Erlotinib In Non-small Cell Lung Cancer Research Articles

Abstract 3290: FTO mediates tumor growth and resistance to erlotinib in NSCLC

Abstract Lung cancer, among the most common cancer types, ranks first in cancer associated deaths. Nearly 85% of the lung cancer cases are non-small cell lung cancer (NSCLC). Epidermal growth factor receptor (EGFR), an established marker for NSCLC, is frequently overexpressed on the cell surface. Amplifying mutations often render these receptors activated in a ligand independent manner, therefore, therapies such as erlotinib, an EGFR tyrosine kinase inhibitor (EGFR-TKI), target these activating mutations and inhibit tumor growth. Nevertheless, patients acquire resistance to EGFR TKI within 9-14 months of treatment. Recent studies show that fat mass and obesity- associated (FTO) gene, initially identified to increase the body mass index, plays a major role in cancer development and acquired therapy resistance in several cancer types. FTO demethylates m6A mRNA and modulates tumorigenesis, and drug resistance. In this study, we explored the functions of FTO in EGFR TKI resistance in NSCLC. To study the expression of FTO in erlotinib resistant cells, we used wildtype-EGFR NSCLC cell lines (H2170, H358) and mutant EGFR NSCLC cell lines (H1975, PC9) that were rendered resistant to erlotinib. Our RT-qPCR results demonstrated that FTO was upregulated by 1.7-3.0 fold in erlotinib resistant H2170, H358, PC9 and H1975 cells. FTO protein was upregulated by 1.3-1.6 fold as seen by western blotting, and by 1.7 to 2.6 fold in the nucleus of erlotinib resistant H1975, H2170 and PC9 cell lines by immunofluorescence. Furthermore, we investigated FTO inhibition with Dac51 (FTOi) on cell viability and cell migration properties. MTT cell viability assay showed a 9-17% decrease in cell viability when treated with a combination of Dac51 and erlotinib compared to Dac51 alone in erlotinib resistant H2170 and PC9 cells, respectively. The combinatory treatment showed an additive effect compared to the Dac51 and erlotinib treatments alone. Wound healing assay revealed an 18% open wound area after 48-hour treatment with both Dac51 and erlotinib compared to 0.1% after treatment with erlotinib alone in erlotinib resistant H2170 cells. To evaluate the effect of siRNA mediated FTO (siFTO) knockdown on cell viability and energy metabolism, MTT and luciferase assays were performed. MTT assay showed a 21-28% decrease in viability in erlotinib resistant H1975, H2170, PC9 cells, demonstrating increase in the erlotinib efficacy, as compared to the mock siRNA treatment. Luciferase assay showed an inverse relationship between the FTO and ATP, where the latter was upregulated by 1.3 to 3.4 folds in erlotinib resistant H1975, H2170 and PC9 cells, when treated with siFTO, suggesting its involvement in the energy requirements of the NSCLC cells. In conclusion, our study suggests FTO was upregulated in erlotinib resistant NSCLC cells on both the gene and protein levels. FTO could mediate resistance to EGFR TKI possibly through cell proliferation and migration and help with prognosis of NSCLC patients. Citation Format: Aayush Rastogi, Rong Qiu, Neelu Puri. FTO mediates tumor growth and resistance to erlotinib in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3290.

LMNA Reduced Acquired Resistance to Erlotinib in NSCLC by Reversing the Epithelial-Mesenchymal Transition via the FGFR/MAPK/c-fos Signaling Pathway.

For patients exhibiting non-small-cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations, epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are a first-line treatment. However, most patients who initially responded to EGFR-TKIs eventually developed acquired resistance, limiting the effectiveness of therapy. It has long been known that epithelial-mesenchymal transition (EMT) leads to acquired resistance to EGFR-TKIs in NSCLC. However, the mechanisms underlying the resistance dependent on EMT are unknown. This research aimed to reveal the effects of LMNA in the regulation of acquired resistance to erlotinib by EMT in NSCLC. The acquired erlotinib-resistant cells (HCC827/ER) were induced by gradual increase of concentrations of erlotinib in erlotinib-sensitive HCC827 cells. RNA sequencing and bioinformatics analysis were performed to uncover the involvement of LMNA in the EMT process that induced acquired resistance to erlotinib. The effect of LMNA on cell proliferation and migration was measured by clone-formation, wound-healing, and transwell assays, respectively. The EMT-related protein, nuclear shape and volume, and cytoskeleton changes were examined by immunofluorescence. Western blot was used to identify the underlying molecular mechanism of LMNA regulation of EMT. HCC827/ER cells with acquired resistance to erlotinib underwent EMT and exhibited lower LMNA expression compared to parental sensitive cells. LMNA negatively regulated the expression of EMT markers; HCC827/ER cells showed a significant up-regulation of mesenchymal markers, such as CDH2, SNAI2, VIM, ZEB1, and TWIST1. The overexpression of LMNA in HCC827/ER cells significantly inhibited EMT and cell proliferation, and this inhibitory effect of LMNA was enhanced in the presence of 2.5 μM erlotinib. Furthermore, a decrease in LMNA expression resulted in a higher nuclear deformability and cytoskeletal changes. In HCC827/ER cells, AKT, FGFR, ERK1/2, and c-fos phosphorylation levels were higher than those in HCC827 cells; Furthermore, overexpression of LMNA in HCC827/ER cells reduced the phosphorylation of AKT, ERK1/2, c-fos, and FGFR. In conclusion, our findings first demonstrated that downregulation of LMNA promotes acquired EGFR-TKI resistance in NSCLC with EGFR mutations by EMT. LMNA inhibits cell proliferation and migration of erlotinib-resistant cells via inhibition of the FGFR/MAPK/c-fos signaling pathway. These findings indicated LMNA as a driver of acquired resistance to erlotinib and provided important information about the development of resistance to erlotinib treatment in NSCLC patients with EGFR mutations.

