact & Figure 2013].
Conventional treatment for advanced NSCLC
has typically consisted of chemotherapy [1]
However, despite proven benefits in an appro-priately selected population, the impact of tra-ditional chemotherapy on progression - free survival (PFS) and overall survival (OS) remains small Currently, the 1 - year survival rate for a patient with stage IIB/IV NSCLC is,
at best, around 30%, with a median survival time of no more than 12 months [2; 3] Recently, targeted cancer therapy has opened
a new window into treatment the of NSCLC This therapy is very effective for patients that 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
Trang 2activating mutations) or a point mutation at
exon 21 (L858R, accounting for about 43% of
EGFR activating mutations) EGFR activating
mutations occur in about 20% of NSCLC
patients, with significantly increased proportions
in those patients with adenocarcinoma on
histology, female patients, patients of Asian
ethnicity, and patients who were never
-smokers [4; 5] Even though sensitizing
mutations in the EGFR gene are associated
with higher response rates to EGFR - TKI
therapies and prolonged progression - free
survival, resistant mutations eventually prevail
via cellular survival pressure [6]
Many studies have postulated various
possible resistance mechanisms, including: a
secondary mutation in the EGFR gene
(a T790M mutation in exon 20, a L747S
mutation, a D761Y mutation in exon 19, or a
T854A mutation in exon 21), amplification of
signaling molecules that bypass EGFR
inhibition (MET and HER2), mutations in other
genes that may substitute as oncogenic
drivers (PIK3CA and B - RAF), epithelial -
to-mesenchymal transition (EMT), and
conver-sion to small - cell lung cancer [7; 8] Among
them, the T790M point mutation in EGFR exon
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
women with NSCLC whose histories showed a
II MATERIALS AND METHODS
1 Materials
Twenty - one re - biopsied formalin - fixed paraffin embedded (FFPE) tissue samples were obtained from NSCLC patients with acquired resistance to EGFR - targeted therapy The patients came from Huu Nghi Hospital, National Bach Mai Hospital, or the National Cancer Hospital All three hospitals are located in Vietnam Patients were diagnosed with NSCLC, adenocarcinoma stage IIIB - IV, based on clinical features and pathology results These patients had all been previously biopsied to identify EGFR sensitizing mutations (11/21 patients had LREA deletion mutations in EGFR exon 19, and 10/21 patients had L858R point mutations
in EGFR exon 21) All patients had been treated with EGFR - TkI, with their responses evaluated using the WHO/RECIST Criteria, and rebiopsied to identify the EGFR - T790M resistance mutation Jackman’s Evaluation Criteria was used to define more precisely the patients’ acquired resistance to EGFR - TkI (Table 1) [9]
2 Methods
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 instruc-tions The DNA obtained was eluted in 50 µL
of sterile bidistilled buffer, and the concentra-tion and purity of the extracted DNA were as-sessed by spectrophotometry The extracted DNA was stored at −20°C until use
Sequencing method: The EGFR gene was
Trang 3directly sequenced using an Advant 3100
automated sequencer (Applied Biosystems
Inc., Foster City, California, USA) Sequences
were aligned and inspected using a reference
sequence from GeneBank (NM_005228).
