MET gene amplification or EGFR mutation activate MET in lung cancers untreated with EGFR tyrosine kinase inhibitors

We analyzed MET protein and copy number in NSCLC with or without EGFR mutations untreated with EGFR tyrosine kinase inhibitors (TKIs). MET copy number was examined in 28 NSCLC and 4 human bronchial epithelial cell lines (HBEC) and 100 primary tumors using quantitative real‐time PCR. Positive results were confirmed by array comparative genomic hybridization and fluorescence in‐situ hybridization. Total and phospho‐MET protein expression was determined in 24 NSCLC and 2 HBEC cell lines using Western blot. EGFR mutations were examined for exon 19 deletions, T790M, and L858R. Knockdown of EGFR with siRNA was performed to examine the relation between EGFR and MET activation. High‐level MET amplification was observed in 3 of 28 NSCLC cell lines and in 2 of 100 primary lung tumors that had not been treated with EGFR‐TKIs. MET protein was highly expressed and phosphorylated in all the 3 cell lines with high MET amplification. In contrast, 6 NSCLC cell lines showed phospho‐MET among 21 NSCLC cell lines without MET amplification (p = 0.042). Furthermore, those 6 cell lines harboring phospho‐MET expression without MET amplification were all EGFR mutant (p = 0.0039). siRNA‐mediated knockdown of EGFR abolished phospho‐MET expression in examined 3 EGFR mutant cell lines of which MET gene copy number was not amplified. By contrast, phospho‐MET expression in 2 cell lines with amplified MET gene was not down‐regulated by knockdown of EGFR. Our results indicated that MET amplification was present in untreated NSCLC and EGFR mutation or MET amplification activated MET protein in NSCLC. © 2008 Wiley‐Liss, Inc.     

Effects of Src inhibitors on cell growth and epidermal growth factor receptor and MET signaling in gefitinib-resistant non-small cell lung cancer cells with acquired MET amplification

The efficacy of epidermal growth factor receptor (EGFR)–tyrosine kinase inhibitors such as gefitinib and erlotinib in non-small cell lung cancer (NSCLC) is often limited by the emergence of drug resistance conferred either by a secondary T790M mutation of EGFR or by acquired amplification of the MET gene. We now show that the extent of activation of the tyrosine kinase Src is markedly increased in gefitinib-resistant NSCLC (HCC827 GR) cells with MET amplification compared with that in the gefitinib-sensitive parental (HCC827) cells. In contrast, the extent of Src activation did not differ between gefitinib-resistant NSCLC (PC9/ZD) cells harboring the T790M mutation of EGFR and the corresponding gefitinib-sensitive parental (PC9) cells. This activation of Src in HCC827 GR cells was largely abolished by the MET-TKI PHA-665752 but was only partially inhibited by gefitinib, suggesting that Src activation is more dependent on MET signaling than on EGFR signaling in gefitinib-resistant NSCLC cells with MET amplification. Src inhibitors blocked Akt and Erk signaling pathways, resulting in both suppression of cell growth and induction of apoptosis, in HCC827 GR cells as effectively as did the combination of gefitinib and PHA-665752. Furthermore, Src inhibitor dasatinib inhibited tumor growth in HCC827 GR xenografts to a significantly greater extent than did treatment with gefitinib alone. These results provide a rationale for clinical targeting of Src in gefitinib-resistant NSCLC with MET amplification. ( Cancer Sci 2009)     

Impact of bevacizumab in combination with erlotinib on EGFR‐mutated non–small cell lung cancer xenograft models with T790M mutation or MET amplification

