Clinica Chimica Acta 444 (2015) 81–85
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Invited critical review
EGFR T790M resistance mutation in non small-cell lung carcinoma Marc G. Denis ⁎, Audrey Vallée, Sandrine Théoleyre Department of Biochemistry, Nantes University Hospital, Nantes, France
a r t i c l e
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Article history: Received 15 December 2014 Received in revised form 25 January 2015 Accepted 27 January 2015 Available online 7 February 2015 Keywords: EGFR T790M Mutation Resistance Non small-cell lung cancer
a b s t r a c t Lung cancer patients carrying sensitive epidermal growth factor receptor (EGFR) mutations show dramatic responses to tyrosine kinase inhibitors (TKIs). However, the majority of patients whose disease responds to drugs eventually develop resistance to these EGFR-TKIs. The T790M gatekeeper mutation in the EGFR tyrosine kinase domain accounts for half of resistance to these drugs. In some patients, this mutation is also detected as a primary event before drug exposure, at a frequency that is highly dependent on the technique used. This review will focus on the methods that have been used to detect the T790M mutation, and its potential clinical applications both in TKI naïve patients and in patients with an acquired resistance. © 2015 Elsevier B.V. All rights reserved.
Contents 1. 2.
EGFR TKI and activating mutations . . . . . . . . . . . . . . . . . . . EGFR alterations and primary resistance to EGFR TKI . . . . . . . . . . . 2.1. Exon 20 insertions . . . . . . . . . . . . . . . . . . . . . . . 2.2. T790M in untreated EGFR-mutant lung cancers . . . . . . . . . . 2.3. Clinical implications of T790M in untreated EGFR-mutant lung cancers 3. Acquired T790M resistance mutation . . . . . . . . . . . . . . . . . . 4. T790M and third generation TKI . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. EGFR TKI and activating mutations Prospective phase III randomized clinical trials showed that tyrosine kinase inhibitors (TKI) gefitinib [1–4], erlotinib [5,6], and more recently afatinib [7,8] improved outcomes compared with chemotherapy as initial treatment in EGFR-mutant non small cell lung cancer (NSCLC). These molecules have thus been approved in many countries worldwide. Therefore, routine molecular analysis of pathological specimens is mandatory in clinical practice to predict patient response. The potential result is an increased likelihood that patients will receive optimal therapy for their tumor and be spared a course of therapy with no or significantly less benefit.
⁎ Corresponding author at: Laboratoire de Biochimie, CHU-Institut de Biologie, 9 quai Moncousu, 44093 Nantes cedex, France. Tel.: +33 240 08 75 90; fax: +33 240 08 39 91. E-mail address: [email protected] (M.G. Denis).
http://dx.doi.org/10.1016/j.cca.2015.01.039 0009-8981/© 2015 Elsevier B.V. All rights reserved.
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Activating EGFR mutations occur in 10–15% of NSCLC patients of Caucasian ethnicity. They are more frequent in never-smokers, females, and adenocarcinomas [9]. EGFR mutations affect the EGFR tyrosine kinase domain, within exons 18–21. The p.L858R substitution in exon 21 and in-frame deletions in exon 19 account for 90% of all EGFR mutations. These are clearly activating mutations. Apart from these two hotspot mutations, other mutations have been described [10]. Substitutions in exon 18 (codon 719) are associated with TKI responses [11–13], although lower than those observed for exon 19 deletions and p.L858R mutation. Exon 19 insertions (in-frame insertions of 6 amino acids in the kinase domain) have been described as EGFR-TKI-sensitizing mutations as well [14]. The p.S768I mutation on exon 20 has initially been associated with low sensitivity to gefitinib and erlotinib in vitro [15], but good clinical responses have been reported in patients presenting an EGFR S768I alone [16] or associated with another EGFR alteration [17]. In current clinical practice, there is no standardized method for the detection of EGFR mutations in NSCLC tumor samples. The samples
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available for detection of somatic mutations in tumors are usually composed of mutant and wild-type DNA from tumor cells and wild-type DNA from non-malignant cells (normal epithelial cells, hematopoietic cells and stromal cells). Therefore there is a need for a sensitive technique and a complete reliable process. Standard dideoxy sequencing has been the “gold standard” for detecting somatic mutations in tissue samples. This method is robust but time-consuming, and it has only moderate sensitivity. Moreover it might suffer from a lack of robustness for the determination of mutations in formalin fixed paraffin embedded tumors [18]. These limitations of direct sequencing for detecting somatic mutations have led to the development of more sensitive, less expensive, and faster methods. A number of alternative procedures have therefore been developed to detect common cancer mutations, such as HRM [19–21], allele-specific amplification [9,22–24], primer extension [25], and pyrosequencing [26]. In most cases, a better sensitivity was obtained using targeted techniques as compared to direct sequencing [27,28] (for a recent review see Ellison et al. [29]).