Polymorphisms of Drug-Metabolizing Enzymes and Transporters Contribute to the Individual Variations of Erlotinib Steady State Trough Concentration, Treatment Outcomes, and Adverse Reactions in Epidermal Growth Factor Receptor-Mutated Non-Small Cell Lung Cancer Patients.

BackgroundErlotinib is presently the first line treatment for non-small cell lung cancer (NSCLC) with epidermal growth factor receptor (EGFR) active mutation. An increasing number of evidences show that the treatment efficacy and toxicities are considerably heterogeneous among individuals. Hence, it is necessary to find biological predictors for further individualized treatment of erlotinib in NSCLC patients.MethodsOur present study enrolled 87 cases of NSCLC patients who had been administrated erlotinib with a fixed dose (150 mg/d). Eleven polymorphisms in seven genes of drug-metabolizing enzymes and transporters were genotyped and the steady state trough concentrations were also determined.ResultsThere were significant variances in the steady-state erlotinib trough plasma concentrations, ranging from 315.6 ng/ml to 4479.83 ng/ml. Erlotinib steady state trough concentration was remarkably lower in current smoking patients. The steady state trough concentration of GG in rs1048943 of CYP1A1 was significantly higher than that of AA allele carriers. The polymorphism of CYP1A2 was significantly associated with the severity of skin rash, and the development of diarrhea was associated with SNPs in ABCB1 and CYP3A5. We also observed that GG allele in CYP1A1 was accompanied with a longer PFS in our study.ConclusionA large variability of erlotinib steady state trough concentration was found among Chinese Han population. SNPs in CYP1A1 appeared to influence the steady state trough concentration of erlotinib. Correlation between CYP1A2 polymorphisms and severity of skin rash was observed, together with the correlation between the development of diarrhea and SNPs in ABCB1 and CYP3A5.

Effects of removing reimbursement restrictions on targeted therapy accessibility for non-small cell lung cancer treatment in Taiwan: an interrupted time series study

InterventionsTargeted therapies have been proven to provide clinical benefits to patients with metastatic non-small cell lung cancer (NSCLC). Gefitinib was initially approved and reimbursed as a third-line therapy for patients...

Down-regulation of miR-214 reverses erlotinib resistance in non-small-cell lung cancer through up-regulating LHX6 expression

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are standard treatments for advanced non-small-cell lung cancer (NSCLC) patients. However, acquired resistance to EGFR-TKIs is widely detected across the world, and the exact mechanisms have not been fully demonstrated until now. This study aimed to examine the role of miR-214 in the acquired resistance to erlotinib in NSCLC, and elucidate the underlying mechanisms. qRT-PCR assay detected higher miR-214 expression in the plasma of NSCLC patients with acquired EGFR-TKI resistance than prior to EGFR-TKI therapy, and in the generated erlotinib-resistant HCC827 (HCC827/ER) cells than in HCC827 cells. Bioinformatics analysis and dual-luciferase reporter assay indentified LHX6 as a direct target gene of miR-214, and LHX6 expression was detected to be down-regulated in erlotinib-resistant HCC827 cells. Transwell invasion assay revealed that overexpressing LHX6 reversed the increase in the invasive ability of HCC827 cells induced by miR-214 overexpression, and the CRISPR-Cas9 system-mediated LHX6 knockdown reversed the reduction in the invasion of erlotinib-resistant HCC827 cells caused by miR-214 down-regulation. The results of the present study demonstrate that down-regulation of miR-214 may reverse acquired resistance to erlotinib in NSCLC through mediating its direct target gene LHX6 expression.