Scorpion ARMS (Scorpion Allele
Refractory Mutation System) method: We
used an EGFR Scorpion TM Kit (EGFR RGQ
PCR Kit, Qiagen, Germany), which combines
two technologies - ARMS and Scorpion - to
detect mutations in real - time PCR reactions,
with an analytical sensitivity of approximately
0.01% All reactions were carried out in 25 µL volumes with 1 µL of template DNA, 10 µL of primer mix and 0.1 µL of Taq polymerase Real - time PCR was carried out under the following conditions: initial denaturation at 95°
C for 10 minutes, 50 cycles of 95°C for 30 seconds and a final 60 seconds at 62°C with fluorescence reading (set to FAM dye (6 - carboxy fluorescein) that allows optical excitation at 480 nm and measurement at 520 nm) at the end of each cycle
Table 1 Jackman’s Evaluation Criteria to define acquired resistance to
EGFR - TKi in NSCLC
1 Previous treatment with a single - agent EGFR TKI (eg, gefitinib or erlotinib)
2 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
4 No intervening systemic therapy between cessation of gefitinib or erlotinib and initiation of new therapy
III RESULTS
Twenty - one patients with NSCLC with
acquired resistance to EGFR - targeted
ther-apy were enrolled in this study Rates of
detection of the T790M mutation in exon 20 of
the EGFR gene using the sequencing method
and the Scorpion ARMS method are shown in
table 2
Our results showed that T790M mutation analysis of the specimens from the 21 patients
in our study detected 10 mutants (47.6%), including 4 mutants that were detected by both sequencing and the Scorpions ARMS method and 6 mutants that were only detected by the Scorpions ARMS method because of the limited cancer tissues obtained after re -biopsy
3 Research Ethics: This research was approved by the ethics committee of the Hanoi
Medical University, decree No.161/HMUIRB, signed on February 15th, 2014
Trang 4Table 2 Patient subtype pathology and EGFR mutation status before
and after treated with TKis
pathology
Stage
Mutation identi-fied before treated with EGFR- TKis
Mutation identified after treated with
EGFR- TKis Sequencing
method
Scorpions ARMS method
1 Adenocarcinoma IIIB LREA LREA LREA, T790M
2 Adenocarcinoma IV LREA LREA LREA
3 Adenocarcinoma IV L858R L858R L858R
4 Adenocarcinoma IV LREA LREA LREA, T790M
5 Adenocarcinoma IIIB L858R L858R, T790M L858R, T790M
6 Adenocarcinoma IV LREA LREA LREA
7 Adenocarcinoma IV L858R L858R L858R, T790M
8 Adenocarcinoma IV L858R L858R L858R
9 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)
Trang 5Figure 1 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 2 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
Trang 6Before 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
detected CT - guided biopsy of the primary
mass revealed a poorly differentiated
non - small - cell carcinoma, consistent with an
adenocarcinoma Point mutation L858R in
EGFR exon 21 was detected in the biopsy
tissue from this patient This EGFR activating
mutation is believed to be a sensitizing
mutation to EGFR tyrosine kinase inhibitors
The patient was started on oral Gefitinib for first - line treatment (250mg daily)
After 6 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 adenocarcinoma The re - biopsied tissue was found to have a T790M mutation in exon 20, which is associated with resistance to first-generation EGFR ‑ TKIs The re - biopsied tissue was also found to have an activating mutation (L858R exon 21) These two mutations were detected by both the sequen-cing method and the Scorpion ARMS method
Trang 7Figure 3 Chest computed tomography (CT) scan of patient no.05
(A) before treatment with EGFR - TKis, (B) 6 months after starting gefitinib, the primary tumor decreased in size (C) 13 months after treatment with gefitinib, disease progression was seen
C
Figure 4 Chest computed tomography (CT) scan and bone scintigraphy of patient no.01
(A) before treatment with EGFR - TKis, (B) 6 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
with no notable occupational exposure, no
family history of lung cancer, and no history of
smoking or exposure to second - hand smoke
The patient visited the National Cancer
Hospi-tal for evaluation of a chronic cough The initial
PET-CT scan showed a 3,5 x 5 cm tumor in
the left middle lobe that was connected to the pleura Left pleural effusion was also seen No affected lymph nodes were detected How-ever, the tumor had already spread to the bone and metastases were detected in the spine (fig 4) The patient was diagnosed with lung adeno-carcinoma by transbronchial lung biopsy and
Trang 8an activating EGFR mutation (exon 19
dele-tion) was detected using both the
sequencing method and the Scorpion ARMS
method The patient was started on Erlotinib
followed by Bisphotphonat for firstline
treatment, and her response to treatment was
evaluated using the RECIST Criteria
Progression - free survival (PFS) was
measured from the first day of target treatment
to the day of disease progression After 6
months, the patient had a complete response
and stable disease At the time of the
15 monthS follow-up after treatment with
er-lotinib was begun, the patient began to
