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1.  EGFR-mutated lung cancer: a paradigm of molecular oncology 
Oncotarget  2010;1(7):497-514.
The development of EGFR tyrosine kinase inhibitors for clinical use in non-small cell lung cancer and the subsequent discovery of activating EGFR mutations have led to an explosion of knowledge in the fields of EGFR biology, targeted therapeutics and lung cancer research. EGFR-mutated adenocarcinoma of the lung has clearly emerged as a unique clinical entity necessitating the routine introduction of molecular diagnostics into our current diagnostic algorithms and leading to the evidence-based preferential usage of EGFR-targeted agents for patients with EGFR-mutant lung cancers. This review will summarize our current understanding of the functional role of activating mutations, key downstream signaling pathways and regulatory mechanisms, pivotal primary and acquired resistance mechanisms, structure-function relationships and ultimately the incorporation of molecular diagnostics and small molecule EGFR tyrosine kinase inhibitors into our current treatment paradigms.
PMCID: PMC3001953  PMID: 21165163
EGFR; tyrosine kinase; mutation; lung cancer; therapy; oncology
2.  EGFR-mutated lung cancer: a paradigm of molecular oncology 
Oncotarget  2010;1(7):497-514.
The development of EGFR tyrosine kinase inhibitors for clinical use in non-small cell lung cancer and the subsequent discovery of activating EGFR mutations have led to an explosion of knowledge in the fields of EGFR biology, targeted therapeutics and lung cancer research. EGFR-mutated adenocarcinoma of the lung has clearly emerged as a unique clinical entity necessitating the routine introduction of molecular diagnostics into our current diagnostic algorithms and leading to the evidence-based preferential usage of EGFR-targeted agents for patients with EGFR-mutant lung cancers. This review will summarize our current understanding of the functional role of activating mutations, key downstream signaling pathways and regulatory mechanisms, pivotal primary and acquired resistance mechanisms, structure-function relationships and ultimately the incorporation of molecular diagnostics and small molecule EGFR tyrosine kinase inhibitors into our current treatment paradigms.
PMCID: PMC3001953  PMID: 21165163
EGFR; tyrosine kinase; lung cancer; therapy; oncology
3.  Epidermal growth factor receptor and K-Ras in non-small cell lung cancer-molecular pathways involved and targeted therapies 
Lung cancer is currently the leading cause of cancer death in Western nations. Non-small cell lung cancer (NSCLC) represents 80% of all lung cancers, and adenocarcinoma is the predominant histological type. Despite the intensive research carried out on this field and therapeutic advances, the overall prognosis of these patients remains unsatisfactory, with a 5-year overall survival rate of less than 15%. Nowadays, pharmacogenetics and pharmacogenomics represent the key to successful treatment. Recent studies suggest the existence of two distinct molecular pathways in the carcinogenesis of lung adenocarcinoma: one associated with smoking and activation of the K-Ras oncogene and the other not associated with smoking and activation of the epidermal growth factor receptor (EGFR). The K-ras mutation is mainly responsible for primary resistance to new molecules which inhibit tyrosine kinase EGFR (erlotinib and gefitinib) and most of the EGFR mutations are responsible for increased tumor sensitivity to these drugs. This article aims to conduct a systematic review of the literature regarding the molecular pathways involving the EGFR, K-Ras and EGFR targeted therapies in NSCLC tumor behavior.
PMCID: PMC3215775  PMID: 22087435
Epidermal growth factor receptor; K-Ras; Non-small-cell lung carcinoma; Pharmacogenomics; p21ras proto-oncogene proteins
4.  The Role of Irreversible HER Family Inhibition in the Treatment of Patients with Non-Small Cell Lung Cancer 
The Oncologist  2011;16(11):1498-1507.
The clinical development of irreversible tyrosine kinase inhibitors that target the human epidermal growth factor receptor family in non-small cell lung cancer is reviewed.
Small-molecule tyrosine kinase inhibitors (TKIs) of the human epidermal growth factor receptor (HER) include the reversible epidermal growth factor receptor (EGFR/HER-1) inhibitors gefitinib and erlotinib. EGFR TKIs have demonstrated activity in the treatment of patients with non-small cell lung cancer (NSCLC) harboring activating EGFR mutations; however, multiple mechanisms of resistance limit the benefit of these drugs. Although resistance to EGFR TKIs can be intrinsic and correlated with molecular lesions such as in Kirsten rat sarcoma viral oncogene homolog (KRAS; generally observed in a wild-type EGFR background), acquired resistance to EGFR TKIs can evolve in the setting of activating EGFR mutations, such as in the case of EGFR T790M mutations. Several irreversible inhibitors that target multiple members of the HER family simultaneously are currently in clinical development for NSCLC and may have a role in the treatment of TKI-sensitive and TKI-resistant disease. These include PF00299804, an inhibitor of EGFR/HER-1, HER-2, and HER-4, and afatinib (BIBW 2992), an inhibitor of EGFR/HER-1, HER-2, and HER-4. Results of large, randomized trials of these agents may help to determine their potential for the treatment of NSCLC.