Tumor marker analyses from the phase III, placebo-controlled, FASTACT-2 study of intercalated erlotinib with gemcitabine/platinum in the first-line treatment of advanced non-small-cell lung cancer

ObjectivesThe FASTACT-2 study of intercalated erlotinib with chemotherapy in Asian patients found that EGFR mutations were the main driver behind the significant progression-free survival (PFS) benefit noted in the overall population. Further exploratory biomarker analyses were conducted to provide additional insight. Materials and methodsThis multicenter, randomized, placebo-controlled, double-blind, phase III study investigated intercalated first-line erlotinib or placebo with gemcitabine/platinum, followed by maintenance erlotinib or placebo, for patients with stage IIIB/IV non-small cell lung cancer (NSCLC). Provision of samples for biomarker analysis was encouraged but not mandatory. The following biomarkers were analyzed (in order of priority): EGFR mutation by cobas® test, KRAS mutation by cobas®KRAS test, HER2 by immunohistochemistry (IHC), HER3 by IHC, ERCC1 by IHC, EGFR gene copy number by fluorescence in-situ hybridization (FISH) and EGFR by IHC. All subgroups were assessed for PFS (primary endpoint), overall survival (OS), non-progression rate and objective response rate. ResultsOverall, 256 patients provided samples for analysis. Considerable overlap was noted among biomarkers, except for EGFR and KRAS mutations, which are mutually exclusive. Other than EGFR mutations (p<0.0001), no other biomarkers were significantly predictive of outcomes in a treatment-by-biomarker interaction test, although ERCC1 IHC-positive status was predictive of improved OS for the erlotinib arm versus placebo in EGFR wild-type patients (median 18.4 vs 9.5 months; hazard ratio [HR] HR=0.32, 95% confidence intervals [CI]: 0.14–0.69, p=0.0024). ConclusionActivating EGFR mutations were predictive for improved treatment outcomes with a first-line intercalated regimen of chemotherapy and erlotinib in NSCLC. ERCC1 status may have some predictive value in EGFR wild-type disease, but requires further investigation.

PTPRF Expression as a Potential Prognostic/Predictive Marker for Treatment with Erlotinib in Non-Small-Cell Lung Cancer

EGFR mutations and anaplastic lymphoma kinase rearrangements are, to date, the only approved biomarkers to select treatment for non-small-cell lung cancer (NSCLC). However, there is considerable interest in identifying other predictive markers. The PTPRF gene has been suggested as a marker of interest in NSCLC and other tumor types. This hypothesis-generating retrospective analysis examined data from two studies of erlotinib in NSCLC, Marker Identification Trial (MERIT; n = 102) and Sequential Tarceva in Unresectable NSCLC (SATURN; n = 262), to determine whether PTPRF expression was prognostic and/or predictive of patient outcomes. Exploratory analyses were conducted using quantitative reverse transcription polymerase chain reaction on existing formalin-fixed paraffin-embedded samples, to assess gene expression levels, including PTPRF. High versus low levels of expression were dichotomized using the median with B2M as a control comparator. Progression-free survival and overall survival were then compared for patients with high versus low levels of PTPRF in the two studies. PTPRF expression was found to be prognostic for shorter overall survival but was also significantly predictive of improved survival with erlotinib versus placebo in SATURN (hazard ratio, 0.45 [95% confidence interval, CI, 0.30-0.69] in PTPRF high versus 0.96 [95% CI, 0.62-1.48] in PTPRF low; interaction p = 0.02), even in the EGFR wild-type subpopulation (adjusted hazard ratio, 0.44 [95% CI, 0.29-0.68] versus 0.96 [95% CI, 0.62-1.48]; interaction p = 0.01). PTPRF may have value as a predictive marker to identify which patients can obtain the greatest benefit from erlotinib in the post-first-line setting. Further research is warranted to determine the potential value of this marker in clinical decision-making.

Abstract 4355: A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1

Abstract Purpose: We endeavored 1) to understand the contribution of TGFβ signaling on expression of a 13-gene microRNA (miRNA) signature and 2) to assess the activity of the signature miRNA genes on mRNA targets that impact the mechanism of activity of erlotinib, the mechanisms by which resistance to erlotinib arises, and the influence of epithelial-to-mesenchymal transition (EMT) on sensitivity to erlotinib. Background: Previous findings from this lab identified a 13-gene miRNA signature that can predict response to the epidermal growth factor receptor (EGFR) inhibitor, erlotinib, in non-small cell lung cancer (NSCLC) cell lines. Bioinformatic analysis of the miRNA signature showed a convergence of the 13 miRNA genes on the TGFβ signaling pathway. We hypothesize that TGFβ signaling controls expression of the miRNAs comprising a signature of response to erlotinib in NSCLC. Methods: Two signature miRNA genes (miR-140-3p and miR-141) were chosen for study because they demonstrate opposing expression levels in erlotinib-resistant and -sensitive NSCLC cell lines. Promoters of both miRNA genes harbor potential Smad Binding Elements (SBEs), binding sites of the TGFβ-responsive Smad transcription factors. TGFβ activity was assessed in erlotinib-resistant (A549) and -sensitive (PC9) NSCLC cell lines using western blot analysis of proteins of interest in the TGFβ signaling cascade and quantitative real-time PCR for expression of miR-140 and -141. The activity of the potential SBEs controlling each of the two miRNA genes was measured by sub-cloning relevant promoter regions into luciferase-reporter constructs. The resulting miR-140 and -141 promoter constructs were transfected into A549 and PC9 cells, and luciferase activity was measured in the presence of TGFβ, the TGFβ inhibitor, SB-431542, and pharmacological inhibitors of EGFR and MAPK. Results: TGFβ acts through the Smad-responsive, canonical signaling pathway in A549 cells. In PC9 cells, TGFβ utilizes the Ras/MAPK pathway for similar signaling endpoints. Analysis of endogenous miRNA expression supports the hypothesis that TGFβ, in the presence and absence of SB-431542 treatment, alters the expression of both microRNAs in both cell lines. Analysis of the influence of the possible SBE sites in each miRNA gene promoter suggests a cell-line specific response. Conclusion: TGFβ can control the expression of signature microRNA genes using different downstream effectors in erlotinib-sensitive and in erlotinib-resistant cell lines. We hypothesize that the erlotinib sensitivity signature miRNA genes can control sensitivity to erlotinib and EMT. Citation Format: Madeline J. Krentz, Rebecca Astley, Andrew Stacy, Esther P. Black. A microRNA signature of response to erlotinib is impacted by the EMT-inducing cytokine TGFβ1. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4355. doi:10.1158/1538-7445.AM2014-4355

