Reliability of the peptide nucleic acid-locked nucleic acid polymerase chain reaction clamp-based test for epidermal growth factor receptor mutations integrated into the clinical practice for non-small cell lung cancers

Gefitinib is an inhibitor of the tyrosine kinase activity of epidermal growth factor receptor (EGFR). Accumulating evidence suggests that gefitinib may provide a survival benefit to EGFR mutation-positive non-small lung cancer patients. We have established a clinical test that can detect EGFR mutations from cytological specimens or paraffin-embedded tissue specimens that are contaminated by normal cells. This test is based on the peptide nucleic acid, locked nucleic acid polymerase chain reaction clamp method that can detect G719S, G719C, L858R, L861Q and seven different exon 19 deletions in the presence of 100-1000-fold wild-type alleles. Consequently, using a small aliquot of samples isolated to establish a cancer diagnosis, the EGFR mutation status is determined soon after the diagnosis of cancer is made. We investigated the EGFR mutation status in 86 patients using a variety of cytological specimens (59 bronchoscopy specimens, 16 pleural effusion, 9 sputum, and 2 pericardial effusion) and in 46 patients who had a disease relapse and paraffin-embedded tissues were available. Forty-five patients (34%) were positive for mutation (29 exon 19 deletions, 16 L858R and 1 L861Q). The sensitivity and the specificity of this test was 97% and 100%, respectively. EGFR mutation status thereby obtained was used to determine each patient's therapeutic regimen. This test is easily integrated into the normal clinical practice for lung cancer, while allowing the medical staff to select therapeutic regimen depending on the EGFR mutation status.     

EGFR mutation status in pleural fluid predicts tumor responsiveness and resistance to gefitinib

It has been reported that the threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene is correlated with acquired resistance to gefitinib. We previously reported that there was some population that harbored the EGFR T790M mutation as a minor clone of tumor cells prior to drug treatment, may be causing resistance to gefitinib during treatment. This fact also suggests that the detection of the EGFR T790M mutation prior to treatment may predict the development of resistance. We also showed that pleural fluid is a useful specimen for detection of EGFR mutation using sensitive assays. In this study, we reported a female patient who was treated with gefitinib because an EGFR L858R mutation was found in her pleural fluid. Our patient showed partial response to gefitinib, but she had progressive disease only 4 months after the start of treatment. Furthermore, the EGFR T790M mutation was detected in the pleural fluid before gefitinib treatment by the mutant-enriched PCR assay. Our findings confirmed that the EGFR T790M mutation was occasionally present as a minor population in tumor cells before treatment and caused resistance after gefitinib administration. The detection of a small fraction of T790M-positive alleles may be useful to predict the clinical course of the gefitinib-treated non-small-cell lung cancer patients.     

Identification of non-small-cell lung cancer with activating EGFR mutations in malignant effusion and cerebrospinal fluid: rapid and sensitive detection of exon 19 deletion E746-A750 and exon 21 L858R mutation by immunocytochemistry

Background

Recently, we have reported that EGFR mutation-specific antibodies performed well in immunohistochemical analysis, with good sensitivity. We investigated whether this method could detect non-small-cell lung cancer (NSCLC) carrying EGFR mutations in malignant effusions and cerebrospinal fluid (CSF), comparable to the peptide nucleic acid–locked nucleic acid (PNA–LNA) PCR clamp assay. Furthermore, we compared activating EGFR mutations between primary and recurrent NSCLC.

Patients and methods

Twenty-four patients with NSCLC effusions and CSF were examined by immunocytochemistry using antibodies specific for the E746-A750 deletion mutation in exon 19 and the L858R point mutation in exon 21. The PNA–LNA PCR clamp assay was used to detect the E746-A750 deletion at exon 19, L858R mutation at exon 21, and T790M mutation at exon 20.

Results

We were able to identify EGFR mutations in NSCLC effusion and CSF with a sensitivity of 100% (5/5) using the anti-delE746-A750 antibody and 100% (8/8) using the anti-L858R antibody. Furthermore, in samples without these EGFR mutations, immunocytochemistry with the two specific antibodies identified 91% (10/11) as negative for both the deletion and the point mutations in EGFR. Activating EGFR mutations decreased in recurrent NSCLC compared with primary NSCLC, and the T790M mutation was detected in recurrent NSCLC of patients receiving gefitinib treatment.