PMCID: PMC3233283  PMID: 22016476
Irreversible EGFR/HER-2 tyrosine kinase inhibitors; Non-small cell lung cancer; Resistance; Afatinib (BIBW 2992); PF00299804
5.  Clinical pharmacogenomic testing of KRAS, BRAF and EGFR mutations by high resolution melting analysis and ultra-deep pyrosequencing 
BMC Cancer  2011;11:406.
Epidermal growth factor receptor (EGFR) and its downstream factors KRAS and BRAF are mutated in several types of cancer, affecting the clinical response to EGFR inhibitors. Mutations in the EGFR kinase domain predict sensitivity to the tyrosine kinase inhibitors gefitinib and erlotinib in lung adenocarcinoma, while activating point mutations in KRAS and BRAF confer resistance to the anti-EGFR monoclonal antibody cetuximab in colorectal cancer. The development of new generation methods for systematic mutation screening of these genes will allow more appropriate therapeutic choices.
We describe a high resolution melting (HRM) assay for mutation detection in EGFR exons 19-21, KRAS codon 12/13 and BRAF V600 using formalin-fixed paraffin-embedded samples. Somatic variation of KRAS exon 2 was also analysed by massively parallel pyrosequencing of amplicons with the GS Junior 454 platform.
We tested 120 routine diagnostic specimens from patients with colorectal or lung cancer. Mutations in KRAS, BRAF and EGFR were observed in 41.9%, 13.0% and 11.1% of the overall samples, respectively, being mutually exclusive. For KRAS, six types of substitutions were detected (17 G12D, 9 G13D, 7 G12C, 2 G12A, 2 G12V, 2 G12S), while V600E accounted for all the BRAF activating mutations. Regarding EGFR, two cases showed exon 19 deletions (delE746-A750 and delE746-T751insA) and another two substitutions in exon 21 (one showed L858R with the resistance mutation T590M in exon 20, and the other had P848L mutation). Consistent with earlier reports, our results show that KRAS and BRAF mutation frequencies in colorectal cancer were 44.3% and 13.0%, respectively, while EGFR mutations were detected in 11.1% of the lung cancer specimens. Ultra-deep amplicon pyrosequencing successfully validated the HRM results and allowed detection and quantitation of KRAS somatic mutations.
HRM is a rapid and sensitive method for moderate-throughput cost-effective screening of oncogene mutations in clinical samples. Rather than Sanger sequence validation, next-generation sequencing technology results in more accurate quantitative results in somatic variation and can be achieved at a higher throughput scale.
PMCID: PMC3192787  PMID: 21943394
6.  KRAS Mutations and Primary Resistance of Lung Adenocarcinomas to Gefitinib or Erlotinib 
PLoS Medicine  2005;2(1):e17.
Somatic mutations in the gene for the epidermal growth factor receptor (EGFR) are found in adenocarcinomas of the lung and are associated with sensitivity to the kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva). Lung adenocarcinomas also harbor activating mutations in the downstream GTPase, KRAS, and mutations in EGFR and KRAS appear to be mutually exclusive.
Methods and Findings
We sought to determine whether mutations in KRAS could be used to further enhance prediction of response to gefitinib or erlotinib. We screened 60 lung adenocarcinomas defined as sensitive or refractory to gefitinib or erlotinib for mutations in EGFR and KRAS. We show that mutations in KRAS are associated with a lack of sensitivity to either drug.
Our results suggest that treatment decisions regarding use of these kinase inhibitors might be improved by determining the mutational status of both EGFR and KRAS.
Mutational analysis of the KRAS gene in lung cancer patients treated with two different kinase inhibitors suggests that tumors with KRAS mutations do not respond to these drugs
PMCID: PMC545207  PMID: 15696205
7.  Oncogenic KRAS-induced interleukin-8 overexpression promotes cell growth and migration and contributes to aggressive phenotypes of non-small cell lung cancer 
The CXC chemokine interleukin-8 (IL-8) is an angiogenic growth factor that is overexpressed in various cancers, including non-small cell lung cancer (NSCLC). Previously, IL-8 was shown as a transcriptional target of RAS signaling, raising the possibility of its role in oncogenic KRAS-driven NSCLC. Using microarray analysis, we identified IL-8 as the most downregulated gene by shRNA-mediated KRAS knockdown in NCI-H1792 NSCLC cells where IL-8 is overexpressed. NSCLC cell lines harboring KRAS or EGFR mutations overexpressed IL-8, while IL-8 levels were more prominent in KRAS mutants compared to EGFR mutants. IL-8 expression was downregulated by shRNA-mediated KRAS knockdown in KRAS mutants or by treatment with EGFR tyrosine kinase inhibitors and EGFR siRNAs in EGFR mutants. In our analysis of the relationship of IL-8 expression with clinical parameters and mutation status of KRAS or EGFR in 89 NSCLC surgical specimens, IL-8 expression was shown to be significantly higher in NSCLCs of males, smokers, and elderly patients and those with pleural involvement and KRAS mutated adenocarcinomas. In KRAS mutant cells, the MEK inhibitor markedly decreased IL-8 expression, while the p38 inhibitor increased IL-8 expression. Attenuation of IL-8 function by siRNAs or a neutralizing antibody inhibited cell proliferation and migration of KRAS mutant/IL-8 overexpressing NSCLC cells. These results indicate that activating mutations of KRAS or EGFR upregulate IL-8 expression in NSCLC; IL-8 is highly expressed in NSCLCs from males, smokers, elderly patients, NSCLCs with pleural involvement, and KRAS-mutated adenocarcinomas; and IL-8 plays a role in cell growth and migration in oncogenic KRAS-driven NSCLC.