CD146 contributes to epithelial-mesenchymal transition of acquired resistance to erlotinib in non-small cell lung cancer in vitro and in vivo

Objective To elucidate the impact of CD146 on epithelial-mesenchymal transition (EMT) of acquired resistance to erlotinib in non-small cell lung cancer (NSCLC),thus to reveal the molecular mechanism of CD146 involved in the EMT and in the metastasis of cancer cells.Methods NSCLC cell line H1650 was used to establish the subline resistant to erlotinib (H1650ER).The expression of E-cadherin,Vimentin and CD146 in the resistant cells was examined using immunostaining and Western blotting.CD146 was silenced by RNA interference in H1650ER,and invasion assay was performed to characterize the resistant cells.The effects of CD146 on cell proliferation and invasion were analyzed.Results The quantity of CD146 expression and Vimentin expression in lung adenocarcinoma cancer cell H1650ER were 0.474 ± 0.013 and 0.465 ± 0.012,significantly higher than before resistance (P ＜ 0.05).The E-cadherin protein expression was 0.180 ± 0.011,significantly lower than before resistance (P ＜ 0.05).As compared with the control group,small interfering RNA (siRNA)-mediated down-regulation of CD146 suppressed the proliferation and invasion ability of H1650ER (P ＜ 0.05).Conclnsion CD146 contributes to EMT of acquired resistance to erlotinib in NSCLC in vitro.It suggests that CD146 is a potential molecular target in the treatment of NSCLC. Key words: Lung adenocarcinoma; Erlotinib; Epithelial-mesenchymal transition ; Small interfering RNA

Erlotinib in the first-line treatment of non-small-cell lung cancer

Non-small-cell lung cancer (NSCLC) is the leading cause of cancer-related deaths. In the last decade the EGF receptor (EGFR) signaling pathway has emerged as one of the most important molecular aberrations in NSCLC. Drugs interfering with the tyrosine kinase domain of the EGFR (EGFR-TKI), such as erlotinib or gefitinib, demonstrated efficacy in patients with advanced NSCLC irrespective of therapy line and particularly in patients harboring activating mutations of the EGFR gene. Results of large Phase III randomized trials clearly demonstrated that an EGFR-TKI is the best front-line option for patients with classical EGFR mutations, while in the EGFR wild-type or EGFR unknown population platinum-based chemotherapy remains the gold standard. In pretreated patients, EGFR-TKIs are considered more effective than standard chemotherapy in the EGFR-mutated population, with no difference in EGFR wild-type NSCLC. Although EGFR-TKIs are certainly particularly effective in patients with EGFR mutations, at present no biomarker, including KRAS mutations, can be recommended in clinical practice for precluding the therapy to any pretreated patient. In this article, the authors analyzed data of erlotinib in NSCLC, focusing on its role in front-line therapy.

OPTIMAL results for erlotinib in NSCLC

Lung cancer is the leading cause of cancer-related mortality, with 5-year survival rates of <20% for patients with non-small-cell lung cancer (NSCLC). Erlotinib improves overall and progression-free survival (PFS) and is approved as a first-line therapy for patients with EGFR-mutation-positive NSCLC. For patients with advanced or metastatic disease, however, quality of life (QoL) is also of vital importance.

The SELECT study: A multicenter phase II trial of adjuvant erlotinib in resected epidermal growth factor receptor (EGFR) mutation-positive non-small cell lung cancer (NSCLC).