A
2. EGFR alterations and primary resistance to EGFR TKI
B
Beside the activating mutations described above, some alterations have been associated with primary resistance to EGFR TKI. They are located on exon 20 of the EGFR gene, which encodes part of the kinase domain. 2.1. Exon 20 insertions Exon 20 insertions account for 4–9% of all EGFR mutant lung tumors [30,31]. Most of these insertions occur between amino acids 767 to 774, within the loop that follows the C-helix of the kinase domain [32]. These alterations have been shown to confer resistance to EGFR TKIs [32–34].
Fig. 1. (A) Detection of the T790M mutation by DNA sequencing. Partial sequences of EGFR exon 20 are presented. The mutated nucleotide is indicated with an arrow. (B) In FFPE tissues, cytosine can be transformed into uracil (transition), which in turn is converted to thymine during DNA synthesis.
2.2. T790M in untreated EGFR-mutant lung cancers The T790M mutation results in an amino acid substitution at position 790 in EGFR, from a threonine to a methionine (Fig. 1). This mutation occurs within exon 20. Threonine 790 is the gatekeeper residue in EGFR. Its key location at the entrance to a hydrophobic pocket in the back of the ATP binding cleft makes it an important determinant of inhibitor specificity in protein kinases. Substitution of this residue in EGFR with a methionine has been thought to cause resistance by steric interference with binding of TKIs [35]. But the T790M mutation has also been shown to increase the ATP affinity of the oncogenic receptor mutant [36]. Germline T790M mutations have been observed at a low frequency (~0.5% of never smokers with lung cancer), sometimes in a context of familial cancer syndrome [37,38]. A prospective study of these patients and their families is under development, in order to define screening and counseling strategies for this rare but potentially high-risk population [39]. Somatic pre-treatment T790M mutations are in most cases associated in cis (i.e. on the same allele) to a sensitizing somatic EGFR alteration such as a L858R substitution or a deletion in exon 19. The most recent studies describing the frequency of baseline T790M mutation in EGFRmutant tumors are reported in Table 1. The rate of pre-treatment T790M mutation appears to be highly dependent on the sensitivity of the method used. T790M is rarely found in untreated EGFR mutant tumors using conventional testing technique. Using Sanger sequencing, a method of relatively low sensitivity, studies report an incidence of baseline EGFR T790M of 0–5.9% in tumors presenting a sensitizing EGFR mutation [33,40–42]. When molecular methods with a higher sensitivity are utilized, the reported incidence of baseline EGFR T790M is greater. These sensitive methods include mass spectrometry-based mutation assays (2% [43]; 25.2% [42]), single allele base extension reaction (7% [44]), mutant-enriched PCR-based methods including the mutation-biased PCR quenching probe method (9.4% [45]), the Scorpion
Amplification Refractory Mutation System (SARMS) technology (38% [40]), and Taqman probes in the presence of a peptide-nucleic acid (34.9% [46]; 65.3% [47]). The highest prevalence (78.9%) was obtained using colony hybridization [48]. This highly sensitive method detects EGFR T790M at concentrations as low as 0.01% of the total DNA present. These highly sensitive assays can detect minor EGFR T790Mcontaining clones. But there is also a potential for false-positive results. Using mass spectrometry-based assays, low DNA content and quality can lead to the generation of mutant peaks that can be indistinguishable from a true mutation [43]. With mutant-enriched PCR assays, the amplification of Taq errors can occur in very low template DNA samples leading to false-positive mutation calls as well. In addition, the use of formalinfixed specimens for molecular testing may result in artificial mutation. Indeed, these fixation conditions have already been shown to induce transitions, i.e. point mutations that change a purine nucleotide to another purine (A N G) or a pyrimidine nucleotide to another pyrimidine (C N T), which is the case for the T790M mutation (Fig. 1). Such artifacts have been observed when performing PCR amplifications of small amounts of DNA, particularly if the DNA is isolated from formalin-fixed paraffin-embedded (FFPE) tissues [49–51]. In the case of the EGFR T790M mutation, this issue has been carefully addressed by Ye et al. [52]. Thirty-six TKI-naïve tumors harboring known sensitizing EGFR mutations with both frozen and FFPE samples were analyzed for EGFR T790M using a mutant-enriched PCR assay (0.1% analytical sensitivity). In the FFPE samples, the EGFR T790Mpositive rate was 42% (15/36) in the tumor tissue. But a similar mutation rate (49%; 16/33) was found in the adjacent normal tissue. When using frozen samples, only 1 of 36 samples (3%) was EGFR T790M positive, and none of the 35 adjacent normal tissue samples were found to harbor T790M. The authors conclude that detection of T790M mutations may in some cases be FFPE-derived artifacts [52]. One way to limit them would be to treat the template with uracil-N-glycosylase, an enzyme that removes uracil from DNA [53].