PMCID: PMC3374723  PMID: 21544811
non-small cell lung cancer; KRAS; interleukin-8; molecular target
8.  A case of lung adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene 
BMC Cancer  2012;12:558.
Lung cancer is the leading cause of cancer-related death worldwide. Epidermal growth factor receptor (EGFR) - tyrosine kinase inhibitor (TKI) is used for the patients with EGFR-mutant lung cancer. Recently, phase III studies in the patients with EGFR-mutant demonstrated that EGFR-TKI monotherapy improved progression-free survival compared with platinum-doublet chemotherapy. The echinoderm microtubule-associated protein-like 4 (EML4) - anaplastic lymphoma kinase (ALK) fusion oncogene represents one of the newest molecular targets in non-small cell lung cancer (NSCLC). Patients who harbor EML4-ALK fusions have been associated with a lack of EGFR or KRAS mutations.
Case presentation
We report a 39-year-old patient diagnosed as adenocarcinoma harboring EGFR mutation and EML4-ALK fusion gene. We treated this patient with erlotinib as the third line therapy, but no clinical benefit was obtained.
We experienced a rare case with EGFR mutation and EML4-ALK. Any clinical benefit using EGFR-TKI was not obtained in our case. The therapeutic choice for the patients with more than one driver mutations is unclear. We needs further understanding of the lung cancer molecular biology and the biomarker infomation.
PMCID: PMC3515412  PMID: 23181703
Lung cancer; EGFR mutation; EML4-ALK; Erlotinib
9.  The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells 
Cell Death & Disease  2013;4(9):e810-.
Epidermal growth factor receptor (EGFR), which promotes cell survival and division, is found at abnormally high levels on the surface of many cancer cell types, including many cases of non-small cell lung cancer. Erlotinib (Tarceva), an oral small-molecule tyrosine kinase inhibitor, is a so-called targeted drug that inhibits the tyrosine kinase domain of EGFR, and thus targets cancer cells with some specificity while doing less damage to normal cells. However, erlotinib resistance can occur, reducing the efficacy of this treatment. To develop more effective therapeutic interventions by overcoming this resistance problem, we combined the histone deacetylase inhibitor, MPT0E028, with erlotinib in an effort to increase their antitumor effects in erlotinib-resistant lung adenocarcinoma cells. This combined treatment yielded significant growth inhibition, induced the expression of apoptotic proteins (PARP, γH2AX, and caspase-3), increased the levels of acetylated histone H3, and showed synergistic effects in vitro and in vivo. These effects were independent of the mutation status of the genes encoding EGFR or K-Ras. MPT0E028 synergistically blocked key regulators of the EGFR/HER2 signaling pathways, attenuating multiple compensatory pathways (e.g., AKT, extracellular signal-regulated kinase, and c-MET). Our results indicate that this combination therapy might be a promising strategy for facilitating the effects of erlotinib monotherapy by activating various networks. Taken together, our data provide compelling evidence that MPT0E028 has the potential to improve the treatment of heterogeneous and drug-resistant tumors that cannot be controlled with single-target agents.
PMCID: PMC3789188  PMID: 24052078
lung cancer; HDAC; synergistic; EGFR; apoptosis; erlotinib
10.  Sorafenib combined with gemcitabine in EGFR-TKI-resistant human lung cancer cells 
Oncology Letters  2012;5(1):68-72.
Sorafenib is a multi-targeted agent and has been reported to have potent antitumor effects against various types of tumors, including human non-small cell lung cancer (NSCLC). In this study, we explored in vitro the antitumor effects of sorafenib alone and in combination with gemcitabine in epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI)-resistant human lung cancer cell lines and the related molecular mechanisms. The NSCLC cell lines A549 (mutant KRAS), H1666 (mutant BRAF) and H1975 (mutant EGFR-T790M) were treated with sorafenib and gemcitabine alone and in combination. The cytotoxicity was assessed by MTT assay, cell cycle distribution was analyzed by flow cytometry, and alterations in signaling pathways were analyzed by western blotting. We found that sorafenib exhibited dose-dependent growth inhibition in all three EGFR-TKI-resistant NSCLC cell lines. When sorafenib was combined with gemcitabine, synergistic activity was observed in the A549 and H1666 cells and antagonistic activity was observed in the H1975 cells. Sorafenib arrested the cell cycle at the G1 phase, whereas gemcitabine arrested the cell cycle at the S phase. Sorafenib inhibited C-RAF and p-ERK in the A549 cells and B-RAF and p-ERK in the H1666 and H1975 cells. The molecular mechanism of this synergism is that RAF/MEK/ERK which are activated by gemcitabine are efficiently suppressed by simultaneously administered sorafenib. By contrast, the mechanism of antagonism may be due to mutual interference with the cell cycle in the H1975 cells. In conclusion, we found that sorafenib exhibits antiproliferative effects in EGFR-TKI-resistant NSCLC cell lines and when combined with gemcitabine demonstrates synergistic activity in A549 and H1666 cells but antagonistic activity in H1975 cells.