Erlotinib (ERL), an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, shows notable efficacy against non–small cell lung cancer (NSCLC) harboring EGFR mutations. Bevacizumab (BEV), a humanized monoclonal antibody to vascular endothelial cell growth factor (VEGF), in combination with ERL (BEV+ERL) significantly extended progression‐free survival in patients with EGFR‐mutated NSCLC compared with ERL alone. However, the efficacy of BEV+ERL against EGFR‐mutated NSCLC harboring T790M mutation or MET amplification, is unclear. Here, we examined the antitumor activity of BEV+ERL in four xenograft models of EGFR‐mutated NSCLC (three harboring ERL resistance mutations). In the HCC827 models (exon 19 deletion: DEL), ERL significantly inhibited tumor growth by blocking EGFR signal transduction. Although there was no difference between ERL and BEV+ERL in maximum tumor growth inhibition, BEV+ERL significantly suppressed tumor regrowth during a drug‐cessation period. In the HCC827‐EPR model (DEL+T790M) and HCC827‐vTR model (DEL+MET amplification), ERL reduced EGFR signal transduction and showed less pronounced but still significant tumor growth inhibition than in the HCC827 model. In these models, tumor growth inhibition was significantly stronger with BEV+ERL than with each single agent. In the NCI‐H1975 model (L858R+T790M), ERL did not inhibit growth or EGFR signal transduction, and BEV+ERL did not inhibit growth more than BEV. BEV alone significantly decreased microvessel density in each tumor. In conclusion, addition of BEV to ERL did not enhance antitumor activity in primarily ERL‐resistant tumors with T790M mutation; however, BEV+ERL enhanced antitumor activity in T790M mutation‐ or MET amplification‐positive tumors as long as their growth remained significantly suppressed by ERL.     

Hypoxia induces gefitinib resistance in non‐small‐cell lung cancer with both mutant and wild‐type epidermal growth factor receptors

Somatic mutations in the epidermal growth factor receptor (EGFR) gene, such as exon 19 deletion mutations, are important factors in determining therapeutic responses to gefitinib in non‐small‐cell lung cancer (NSCLC). However, some patients have activating mutations in EGFR and show poor responses to gefitinib. In this study, we examined three NSCLC cell lines, HCC827, PC9, and HCC2935, that expressed an EGFR exon 19 deletion mutation. All cells expressed mutant EGFR, but the PC9 and HCC2935 cells also expressed wild‐type EGFR. The HCC827 cells were highly sensitive to gefitinib under both normoxia and hypoxia. However, the PC9 and HCC2935 cells were more resistant to gefitinib under hypoxic conditions compared to normoxia. Phosphorylation of EGFR and ERK was suppressed with gefitinib treatment to a lesser extent under hypoxia. The expression of transforming growth factor‐α (TGFα) was dramatically upregulated under hypoxia, and the knockdown of TGFα or hypoxia‐inducible factor‐1α (HIF1α) reversed the resistance to gefitinib in hypoxic PC9 and HCC2935 cells. Finally, introduction of the wild‐type EGFR gene into the HCC827 cells caused resistance to gefitinib under hypoxia. This phenomenon was also reversed by the knockdown of TGFα or HIF1α. Our results indicate that hypoxia causes gefitinib resistance in EGFR‐mutant NSCLC through the activation of wild‐type EGFR mediated by the upregulation of TGFα. The presence of wild‐type and mutant EGFR along with tumor hypoxia are important factors that should be considered when treating NSCLC patients with gefitinib.     

MicroRNA-214 regulates the acquired resistance to gefitinib via the PTEN/AKT pathway in EGFR-mutant cell lines

Patients with non-small cell lung cancer (NSCLC) who have activating epidermal growth factor receptor (EGFR) mutations derive clinical benefit from treatment with EGFR-tyrosine kinase inhibitors ((EGFR-TKIs)- namely gefitinib and erlotinib. However, these patients eventually develop resistance to EGFR-TKIs. Despite the fact that this acquired resistance may be the result of a secondary mutation in the EGFR gene, such as T790M or amplification of the MET proto-oncogene, there are other mechanisms which need to be explored. MicroRNAs (miRs) are a class of small non-coding RNAs that play pivotal roles in tumorigenesis, tumor progression and chemo-resistance. In this study, we firstly successfully established a gefitinib resistant cell line-HCC827/GR, by exposing normal HCC827 cells (an NSCLC cell line with a 746E-750A in-frame deletion of EGFR gene) to increasing concentrations of gefitinib. Then, we found that miR-214 was significantly up-regulated in HCC827/ GR. We also showed that miR-214 and PTEN were inversely expressed in HCC827/GR. Knockdown of miR-214 altered the expression of PTEN and p-AKT and re-sensitized HCC827/GR to gefitinib. Taken together, miR-214 may regulate the acquired resistance to gefitinib in HCC827 via PTEN/AKT signaling pathway. Suppression of miR-214 may thus reverse the acquired resistance to EGFR-TKIs therapy.     