Conclusions

Identification of EGFR mutations is important for patients with primary and recurrent NSCLC. Rapid and sensitive immunocytochemistry using mutation-specific antibodies to detect EGFR mutations will be useful for diagnosing responsiveness to EGFR-targeted drugs.     

Sustained response to standard dose osimertinib in a patient with plasma T790M‐positive leptomeningeal metastases from primary lung adenocarcinoma

A 44‐year‐old male, never smoker, suffers from stage IV adenocarcinoma of the right lung with epidermal growth factor receptor (EGFR) exon‐21 L858R point mutation on initial presentation. After 23 months of treatment with gefitinib, intercalated with multiple courses of radiotherapy, leptomeningeal metastases (LMs) developed. Acquired T790M mutation was confirmed by the droplet digital polymerase chain reaction plasma EGFR test. After switching to osimertinib at the standard dose, his neurocognitive function improved clinically, coupled with sustained radiological improvement. As this clinical entity is underrepresented in clinical trials, the practicability of plasma EGFR testing and the optimal dose–response relationship of osimertinib in T790M‐positive lung cancer complicated with LM deserves further exploration.     

An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor

Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in non-small cell lung cancer specimens from patients responding to anilinoquinazoline EGFR inhibitors. However, clinical resistance to EGFR inhibitor therapy is commonly observed. Previously, we showed that such resistance can be caused by a second mutation of the EGFR gene, leading to a T790M amino acid change in the EGFR tyrosine kinase domain and also found that CL-387,785, a specific and irreversible anilinoquinazoline EGFR inhibitor, was able to overcome this resistance on the biochemical level. Here, we present the successful establishment of a stable Ba/F3 cell line model system for the study of oncogenic EGFR signaling and the functional consequences of the EGFR T790M resistance mutation. We show the ability of gefitinib to induce growth arrest and apoptosis in cells transfected with wild-type or L858R EGFR, whereas the T790M mutation leads to high-level functional resistance against gefitinib and erlotinib. In addition, CL-387,785 is able to overcome resistance caused by the T790M mutation on a functional level, correlating with effective inhibition of downstream signaling pathways. Similar data was also obtained with the use of the gefitinib-resistant H1975 lung cancer cell line. The systems established by us should prove useful for the large-scale screening of alternative EGFR inhibitor compounds against the T790M or other EGFR mutations. These data also support the notion that clinical investigations of compounds similar to CL-387,785 may be useful as a treatment strategy for patients with resistance to EGFR inhibitor therapy caused by the T790M mutation.     

Double mutation and gene copy number of EGFR in gefitinib refractory non-small-cell lung cancer

Mutations of the epidermal growth factor receptor (EGFR) gene have been reported in non-small-cell lung cancer (NSCLC), especially in patients with adenocarcinoma and never smokers. Some common somatic mutations in EGFR, including deletion mutations in exon 19 and leucine-to-arginine substitution at amino acid position 858 (L858R) in exon 21, have been examined for their ability to predict sensitivity to gefitinib or erlotinib, which are selective EGFR tyrosine kinase inhibitors (EGFR-TKIs). On the other hand, reports have shown that the threonine-to-methionine substitution at amino acid position 790 (T790M) in exon 20 is related to gefitinib resistance. Some studies have indicated that high copy numbers of the EGFR gene may be a more effective molecular predictor to responsiveness and prolonged survival in patients treated with EGFR-TKIs. Here, we describe two NSCLC patients with the L858R mutation who did not respond to gefitinib. Case 1 harbored both the T790M and L858R mutations, and fluorescence in situ hybridization showed EGFR gene amplification. Case 2 harbored both the L858R and aspartic acid-to-tyrosine substitution at amino acid position 761 in exon 19 of EGFR mutations and had a high polysomy status for EGFR. In these two cases, tumors showed resistance to gefitinib treatment despite the presence of EGFR L858R mutation and increased copy number. Our findings encourage further molecular analysis to elucidate the relationship between the EGFR status, including mutations and amplifications, and the responsiveness of NSCLC to gefitinib.     