PMCID: PMC3525450  PMID: 23255896
sorafenib; gemcitabine; non-small cell lung cancer; epidermal growth factor-tyrosine kinase inhibitor-resistance
11.  The Role of MET Receptor Tyrosine Kinase in Non-Small Cell Lung Cancer and Clinical Development of Targeted Anti-MET Agents 
The Oncologist  2013;18(2):115-122.
The role of MET in the pathophysiology of non-small cell lung cancer and in acquired resistance to epidermal growth factor receptor inhibitors is summarized. An update on progress in the clinical development of inhibitors of MET for treatment of non-small cell lung cancer is provided.
A better understanding of the pathophysiology and evolution of non-small cell lung cancer (NSCLC) has identified a number of molecular targets and spurred development of novel targeted therapeutic agents. The MET receptor tyrosine kinase and its ligand hepatocyte growth factor (HGF) are implicated in tumor cell proliferation, migration, invasion, and angiogenesis in a broad spectrum of human cancers, including NSCLC. Amplification of MET has been reported in approximately 5%–22% of lung tumors with acquired resistance to small-molecule inhibitors of the epidermal growth factor receptor (EGFR). Resistance to EGFR inhibitors is likely mediated through downstream activation of the phosphoinositide 3-kinase /AKT pathway. Simultaneous treatment of resistant tumors with a MET inhibitor plus an EGFR inhibitor can abrogate activation of downstream effectors of cell growth, proliferation, and survival, thereby overcoming acquired resistance to EGFR inhibitors. Development and preclinical testing of multiple agents targeting the HGF–MET pathway, including monoclonal antibodies targeting HGF or the MET receptor and small-molecule inhibitors of the MET tyrosine kinase, have confirmed the crucial role of this pathway in NSCLC. Several agents are now in phase III clinical development for the treatment of NSCLC. This review summarizes the role of MET in the pathophysiology of NSCLC and in acquired resistance to EGFR inhibitors and provides an update on progress in the clinical development of inhibitors of MET for treatment of NSCLC.
PMCID: PMC3579594  PMID: 23345546
Epidermal growth factor receptor tyrosine kinase inhibitor; Hepatocyte growth factor; MET; Non-small cell lung cancer; Tyrosine kinase inhibitor
12.  Inhibition of TWIST1 Leads to Activation of Oncogene-Induced Senescence in Oncogene Driven Non-Small Cell Lung Cancer 
Molecular cancer research : MCR  2013;11(4):329-338.
A large fraction of non-small cell lung cancers (NSCLC) are dependent on defined oncogenic driver mutations. Although targeted agents exist for EGFR- and EML4-ALK-driven NSCLC, no therapies target the most frequently found driver mutation, KRAS. Furthermore, acquired resistance to the currently targetable driver mutations is nearly universally observed. Clearly a novel therapeutic approach is needed to target oncogene driven NSCLC. We recently demonstrated that the basic helix-loop-helix transcription factor Twist1 cooperates with mutant Kras to induce lung adenocarcinoma in transgenic mouse models and that inhibition of Twist1 in these models led to Kras-induced senescence. In the current study, we examine the role of TWIST1 in oncogene driven human NSCLC. Silencing of TWIST1 in KRAS mutant human NSCLC cell lines resulted in dramatic growth inhibition and either activation of a latent oncogene-induced senescence program or in some cases, apoptosis. Similar effects were observed in EGFR mutation driven and c-Met amplified NSCLC cell lines. Growth inhibition by silencing of TWIST1 was independent of p53 or p16 mutational status and did not require previously defined mediators of senescence, p21 and p27, nor could this phenotype be rescued by overexpression of SKP2. In xenograft models, silencing of TWIST1 resulted in significant growth inhibition of KRAS mutant, EGFR mutant and c-Met amplified NSCLC. Remarkably, inducible silencing of TWIST1 resulted in significant growth inhibition of established KRAS mutant tumors. Together these findings suggest that silencing of TWIST1 in oncogene driver dependent NSCLC represents a novel and promising therapeutic strategy.
PMCID: PMC3631276  PMID: 23364532
13.  Clinical Implications of KRAS Mutations in Lung Cancer Patients Treated with Tyrosine Kinase Inhibitors: An Important Role for Mutations in Minor Clones1 
Neoplasia (New York, N.Y.)  2009;11(10):1084-1092.