Absence of copy number gain of EGFR: A possible predictive marker of long-term response to afatinib

Treatment efficacy of epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) is diverse even in non-small cell lung cancer (NSCLC) patients with EGFR activating mutations. Extraordinary long-term responses sustained over 3 years among NSCLC patients treated with afatinib, an EGFR-TKI, have been reported, but how to predict such long survivors has not been clarified. A multi-institutional prospective observational study, based on comprehensive genomic examination performed with next-generation sequencing of circulating tumor DNA (ctDNA), was conducted to identify potential predictive markers of long-term response to afatinib. Twenty-nine patients with advanced stage NSCLC and EGFR driver mutations detected by standard techniques were enrolled in the study. ctDNA from plasma collected before afatinib treatment was analyzed by Guardant360. ctDNA was detected in 25 of the 29 samples. Median progression-free survival was shorter in patients whose tumors had EGFR copy number gain (7.0 vs 23.0 months, p = 0.022). The impact of EGFR copy number on cell proliferation and the antitumor effect of afatinib were evaluated using genome-editing lung cancer cell lines. HCC827 with EGFR amplification was relatively resistant to afatinib at concentrations below 0.5 nM, but genome-edited derivatives of HCC827 with decreased EGFR copy number demonstrated growth inhibition with 0.1 nM afatinib. The absence of EGFR copy number gain detected in ctDNA may be a predictive marker of long-term response to afatinib. Comprehensive genomic analysis could lead to a more accurate prediction of EGFR-TKI efficacy.     

Oncogene swap as a novel mechanism of acquired resistance to epidermal growth factor receptor‐tyrosine kinase inhibitor in lung cancer

Mutant selective epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs), such as rociletinib and AZD9291, are effective for tumors with T790M secondary mutation that become refractory to first‐generation EGFR‐TKI. However, acquired resistance to these prospective drugs is anticipated considering the high adaptability of cancer cells and the mechanisms remain largely obscure. Here, CNX‐2006 (tool compound of rociletinib) resistant sublines were established by chronic exposure of HCC827EPR cells harboring exon 19 deletion and T790M to CNX‐2006. Through the analyses of these resistant subclones, we identified two resistant mechanisms accompanied by MET amplification. One was bypass signaling by MET amplification in addition to T790M, which was inhibited by the combination of CNX‐2006 and MET‐TKI. Another was loss of amplified EGFR mutant allele including T790M while acquiring MET amplification. Interestingly, MET‐TKI alone was able to overcome this resistance, suggesting that oncogenic dependence completely shifted from EGFR to MET. We propose describing this phenomenon as an “oncogene swap.” Furthermore, we analyzed multiple lesions from a patient who died of acquired resistance to gefitinib, then found a clinical example of an oncogene swap in which the EGFR mutation was lost and a MET gene copy was gained. In conclusion, an “oncogene swap” from EGFR to MET is a novel resistant mechanism to the EGFR‐TKI. This novel mechanism should be considered in order to avoid futile inhibition of the original oncogene.     

大黄素逆转非小细胞肺癌EGFR-TKI耐药的机制研究

目的 探讨大黄素逆转非小细胞肺癌（NSCLC）表皮生长因子受体酪氨酸激酶抑制剂（EGFR TKI）耐药的作用机制。方法 应用持续诱导的方法构建NSCLC EGFR-TKI耐药细胞株HCC827／GR;应用MTS法检测大黄素（30μmol／L）、吉非替尼（1μmol／L）及两药联合处理HCC827和HCC827／GR细胞48h后细胞增殖能力的变化;应用Western blotting法检测HCC827和 HCC827／GR细胞中p EGFR、p-AKT、p-ERK1／2及p-MET蛋白表达水平的变化。结果 MTS法检测结果显示,经单药吉非替尼或大黄素处理后,HCC827／GR细胞增殖能力未减弱,而两药联合处理组的细胞增殖能力明显下降,差异有统计学意义（P＜0.05）。Western blotting检测结果显示,HCC827、HCC827／GR细胞中p-EGFR、p-ERK1/2明显表达,而p-AKT表达微弱;HCC827／GR 中p-MET表达水平较HCC827明显上调。经单药吉非替尼处理后,HCC827细胞株p-EGFR、p-ERK1/2表达水平下调,HCC827／GR细胞株p-EGFR表达明显下调;大黄素可显著下调HCC827／GR细胞株p-MET表达,但对p-EGFR、p-ERK1/2的表达无影响;而大黄素与吉非替尼两药联用可明显抑制HCC827／GR细胞株p-EGFR、p-ERK1/2以及p-MET的表达。结论 大黄素可以逆转NSCLC EGFR-TKI耐药,可能是通过抑制c-Met的活化来实现。     