7010 Background: Cancers with activating EGFR mutations are exquisitely sensitive to EGFR tyrosine kinase inhibitors (TKIs) and retrospective data suggests adjuvant TKIs may improve outcomes in EGFR mutants. This prospective trial investigates the safety and efficacy of adjuvant erlotinib in EGFR mutation-positive NSCLC. Methods: Patients (pts) with surgically resected stage IA-IIIA NSCLC harboring activating EGFR mutations were treated with 150 mg/day of erlotinib for 2 years (y) after completion of any standard adjuvant chemotherapy and/or radiotherapy. The trial was designed to enroll 36 patients, and powered to demonstrate a primary endpoint of 2 y disease free survival (DFS) exceeding 85%, which would suggest improvement over the historically expected 70% 2 y DFS in early stage EGFR-mutant NSCLC (J Thorac Oncol 6:569). Results: Thirty-six pts were enrolled at five sites between 1/08 and 11/09; 53% stage I; 19% stage II; 28% stage IIIA. Toxicities were typical of erlotinib; no grade 4 or 5 events or pneumonitis occurred. 8 pts (22%) required one dose reduction to 100 mg/day and 5 (14%) two reductions to 50 mg/day for grade 3 or persistent grade 2 toxicities. 11 pts discontinued before 2 full years (&lt;1 month (mo) [4], 1-12 mo [2] and 12-23 mo [5]) for toxicities [6], patient preference [3], prostate cancer [1] and recurrence [1]. After a median follow-up of 2.5 y, the 2 y DFS from enrollment is 94% (95% CI 80%, 99%). 10 patients have recurred, 1 during erlotinib treatment and the others after stopping erlotinib (interval before recurrence 2 mo [1], 6-12 mo [4], &gt;12 mo [4]). Genotyping on repeat biopsies from seven of the recurrent cases is underway, as is assessment of response to subsequent erlotinib therapy. Two pts have died of recurrence: one at 1.5 y who stopped erlotinib after 1 mo for toxicity, and one at 2 y who progressed while on erlotinib. Conclusions: This is the first prospective study to report the efficacy of adjuvant erlotinib in NSCLC pts with EGFR mutations. This approach is feasible and yields excellent 2y DFS compared to historical genotype-matched controls. This trial was subsequently expanded to 100 pts to permit subgroup analysis by stage.

Complex Role of Histone Deacetylase Inhibitors in the Treatment of Non–Small-Cell Lung Cancer

Although cancer was historically believed to be a result of simple genetic mutations in tumor DNA, it has since become apparent that epigenetic alteration of chromosomes, or modifications that do not involve a change in the DNA nucleotide sequence, can also result in the expression of oncogenes and tumor suppressors. Eukaryotic DNA is wound around nucleosome spools, which are composed of eight individual histone proteins. Posttranslational epigenetic modification of histones, via acetylation, methylation, and phosphorylation, increases the accessibility of gene promoter regions to transcription factors and globally upregulates gene transcription. Histone deacetylases (HDACs) oppose the acetylation of histones, promoting the condensation of chromatin and gene repression; HDACs also deacetylate a plethora of other proteins important in oncogenesis, including p53, c-Myc, NFB, HIF-1 , HSP90, and others. HDAC expression seems to be a general feature of malignancy, because many cancers display sensitivity to HDAC inhibitors, prompting the exploration of HDAC inhibitor drugs in the treatment of cancer. Two HDAC inhibitors—vorinostat (suberoylanilide hydroxamic acid [SAHA]) and romidepsin (depsipeptide)—have clinically proven antitumor activity in the treatment of cutaneous T-cell lymphoma. However, the utility of HDAC inhibition in other cancers such as non–small-cell lung cancer (NSCLC) is less established. A randomized phase II trial in NSCLC showed improved response rates when vorinostat was added to first-line carboplatin and paclitaxel. However, a subsequent phase III randomized trial was prematurely terminated because of no anticipated improvement in response rate, progressionfree survival, or overall survival. Current research on incorporating HDAC inhibitors into NSCLC treatment is focusing on their use in combination with epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs). The EGFR TKI erlotinib is a standard treatment for NSCLC and has particularly striking activity against tumors with EGFR mutations. Multiple lines of preclinical evidence suggest a complex interplay between the sensitivity of NSCLCs to EGFR TKIs, HDAC inhibitors, and the epithelial-mesenchymal transition (EMT) state. Although all lung cancers initially arise from epithelial cells, activation of the innate EMT developmental regulatory program triggers acquisition of mesenchymal characteristics along with a propensity to invade, metastasize, and resist apoptosis. Baseline sensitivity of many EGFR wild-type NSCLC cell lines to EGFR TKIs is highly correlated with an epithelial phenotype characterized by high e-cadherin expression and low vimentin expression. Cells with a mesenchymal phenotype, characterized by high vimentin and ZEB1 transcription factor activity, can be resensitized to EGFR TKIs with HDAC inhibitors, particularly entinostat, which is thought to cause reversion to an epithelial phenotype. These experiments formed the rationale for the clinical trial published in Journal of Clinical Oncology, in which Witta et al report a randomized phase II study using the HDAC inhibitor entinostat and the EGFR TKI erlotinib in NSCLC. In contrast to pan-HDAC inhibitors like vorinostat, the oral small-molecule entinostat (SNDX-275/ MS-275) belongs to a pharmacologically distinct benzamide class of compounds and inhibits only the class I enzymes HDAC1 to HDAC3, which are highly expressed in a number of cancers. Witta et al found no additional efficacy with the addition of entinostat to erlotinib. However, a preplanned biomarker analysis revealed that the subset of patients with EGFR wild-type tumors and high e-cadherin protein expression had a significantly longer overall survival (12.2 v 5.4 months; P .03) when treated with entinostat and erlotinib compared with erlotinib alone. The clinical data thus partially recapitulate the laboratory work, suggesting that HDAC inhibitors can preserve a pre-existing epithelial phenotype and that such patients will demonstrate sensitivity to erlotinib for a longer period of time. As of yet, we have not seen evidence that HDAC inhibitors can convert a mesenchymal phenotype back to the epithelial state. There is an important distinction between lung cancers harboring EGFR mutations, which confer exquisite sensitivity to EGFR TKI treatment, and tumors that are sensitive to EGFR TKI treatment without a known EGFR mutation. Although much of the preclinical work examining EMT, HDAC inhibitors, and EGFR TKIs has been performed in EGFR wild-type models, newer data suggest analogous relationships in EGFR mutant tumors. Most importantly, in patients with EGFR mutations, emergence of EMT has been directly observed by multiple groups as a mechanism of acquired resistance to EGFR inhibitors. Because EMT is a plastic state, the driving force is unlikely to be irreversible genetic mutations but rather changes in chromatin state or another modulation in cellular signaling pathways. Additionally, there is mounting evidence that autocrine or paracrine signals can promote EMT. In one recent example, treatment of A549 (EGFR wild type) JOURNAL OF CLINICAL ONCOLOGY U N D E R S T A N D I N G T H E P A T H W A Y VOLUME 30 NUMBER 18 JUNE 2