Mutations inducing resistance to anti-epidermal growth factor receptor (EGFR) therapy may have a clinical impact even if present in minor cell clones which could expand during treatment. We tested this hypothesis in lung cancer patients treated with tyrosine kinase inhibitors (TKIs). Eighty-three patients with lung adenocarcinoma treated with erlotinib or gefitinib were included in this study. The mutational status of KRAS and EGFR was investigated by direct sequencing (DS). KRAS mutations were also assessed by mutant-enriched sequencing (ME-sequencing). DS detected KRAS mutations in 16 (19%) of 83 tumors; ME-sequencing identified all the mutations detected by DS but also mutations in minor clones of 14 additional tumors, for a total of 30 (36%) of 83. KRAS mutations assessed by DS and ME-sequencing significantly correlated with resistance to TKIs (P = .04 and P = .004, respectively) and significantly affected progression-free survival (PFS) and overall survival (OS). However, the predictive power of mutations assessed by ME-sequencing was higher than that obtained by DS (hazard ratio [HR] = 2.82, P = .0001 vs HR = 1.98, P = .04, respectively, for OS; HR = 2.52, P = .0005 vs HR = 2.21, P = .007, respectively, for PFS). Survival outcome of patients harboring KRAS mutations in minor clones, detected only by ME-sequencing, did not differ from that of patients with KRAS mutations detected by DS. Only KRAS mutations assessed by ME-sequencing remained an independent predictive factor at multivariate analysis. KRAS mutations in minor clones have an important impact on response and survival of patients with lung adenocarcinoma treated with EGFR-TKI. The use of sensitive detection methods could allow to more effectively identify treatment-resistant patients.
PMCID: PMC2745674  PMID: 19794967
14.  Novel agents and strategies for overcoming EGFR TKIs resistance 
Since the recognition of epidermal growth factor receptor (EGFR) as a therapeutic target, EGFR tyrosine kinase inhibitors (TKIs) have been used in lung cancer patients with EGFR mutations, which has been a major breakthrough for lung cancer treatment.. The progression-free survival (PFS) of patients with EGFR mutations treated with EGFR TKIs is significantly prolonged compared with that of patients who underwent standard chemotherapy. However, all patients who initially respond to EGFR TKIs eventually develop acquired resistance (AR). Many small molecule agents and monoclonal antibodies (McAb) targeting signaling pathways are potential therapeutic regimens for overcoming resistance, and various therapeutic strategies are used in clinical practice. Here we review the novel agents and therapeutic strategies for overcoming AR to EGFR TKIs.
PMCID: PMC3898214  PMID: 24410791
Non-small cell lung cancer; EGFR TKI acquired resistance; New agents; IMPRESS; ASPIRATION
15.  Targeted therapy against VEGFR and EGFR with ZD6474 enhances the therapeutic efficacy of irradiation in an orthotopic model of human non-small cell lung cancer 
Conventional therapies for lung cancer have reached a therapeutic plateau. We therefore evaluated the feasibility of combined VEGFR2 and EGFR targeting with radiation therapy in an orthotopic model that closely recapitulates the clinical presentation of human lung cancer.
Methods and Materials
The effects of irradiation and/or ZD6474, a small molecule inhibitor of VEGFR2 and EGFR tyrosine kinases, were studied in vitro for human lung adenocarcinoma cells using proliferation and clonogenic assays. The feasibility of combining ZD6474 with radiation therapy was then evaluated in an orthotopic model of human lung adenocarcinoma. Lung tumor burden and spread within the thorax were assessed and tumor and adjacent tissues were analyzed by immunohistochemical staining for multiple parameters including CD31, VEGF, VEGFR2, EGF, EGFR, MMP2, MMP9 and bFGF.
ZD6474 enhanced the radioresponse of NCI-H441 human lung adenocarcinoma cells by a factor of 1.37 and markedly inhibited sublethal damage repair. In vivo, the combined blockade of VEGFR2 and EGFR by ZD6474 blocked pleural effusion formation and angiogenesis and enhanced the anti-vascular and anti-tumor effects of radiation therapy in the orthotopic human lung cancer model and was superior to chemoradiotherapy.
When radiation therapy is combined with VEGFR2 and EGFR blockade a significant enhancement of antiangiogenic, anti-vascular and anti-tumor effects are seen in an orthotopic model of lung cancer. These data provide support for clinical trials of biologically targeted and conventional therapies for human lung cancer.
PMCID: PMC2151850  PMID: 17889445
Angiogenesis; Lung cancer; Antiangiogenesis; Radiotherapy; Targeted therapy
16.  Biomarkers that currently affect clinical practice: EGFR, ALK, MET, KRAS 
Current Oncology  2012;19(Suppl 1):S33-S44.
New drugs such as pemetrexed, the epidermal growth factor receptor (egfr) tyrosine kinase inhibitors, and the Alk inhibitor crizotinib have recently enabled progress in the management of advanced non-small-cell lung cancer (nsclc). More drugs, especially Met inhibitors, will follow. However, the benefits of these agents are not uniform across the spectrum of nsclc, and optimizing their utility requires some degree of subgrouping of nsclc by the presence or absence of certain biomarkers.
The biomarkers of current or imminent value are EGFR and KRAS mutational status, ALK rearrangements, and MET immunohistochemistry. As a predictor of benefit for anti-egfr monoclonal antibodies, EGFR immunohistochemistry is also of potential interest.