Digital PCR analysis of plasma cell-free DNA for non-invasive detection of drug resistance mechanisms in EGFR mutant NSCLC: Correlation with paired tumor samples

As the development of resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has become an issue of concern, identification of the mechanisms responsible has become an urgent priority. However, for research purposes, it is not easy to obtain tumor samples from patients with EGFR mutation-positive non-small-cell lung cancer (NSCLC) that has relapsed after treatment with EGFR-TKIs. Here, using digital PCR assay as an alternative and noninvasive method, we examined plasma and tumor samples from patients with relapsed NSCLC to establish the inter-relationships existing among T790M mutation, activating EGFR mutations, HER2 amplification, and MET amplification. Paired samples of tumor and blood were obtained from a total of 18 patients with NSCLC after they had developed resistance to EGFR-TKI treatment, and the mechanisms of resistance were analyzed by digital PCR. Digital PCR analysis of T790M mutation in plasma had a sensitivity of 81.8% and specificity of 85.7%, the overall concordance between plasma and tissue samples being 83.3%. MET gene copy number gain in tumor DNA was observed by digital PCR in three patients, of whom one exhibited positivity for MET amplification by FISH, whereas no patient demonstrated MET and HER2 copy number gain in plasma DNA. Digital PCR analysis of plasma is feasible and accurate for detection of T790M mutation in NSCLC that becomes resistant to treatment with EGFR-TKIs.     

Co-existence of positive MET FISH status with EGFR mutations signifies poor prognosis in lung adenocarcinoma patients

MET, a receptor tyrosine kinase for hepatocyte growth factor, is associated with tumor progression and acquired resistance to epidermal growth factor tyrosine kinase inhibitors (EGFR-TKI). Therefore, MET gene alterations could be both prognostic and predictive. Fluorescence in situ hybridization (FISH) is one method for assessing gene alteration, but the frequency of positive cases varies due to a lack of standardized criteria. We evaluated MET gene copy number in lung adenocarcinoma and its association with clinicopathological characteristics. FISH was applied to evaluate high MET gene copy number and true amplification in 138 lung adenocarcinoma patients using two criteria: the Cappuzzo scoring system and PathVysion. MET positive cases according to the Cappuzzo scoring system evidenced both aneuploidy and true amplification, whereas PathVysion revealed only amplification. Proportion of MET FISH positive cases was 15% and 4% determined by the Cappuzzo system and PathVysion, respectively. PathVysion demonstrated higher frequencies of MET FISH positives among men and smokers and evidenced no MET FISH positives in patients with bronchioloalveolar carcinoma. Prognosis was significantly associated with MET FISH positive only as defined by the PathVysion system (gene amplification), not by the Cappuzzo system. However, progression-free survival time of patients with both EGFR mutations and MET FISH positive defined by the Cappuzzo scoring system was significantly shorter than with EGFR mutations alone. These results suggest that MET FISH is a potential prognostic factor and coexistence of MET FISH with EGFR mutations signifies worse prognosis.     

MicroRNA-34a overcomes HGF-mediated gefitinib resistance in EGFR mutant lung cancer cells partly by targeting MET

In non-small-cell lung cancer (NSCLC) that harbours an activating epidermal growth factor receptor (EGFR) mutation, over-expression of hepatocyte growth factor (HGF) is an important mechanism involved in the acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs) by restoring activity of the PI3K/Akt pathway via phosphorylation of MET. In our study, we found that the forced expression of miR-34a inhibited cell growth and induced apoptosis partly by targeting MET in HGF-induced gefitinib-resistant HCC827 and PC-9 cells. Furthermore, dramatic tumour regression was observed in the miR-34a plus gefitinib group in HGF-induced gefitinib resistant mouse xenograft models. This study demonstrates for the first time that miR-34a rescues HGF-induced gefitinib resistance in EGFR mutant NSCLC cells.     

