Low-dose Erlotinib Research Articles

Abstract 5276: Immunomodulatory activity of intermittent low-dose erlotinib plus sulindac: Implications for hereditary and sporadic colorectal cancer

Abstract A once-weekly optimized dosing regimen of erlotinib (ERL) in the polyposis in rat colon (Pirc) model [1] was a reliable predictor of antitumor efficacy in familial adenomatous polyposis (FAP) patients [2]. Pirc rats are increasingly used as a translational tool for FAP studies. Based on transcriptomic data from FAP patients receiving ERL plus sulindac (SUL) vs. placebo [3], we determined the timing of changes in interferon-γ signaling in Pirc adenomas, while performing additional dose titration. In rats that were given ERL administered at 5 mg per kilogram of body weight through oral gavage (two times a week) and that received a diet containing 125 parts per million of SUL, a regimen referred to as SUL125+ERL5x2, significant suppression of tumor growth was observed. This combination represented half of the standard of care SUL dose and about one-quarter of the ERL dose in FAP patients. In adenomas collected after one year of treatment from SUL125+ERL5x2 treated rats, the expression of genes related to the major histocompatibility complex (MHC) class I (β2m, Cnx, Psmb8 and Tap1) and MHC class II (RT1-A2, Cd74, RT-1Bb, Cd74, and Ciita) was increased, compared to vehicle controls, excluding a subset of adenomas that remained resistant. Through the use of multiplex mass cytometry (CyTOF), an increase in tumor-infiltrating CD4+ T cells was observed in adenomas from the SUL125+ERL5x2 treatment group. Administration of single or combined agents increased CD68+ cells and reduced the presence of Foxp3+ and Arg1+ cells in Pirc adenomas. The natural history of adenomatous colon polyps indicated an increase in MHC gene expression as tumors increased in size; however, this trend reversed markedly in fully occluding lesions. Additionally, expression of MHC-related factors increased in Pirc colon adenoma and murine colon carcinoma cells treated with ERL±SUL, resulting in enhanced CD8+ T-cell activation and IL-2 secretion. In conclusion, treatment with intermittent low doses of ERL±SUL increased MHC gene expression and induced changes in the immune cell component in the tumor microenvironment of Pirc rat adenomas. A subset of non-responsive adenomas could benefit from additional immunopreventive approaches, with implications for possible clinical applications in both hereditary and sporadic colorectal cancer.

Low-Dose Erlotinib Treatment in Elderly or Frail Patients With EGFR Mutation–Positive Non–Small Cell Lung Cancer

Although the efficacy of epidermal growth factor receptor tyrosine kinase inhibitors for EGFR gene mutation-positive non-small cell lung cancer is well established, optimal dosing remains to be established, especially in elderly or frail patients. To investigate the efficacy and safety of low-dose erlotinib in elderly or frail patients with EGFR mutation-positive non-small cell lung cancer. Single-arm phase 2 trial with the Southwest Oncology Group (SWOG) 2-stage design that enrolled frail patients from 21 Japanese institutions after meeting the inclusion criteria. Chemotherapy-naive patients with EGFR-activating mutation-positive non-small cell lung cancer who were considered frail based on age, the Charlson Comorbidity Index, and Eastern Cooperative Oncology Group performance status were eligible for the study. Patients were initially administered 50 mg/d erlotinib for 4 weeks, which was modified based on response or adverse events. Dose increase was permitted for patients with stable disease after 4 weeks. The primary end point was the independent review committee-confirmed objective response rate (ORR) at the dose of 50 mg/d. The study also evaluated the pharmacokinetics of low-dose erlotinib and influence of ABCB1 gene polymorphisms. Eighty patients were enrolled, with a median (range) age of 80 (49-90) years; 54 (68%) were men. An independent review committee confirmed a significant ORR of 60.0% (90% CI, 50.2%-69.2%). The disease control rate was 90.0% (90% CI, 82.7%-94.9%), median progression-free survival was 9.3 months (95% CI, 7.2-11.4 months), and median overall survival was 26.2 months (95% CI, 21.9-30.4 months). Mild adverse events were observed in some participants, with few patients exhibiting grade 3 or greater adverse events. Low-dose erlotinib treatment was temporarily suspended for 10 patients owing to adverse events. Five of 80 patients (6%) had their erlotinib dose reduced to 25 mg because of oral mucositis, paronychia, erythema multiforme, diarrhea, and anorexia. Two patients discontinued treatment because of adverse events (cutaneous ulcer and bone infection, and oral mucositis, respectively). There were no cases of interstitial lung disease or treatment-related deaths. The median (range) erlotinib plasma concentration was measured at 685 (153-1950) ng/mL. Seventy-three patients discontinued study treatment owing to disease progression (n = 60), death (n = 3), AEs (n = 4), and patient requests (n = 6). No clear association was observed between the pharmacokinetics of low-dose erlotinib and the treatment outcome. Low-dose erlotinib appears to be safe and effective in elderly or frail patients with EGFR mutation-positive non-small cell lung cancer and can be a valid treatment option. UMIN-CTR Identifier: UMIN000015949.