Some of the foregoing biomarkers (EGFR, ALK, MET) are direct drivers of the malignant phenotype. As such, they are, quite rationally, the direct targets of inhibitory drugs. However, KRAS, while definitely a driver, has resisted attempts at direct pharmacologic manipulation, and its main value might lie in its role as part of an efficient testing algorithm, because KRAS mutations appear to exclude EGFR and ALK mutations. The indirect value of KRAS in determining sensitivity to other targeted agents or to pemetrexed remains controversial. The other biomarkers (EGFR, ALK, MET) may also have indirect value as predictors of sensitivity to chemotherapy in general, to pemetrexed specifically, and to radiotherapy and molecularly targeted agents.
These biomarkers have all enabled the co-development of new drugs with companion diagnostics, and they illustrate the paradigm that will govern progress in oncology in the immediate future. However, in nsclc, the acquisition of sufficient biopsy material remains a stubborn obstacle to the evolution of novel targeted therapies.
PMCID: PMC3377752  PMID: 22787409
nsclc; lung cancer; EGFR; ALK; KRAS; prognosis; prediction
17.  BIBW2992, an irreversible EGFR/HER2 inhibitor highly effective in preclinical lung cancer models 
Oncogene  2008;27(34):4702-4711.
Genetic alterations in the kinase domain of the epidermal growth factor receptor (EGFR) in non-small cell lung cancer (NSCLC) patients are associated with sensitivity to treatment with small molecule tyrosine kinase inhibitors. Although first-generation reversible, ATP-competitive inhibitors showed encouraging clinical responses in lung adenocarcinoma tumors harboring such EGFR mutations, almost all patients developed resistance to these inhibitors over time. Such resistance to first-generation EGFR inhibitors was frequently linked to an acquired T790M point mutation in the kinase domain of EGFR, or upregulation of signaling pathways downstream of HER3. Overcoming these mechanisms of resistance, as well as primary resistance to reversible EGFR inhibitors driven by a subset of EGFR mutations, will be necessary for development of an effective targeted therapy regimen. Here, we show that BIBW2992, an anilino-quinazoline designed to irreversibly bind EGFR and HER2, potently suppresses the kinase activity of wild-type and activated EGFR and HER2 mutants, including erlotinib-resistant isoforms. Consistent with this activity, BIBW2992 suppresses transformation in isogenic cell-based assays, inhibits survival of cancer cell lines and induces tumor regression in xenograft and transgenic lung cancer models, with superior activity over erlotinib. These findings encourage further testing of BIBW2992 in lung cancer patients harboring EGFR or HER2 oncogenes.
PMCID: PMC2748240  PMID: 18408761
EGFR; HER2; lung cancer; BIBW2992; therapeutics
18.  Genetic and epigenetic analysis of erbB signaling pathway genes in lung cancer 
Prognosis for patients with non-small cell lung cancer (NSCLC) is poor. The potential value of modulating EGFR for treatment is reflected by the recent approval of specific drugs that inhibit its activity. Mutations in EGFR were reported in lung cancer and generated interest, once they enable the identification of lung cancers likely to respond to various targeted small molecules.
We tested 3 key genetic and epigenetic alterations (EGFR, RASSF1A, and BRAF) of this pathway on a series of primary NSCLC [Total 111; adenocarcinoma 49, squamous cell carcinoma (SCC) 48 and others 14]. The mutational status of KRAS (and p53) was known for these samples. The purpose of this study was to define the pattern of erbB pathway alterations in NSCLC and to test for associations with clinical parameters.
Five EGFR mutations were identified: 3 in adenocarcinoma (6 %), 1 in SCC (2%) and 1 in adenocarcinoma with bronchoalveolar component tumor (7%). EGFR mutations included 3 in-frame deletions in exon 19 and 2 point mutations in exon 21. Promoter methylation of RASSF1A was detected in 25 of 45 adenocarcinomas and 18 of 46 SCC. Mutations of EGFR, BRAF and KRAS in adenocarcinoma were mutually exclusive and inversely correlated with RASSF1A methylation (p = −0.394; p=0.007). Overall, genetic and/or epigenetic alterations of erbB pathway genes were detected in 80% (39/49) of adenocarcinomas.
Nearly half of primary adenocarcinoma harbor molecular alterations of the erbB pathway. Careful characterization of these alterations and response to anti-EGFR therapies is warranted to determine better and accurate determinants of clinical response.
PMCID: PMC3057519  PMID: 21102258
EGFR mutation; DNA methylation/epigenetics; RASSF1A
19.  EGFR Inhibition in the Treatment of Non-Small Cell Lung Cancer 
Drug development research  2008;69(6):359-372.