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Table 1 Baseline T790M mutation frequency among EGFR mutated NSCLC tumors. Reference
Method
Baseline T790M mutation (among EGFR mutated)
Analytical sensitivity
Maheswaran et al. 2008 [40]
Direct sequencing Scorpion ARMS Direct sequencing MBQ-QP TaqMan assay + PNA Direct sequencing Scorpion ARMS Colony hybridization Direct sequencing MALDI-TOF MS SABER Taqman probe + PNA MALDI-TOF MS
0 / 26 (0%) 10 / 26 (38%) 2 / 34 (5.9%) 3 / 32 (9.4%) 45 / 129 (34.9%) 6 / 627 (1%) 0 / 38 (0%) 30 / 38 (78.9%) 0 / 23 (0%) 7 / 28 (25%) 2 / 28 (7%) 62 / 95 (65.3%) 11 / 579 (2%)
NR 0.2% NR 0.4% 0.02% NR
Sequist et al. 2008 [41] Nakamura et al. 2011 [45] Rosell et al. 2011 [46] Wu et al. 2011 [33] Fujita et al. 2012 [48] Su et al. 2012 [42] Sakai et al. 2013 [44] Costa et al. 2014 [47] Yu et al. 2014 [43]
0.01% NR 2.2% 0.3% 0.02% NR
NR, not reported; ARMS, amplification refractory mutation system; MBQ-QP, mutation-biased polymerase chain reaction — quenching probe; PNA, peptide-nucleic acid; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; SABER, single allele base extension reaction.
2.3. Clinical implications of T790M in untreated EGFR-mutant lung cancers
4. T790M and third generation TKI
In order to determine whether the existence of T790M mutation before TKI therapy was associated with clinical outcome, Su et al. [42] analyzed 107 EGFR-mutant NSCLC patients. A T790M mutation was detected in 27 (25.2%) patients. They clearly demonstrated that the presence of a pre-treatment T790M mutation was not associated with the response rate and the overall survival of patients treated with an EGFR TKI, but that it was associated with a shorter progression free survival (PFS) compared to patients with activation EGFR mutations but without the T790M mutation. However, PFS was still significantly longer than for patients with wild-type EGFR. Therefore, detection of the T790M mutation in tumors of TKI-naive patients should not prevent them from being treated with an EGFR TKI, and has thus no clinical consequence at the moment.
Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR. These inhibitors have not provided compelling clinical benefit in T790Mpositive patients, apparently because of dose-limiting toxicities associated with inhibition of wild type EGFR. Third generation EGFR TKIs represent a new class of drugs that irreversibly inhibit mutant EGFR, including EGFR T790M, with much less activity against wild type EGFR. The most studied molecules are AZD9291 and CO-1686. AZD9291 is an oral, irreversible and selective inhibitor of both EGFR sensitizing and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition of this mono-anilino-pyrimidine compound in animal models, early efficacy has been observed when administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC, accompanied by an encouraging safety profile [61,62]. CO-1686 is an irreversible 2,4-diaminopyrimidine compound that is also EGFR mutant selective and has been designed to have low affinity for wild type EGFR. Pharmacokinetic and pharmacodynamic studies in H1975 (EGFR L858R/T790M) tumor-bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. As indicated for AZD9291, this compound showed no inhibition of wild type EGFR in animals [63]. CO-1686 is currently in a phase I/II clinical trial in patients with EGFR mutated-advanced NSCLC that have received prior EGFR-directed therapy. If the clinical benefit of these compounds is confirmed, T790M testing will become mandatory. In some cases, it will be difficult to collect additional biopsies at progression. Molecular testing could then be performed on circulating DNA. Recent works have demonstrated that circulating DNA is a powerful source of DNA that can be successfully used to detect activating EGFR mutations [64–66]. This strategy can also be applied to the T790M mutation [45,67]. This has 2 main advantages: no additional biopsy is required, and since these samples do not require formaldehyde fixation, the risk of false positive result is very limited. However, the sensitivity of these assays will probably not reach 100%. But it is tempting to speculate that a first screening test can be performed on circulating DNA, and that a re-biopsy will be necessary only when the screening test will be negative.
3. Acquired T790M resistance mutation Most NSCLCs with activating EGFR alterations respond dramatically to TKIs. However, all patients with EGFR-mutant NSCLC treated with EGFR TKIs will ultimately relapse. Acquired, or secondary, resistance refers to patients who have progressive disease following an initial objective response or prolonged stable disease [16]. Several mechanisms of acquired resistance to EGFR TKIs in EGFRmutant NSCLC have been described. A detailed genetic and histological analysis of NSCLC tumor biopsies from 37 patients at the time they acquired resistance has been performed by Sequist and colleagues [54]. Their first observation was that the tumors retained their original activating EGFR mutation. Some patients became resistant because they developed amplification of the MET gene or of the EGFR gene, or mutation of the PIK3CA gene. A few other lung cancers transitioned from epithelial cell morphology to mesenchymal cell-like appearance. In 5 other cases a conversion of NSCLC into small cell lung cancers was observed. But the most frequent alteration associated with TKI resistance was the EGFR T790M mutation. The T790M mutation was first discovered in patients who acquired resistance to gefitinib or erlotinib [55,56]. It has then been shown to be a “second-site mutation” in more than 50% of EGFR-mutant lung cancers that have developed acquired resistance to EGFR TKI [56–58]. Interestingly, case reports have shown that this resistance mutation can be lost after an EGFR TKI free period [54]. These patients then responded to a re-challenge with EGFR TKI. Other reports have also shown that patients can re-respond to TKI treatment after a hiatus from targeted therapy [59]. Finally, patients with secondary T790M mutation at the time of gradual or local progression benefit more from EGFR-TKI treatment beyond progression compared to those without T790M mutation [60].
5. Conclusions It is still unclear whether the majority of T790M mutations emerge from preexisting T790M mutation in TKI-naïve patients as a minor clone or is acquired during disease progression. The highest frequencies of T790M mutations reported in TKI-naïve patients are most likely overestimated, because of artificial DNA alterations occurring in FFPE
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samples. But this has no practical clinical consequence at the moment in TKI-naïve patients. By contrast, with the very promising results recently reported with third generation TKI, detection of this resistance T790M mutation will probably be mandatory in the near future. The analysis could be performed on re-biopsied tumor tissues, but also on circulating cell free DNA.
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[63] Tjin Tham Sjin R, Lee K, Walter AO, et al. In vitro and in vivo characterization of irreversible mutant-selective EGFR inhibitors that are wild-type sparing. Mol Cancer Ther 2014;13:1468–79. [64] Luo J, Shen L, Zheng D. Diagnostic value of circulating free DNA for the detection of EGFR mutation status in NSCLC: a systematic review and meta-analysis. Sci Rep 2014;4:6269. [65] Douillard JY, Ostoros G, Cobo M, et al. First-line gefitinib in Caucasian EGFR mutationpositive NSCLC patients: a phase-IV, open-label, single-arm study. Br J Cancer 2014; 110:55–62. [66] Vallee A, Marcq M, Bizieux A, et al. Plasma is a better source of tumor-derived circulating cell-free DNA than serum for the detection of EGFR alterations in lung tumor patients. Lung Cancer 2013;82:373–4. [67] Marcq M, Vallee A, Bizieux A, Denis MG. Detection of EGFR mutations in the plasma of patients with lung adenocarcinoma for real-time monitoring of therapeutic response to tyrosine kinase inhibitors? J Thorac Oncol 2014;9:e49–50.