MA21.11 A Multicenter Phase II Study of Low-Dose Erlotinib in Frail Patients with EGFR Mutation-Positive, Non-Small Cell Lung Cancer: TORG1425

MA21.11 A Multicenter Phase II Study of Low-Dose Erlotinib in Frail Patients with EGFR Mutation-Positive, Non-Small Cell Lung Cancer: TORG1425

O2-11-2 - A phase II study of low-dose erlotinib in frail patients with EGFR-mutant non-small cell lung cancer: TORG1425

O2-11-2 - A phase II study of low-dose erlotinib in frail patients with EGFR-mutant non-small cell lung cancer: TORG1425

A multicenter phase II study of low-dose erlotinib in frail patients with EGFR mutation-positive, non-small cell lung cancer: Thoracic oncology research group (TORG) trial 1425.

9063Background: We previously reported that low-dose erlotinib has a certain degree of efficacy with lower toxicity in patients with non-small cell lung cancer (NSCLC) harboring epidermal growth fa...

P2.03-005 Overall Survival Results from a Prospective, Multicenter Phase II Trial of Low-Dose Erlotinib as Maintenance in NSCLC Harboring EGFR Mutation

Maintenance therapy with full-dose erlotinib for patients with advanced non-small cell lung cancer (NSCLC) has demonstrated a significant overall survival (OS) benefit. However, 150 mg/day of erlotinib seems too toxic as maintenance therapy. This study aimed to evaluate the efficacy and safety of low-dose erlotinib (25 mg/day) as maintenance treatment after platinum doublet chemotherapy in NSCLC harboring epidermal growth factor receptor (EGFR) mutation.

Abstract B45: Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: Rationale for combination therapies

Abstract The epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor erlotinib has been approved for years as a first-line therapy for patients harboring EGFR sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial-mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Utilizing a panel of lung carcinoma cell lines with mutated EGFR, we have evaluated the ability of erlotinib to modulate tumor phenotype and to modulate cytotoxic responses to effector NK cells and T cells specific for brachyury and MUC-1. The effect of erlotinib treatment was also evaluated with single cell clonal populations of lung cancer cells isolated based on their expression of epithelial and mesenchymal markers. Our data shows that short-term exposure of tumor cells to low dose erlotinib leads to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as altered apoptotic signaling through enhanced caspase cleavage. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Additionally, with the utilization of single cell clones, it was evident that there was a selection of mesenchymal populations during erlotinib treatment, which could explain the acquired resistance to immune attack seen at longer time points. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, prior to the establishment of tumor relapse with long-term EGFR inhibition. Citation Format: Charli Dominguez, Kwong-Yok Tsang, Claudia Palena. Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: Rationale for combination therapies. [abstract]. In: Proceedings of the AACR Special Conference on Tumor Immunology and Immunotherapy; 2016 Oct 20-23; Boston, MA. Philadelphia (PA): AACR; Cancer Immunol Res 2017;5(3 Suppl):Abstract nr B45.

Phase 1 study of twice weekly pulse dose and daily low-dose erlotinib as initial treatment for patients with EGFR-mutant lung cancers