Epidermal growth factor receptor (EGFR) inhibitors have introduced the concept of targeted therapy to the treatment of non-small cell lung cancer (NSCLC). These agents appear most effective in patients with tumors that are highly dependent on EGFR signaling pathways, a population that disproportionately includes females, nonsmokers, individuals of East Asian origin, and patients with adenocarcinoma histology. Currently available EGFR-inhibiting drugs include the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib, and lapatinib, which are administered orally and interfere with the intracellular tyrosine kinase domain, and the monoclonal antibodies (mAbs) cetuximab and panitumumab, which are administered intravenously and interfere with extracellular ligand binding. While the use of EGFR TKIs as monotherapy prolongs survival in metastatic NSCLC, they have demonstrated no benefit when added to conventional, cytotoxic chemotherapy. In contrast, the anti-EGFR mAb cetuximab appears most effective when combined with chemotherapy or radiation. Despite dramatic initial responses to treatment in some cases, NSCLC eventually becomes resistant to EGFR inhibition. Possible mechanisms include secondary mutations that interfere with drug binding, oncogenic pathways driven by other receptor tyrosine kinases, and independent activity of downstream signaling molecules. Efforts to overcome such resistance include irreversibly binding EGFR TKIs, multi-targeted TKIs, and combinations with chemotherapy, radiation, and other targeted therapies.
PMCID: PMC2701650  PMID: 19562083
non-small cell lung cancer; epidermal growth factor receptor; monoclonal antibodies; tyrosine kinase inhibitors; targeted therapy
20.  Mutations within the tyrosine kinase domain of EGFR gene specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking 
British Journal of Cancer  2006;94(6):896-903.
Somatically acquired mutations in the epidermal growth factor receptor (EGFR) gene in lung cancer are associated with significant clinical responses to gefitinib, a tyrosine kinase inhibitor that targets EGFR. We screened the EGFR in 469 resected tumours of patients with lung cancer, which included 322 adenocarcinomas, 102 squamous cell carcinomas, 27 large cell carcinomas, 13 small cell carcinomas, and five other cell types. PCR with a specific condition was performed to identify any deletion in exon 19, while mutant-allele-specific amplification was performed to identify a mutation in codon 858 of exon 21. EGFR mutations were found in 136 cases (42.2%) with adenocarcinoma, in one case with large cell carcinoma, and in one case with pleomorphic carcinoma. An in-frame deletion in exon 19 was found in 62 cases while an L858R mutation was found in 77 cases. In the 322 cases with adenocarcinoma, these mutations were more frequently found in women than in men (P=0.0004), in well differentiated tumours than in poorly differentiated tumours (P=0.0014), and in patients who were never smokers than in patients who were current/former smokers (P<0.0001). The mutation was more frequently observed in patients who smoked ⩽20 pack-year, and in patients who quit at least 20 years before the date of diagnosis for lung cancer. The K-ras mutations were more frequently found in smokers than in never smokers, and in high-dose smokers than in low-dose smokers. In conclusion, the mutations within the tyrosine kinase domain of EGFR were found to specifically occur in lung adenocarcinoma patients with a low exposure of tobacco smoking.
PMCID: PMC3216424  PMID: 16552419
EGFR; mutation; lung cancer; adenocarcinoma; smoking; screening; K-ras
21.  Tankyrase and the canonical Wnt pathway protect lung cancer cells from EGFR inhibition 
Cancer research  2012;72(16):4154-4164.
Lung cancer is the leading cause of death worldwide. Adenocarcinomas, the most common histological subtype of non-small cell lung cancer (NSCLC), are frequently associated with activating mutations in the epidermal growth factor receptor (EGFR) gene. Although these patients often respond clinically to the EGFR tyrosine kinase inhibitors erlotinib and gefitinib, relapse inevitably occurs, suggesting the development of escape mechanisms that promote cell survival. Using a loss-of-function, whole genome shRNA screen, we identified that the canonical Wnt pathway contributes to the maintenance of NSCLC cells during EGFR inhibition, particularly the poly-ADP-ribosylating enzymes tankyrase 1 and 2 that positively regulate canonical Wnt signaling. Inhibition of tankyrase and various other components of the Wnt pathway with shRNAs or small molecules significantly increased the efficacy of EGFR inhibitors both in vitro and in vivo. Our findings therefore reveal a critical role for tankyrase and the canonical Wnt pathway in maintaining lung cancer cells during EGFR inhibition. Targeting the Wnt-tankyrase-β-catenin pathway together with EGFR inhibition may improve clinical outcome in patients with NSCLC.
PMCID: PMC3673784  PMID: 22738915
tankyrase; β-catenin; EGFR; casein kinase; CK1; CK2; synthetic lethal
22.  Oncogenic KRAS Desensitizes Colorectal Tumor Cells to Epidermal Growth Factor Receptor Inhibition and Activation12 
Neoplasia (New York, N.Y.)  2010;12(6):443-452.