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Invited critical review
EGFR T790M resistance mutation in non small-cell lung carcinoma Marc G. Denis ⁎, Audrey Vallée, Sandrine Théoleyre Department of Biochemistry, Nantes University Hospital, Nantes, France
a r t i c l e
i n f o
Article history: Received 15 December 2014 Received in revised form 25 January 2015 Accepted 27 January 2015 Available online 7 February 2015 Keywords: EGFR T790M Mutation Resistance Non small-cell lung cancer
a b s t r a c t Lung cancer patients carrying sensitive epidermal growth factor receptor (EGFR) mutations show dramatic responses to tyrosine kinase inhibitors (TKIs). However, the majority of patients whose disease responds to drugs eventually develop resistance to these EGFR-TKIs. The T790M gatekeeper mutation in the EGFR tyrosine kinase domain accounts for half of resistance to these drugs. In some patients, this mutation is also detected as a primary event before drug exposure, at a frequency that is highly dependent on the technique used. This review will focus on the methods that have been used to detect the T790M mutation, and its potential clinical applications both in TKI naïve patients and in patients with an acquired resistance. © 2015 Elsevier B.V. All rights reserved.
Contents 1. 2.
EGFR TKI and activating mutations . . . . . . . . . . . . . . . . . . . EGFR alterations and primary resistance to EGFR TKI . . . . . . . . . . . 2.1. Exon 20 insertions . . . . . . . . . . . . . . . . . . . . . . . 2.2. T790M in untreated EGFR-mutant lung cancers . . . . . . . . . . 2.3. Clinical implications of T790M in untreated EGFR-mutant lung cancers 3. Acquired T790M resistance mutation . . . . . . . . . . . . . . . . . . 4. T790M and third generation TKI . . . . . . . . . . . . . . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1. EGFR TKI and activating mutations Prospective phase III randomized clinical trials showed that tyrosine kinase inhibitors (TKI) gefitinib [1–4], erlotinib [5,6], and more recently afatinib [7,8] improved outcomes compared with chemotherapy as initial treatment in EGFR-mutant non small cell lung cancer (NSCLC). These molecules have thus been approved in many countries worldwide. Therefore, routine molecular analysis of pathological specimens is mandatory in clinical practice to predict patient response. The potential result is an increased likelihood that patients will receive optimal therapy for their tumor and be spared a course of therapy with no or significantly less benefit.
⁎ Corresponding author at: Laboratoire de Biochimie, CHU-Institut de Biologie, 9 quai Moncousu, 44093 Nantes cedex, France. Tel.: +33 240 08 75 90; fax: +33 240 08 39 91. E-mail address: [email protected] (M.G. Denis).
http://dx.doi.org/10.1016/j.cca.2015.01.039 0009-8981/© 2015 Elsevier B.V. All rights reserved.
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Activating EGFR mutations occur in 10–15% of NSCLC patients of Caucasian ethnicity. They are more frequent in never-smokers, females, and adenocarcinomas [9]. EGFR mutations affect the EGFR tyrosine kinase domain, within exons 18–21. The p.L858R substitution in exon 21 and in-frame deletions in exon 19 account for 90% of all EGFR mutations. These are clearly activating mutations. Apart from these two hotspot mutations, other mutations have been described [10]. Substitutions in exon 18 (codon 719) are associated with TKI responses [11–13], although lower than those observed for exon 19 deletions and p.L858R mutation. Exon 19 insertions (in-frame insertions of 6 amino acids in the kinase domain) have been described as EGFR-TKI-sensitizing mutations as well [14]. The p.S768I mutation on exon 20 has initially been associated with low sensitivity to gefitinib and erlotinib in vitro [15], but good clinical responses have been reported in patients presenting an EGFR S768I alone [16] or associated with another EGFR alteration [17]. In current clinical practice, there is no standardized method for the detection of EGFR mutations in NSCLC tumor samples. The samples
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available for detection of somatic mutations in tumors are usually composed of mutant and wild-type DNA from tumor cells and wild-type DNA from non-malignant cells (normal epithelial cells, hematopoietic cells and stromal cells). Therefore there is a need for a sensitive technique and a complete reliable process. Standard dideoxy sequencing has been the “gold standard” for detecting somatic mutations in tissue samples. This method is robust but time-consuming, and it has only moderate sensitivity. Moreover it might suffer from a lack of robustness for the determination of mutations in formalin fixed paraffin embedded tumors [18]. These limitations of direct sequencing for detecting somatic mutations have led to the development of more sensitive, less expensive, and faster methods. A number of alternative procedures have therefore been developed to detect common cancer mutations, such as HRM [19–21], allele-specific amplification [9,22–24], primer extension [25], and pyrosequencing [26]. In most cases, a better sensitivity was obtained using targeted techniques as compared to direct sequencing [27,28] (for a recent review see Ellison et al. [29]).