Patients with EGFR-mutant lung cancers treated with EGFR tyrosine kinase inhibitors (TKIs) develop clinical resistance, most commonly with acquisition of EGFR T790M. Evolutionary modeling suggests that a schedule of twice weekly pulse and daily low-dose erlotinib may delay emergence of EGFR T790M. Pulse dose erlotinib has superior central nervous system (CNS) penetration and may result in superior CNS disease control. We evaluated toxicity, pharmacokinetics, and efficacy of twice weekly pulse and daily low-dose erlotinib. We assessed six escalating pulse doses of erlotinib. We enrolled 34 patients; 11 patients (32%) had brain metastases at study entry. We observed 3 dose-limiting toxicities in dose escalation: transaminitis, mucositis, and rash. The MTD was erlotinib 1200 mg days 1-2 and 50 mg days 3-7 weekly. The most frequent toxicities (any grade) were rash, diarrhea, nausea, fatigue, and mucositis. 1 complete and 24 partial responses were observed (74%, 95% CI 60-84%). Median progression-free survival was 9.9months (95% CI 5.8-15.4months). No patient had progression of an untreated CNS metastasis or developed a new CNS lesion while on study (0%, 95% CI 0-13%). Of the 18 patients with biopsies at progression, EGFR T790M was identified in 78% (95% CI 54-91%). This is the first clinical implementation of an anti-cancer TKI regimen combining pulse and daily low-dose administration. This evolutionary modeling-based dosing schedule was well-tolerated but did not improve progression-free survival or prevent emergence of EGFR T790M, likely due to insufficient peak serum concentrations of erlotinib. This dosing schedule prevented progression of untreated or any new central nervous system metastases in all patients.

Short-term EGFR blockade enhances immune-mediated cytotoxicity of EGFR mutant lung cancer cells: rationale for combination therapies.

The epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI) erlotinib has been approved for years as a first-line therapy for patients harboring EGFR-sensitizing mutations. With the promising implementation of immunotherapeutic strategies for the treatment of lung cancer, there is a growing interest in developing combinatorial therapies that could utilize immune approaches in the context of conventional or targeted therapies. Tumor cells are known to evade immune attack by multiple strategies, including undergoing phenotypic plasticity via a process designated as the epithelial–mesenchymal transition (EMT). As signaling through EGFR is a major inducer of EMT in epithelial cells, we have investigated the effect of EGFR inhibition with erlotinib on tumor phenotype and susceptibility to immune attack. Our data shows that short-term exposure of tumor cells to low-dose erlotinib modulates tumor plasticity and immune-mediated cytotoxicity in lung cancer cells harboring a sensitizing EGFR mutation, leading to a remarkable enhancement of tumor lysis mediated by innate NK cells and antigen-specific T cells. This effect positively correlated with the ability of short-term EGFR blockade to modulate tumor phenotype towards a more epithelial one, as well as to increase susceptibility to caspase-mediated apoptosis. The effect, however, was lost when erlotinib was utilized for long periods of time in vitro or in vivo, which resulted in gain of mesenchymal features and decreased (rather than increased) tumor lysis in response to immune effector mechanisms. Our data provides rationale for potential combinations of erlotinib and immunotherapies for the treatment of lung carcinomas in the early setting, before the establishment of tumor relapse with long-term EGFR inhibition.

A prospective, multicentre phase II trial of low-dose erlotinib in non-small cell lung cancer patients with EGFR mutations pretreated with chemotherapy: Thoracic Oncology Research Group 0911

BackgroundLow-dose erlotinib may be as effective as gefitinib or erlotinib at full dose in non-small cell lung cancer (NSCLC) patients with activating mutations of the epidermal growth factor receptor (EGFR) gene. MethodsPatients with chemotherapy pretreated NSCLC harbouring EGFR mutations received erlotinib at 50mg/d until disease progression or unacceptable toxicities. The dose was escalated to 150mg/d in patients showing no response (i.e. without major tumour shrinkage according to Response Evaluation Criteria in Solid Tumours (RECIST)) to the initial dose during the first 4weeks. The primary end-point was the objective response rate at the dose of 50mg/d. ResultsThirty-four patients from seven institutes were enrolled. The study was closed early when no response was confirmed in 15 patients, excluding the possibility that the primary end-point would be met. The objective response and disease control rates at the dose of 50mg/d as determined by an independent review committee were 54.5% and 84.8%, respectively. Four additional patients achieved partial response with increased 150mg/d dose. Progression-free survival and median survival times during the entire period of the study were 9.5 and 28.5months, respectively. Treatment-related toxicities were generally mild, the most common being skin disorders and diarrhoea. Only one case experienced grade 3 toxicity, which was transient increase of hepatic enzymes. ConclusionThe primary end-point was not met; low-dose erlotinib is not recommended for fit patients with NSCLC harbouring EGFR mutations. However, it may merit further evaluation for elderly or frail patients.

A phase I study of twice weekly pulse dose and daily low dose erlotinib as initial treatment for patients (pts) with EGFR-mutant lung cancers.