Epidermal growth factor receptor (EGFR)-targeting therapeutics have shown efficacy in the treatment of colorectal cancer patients. Clinical studies have revealed that activating mutations in the KRAS protooncogene predict resistance to EGFR-targeted therapy. However, the causality between mutant KRAS and resistance to EGFR inhibition has so far not been demonstrated. Here, we show that deletion of the oncogenic KRAS allele from colorectal tumor cells resensitizes those cells to EGFR inhibitors. Resensitization was accompanied by an acquired dependency on the EGFR for maintaining basal extracellular signal-regulated kinase (ERK) activity. Deletion of oncogenic KRAS not only resensitized tumor cells to EGFR inhibition but also promoted EGF-induced NRAS activation, ERK and AKT phosphorylation, and c-FOS transcription. The poor responsiveness of mutant KRAS tumor cells to EGFR inhibition and activation was accompanied by a reduced capacity of these cells to bind and internalize EGF and by a failure to retain EGFR at the plasma membrane. Of 16 human colorectal tumors with activating mutations in KRAS, 15 displayed loss of basolateral EGFR localization. Plasma membrane localization of the EGFR could be restored in vitro by suppressing receptor endocytosis through Rho kinase inhibition. This caused an EGFR-dependent increase in basal and EGF-stimulated ERK phosphorylation but failed to restore tumor cell sensitivity to EGFR inhibition. Our results demonstrate a causal role for oncogenic KRAS in desensitizing tumor cells not only to EGFR inhibitors but also to EGF itself.
PMCID: PMC2887497  PMID: 20563247
23.  Molecularly targeted approaches herald a new era of non-small-cell lung cancer treatment 
The discovery of activating mutations in the epidermal growth-factor receptor (EGFR) gene in 2004 opened a new era of personalized treatment for non-small-cell lung cancer (NSCLC). EGFR mutations are associated with a high sensitivity to EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib. Treatment with these agents in EGFR-mutant NSCLC patients results in dramatically high response rates and prolonged progression-free survival compared with conventional standard chemotherapy. Subsequently, echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK), a novel driver oncogene, has been found in 2007. Crizotinib, the first clinically available ALK tyrosine kinase inhibitor, appeared more effective compared with standard chemotherapy in NSCLC patients harboring EML4-ALK. The identification of EGFR mutations and ALK rearrangement in NSCLC has further accelerated the shift to personalized treatment based on the appropriate patient selection according to detailed molecular genetic characterization. This review summarizes these genetic biomarker-based approaches to NSCLC, which allow the instigation of individualized therapy to provide the desired clinical outcome.
PMCID: PMC3682814  PMID: 23785245
non-small-cell lung cancer; epidermal growth factor receptor; ALK rearrangement; gefitinib; erlotinib; crizotinib
24.  Knockdown of Oncogenic KRAS in Non-Small Cell Lung Cancers Suppresses Tumor Growth and Sensitizes Tumor Cells to Targeted Therapy 
Molecular cancer therapeutics  2011;10(2):336-346.
Oncogenic KRAS is found in >25% of lung adenocarcinomas, the major histologic subtype of non-small cell lung cancer (NSCLC), and is an important target for drug development. To this end, we generated four NSCLC lines with stable knockdown selective for oncogenic KRAS. As expected, stable knockdown of oncogenic KRAS led to inhibition of in vitro and in vivo tumor growth in the KRAS mutant NSCLC cells, but not in NSCLC cells that have wild-type KRAS (but mutant NRAS). Surprisingly, we did not see large-scale induction of cell death and the growth inhibitory effect was not complete. To further understand the ability of NSCLCs to grow despite selective removal of mutant KRAS expression, we performed microarray expression profiling of NSCLC cell lines with or without mutant KRAS knockdown and isogenic human bronchial epithelial cell lines (HBECs) with and without oncogenic KRAS. We found that while the MAPK pathway is significantly down-regulated after mutant KRAS knockdown, these NSCLCs showed increased levels of phospho-STAT3 and phospho-EGFR, and variable changes in phospho-Akt. In addition, mutant KRAS knockdown sensitized the NSCLCs to p38 and EGFR inhibitors. Our findings suggest that targeting oncogenic KRAS by itself will not be sufficient treatment but may offer possibilities of combining anti-KRAS strategies with other targeted drugs.
PMCID: PMC3061393  PMID: 21306997
25.  Development of New Mouse Lung Tumor Models Expressing EGFR T790M Mutants Associated with Clinical Resistance to Kinase Inhibitors 
PLoS ONE  2007;2(8):e810.
The EGFR T790M mutation confers acquired resistance to kinase inhibitors in human EGFR mutant lung adenocarcinoma, is occasionally detected before treatment, and may confer genetic susceptibility to lung cancer.
Methodology/Principal Findings
To study further its role in lung tumorigenesis, we developed mice with inducible expression in type II pneumocytes of EGFRT790M alone or together with a drug-sensitive L858R mutation. Both transgenic lines develop lung adenocarcinomas that require mutant EGFR for tumor maintenance but are resistant to an EGFR kinase inhibitor. EGFRL858R+T790M-driven tumors are transiently targeted by hsp90 inhibition. Notably, EGFRT790M-expressing animals develop tumors with longer latency than EGFRL858R+T790M-bearing mice and in the absence of additional kinase domain mutations.
These new mouse models of mutant EGFR-dependent lung adenocarcinomas provide insight into clinical observations. The models should also be useful for developing improved therapies for patients with lung cancers harboring EGFRT790M alone or in conjunction with drug-sensitive EGFR kinase domain mutations.
PMCID: PMC1950079  PMID: 17726540

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