Anti‐tumor activity of WK88‐1, a novel geldanamycin derivative,in gefitinib‐resistant non‐small cell lung cancers with Met amplification

Although epidermal growth factor receptor‐tyrosine kinase inhibitors (EGFR‐TKIs) have been introduced for the treatment of non‐small cell lung cancer (NSCLC), the emergence of secondary T790M mutation in EGFR or amplification of the Met proto‐oncogene restrain the clinical success of EGFR‐TKIs. Since heat shock protein‐90 (Hsp90) stabilizes various oncoproteins including EGFR and c‐Met, the inhibition of Hsp90 activity appears as a rational strategy to develop anticancer drugs. Despite preclinical efficacy of geldanamycin‐anasamycin (GA)‐derivatives containing benzoquinone moiety as Hsp90 inhibitors, the hepatotoxicity of these GA‐derivatives restricts their therapeutic benefit. We have prepared WK‐88 series of GA‐derivatives, which lack the benzoquinone moiety. In this study, we have examined the anticancer effects of WK88‐1 in Met‐amplified‐ and gefitinib‐resistant (HCC827GR) NSCLC cells and its parental HCC827 cells. Treatment with WK88‐1 reduced the cell viability in both HCC827 and HCC827GR cells, which was associated with marked decrease in the constitutive expression of Hsp90 client proteins, such as EGFR, ErbB2, ErbB3, Met and Akt. Moreover, WK88‐1 attenuated phosphorylation of these Hsp90 client proteins and reduced the anchorage‐independent growth of HCC827GR cells. Administration of WK88‐1 did not cause hepatotoxicity in animals and significantly reduced the growth of HCC827GR cells xenograft tumors in nude mice. Our study provides evidence that ErbB3 might be a client for Hsp90 in Met‐amplified NSCLCs. In conclusion, we demonstrate that inhibition of Hsp90 dampens the activation of EGFR‐ or c‐Met‐mediated survival of Met‐amplified NSCLCs and that WK88‐1 as a Hsp90 inhibitor alleviates gefitinib resistance in HCC827GR cells.     

Acquisition of cancer stem cell‐like properties in non‐small cell lung cancer with acquired resistance to afatinib

Afatinib is an irreversible epidermal growth factor receptor (EGFR)‐tyrosine kinase inhibitor (TKI) that is known to be effective against the EGFR T790M variant, which accounts for half of the mechanisms of acquired resistance to reversible EGFR‐TKIs. However, acquired resistance to afatinib was also observed in clinical use. Thus, elucidating and overcoming the mechanisms of resistance are important issues in the treatment of non‐small cell lung cancer. In this study, we established various afatinib‐resistant cell lines and investigated the resistance mechanisms. EGFR T790M mutations were not detected using direct sequencing in established resistant cells. Several afatinib‐resistant cell lines displayed MET amplification, and these cells were sensitive to the combination of afatinib plus crizotinib. As a further investigation, a cell line that acquired resistance to afatinib plus crizotinib, HCC827‐ACR, was established from one of the MET amplified‐cell lines. Several afatinib‐resistant cell lines including HCC827‐ACR displayed epithelial‐to‐mesenchymal transition (EMT) features and epigenetic silencing of miR‐200c, which is a suppresser of EMT. In addition, these cell lines also exhibited overexpression of ALDH1A1 and ABCB1, which are putative stem cell markers, and resistance to docetaxel. In conclusion, we established afatinib‐resistant cells and found that MET amplification, EMT, and stem cell‐like features are observed in cells with acquired resistance to EGFR‐TKIs. This finding may provide clues to overcoming resistance to EGFR‐TKIs.     

Asian Thoracic Oncology Research Group (ATORG) Expert Consensus Statement on MET Alterations in NSCLC: Diagnostic and Therapeutic Considerations

Non-small cell lung cancer (NSCLC) is a heterogeneous disease, with many oncogenic driver mutations, including de novo mutations in the Mesenchymal Epithelial Transition (MET) gene (specifically in Exon 14 [ex14]), that lead to tumourigenesis. Acquired alterations in the MET gene, specifically MET amplification is also associated with the development of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor (TKI) resistance in patients with EGFR-mutant NSCLC. Although MET has become an actionable biomarker with the availability of MET-specific inhibitors in selected countries, there is differential accessibility to diagnostic platforms and targeted therapies across countries in Asia-Pacific (APAC).The Asian Thoracic Oncology Research Group (ATORG), an interdisciplinary group of experts from Australia, Hong Kong, Japan, Korea, Mainland China, Malaysia, the Philippines, Singapore, Taiwan, Thailand and Vietnam, discussed testing for MET alterations and considerations for using MET-specific inhibitors at a consensus meeting in January 2022, and in subsequent offline consultation.Consensus recommendations are provided by the ATORG group to address the unmet need for standardised approaches to diagnosing MET alterations in NSCLC and for using these therapies. MET inhibitors may be considered for first-line or second or subsequent lines of treatment for patients with advanced and metastatic NSCLC harbouring MET ex14 skipping mutations; MET ex14 testing is preferred within multi-gene panels for detecting targetable driver mutations in NSCLC. For patients with EGFR-mutant NSCLC and MET amplification leading to EGFR TKI resistance, enrolment in combination trials of EGFR TKIs and MET inhibitors is encouraged.     