Simple method to detect important epidermal growth factor receptor gene mutations with bronchoscopic specimens of lung cancer patients for gefitinib treatment

Recent studies have demonstrated that epidermal growth factor receptor (EGFR) mutations are a predictive molecular marker of tumor response to gefitinib treatment in non-small-cell lung cancer (NSCLC). Early studies of EGFR mutations have been analyzed by direct sequencing with surgically resected specimens. However, it is often difficult to obtain sufficient and tumor-enriched tissue specimens from inoperative NSCLC patients for mutation analysis. Therefore, we set out to develop a simple, sensitive and reliable method based on polymerase chain reaction (PCR) and restriction endonuclease treatment to detect important EGFR mutations for gefitinib treatment, including G719S, deletion or insertion in exons 19 and 20, T790M, L858R and L861Q using bronchoscopic specimens. Two hundred and eleven bronchoscopic samples cytologically diagnosed as malignant were analyzed and mutations detected from 60 patients (28.4%). The present assay could detect deletion in exon 19 and L858R mutations in the presence of at least 1% and 5% mutant cells, respectively, from a background of normal cells. Comparison of mutation detection between bronchoscopic and surgical specimens from 42 patients indicated that the sensitivity of this assay using bronchoscopic specimens was 95% (18/19) with a specificity of 100% (23/23). Furthermore, deletion variants in exon 19 could be distinguished by native polyacrylamide gel electrophoresis. Consequently, this simple, sensitive and reliable assay can provide important information pertaining to optimal therapeutic strategies.     

244-MPT overcomes gefitinib resistance in non-small cell lung cancer cells

The epidermal growth factor receptor (EGFR) is known to play a critical role in non-small cell lung cancer(NSCLC). Several EGFR tyrosine kinase inhibitors(TKIs), such as gefitinib, have been used as effective clinical therapies for patients with NSCLC. Unfortunately, acquired resistance to gefitinib commonly occurs after 6–12 months of treatment. The resistance is associated with the appearance of the L858R/T790M double mutation of the EGFR. In our present study, we discovered a compound,referred to as 244-MPT, which could suppress either gefitinib-sensitive or -resistant lung cancer cell growth and colony formation, and also suppressed the kinase activity of both wildtype and double mutant (L858R/T790M) EGFR. The underlying mechanism reveals that 244-MPT could interact with either the wildtype or double-mutant EGFR in an ATP-competitive manner and inhibit activity. Treatment with 244-MPT could substantially reduce the phosphorylation of EGFR and its downstream signaling pathways, including Akt and ERK1/2 in gefitinib-sensitive and -resistant cell lines. It was equally effective in suppressing EGFR phosphorylation and downstream signaling in NL20 cells transfected with wildtype, single-mutant (L858R) or mutant (L858R/T790M) EGFR. 244-MPT could also induce apoptosis in a gefitinib-resistant cell line and strongly suppress gefitinib-resistant NSCLC tumor growth in a xenograft mouse model. In addition, 244-MPT could effectively reduce the size of tumors in a gefitinib-resistant NSCLC patient-derived xenograft (PDX) SCID mouse model. Overall, 244-MPT could overcome gefitinib-resistance by directly targeting the EGFR.     

Analysis of epidermal growth factor receptor gene mutation in patients with non-small cell lung cancer and acquired resistance to gefitinib.

PURPOSE: Non-small cell lung cancers carrying activating mutations in the gene for the epidermal growth factor receptor (EGFR) are highly sensitive to EGFR-specific tyrosine kinase inhibitors. However, most patients who initially respond subsequently experience disease progression while still on treatment. Part of this "acquired resistance" is attributable to a secondary mutation resulting in threonine to methionine at codon 790 (T790M) of EGFR. EXPERIMENTAL DESIGN: We sequenced exons 18 to 21 of the EGFR gene to look for secondary mutations in tumors with acquired resistance to gefitinib in 14 patients with adenocarcinomas. Subcloning or cycleave PCR was used in addition to normal sequencing to increase the sensitivity of the assay. We also looked for T790M in pretreatment samples from 52 patients who were treated with gefitinib. We also looked for secondary KRAS gene mutations because tumors with KRAS mutations are generally resistant to tyrosine kinase inhibitors. RESULTS: Seven of 14 tumors had a secondary T790M mutation. There were no other novel secondary mutations. We detected no T790M mutations in pretreatment specimens from available five tumors among these seven tumors. Patients with T790M tended to be women, never smokers, and carrying deletion mutations, but the T790M was not associated with the duration of gefitinib administration. None of the tumors had an acquired mutation in the KRAS gene. CONCLUSIONS: A secondary T790M mutation of EGFR accounted for half the tumors with acquired resistance to gefitinib in Japanese patients. Other drug-resistant secondary mutations are uncommon in the EGFR gene.     