8017 Background: Pts with EGFR-mutant lung cancers treated with EGFR tyrosine kinase inhibitors (TKI) develop clinical resistance, most frequently due to acquisition of EGFR T790M. Modeling suggest...

A prospective, multicenter phase II trial of low-dose erlotinib monotherapy for patients with previously treated non-small cell lung cancer (NSCLC) with activating mutation of epidermal growth factor receptor (EGFR): Thoracic Oncology Research Group (TORG) 0911.

8080 Background: Erlotinib and gefitinib have been shown to have similar activity for EGFR mutation-positive (EGFRm+) NSCLC. Since the steady-state plasma trough concentration of erlotinib is appro...

Abstract 2068: Combination erlotinib and cisplatin causes decrease angiogenesis in lung adenocarcinoma.

Abstract Introduction: Platinum based agents such as cisplatin have been traditionally used to for the treatment of lung cancer, and tyrosine kinase inhibitors such as erlotinib are being used more readily. Combinations of anti-cancer agents are becoming more popular due to the anticipation that combination treatment is more effective than individual drug treatments. In this study, we evaluate the anti-cancer effects of combination erlotinib and cisplatin in in vitro cell lines and in in vivo mice models. In addition, as studies of cisplatin and erlotinib in other cancer types show potential to inhibit angiogenesis, we examine whether these agents are able to target angiogenesis in lung adenocarcinoma. Method: Three individual lung adenocarcinoma cell lines (PC9, A549, and H292) were utilized. Each cell line had four treatment groups: control, erlotinib only, cisplatin only, and combination erlotinib and cisplatin. Alamar Blue Assay was utilized to evaluate for in vitro cell death following two day treatments. PC9 cells were injected into the left flank of nude mice for in vivo experiments. Erlotinib and/or cisplatin treatments were injected directly into the tumor and tumor measurements were made every 2 days for a total of 14 days. Western blot analysis of VEGFR, p-VEGFR, and CD31was performed for both in vitro and in vivo samples. Hematoxylin and eosin (H&E) staining and 3,3′-Diaminobenzidine (DAB) staining for CD31 were done on tumor samples. Results: Combination treatment in EGFR mutated PC9 cells led to significantly higher cell death compared to other treatment groups (P<0.0001); the effect was synergistic. In EGFR wild type A549 and H292 cell lines there were no significant differences. Similar to results from cell cultures, in vivo studies of combination treatment showed significant inhibition of tumor growth compared to other treatment groups (p=0.001). On western blot analysis, there were significantly lower levels of p-VEGFR (p<0.0001) and CD31 (p=0.0291) in the combination group compared to all other groups. H&E staining shows significantly lower density of blood vessel and DAB staining of CD31in the combination group compared to other groups. Conclusions: Combination low dose erlotinib and cisplatin is able to induce synergistic cell death. This effect, however, may be specific to EGFR mutated lung adenocarcinoma. Combination treatment also demonstrated the ability to inhibit tumor growth in mice. The inhibition of tumor growth may be secondary to inhibition of angiogenesis. Citation Format: Jasmine G. Lee, Reen Wu. Combination erlotinib and cisplatin causes decrease angiogenesis in lung adenocarcinoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2068. doi:10.1158/1538-7445.AM2013-2068

Abstract 1895: The role of autophagy and Atg3 in erlotinib-resistant lung cancer and synergistic cell death with combination of erlotinib and cisplatin