An open-label study of vandetanib with pemetrexed in patients with previously treated non-small-cell lung cancer

Background: Vandetanib (ZACTIMA^TM; ZD6474) is a once-daily,oral inhibitor of vascular endothelial growth factor receptorand epidermal growth factor receptor signaling. The safety andtolerability of vandetanib plus pemetrexed was assessed in patientswith advanced non-small-cell lung cancer (NSCLC). Patients and methods: Patients with previously treated NSCLC(stage IIIB/IV) received once-daily oral vandetanib (100 or300 mg) with pemetrexed (500 mg/m² i.v. infusion every 21 days). Results: Patients received vandetanib 100 mg + pemetrexed (n= 10) or vandetanib 300 mg + pemetrexed (n = 11). The protocoldefinition of a tolerable dose [vandetanib-related dose-limitingtoxicity (DLT) in less than 2 patients] was met in both dosecohorts, with one DLT reported in each: asymptomatic QTc prolongation(>100 ms increase from baseline, but absolute QTc <500ms) in the 100 mg cohort and interstitial lung disease, whichresolved after steroid therapy, in the 300 mg cohort. The mostcommon adverse events were rash, anorexia, fatigue and diarrhea(all n = 10). Conclusion: Vandetanib and pemetrexed in combination were generallywell tolerated in patients with advanced NSCLC. Key words: Angiogenesis, EGFR, advanced NSCLC, pemetrexed, vandetanib Received for publication June 22, 2008. Revision received September 9, 2008. Accepted for publication September 10, 2008.     

Aberrant epidermal growth factor receptor signaling and enhanced sensitivity to EGFR inhibitors in lung cancer

Epidermal growth factor receptor (EGFR) is occasionally amplified and/or mutated in non-small cell lung cancer (NSCLC) and can be coexpressed with other members of the HER receptor family to form functional heterodimers. We therefore investigated lung cancer cell lines for alterations in EGFR gene copy number, enhanced expression of EGFR and other HER family members, and EGFR coding sequence mutations and correlated these findings with response to treatment with the EGFR inhibitors and the kinetics of ligand-induced signaling. We show here that somatic deletions in the tyrosine kinase domain of EGFR were associated with increased EGFR gene copy number in NSCLC. Treatment with the specific EGFR tyrosine kinase inhibitors (TKI) gefitinib or erlotinib or the EGFR inhibitory antibody cetuximab induced apoptosis of HCC827, a NSCLC cell line with EGFR gene amplification and an exon 19 deletion. H1819, a NSCLC cell line that expresses high levels of EGFR, ErbB2, and ErbB3 but has wild-type EGFR, showed intermediate sensitivity to TKIs. In both cell lines, ligand-induced receptor tyrosine phosphorylation was delayed and prolonged and AKT was constitutively phosphorylated (but remained inhibitable by EGFR TKI). Thus, in addition to EGFR mutations, other factors in NSCLC cells, such as high expression of ErbB family members, may constitutively activate AKT and sensitize cells to EGFR inhibitors.     

Molecular predictors of response to tyrosine kinase inhibitors in patients with Non-Small-Cell Lung Cancer

Introduction

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have become a treatment option in non-small-cell lung cancer (NSCLC) patients. However, despite their use in this disease, a significant number of patients will eventually develop resistance and relapse. In this study, we aimed to characterize several molecular events involved in potential resistance mechanisms to anti-EGFR treatment and correlate our findings with clinical outcome.

Material and methods

The medical records of patients with NSCLC who received anti-EGFR TKIs in any line within the participating centers were reviewed and available paraffin embedded tissue was retrieved. Mutational analysis for EGFR, KRAS, BRAF and intron-exon 14 deletions of MET; FISH analysis for chromosomal gain or amplification for EGFR, MET and the deletion marker D7S486 were performed. Furthermore, the expression of EGFR and MET were analysed by immunohistochemistry. All results were correlated with treatment outcomes.