EGFR-independent mechanisms of acquired resistance to AZD9291 in EGFR T790M-positive NSCLC patients

BackgroundAZD9291 is an oral, irreversible, mutant-selective epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR-TKI), which specifically targets both sensitizing and resistant T790M mutations. This compound has shown outstanding activity, in a phase I/II (AURA) trial. However, despite impressive tumor responses in T790M-positive patients, acquired resistance to this drug limits the benefit of this compound. Mutations at the EGFR C797 codon, located within the kinase-binding site, were very recently reported to be a potential mechanism of resistance to AZD9291 in T790M-positive patients.Patients and methodsTo identify potential mechanisms of resistance to AZD9291, we report here on two patients with resistant biopsy specimens that had been treated with AZD9291.ResultsWe identified in two distinct cases, HER2 and MET amplification by FISH and CGH as a potential mechanism of acquired resistance to third-generation EGFR-TKI. Interestingly, this event occurred with complete loss of the T790M mutation. In one case, we observed a different molecular status at two biopsy sites (the T790M mutation at the primary site and wild-type T790M at the metastatic site with different pathways of acquired resistance to AZD9291).ConclusionOur observations suggest that T790M-positive and wild-type T790M clones may coexist at baseline. AZD9291 efficiently suppresses the growth of T790M-positive cells, but a population of wild-type T790M cells at baseline will mediate the development of resistance, here via a by-pass pathway activating either HER2 or MET.     

Incidence of T790M in Patients With NSCLC Progressed to Gefitinib,Erlotinib, and Afatinib: A Study on Circulating Cell-free DNA

BackgroundInsights into the mechanism of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) could provide important information for further patient management, including the choice of second-line treatment. The EGFR T790M mutation is the most common mechanism of resistance to first- and second-generation EGFR TKIs. Owing to its biologic relevance in the response of non–small-cell lung cancer (NSCLC) to the selective pressure of treatment, the present study investigated whether the occurrence of T790M at progression differed among patients receiving gefitinib, erlotinib, or afatinib.Patients and MethodsThe present retrospective study included patients with NSCLC with an EGFR activating mutation, who had received gefitinib, erlotinib, or afatinib as first-line treatment. Plasma samples for the analysis of cell-free DNA were taken at disease progression and analyzed using a digital droplet polymerase chain reaction EGFR mutation assay.ResultsA total of 83 patients were enrolled; 42 had received gefitinib or erlotinib and 41afatinib. The patient characteristics were comparable across the 2 groups. The median time to progression (TTP) was 14.4 months for the gefitinib and erlotinib group and 10.2 months for the afatinib group (P = .09). Of the 83 patients, 47 (56.6%) were positive for the T790M in plasma. A greater incidence of T790M was observed in patients with progression during gefitinib or erlotinib therapy compared with patients treated with afatinib (33 [79%] vs. 14 [34%], respectively; odds ratio, 7.1; 95% confidence interval, 2.7-18.5; P = .0001).ConclusionsAlthough gefitinib, erlotinib, and afatinib showed a comparable TTP in patients receiving first-line therapy, the incidence of T790M differed among them, as demonstrated by the present study, which could have implications for the choice of second-line treatment.     

Hsp90 inhibition suppresses mutant EGFR-T790M signaling and overcomes kinase inhibitor resistance

The epidermal growth factor receptor (EGFR) secondary kinase domain T790M non-small cell lung cancer (NSCLC) mutation enhances receptor catalytic activity and confers resistance to the reversible tyrosine kinase inhibitors gefitinib and erlotinib. Currently, irreversible inhibitors represent the primary approach in clinical use to circumvent resistance. We show that higher concentrations of the irreversible EGFR inhibitor CL-387,785 are required to inhibit EGFR phosphorylation in T790M-expressing cells compared with EGFR mutant NSCLC cells without T790M. Additionally, CL-387,785 does not fully suppress phosphorylation of other activated receptor tyrosine kinases (RTK) in T790M-expressing cells. These deficiencies result in residual Akt and mammalian target of rapamycin (mTOR) activities. Full suppression of EGFR-mediated signaling in T790M-expressing cells requires the combination of CL-387,785 and rapamycin. In contrast, Hsp90 inhibition overcomes these limitations in vitro and depletes cells of EGFR, other RTKs, and phospho-Akt and inhibits mTOR signaling whether or not T790M is present. EGFR-T790M-expressing cells rendered resistant to CL-387,785 by a kinase switch mechanism retain sensitivity to Hsp90 inhibition. Finally, Hsp90 inhibition causes regression in murine lung adenocarcinomas driven by mutant EGFR (L858R) with or without T790M. However, efficacy in the L858R-T790M model requires a more intense treatment schedule and responses were transient. Nonetheless, these findings suggest that Hsp90 inhibitors may be effective in T790M-expressing cells and offer an alternative therapeutic strategy for this subset of lung cancers.     