A
2. EGFR alterations and primary resistance to EGFR TKI
B
Beside the activating mutations described above, some alterations have been associated with primary resistance to EGFR TKI. They are located on exon 20 of the EGFR gene, which encodes part of the kinase domain. 2.1. Exon 20 insertions Exon 20 insertions account for 4–9% of all EGFR mutant lung tumors [30,31]. Most of these insertions occur between amino acids 767 to 774, within the loop that follows the C-helix of the kinase domain [32]. These alterations have been shown to confer resistance to EGFR TKIs [32–34].
Fig. 1. (A) Detection of the T790M mutation by DNA sequencing. Partial sequences of EGFR exon 20 are presented. The mutated nucleotide is indicated with an arrow. (B) In FFPE tissues, cytosine can be transformed into uracil (transition), which in turn is converted to thymine during DNA synthesis.
2.2. T790M in untreated EGFR-mutant lung cancers The T790M mutation results in an amino acid substitution at position 790 in EGFR, from a threonine to a methionine (Fig. 1). This mutation occurs within exon 20. Threonine 790 is the gatekeeper residue in EGFR. Its key location at the entrance to a hydrophobic pocket in the back of the ATP binding cleft makes it an important determinant of inhibitor specificity in protein kinases. Substitution of this residue in EGFR with a methionine has been thought to cause resistance by steric interference with binding of TKIs [35]. But the T790M mutation has also been shown to increase the ATP affinity of the oncogenic receptor mutant [36]. Germline T790M mutations have been observed at a low frequency (~0.5% of never smokers with lung cancer), sometimes in a context of familial cancer syndrome [37,38]. A prospective study of these patients and their families is under development, in order to define screening and counseling strategies for this rare but potentially high-risk population [39]. Somatic pre-treatment T790M mutations are in most cases associated in cis (i.e. on the same allele) to a sensitizing somatic EGFR alteration such as a L858R substitution or a deletion in exon 19. The most recent studies describing the frequency of baseline T790M mutation in EGFRmutant tumors are reported in Table 1. The rate of pre-treatment T790M mutation appears to be highly dependent on the sensitivity of the method used. T790M is rarely found in untreated EGFR mutant tumors using conventional testing technique. Using Sanger sequencing, a method of relatively low sensitivity, studies report an incidence of baseline EGFR T790M of 0–5.9% in tumors presenting a sensitizing EGFR mutation [33,40–42]. When molecular methods with a higher sensitivity are utilized, the reported incidence of baseline EGFR T790M is greater. These sensitive methods include mass spectrometry-based mutation assays (2% [43]; 25.2% [42]), single allele base extension reaction (7% [44]), mutant-enriched PCR-based methods including the mutation-biased PCR quenching probe method (9.4% [45]), the Scorpion
Amplification Refractory Mutation System (SARMS) technology (38% [40]), and Taqman probes in the presence of a peptide-nucleic acid (34.9% [46]; 65.3% [47]). The highest prevalence (78.9%) was obtained using colony hybridization [48]. This highly sensitive method detects EGFR T790M at concentrations as low as 0.01% of the total DNA present. These highly sensitive assays can detect minor EGFR T790Mcontaining clones. But there is also a potential for false-positive results. Using mass spectrometry-based assays, low DNA content and quality can lead to the generation of mutant peaks that can be indistinguishable from a true mutation [43]. With mutant-enriched PCR