Abstract C27: Targeting ErbB3 and EGFR in lung cancer patients: A phase I trial of MM-121 in combination with erlotinib in patients with non-small cell lung cancer (NSCLC).

Abstract Background: The benefit of EGFR tyrosine kinase inhibitors (TKIs) is largely restricted to EGFR mutation-positive cancers and resistance invariably develops. A central theme of acquired resistance is persistent ErbB3 signaling, resulting in activation of the PI3K/AKT survival pathway. MM-121 is a fully human IgG1 monoclonal antibody (mAb) to ErbB3 with pre-clinical activity as a single agent and in combination with erlotinib in NSCLC, particularly in cancers with ligand-dependent activation of EGFR. This phase 1 study evaluated the safety and tolerability of MM-121 and erlotinib in NSCLC, as well as PK, immunogenicity, efficacy endpoints and exploratory biomarker evaluation. Methods: Patients with advanced NSCLC, good performance status and adequate organ function were enrolled. Patients were EGFR TKI-naïve, unless they were EGFR mutant, in which case acquired resistance was allowed. MM-121 was administered weekly and erlotinib was administered daily. Seven cohorts were enrolled, evaluating varying dose levels of the combination, as well as alternate MM-121 infusion schedules. Dose levels were determined by safety and pharmacokinetic (PK) data. Results: Between February 2010 and July 2011, 33 patients were enrolled. Median age was 64 years and there were 19 (57.5%) women. Twenty-four patients were erlotinib-naïve and 1 patient was an EGFR mutant. The most frequent adverse events were rash, diarrhea, nausea and fatigue. As of 31 July 2001, 16 patients remain on study. Full results will be presented at the meeting. Conclusions: In this phase 1 dose escalation study, MM-121 plus erlotinib was well tolerated by the majority of patients. A phase 2 study is planned. Reference: NCT00994123 Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C27.

DAB2 interactive protein (DAB2IP) methylation in serum DNA of non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations.

7593 Background: DAB2IP loss promotes primary tumor growth by activating Ras and drives metastasis through NFkB, serving as a signaling scaffold to coordinately regulate these pathways. DAB2IP is frequently methylated in lung cancer, and methylation in the m2a region is a key regulatory factor for DAB2IP expression in prostate cancer. We examined DAB2IP methylation in cell lines and in serum from erlotinib-treated NSCLC p with EGFR mutations. Methods: In human lung, breast and colorectal cancer cell lines, we analyzed DAB2IP promoter methylation in regions m2a and m2b by methylation-specific PCR (MSP) and bisulfite genomic sequencing. In circulating serum DNA from 152 erlotinib-treated NSCLC p with EGFR mutations, we analyzed methylation in the m2a and m2b promoter regions of DAB2IP by MSP. Methylation status was correlated with clinical outcome. Results: Methylation was detected in the m2a region of 42 (27.63%) p, and in the m2b region in 51 (33.55%) p. There were no major differences in clinical characteristics (age, gender, smoking history, EGFR mutation type, metastatic sites) between p with methylation in the m2a region and p with methylation in the m2b region. Overall progression-free survival (PFS) was 15 months (m), and median survival (MS) 28 m for all 152 p. For the 41 p with bone metastases (mets), PFS was 14 m for 30 p without methylation in the m2a region vs 8 m for 11 p with methylation in the m2a region (P=0.01), and MS was 23 m vs 10 m, respectively (P=0.19). For the 57 p with distant mets but no lung mets, PFS was 18 m for 36 p without methylation in the m2a region vs 10 m for 21 p with methylation in the m2a region (P=0.01), and MS was 24 m vs 16 m, respectively (P=0.03). No differences in either PFS or MS were observed according to the methylation status of the m2b region. Conclusions: Methylation in the m2a region of DAB2IP in serum DNA correlates with PFS and MS to erlotinib in NSCLC p with EGFR mutations with non-lung mets. Surveillance of DAB2IP methylation status in circulating DNA could be a useful tool to predict outcome to erlotinib in EGFR-mutated NSCLC p with non-lung mets.