Challenges of detecting EGFR T790M in gefitinib/erlotinib-resistant tumours

For patients with advanced non-small cell lung cancer (NSCLC), the introduction of the epi- dermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), gefitinib and erlotinib, into clinical practice was promising. Treatment with either of these agents is associated with an objective response in 10-20% of patients. Subsequent studies have shown that patients responsive to gefitinib/erlotinib have tumours containing somatic activating mutations in the EGFR gene. Although impressive clinical and radiological responses have been observed in these patients, tumour progression occurs after the prolonged administration of gefitinib/erlotinib as acquired resistance develops. In order to combat acquired resistance, research has been largely focused on determining the factors underlying it. Two resistance mechanisms have so far been identified: a secondary mutation in the EGFR gene, T790M, and amplification of the MET proto-oncogene. This review will centre on T790M, which is thought to cause steric hindrance and impair the binding of gefitinib/erlotinib. A novel class of irreversible TKIs currently under development may retain activity against some common resistance mechanisms, including T790M. The next challenge is to identify accurately the subgroup of patients with NSCLC whose tumours harbour EGFR T790M. To this end, post-treatment tumour specimens will be needed to establish molecular profiles for each patient. In addition, novel, highly sensitive technology will be required to detect these mutations. This is because allelic dilution, whereby the EGFR gene is amplified but only a few copies of the T790M allele are needed to confer resistance, may obscure results of conventional sequencing methods. The importance of identifying patients who harbour T790M cannot be overstated; the development of irreversible TKIs will have profound implications for their treatment. In this way, treatment strategies in NSCLC are becoming increasingly tailored to the individual, and may set an example for other areas of oncology.     

Novel D761Y and common secondary T790M mutations in epidermal growth factor receptor-mutant lung adenocarcinomas with acquired resistance to kinase inhibitors.

PURPOSE: In patients whose lung adenocarcinomas harbor epidermal growth factor receptor (EGFR) tyrosine kinase domain mutations, acquired resistance to the tyrosine kinase inhibitors (TKI) gefitinib (Iressa) and erlotinib (Tarceva) has been associated with a second-site EGFR mutation, which leads to substitution of methionine for threonine at position 790 (T790M). We aimed to elucidate the frequency and nature of secondary EGFR mutations in patients with acquired resistance to TKI monotherapy. EXPERIMENTAL DESIGN: Tumor cells from patients with acquired resistance were examined for secondary EGFR kinase domain mutations by molecular analyses. RESULTS: Eight of 16 patients (50% observed rate; 95% confidence interval, 25-75%) had tumor cells with second-site EGFR mutations. Seven mutations were T790M and one was a novel D761Y mutation found in a brain metastasis. When combined with a drug-sensitive L858R mutation, the D761Y mutation modestly reduced the sensitivity of mutant EGFR to TKIs in both surrogate kinase and cell viability assays. In an autopsy case, the T790M mutation was found in multiple visceral metastases but not in a brain lesion. CONCLUSIONS: The T790M mutation is common in patients with acquired resistance. The limited spectrum of TKI-resistant mutations in EGFR, which binds to erlotinib in the active conformation, contrasts with a wider range of second-site mutations seen with acquired resistance to imatinib, which binds to ABL and KIT, respectively, in closed conformations. Collectively, our data suggest that the type and nature of kinase inhibitor resistance mutations may be influenced by both anatomic site and mode of binding to the kinase target.     