assays, the amplification of Taq errors can occur in very low template DNA samples leading to false-positive mutation calls as well. In addition, the use of formalinfixed specimens for molecular testing may result in artificial mutation. Indeed, these fixation conditions have already been shown to induce transitions, i.e. point mutations that change a purine nucleotide to another purine (A N G) or a pyrimidine nucleotide to another pyrimidine (C N T), which is the case for the T790M mutation (Fig. 1). Such artifacts have been observed when performing PCR amplifications of small amounts of DNA, particularly if the DNA is isolated from formalin-fixed paraffin-embedded (FFPE) tissues [49–51]. In the case of the EGFR T790M mutation, this issue has been carefully addressed by Ye et al. [52]. Thirty-six TKI-naïve tumors harboring known sensitizing EGFR mutations with both frozen and FFPE samples were analyzed for EGFR T790M using a mutant-enriched PCR assay (0.1% analytical sensitivity). In the FFPE samples, the EGFR T790Mpositive rate was 42% (15/36) in the tumor tissue. But a similar mutation rate (49%; 16/33) was found in the adjacent normal tissue. When using frozen samples, only 1 of 36 samples (3%) was EGFR T790M positive, and none of the 35 adjacent normal tissue samples were found to harbor T790M. The authors conclude that detection of T790M mutations may in some cases be FFPE-derived artifacts [52]. One way to limit them would be to treat the template with uracil-N-glycosylase, an enzyme that removes uracil from DNA [53].
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Table 1 Baseline T790M mutation frequency among EGFR mutated NSCLC tumors. Reference
Method
Baseline T790M mutation (among EGFR mutated)
Analytical sensitivity
Maheswaran et al. 2008 [40]
Direct sequencing Scorpion ARMS Direct sequencing MBQ-QP TaqMan assay + PNA Direct sequencing Scorpion ARMS Colony hybridization Direct sequencing MALDI-TOF MS SABER Taqman probe + PNA MALDI-TOF MS
0 / 26 (0%) 10 / 26 (38%) 2 / 34 (5.9%) 3 / 32 (9.4%) 45 / 129 (34.9%) 6 / 627 (1%) 0 / 38 (0%) 30 / 38 (78.9%) 0 / 23 (0%) 7 / 28 (25%) 2 / 28 (7%) 62 / 95 (65.3%) 11 / 579 (2%)
NR 0.2% NR 0.4% 0.02% NR
Sequist et al. 2008 [41] Nakamura et al. 2011 [45] Rosell et al. 2011 [46] Wu et al. 2011 [33] Fujita et al. 2012 [48] Su et al. 2012 [42] Sakai et al. 2013 [44] Costa et al. 2014 [47] Yu et al. 2014 [43]
0.01% NR 2.2% 0.3% 0.02% NR
NR, not reported; ARMS, amplification refractory mutation system; MBQ-QP, mutation-biased polymerase chain reaction — quenching probe; PNA, peptide-nucleic acid; MALDI-TOF MS, matrix-assisted laser desorption ionization time-of-flight mass spectrometry; SABER, single allele base extension reaction.
2.3. Clinical implications of T790M in untreated EGFR-mutant lung cancers
4. T790M and third generation TKI
In order to determine whether the existence of T790M mutation before TKI therapy was associated with clinical outcome, Su et al. [42] analyzed 107 EGFR-mutant NSCLC patients. A T790M mutation was detected in 27 (25.2%) patients. They clearly demonstrated that the presence of a pre-treatment T790M mutation was not associated with the response rate and the overall survival of patients treated with an EGFR TKI, but that it was associated with a shorter progression free survival (PFS) compared to patients with activation EGFR mutations but without the T790M mutation. However, PFS was still significantly longer than for patients with wild-type EGFR. Therefore, detection of the T790M mutation in tumors of TKI-naive patients should not prevent them from being treated with an EGFR TKI, and has thus no clinical consequence at the moment.