Mutations of the catalytic subunit a of PI3K (PIK3CA) in erlotinib-treated non-small cell lung cancer (NSCLC) patients (p) with epidermal growth factor receptor (EGFR) mutations.

7588 Background: Stage IV NSCLC p with EGFR mutations treated with erlotinib had a median progression-free survival (PFS) of 14 months (m) and median survival (MS) of 27 m in our experience. However, EGFR mutations can only imperfectly predict outcome. PIK3CA mutations occur in several tumors and have prognostic and therapeutic implications. We examined the presence and potential influence of PIK3CA mutations on outcome to erlotinib in NSCLC p with EGFR mutations. Methods: PIK3CA mutation status was assessed in pretreatment paraffin-embedded tumor samples from 118 advanced NSCLC p with EGFR mutations treated with erlotinib. E542K, E545K and H1047R mutations were screened by 5’-nuclease PCR (Taqman) assay and confirmed by standard PCR followed by sequencing. Results: PIK3CA mutations were detected in 6 of 118 patients (5.1%). 84% of p with PIK3CA mutations had adenocarcinomas vs 50% of p with wild-type (wt) PIK3CA (P=0.01). Response rate was 50% for p with mutations vs 70% for p with wt PIK3CA (P=0.03). There were no other differences between p with and without PIK3CA mutations. 2 of the 6 p with PIK3CA mutations had the uncommon L861Q mutation in exon 21 of EGFR. 1 of the 6 p had complete response; 2 had partial response; 3 had progressive disease. There was a non-significant trend towards shorter PFS for the 6 p with PIK3CA mutations (9 vs 16 m; P=0.59). MS was not reached for the 6 p with PIK3CA mutations, while it was 29 m for p with wt PIK3CA (P=0.09). Conclusions: PIK3CA mutations can be present in some EGFR-mutant NSCLC p, with lower response and a trend towards shorter PFS. Treatment with EGFR tyrosine kinase inhibitors plus PIK3CA inhibitors should be tested in this small subset of p with double mutations.

Expression of MET, IGF-1R, IL-6/gp130, and AXL in erlotinib-treated non-small cell lung cancer (NSCLC) patients (p) with EGFR mutations.

7555 Background: We have observed a 70% response rate (RR) in NSCLC p with EGFR mutations treated with erlotinib. Primary resistance to erlotinib could result from the absence of an EGFR-organized network. Crosstalk and transactivation between other receptor tyrosine kinases, such as EGFR, IGF-1R and MET, is recognized as a mechanism of growth in several tumors. Mutant EGFR could activate the IL-6/gp130 pathway. Overexpression of AXL has been observed in NSCLC and in GIST imatinib-resistant tumors. Methods: The expression of MET, IGF-1R, IL-6/gp130 and AXL was examined by QPCR in the original pretreatment tumor biopsies of 98 NSCLC p with EGFR mutations. Expression levels by terciles were correlated with outcome to erlotinib. Results: There was a good correlation between the expression of IL-6/gp130 and AXL (r = 0.33; p = 0.001). No differences were observed in expression of the four genes according to gender, smoking history, type of EGFR mutation, or other clinical characteristics. The highest tercile of AXL expression was associated with a higher incidence of lung metastases (87.5%; p = 0.02). No other association between any of the genes and metastatic sites (including brain and bone) was observed. Overall RR was 70%, progression-free survival (PFS) was 14 months (m), and median survival (MS) was 27 m. No differences in RR, PFS or MS were observed according to the expression of any of the four genes. Conclusions: The coactivation of other receptor tyrosine kinases seems not to affect the initial outcome to erlotinib in NSCLC p with EGFR mutations. The association of high levels of AXL with lung metastases should be further examined, since AXL expression has been correlated with metastasis to the lung in a breast cancer model. The addition of AXL inhibition could be a novel treatment approach for enhancing the effect of erlotinib in p with EGFR mutations. No significant financial relationships to disclose.

Single nucleotide polymorphisms in FAS and FASL and sensitivity to gefitinib in patients with advanced non-small cell lung cancer.

e13529 Background: Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) play a key role in the management of advanced non-small cell lung cancer (NSCLC). Numerous investigations have demonstrated an association between the presence of somatic mutations in EGFR and the sensitivity to gefitinib or erlotinib in NSCLC. Since genetic polymorphisms are more prevalent than mutations in human genome, some of them may be more practical as genetic biomarkers to predict the sensitivity to EGFR-TKIs. The FAS-FASL system plays crucial role in apoptotic signaling in many tumor cells. Here, we assessed the possibility that single nucleotide polymorphisms (SNPs) in FAS and FASL could be used to predict the sensitivity to gefitinib in patients with advanced NSCLC. Methods: Candidate polymorphic loci approach was used to determine the functional polymorphisms. SNPs in the promoter region of the FAS (−1377G/A and −670A/G) and FASL (−844T/C) were assessed in 88 advanced NSCLC patients treated with gefitinib. The Genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The association between genotypes and sensitivity to gefitinib was validated by statistical tests. Results: For FAS -1377G/A, the clinical benefit rates (CBR) of gefitinib in advanced NSCLC patients were 31.3%, 61.1% or 74.3% (p = 0.014) in AA, GA or GG genotypes, respectively. For FAS −670A/G, the CBR were 32.0%, 78.0% or 57.9% (p = 0.001) in GG, AG or AA genotypes. For FASL - 844T/C, the CBR were 65.2%, 57.1% or 50% (p = 0.276) in CC, CT or TT genotypes. FAS -670 GG genotype carriers also had a significantly shorter survival time after the treatment of gefitinib than variation allele carriers (AG or AA genotypes), the overall survival time were 26 and 47 months (p = 0.012), respectively. Conclusions: FAS -1377G/A and -670A/G polymorphisms may be predictive for sensitivity to gefitinib in patients with advanced NSCLC. No significant financial relationships to disclose.