Different characteristics and survival in non-small cell lung cancer patients with primary and acquired EGFR T790M mutation

Primary epidermal growth factor receptor (EGFR) T790M mutation can be occasionally identified in previous untreated nonsmall cell lung cancer (NSCLC) patients. To compare clinical characteristics and outcomes in patients with primary and acquired EGFR T790M mutation, we collected the data of patients diagnosed with EGFR mutation from 2012 to 2017 in Shanghai Chest Hospital. Primary EGFR T790M mutation was identified in 61 patients (1.1%; 95% confidence interval (CI): 0.8%–1.3%) of 5685 TKI-naive EGFR mutant patients. Acquired T790M mutation was detected in 98 patients (50.3%; 95%CI: 43.2%–57.3%) of 195 TKI-treated patients. T790M mutation always coexisted with sensitizing EGFR mutations. Primary EGFR T790M always coexisted with 21L858R (46/61) whereas acquired T790M coexisted with 19del (68/98), (p < 0.001). Among them, 18 patients with primary T790M mutation received osimertinib and 72 patients with acquired T790M mutation received osimertinib. The median progression-free survival (PFS) of osimertinib was significantly longer in primary T790M group (17.0 months, 95%CI:14.0–20.0 months) compared to acquired T790M group (10.0 months, 95%CI:8.6–11.4 months, p = 0.022). However, the median overall survival (OS) of acquired T790M mutation patients was significantly longer compared to that of primary T790M mutation patients who received osimertinib (50.4 months vs. 29.9 months, p = 0.016). Our findings suggest that primary T790M mutation likely coexists with 21L858R while acquired mutation likely coexists with 19del. Both mutations showed good response to osimertinib. Patients with primary T790M mutation experienced greater benefits from osimertinib. However, patients with acquired T790M mutation had a better overall survival during the entire clinical treatment.     

Oncogenic activity of epidermal growth factor receptor kinase mutant alleles is enhanced by the T790M drug resistance mutation

Activating mutations in the epidermal growth factor receptor (EGFR) characterize a subset of non-small cell lung cancers (NSCLC) with extraordinary sensitivity to targeted tyrosine kinase inhibitors (TKI). A single secondary EGFR mutation, T790M, arising in cis with the primary activating mutation, confers acquired resistance to these drugs. However, the T790M mutation is also detected in the absence of drug selection, suggesting that it may provide a growth advantage. We show here that although T790M alone has only a modest effect on EGFR function, when combined with the characteristic activating mutations L858R or del746-750, it results in a dramatic enhancement of EGFR activity. The double mutants show potent ligand-independent receptor autophosphorylation associated with altered cellular phenotypes, soft agar colony formation, and tumorigenesis in nude mice. The significant gain-of-function properties of these double mutants may explain their initial presence before drug selection and their rapid selection as the single drug resistance mutation during therapy with gefitinib/erlotinib, and suggests that they may contribute to the adverse clinical course of TKI-resistant NSCLC.     

Presence of epidermal growth factor receptor gene T790M mutation as a minor clone in non-small cell lung cancer

The threonine-to-methionine substitution at amino acid position 790 (T790M) of the epidermal growth factor receptor (EGFR) gene has been reported in progressing lesions after gefitinib treatment in non-small cell lung cancer (NSCLC) that causes sensitive tumors to become resistant to gefitinib. Alternatively, the EGFR T790M mutation might be present in small fractions of tumor cells before drug treatment, and the tumor cells harboring the T790M mutation might be enriched during the proliferation after drug treatment. We developed a mutant-enriched PCR assay to detect small fractions of cells with T790M mutation and used this technique to detect mutations in 280 NSCLCs, including gefitinib-treated 95 cases. Although the direct sequencing detected only 1 T790M mutant case, the mutant-enriched PCR (confirmed to enrich one mutant out of 1 x 10(3) wild-type alleles) detected 9 additional cases among 280 cases. As linkage to clinicopathologic factors, the T790M mutation showed no bias for sex, smoking status, or histology but was significantly more frequent in advanced tumors (9 of 111 cases) than in early-stage tumors (1 of 169 cases; P = 0.0013). Among gefitinib-treated cases, gefitinib-sensitive mutations were found in 30 cases. The T790M mutation was present in 3 of 7 no-responders with the gefitinib-sensitive mutation and was not present in 19 responders (P = 0.014). Our results indicate that the T790M mutation is sometimes present in a minor population of tumor cells during the development of NSCLC and suggest that the detection of small fractions of T790M mutant alleles may be useful for predicting gefitinib resistance of NSCLCs with sensitive EGFR mutations.     