Second-generation irreversible EGFR inhibitors are effective against T790M mutations in vitro, but retain affinity for wild-type EGFR. These inhibitors have not provided compelling clinical benefit in T790Mpositive patients, apparently because of dose-limiting toxicities associated with inhibition of wild type EGFR. Third generation EGFR TKIs represent a new class of drugs that irreversibly inhibit mutant EGFR, including EGFR T790M, with much less activity against wild type EGFR. The most studied molecules are AZD9291 and CO-1686. AZD9291 is an oral, irreversible and selective inhibitor of both EGFR sensitizing and T790M resistance mutations with selectivity over the wild type form of the receptor. Following observations of significant tumor inhibition of this mono-anilino-pyrimidine compound in animal models, early efficacy has been observed when administered clinically to patients with T790M positive EGFR-TKI resistant NSCLC, accompanied by an encouraging safety profile [61,62]. CO-1686 is an irreversible 2,4-diaminopyrimidine compound that is also EGFR mutant selective and has been designed to have low affinity for wild type EGFR. Pharmacokinetic and pharmacodynamic studies in H1975 (EGFR L858R/T790M) tumor-bearing mice showed that exposure was dose proportional resulting in dose-dependent EGFR modulation. As indicated for AZD9291, this compound showed no inhibition of wild type EGFR in animals [63]. CO-1686 is currently in a phase I/II clinical trial in patients with EGFR mutated-advanced NSCLC that have received prior EGFR-directed therapy. If the clinical benefit of these compounds is confirmed, T790M testing will become mandatory. In some cases, it will be difficult to collect additional biopsies at progression. Molecular testing could then be performed on circulating DNA. Recent works have demonstrated that circulating DNA is a powerful source of DNA that can be successfully used to detect activating EGFR mutations [64–66]. This strategy can also be applied to the T790M mutation [45,67]. This has 2 main advantages: no additional biopsy is required, and since these samples do not require formaldehyde fixation, the risk of false positive result is very limited. However, the sensitivity of these assays will probably not reach 100%. But it is tempting to speculate that a first screening test can be performed on circulating DNA, and that a re-biopsy will be necessary only when the screening test will be negative.
3. Acquired T790M resistance mutation Most NSCLCs with activating EGFR alterations respond dramatically to TKIs. However, all patients with EGFR-mutant NSCLC treated with EGFR TKIs will ultimately relapse. Acquired, or secondary, resistance refers to patients who have progressive disease following an initial objective response or prolonged stable disease [16]. Several mechanisms of acquired resistance to EGFR TKIs in EGFRmutant NSCLC have been described. A detailed genetic and histological analysis of NSCLC tumor biopsies from 37 patients at the time they acquired resistance has been performed by Sequist and colleagues [54]. Their first observation was that the tumors retained their original activating EGFR mutation. Some patients became resistant because they developed amplification of the MET gene or of the EGFR gene, or mutation of the PIK3CA gene. A few other lung cancers transitioned from epithelial cell morphology to mesenchymal cell-like appearance. In 5 other cases a conversion of NSCLC into small cell lung cancers was observed. But the most frequent alteration associated with TKI resistance was the EGFR T790M mutation. The T790M mutation was first discovered in patients who acquired resistance to gefitinib or erlotinib [55,56]. It has then been shown to be a “second-site mutation” in more than 50% of EGFR-mutant lung cancers that have developed acquired resistance to EGFR TKI [56–58]. Interestingly, case reports have shown that this resistance mutation can be lost after an EGFR TKI free period [54]. These patients then responded to a re-challenge with EGFR TKI. Other reports have also shown that patients can re-respond to TKI treatment after a hiatus from targeted therapy [59]. Finally, patients with secondary T790M mutation at the time of gradual or local progression benefit more from EGFR-TKI treatment beyond progression compared to those without T790M mutation [60].
5. Conclusions It is still unclear whether the majority of T790M mutations emerge from preexisting T790M mutation in TKI-naïve patients as a minor clone or is acquired during disease progression. The highest frequencies of T790M mutations reported in TKI-naïve patients are most likely overestimated, because of artificial DNA alterations occurring in FFPE
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samples. But this has no practical clinical consequence at the moment in TKI-naïve patients. By contrast, with the very promising results recently reported with third generation TKI, detection of this resistance T790M mutation will probably be mandatory in the near future. The analysis could be performed on re-biopsied tumor tissues, but also on circulating cell free DNA.
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