Roles of MKK1/2-ERK1/2 and Phosphoinositide 3-Kinase–AKT Signaling Pathways in Erlotinib-Induced Rad51 Suppression and Cytotoxicity in Human Non–Small Cell Lung Cancer Cells

Erlotinib (Tarceva) is a selective epidermal growth factor receptor tyrosine kinase inhibitor in the treatment of human non-small cell lung cancer (NSCLC). In this study, we investigated the roles of ERK1/2 and AKT signaling pathways in regulating Rad51 expression and cytotoxic effects in different NSCLC cell lines treated with erlotinib. Erlotinib decreased cellular levels of phosphorylated ERK1/2, phosphorylated AKT, Rad51 protein, and mRNA in erlotinib-sensitive H1650, A549, and H1869 cells, leading to cell death via apoptosis, but these results were not seen in erlotinib-resistant H520 and H1703 cells. Erlotinib decreased Rad51 protein levels by enhancing Rad51 mRNA and protein instability. Enforced expression of constitutively active MKK1 or AKT vectors could restore Rad51 protein levels, which were inhibited by erlotinib, and decrease erlotinib-induced cytotoxicity. Knocking down endogenous Rad51 expression by si-Rad51 RNA transfection significantly enhanced erlotinib-induced cytotoxicity. In contrast, overexpression of Rad51 by transfection with Rad51 vector could protect the cells from cytotoxic effects induced by erlotinib. Blocking the activations of ERK1/2 and AKT by MKK1/2 inhibitor (U0126) and phosphoinositide 3-kinase inhibitor (wortmannin) suppressed the expression of Rad51 and enhanced the erlotinib-induced cell death in erlotinib-resistant cells. In conclusion, suppression of Rad51 may be a novel therapeutic modality in overcoming drug resistance of erlotinib in NSCLC.

Tumor regression and pharmacodynamic (PD) biomarker validation in non-small cell lung cancer (NSCLC) patients treated with the ErbB/VEGFR inhibitor BMS-690514

8098 Background: BMS-690514 is an oral selective inhibitor of EGFR, HER2, and VEGFR1–3. Previous results from the phase I portion of this phase I/II study established 200 mg/day as safe and tolerable (ASCO 2008; abstr 2564). Methods: Erlotinib-naïve and erlotinib-resistant adult patients with advanced/metastatic, measurable NSCLC received BMS-690514 200 mg/d. Eligible patients had an ECOG PS ≤1 and adequate organ function. Objectives were to assess disease control rate (DCR; CR, PR, SD ≥4 months), safety, PK and potential predictive and PD biomarkers of BMS-690514. Response was assessed every 8 weeks (modified WHO criteria). Predictive biomarkers included EGFR copy number, and EGFR and KRAS mutations. PD biomarkers included immunohistochemistry of EGFR signaling proteins in skin biopsies, circulating sVEGFR2, blood pressure, skin rash and diarrhea. Results: For 60 patients treated, DCR were 11/28 (39%) and 7/32 (22%) for erlotinib-naive and -resistant patients, respectively. DCR was significantly higher among patients harboring an EGFR mutation (6/8) than those with WT EGFR (5/18). One erlotinib-naive patient had PR (57 wks) and subsequent surgical removal of remaining tumor. Regression (48%) was seen in one erlotinib-naive patient harboring a KRAS G13D mutation. One erlotinib-resistant patient had PR (66%, 31 wks). Two erlotinib-resistant patients with EGFR T790M mutations had SD with 6% and 31% decrease in tumor burden. Most frequent treatment-related AES were diarrhea (90%), skin rash (31%), asthenia (29%), anorexia (27%), hypertension (26%), and reversible acute renal insufficiency (11%). sVEGFR2 (14% decrease from baseline, n=14) and decreased pMAPK levels from skin biopsies (14 of 18 pts) were consistent with EGFR and VEGFR2 inhibition. Conclusions: BMS-690514 200 mg/d showed evidence of anti-tumor activity and disease control in patients with NSCLC, including erlotinib-resistant and those with WT EGFR, EGFR T790M or KRAS mutations. Predictive and PD clinical biomarkers confirmed inhibition of both EGFR and VEGFR signaling pathways by BMS-690514. A randomized phase II trial versus erlotinib in NSCLC is underway. [Table: see text]