Abstract Objective: Erlotinib is a mainstay anti-cancer agent for non-small-cell lung cancer (NSCLC) but resistance to erlotinib is a major problem, clinically. The combination of cisplatin and erlotinib has been suggested to inhibit tumor growth more effectively than a single drug alone in sensitive NSCLC. However, the mechanism underlying this finding remains unclear. The specific role of autophagy in cancer and chemotherapy resistance continues to be unclear (promotion vs. prevention of cancer). In this study, we investigate whether combination low dose erlotinib and cisplatin is effective against erlotinib resistant NSCLC and examined the role of autophagy in erlotinib resistance. Methods: PC9, a NSCLC cell line, was used to develop erlotinib resistant PC9 cells (PC9ER). Cell survival was quantified with Alamar blue assay. LC3II and regulatory proteins of autophagy were measured by western blot. Small interfering RNA (siRNA) transfection was used to block translation of protein of interest. Results: Combination low dose erlotinib and cisplatin resulted in synergistic cell death in PC9ER cells (p<0.0001) and a significant decrease in autophagy (p=0.011). At baseline, there was a significantly higher level of LC3II in PC9ER cells compared to sensitive PC9 (p=0.030) suggesting autophagy may play a role in resistance. Rapamycin significantly increased ER (p=0.0004) and 3-MA significantly sensitized PC9ER cells to treatment (p=0.037). Upon examination of autophagy regulatory proteins between PC9ER and PC9 cells, there was significantly lower levels in p-mTOR (p=0.021) and higher levels of Atg3 in PC9ER at baseline (p=0.018). Among the four treatment groups (control, erlotinib-alone, cisplatin-alone, and erlotinib-cisplatin combination), there was a specific decrease in Atg3 with combination treatment (p=0.005). siRNA transfection of Atg3 resulted in the reversal of ER: 42% more cell death with erlotinib-alone (p<0.0001), 18% more cell death (p=0.002) in cisplatin treatment, and 9% more cell death in erlotinib-cisplatin combination (p=0.047) compared to the counterpart treatment groups without siRNA transfection. Conclusions: Our data may suggest that autophagy may promote cancer survival and ER in NSCLC, specifically by up regulation of Atg3 and targeting of Atg3 may be the key to re-sensitizing erlotinib resistant cancers. In addition, it seems erlotinib-cisplatin combination is an effective therapy by down modulation of autophagy by targeting Atg3. Future pharmacologic therapies specifically targeting Atg3 in complement to erlotinib therapy may be effective in preventing and treating ER. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1895. doi:1538-7445.AM2012-1895

The role of autophagy in erlotinib‐resistant lung cancer and the synergistic induction of cell death with combination cisplatin and erlotinib treatment by Atg3 modulation

ObjectiveErlotinib resistance (ER) in non‐small cell lung cancer (NSCLC) is a major problem clinically. Therefore, in this study we tested whether combination of low dose erlotinib and cisplatin is effective against ER and specifically examined the role of autophagy in ER.MethodsPC9 cells were used to establish erlotinib resistant cells (PC9ER). Cell survival was quantified with Alamar blue assay. LC3II and regulatory proteins of autophagy were measured by western blot. SiRNA was used to modulate protein translation.ResultsCombination of low dose erlotinib‐cisplatin induced synergistic cell death in PC9ER cells and this response was related to a decrease in autophagy. Concordantly, rapamycin increased ER and 3‐MA sensitized cell death response in PC9ER cells. Western blot analysis of autophagy regulatory proteins showed significantly higher baseline levels of LC3II and Atg3 in ER cells as compared to the sensitive PC9 and a significant decrease in Atg3 level was seen with combination treatment. Atg3 SiRNA transfection resulted in the reversal of the resistance in PC9ER cells to both erlotinib‐alone and combination treatment.ConclusionsOur data suggests that ER lung cancer cells are responsive to combined low dose erlotinib‐cisplatin treatment by targeting the baseline over production of Atg3 and that Atg3 is a key regulator/promoter of ER in NSCLC.This research was supported by NIH‐T32 grant.

Abstract C59: The effects of combined treatment of erlotinib and cisplatin in resistant lung cancer and the role of autophagy in response to chemotherapy