ex-perience severe thoracic pain, shortness of
breath, and analgesia refractory to medication
A PET - CT scan revealed new metastatic
lesions in the contralateral lung and spine (fig
4C) A new biopsy was taken Besides the
previously identified activating mutation in
exon 19 of the EGFR gene, an additional
secondary T790M point mutation in exon 20 was
now seen, conferring resistance to erlotinib In
this case, the secondary T790M mutation was
confirmed only by the Scorpion ARMS method
with an analytical sensitivity of approximately
0.01% This was because of the limited cancer
tissues obtained after re - biopsy
IV DISCUSSION
Many previous studies have found that a
secondary mutation in the EGFR gene
(T790M mutation) and amplification of the
MET proto - oncogene could be the causes of
resistance mechanisms in small cell lung
cancer transformation Moreover, the T790M
point mutation in exon 20 of the EGFR gene is
the main mutation believed to cause
tumor cells’ acquired resistance to TKIs: i) the T790M mutation can change the kinase domain’s interactive region and block the binding of the EGFR TKI, so the transduction pathway can still be activated and cancer cells can continue with proliferation, migration, invasion and angiogenesis; ii) changing amino acid 790 from tyrosine to methionine physically impedes access to the ATP binding site by TKI drugs such as Erlotinib and Gefitinib [12] In this study, both the sequencing method and the Scorpion ARMS method was used to look for mutations However, the detection rate of the T790M mutation was lower than that found in previous studies, possibly due to sample size limitations [11; 13] Overall, our results suggest that the scorpion ARMS method should be used to detect secondary T790M -EGFR mutations instead of the direct sequencing method The direct sequencing method can be used initially as a screening method to detect EGFR mutations
V CONCLUSION
In this study, secondary EGFR-T790M mutation analysis of specimens from
21 Vietnamese NSCLC patients detected 10 mutants (47.6%), including 4 mutants that were detected by both sequencing and the Scorpions ARMS method and 6 mutants that were only detected by the Scorpions ARMS method because of the limited cancer tissues obtained after re - biopsy Further studies should be conducted better elucidate rates of EGFR mutations in NSCLC patients in Vietnam
Acknowledgements
Trang 9their voluntary involvement in this study This
work was supported by Research Grant
KC.04.16/11-15 from the Ministry of Science
and Technology in Vietnam
REFERENCE
1 Coudert B, Ciuleanu T, Park K et al
(2012) Survival benefit with erlotinib
mainte-nance therapy in patients with advanced
non-small-cell lung cancer (NSCLC) according to
response to first-line chemotherapy Ann
On-col, 23(2), 388 - 3894.
2 Sharma SV, Bell DW, Settleman J and
Haber DA (2007) Epidermal growth factor
receptor mutations in lung cancer Nat Rev
Cancer, 7(3), 169 - 181.
3 Riely GJ, Pao W et al (2006) Clinical
course of patients with non-small cell lung
cancer and epidermal growth factor receptor
exon 19 and exon 21 mutations treated with
gefitinib or erlotinib Clin Cancer Res, 12, 839
- 844
4 Paez JG (2004) EGFR mutations in
lung cancer: correlation with clinical response
to gefitinib therapy Science, 304(5676), 1497
- 1500
5 Mitsudomi T, Kosaka T et al (2005).
Mutations of the epidermal growth factor
re-ceptor gene predict prolonged survival after
gefitinib treatment in patients with
non-small-cell lung cancer with postoperative recurrence
J Clin Oncol, 23(11), 2513 - 2520.
6 Costa DB, Schumer ST, Tenen DG et
al (2008) Differential responses to erlotinib in
epidermal growth factor receptor (EGFR)
-mutated lung cancers with acquired resistance
to gefitinib carrying the L747S or T790M
secondary mutations J Clin Oncol, 26, 1182
-1184
7 Bean J., Brennan C., Shih JY et al (2007) MET amplification occurs with or
wi-thout T790M mutations in EGFR mutant lung tumors with acquired resistance to gefitinib or
erlotinib Proc Natl Acad Sci USA, 104, 20932
- 20937
8 Oxnard GR, Miller VA, Robson ME (2012) Screening for germline EGFR T790M
mutations through lung cancer genotyping J
Thorac Oncol, l7, 1049 - 1052.
9 Jackman D., Pao W., Riely G.J (2010).
Clinical definition of acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors in non-small-cell lung cancer
J Clin Oncol, 28(2), 357 - 360.
10 Kobayashi S, Boggon TJ, Dayaram T (2005) EGFR mutation and resistance of non
small-cell lung cancer to gefitinib N Engl J
Med, 352, 786 - 792.
11 Pao W, Miller VA, Politi KA (2005).
Acquire of resistance of lung adenocarcinomas to gefitinib or erlotinib is associated with a second mutation in the
EGFR kinase domain PLoS Med, 2, 73.
12 Yun C.H., Mengwasser K.E., Toms A.V et al (2008) The T790Mmutation in
EGFR kinase causes drug resistance by increasing the affinity for ATP Proceedings of the National Academy of Sciences of the
United States of America, 105(6), 2070 - 2075.
13 Takamitsu O, Hidetaka U (2010)
Acquired resistance to gefitinib: The contribution of mechanisms other than the T790M, MET, and HGF status Lung Cancer
68(2), 198 – 203.