The safety and efficacy of osimertinib for the treatment of EGFR T790M mutation positive non-small-cell lung cancer

First- and second-generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are the evidence-based first-line treatment for metastatic non-small-cell lung cancers (NSCLCs) that harbor sensitizing EGFR mutations (i.e. exon 19 deletions or L858R). However, acquired resistance to EGFR TKI monotherapy occurs invariably within a median time frame of one year. The most common form of biological resistance is through the selection of tumor clones harboring the EGFR T790M mutation, present in >50% of repeat biopsies. The presence of the EGFR T790M mutation negates the inhibitory activity of gefitinib, erlotinib, and afatinib. A novel class of third-generation EGFR TKIs has been identified by probing a series of covalent pyrimidine EGFR inhibitors that bind to amino-acid residue C797 of EGFR and preferentially inhibit mutant forms of EGFR versus the wild-type receptor. We review the rapid clinical development and approval of the third-generation EGFR TKI osimertinib for treatment of NSCLCs with EGFR-T790M.     

Non-small-cell lung cancer harbouring mutations in the EGFR kinase domain

Key “driver” mutations have been discovered in specific subgroups of non-small-cell lung cancer (NSCLC) patients. Activating mutations in the form of deletions in exon 19 (del 19) or the missense mutation L858R in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) predict outcome to EGFR tyrosine kinase inhibitors (TKIs) such as gefitinib and erlotinib. Pooled data from several phase II studies show that gefitinib and erlotinib induce responses in over 70% of NSCLC patients harbouring EGFR mutations, with progression-free survival (PFS) ranging from 9 to 13 months and median survival of around 23 months. Two studies in Caucasian and Asian patients have confirmed that these subgroups of patients attain response rates of 70% with erlotinib and gefitinib, including complete responses, PFS up to 14 months and median survival up to 27 months. These landmark outcomes have been accompanied by new challenges: the additional role of chemotherapy and the management of tumours with the secondary T790M mutation that confers resistance to EGFR TKIs. Mechanisms of resistance to reversible EGFR TKIs should be further clarified and could be related to modifications in DNA repair. The presence of double mutations (T790M plus either L858R or del 19) at the time of diagnosis could be much more frequent than originally thought. The sensitivity to EGFR TKIs could be greatly influenced by the expression of genes involved in the repair of DNA double-strand breaks by homologous recombination and non-homologous end joining.     

Indication of EGFR kinase inhibitors should be refined

Even though lung cancer incidence began to decline in the majority of industrialized countries, is still belong to cancers with one of the highest incidence and mortality rates. In the Czech Republic, epidermal growth factor receptor (EGFR) kinase activity inhibitors erlotinib and gefitinib are approved for the use as the second- and third-line treatment of non-small-cell lung cancer. In a cohort of non-small-cell lung cancer patients, erlotinib administration led to tumour regression in less than 20% of patients. However, when used in patients with EGFR-activating mutations, e.g. L858R or delE746-A750, the response rate increased to 75-82% in several parallel clinical studies. Similarly, improved response rate was reported in patients bearing amplified wild-type EGFR gene. In contrary, patients with T790M, D761Y, L747S, and T854A mutations (and some other rare abberations) were found to be resistant to treatment with small-molecule inhibitors targeting the active site of the kinase domain. These mutations do not change the EGFR affinity to gefitinib or erlotinib but the mutated receptor is able to bind ATP into its active site even in the presence of erlotinib or gefitinib, similar to a wild-type receptor without an inhibitor. Besides that, when the EGFR molecule bears both the activating (e.g. L858R) and resistance-inducing mutation (e.g. T790M), the tumour acquires resistance to both erlotinib and gefitinib treatment. Currently, research focuses on a development of new strategies that would allow treatment of patients bearing mutations inducing resistance to the small-molecule inhibitors targeted on the active site of EGFR kinase domain. Contrary to the current guidelines for Czech oncologists, identification of EGFR with any of the above mentioned resistance-inducing somatic mutations should be considered as an explicit contraindication for non-small-cell cancer treatment using small-molecule EGFR kinase activity inhibitors erlotinib or gefitinib. This should also include patients in whom a resistance-inducing mutation is detected together with any of the activating mutations or deletions.