Abstract Introduction: Non-small cell lung cancer (NSCLC) accounts for about 80% of all cases of lung cancer. The treatment of choice for NSCLC is complete surgical resection. However, in advance stages with metastasis and/or aggressive local infiltrate chemotherapy becomes the only option. At high therapeutic doses, chemotherapy can cause significant side effects, and the development of resistance may arise if initial treatment doses of therapy become ineffective. In order to prevent serious side effects and resistance, low-dose combination regimens that offer comparable results to current high-dose regimens are needed. The combination of cisplatin and erlotinib has been shown to inhibit tumor growth more effectively than a single drug in NSCLC cells. However, the mechanism underlying this finding remains unclear. Autophagy has been suggested to play a critical role in cancer and chemotherapy, but there is ongoing controversy among its role (promotion vs. prevention of cancer). In this study, we examined whether the combination of low-dose cisplatin and erlotinib is able to induce higher levels of cancer cell death than each drug alone in both sensitive and erlotinib-resistant lung adenocarcinoma. In addition, we looked into the role of autophagy in cancer cells and its association with chemotherapy. Methods: Alamar blue assay was used to construct dose response curves in which IC 50 values of cisplatin and erlotinib were determined. Erlotinib-resistant PC9 (PC9/ER) cells were developed by culturing PC9 (EGFR overexpression adenocarcinoma) cells in media containing an erlotinib dose 1000 times the IC50 value over a two week period; PC9/ER cells were verified to be 26-fold more resistant than PC9 sensitive cancer cells. Baseline levels of autophagy of normal human bronchial epithelial (NHBE) cells, PC9, and PC9/ER were examined by Western blot with antibodies against LC3I/II. The IC 25 of each drug was chosen as the low-dose treatments (3 uM cisplatin and 10 nM erlotinib). Cells were treated accordingly: control, cisplatin, erlotinib, and combination. Cell death and autophagy levels were measured. Next, cells were pretreated in one day in advance with rapamycin and cell death and changes in autophagy were assessed. Results: At baseline, there were higher levels of LC3II in PC9/ER compared to PC9 and NHBE. Combined treatment of cisplatin and erlotinib was able to induce significantly more cell death than individual drugs at the same dose (p<0.001). In fact, combination treatment provided synergistic killing effects in PC9 and PC9/ER cells. In PC9 cells, erlotinib alone had 79.9% survival, cisplatin 76.3%, and combination 45.0%. In PC9/ER, erlotinib alone had 96.6% survival, cisplatin 89.3%, and combination 61.1%. Levels of autophagy decreased in a similar pattern to killing-effects in both PC9 and PC9/ER cells. In addition, a significant increase in cell survival of about 20% was observed when pretreated with rapamycin (p<0.001). NHBE demonstrated no significant killing and no changes in autophagy levels. Conclusion: Our data is consistent for autophagy's role in promoting cancer cell survival, work as a protective mechanism for cancer-resistant cells, and a critical mechanism in chemotherapy-induced cancer cell death. The combination of erlotinib and cisplatin offers synergistic killing properties in vitro and may be a potential regimen against resistant lung cancer. In addition, the combination treatment does not induce significant cytotoxic effects and death in NHBE cells indicating its safe dosing. We plan to further characterize autophagy with 3-methyladenine pretreatment, changes in apoptosis, and invasiveness of cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr C59.

Establishment and molecular characteristics of PC-9 derived erlotinib resistant cell lines

e22193 Background: EGFR mutation is closely associated with tumor responsiveness to EGFR tyrosine kinase inhibitors (TKIs), gefitinib or erlotinib. However, many non-small cell lung cancer (NSCLC) cases with EGFR mutation that had responded to EGFR-TKIs finally acquire resistance to EGFR-TKIs. It has been reported that T790M mutation and MET amplification caused resistance to EGFR-TKIs . Recent study suggested that overexpression of HGF was also related to resistance to EGFR-TKIs. However, the whole mechanism of acquired resistance to TKIs is still not fully known. Methods: We established 5 clones of elrotinib resistant PC-9 cell line (harboring EGFR exon 19 deletion) (designated as PC-9ER1–5) by exposing PC-9 to low-dose erlotinib. The protein expression of EGFR-related molecules was examined by Western blotting. Array comparative genomic hybridization (aCGH) or real time quantitative PCR (qPCR) assay was performed to examine copy numbers of EGFR-related genes. Results: In MTS assay, the IC50 value for the parental PC-9 cells was 0.02 μmol/L. By contrast, those for PC-9ER1–5 were more than 33μmol/L. All 5 resistant cell lines retained exon19 deletions and did not obtain T790M mutation. The qPCR assay and aCGH showed no MET amplification in PC-9 or PC-9ER1–5. HGF protein was not overexpressed in PC-9ER1–5 by ELISA. The protein expressions of several molecules in the EGFR-Akt signaling pathway were examined by western blotting after erlotinib treatment. Although phospho-EGFR was suppressed in both PC-9 and PC-9ER1–5 with 2-μmol/L erlotinib, phospho-Akt was not suppressed in PC-9ER1–5. PTEN expression was not down-regulated in PC-9ER1–5. The combination of erlotinib and PHA-665752, MET tyrosine kinase inhibitor, did not suppress phospho-Akt or cell proliferation in PC-9ER1–5. Conclusions: As common molecular features of our EGFR-TKI resistant cell lines, phospho-Akt was not suppressed with exposure to erlotinib. These cell lines did not show previously reported features of resistant cell lines including T790M mutation, MET amplification or HGF overexpression. Our results indicated that other mechanisms leading to Akt activation caused resistance to EGFR-TKIs. No significant financial relationships to disclose.