Results

Between 10/2001 and 12/2009 from an initial cohort of 72 treated patients, 59 cases (28 gefitinib/ 31 erlotinib) were included in the analysis. The majority had adenocarcinoma histology (68%), and received treatment in the second line setting (56%). Disease control rate (DCR) was 25.4% for all patients. EGFR and RAS mutational rates were 33% and 10% respectively, no other mutations were identified. High EGFR expressing tumors were found in 7 of 45 cases and pEGFR positivity (IHC) was found in 56% of the cases; MET expression was found in 48% of tumors. EGFR gene amplification was found in 4 cases, two cases showed high polysomy; overall, 13% cases were FISH positive for EGFR. High polysomy of MET gene was detected in 1/43 cases tested. D7S486 locus deletion was detected in 15/37 (40%) of cases. EGFR mutational status and gene gain were both associated with more favorable DCR. No other associations between examined biomarkers and DCR or survival were noted.

Conclusions

EGFR mutational status is a predictor for disease control in patients with NSCLC treated with anti-EGFR TKIs. The predictive role of several other molecules involved in potential resistance to anti-EGFR TKIs is worthy of additional investigation.     

Acquired resistance to cetuximab is mediated by increased PTEN instability and leads cross-resistance to gefitinib in HCC827 NSCLC cells

EGFR inhibitors, including the small-molecule tyrosine kinase inhibitors such as gefitinib, and the monoclonal antibodies directed at the receptor such as cetuximab, have demonstrated promising effects in non-small cell lung cancer (NSCLC). In this study, we generated cetuximab-resistant cell lines (HCC827-CR) from HCC827 NSCLC cells to investigate acquired resistance mechanisms to cetuximab. In HCC827-CR cells, Akt was hyperactivated and its activity was persistent upon cetuximab treatment. Blockade of PI3K/Akt activity restored cetuximab sensitivity in HCC827-CR cells. Further investigation revealed that increased PTEN instability mediates constitutive Akt activation. By 1 μM proteosomal inhibitor, MG-132, PTEN protein levels were restored and Akt activity was dramatically reduced. Overexpression of PTEN by transfection could not restore cetuximab sensitivity in HCC827-CR because overexpressed PTEN was degraded rapidly (∼72 h). The increased PTEN instability was confirmed by the treatment of HCC827-CR with a protein synthesis inhibitor, cycloheximide. In the presence of cycloheximide, overexpressed PTEN was degraded more rapidly (∼12 h) in HCC827-CR cells. Interestingly, HCC827-CR cells also revealed de novo resistance to gefitinib. Inhibition of PI3K/Akt signaling pathway restored sensitivity to gefitinib in HCC827-CR cells. Taken together, these data show that PTEN instability-mediated constitutive Akt activation is involved in acquired resistance mechanisms to cetuximab and also induces de novo resistance to gefitinib. Importantly, these findings suggest emergence of cross-resistance between two agents as a potential serious problem in the clinical setting.     

Targeting MET in Lung Cancer: Will Expectations Finally Be MET?

The hepatocyte growth factor receptor (MET) is a potential therapeutic target in a number of cancers, including NSCLC. In NSCLC, MET pathway activation is thought to occur through a diverse set of mechanisms that influence properties affecting cancer cell survival, growth, and invasiveness. Preclinical and clinical evidence suggests a role for MET activation as both a primary oncogenic driver in subsets of lung cancer and as a secondary driver of acquired resistance to targeted therapy in other genomic subsets. In this review, we explore the biology and clinical significance behind MET proto-oncogene receptor tyrosine kinase (MET) exon 14 alterations and MET amplification in NSCLC, the role of MET amplification in the setting of acquired resistance to EGFR tyrosine kinase inhibitor therapy in EGFR-mutant NSCLC, and the history of MET pathway inhibitor drug development in NSCLC, highlighting current strategies that enrich for biomarkers likely to be predictive of response. Whereas previous trials that focused on MET pathway–directed targeted therapy in unselected or MET-overexpressing NSCLC yielded largely negative results, more recent investigations focusing on MET exon 14 alterations and MET amplification have been notable for meaningful clinical responses to MET inhibitor therapy in a substantial proportion of patients.