EGFR-mutant Tumors Research Articles

Small-cell lung cancer with a rare epidermal growth factor receptor gene mutation showing “wax-and-wane” transformation

BackgroundSmall-cell lung cancer with epidermal growth factor receptor (EGFR) gene mutation typically manifests as a transformation occurring after EGFR tyrosine kinase inhibitor therapy for adenocarcinoma with EGFR mutation, whereas primary small-cell lung cancer showing EGFR mutation is extremely rare. Second biopsy of EGFR-mutated tumor has been broadly recognized as necessary, but is not always performed in daily practice, mainly due to the imbalance between the potential risk of the diagnostic procedure and the therapeutic impact of the biopsy result.Case presentationA 70-year-old woman who had never smoked was referred to our hospital with chief complaints of cough and back pain. Transbronchial lung biopsy from the primary tumor of the left upper lobe revealed combined small-cell lung cancer and adenocarcinoma, a subtype of small-cell lung cancer. EGFR L861Q mutation was detected in both small-cell lung cancer and adenocarcinoma components. Given the staging of cT2aN3M1b (Stage IV) and histological diagnosis, first-line chemotherapy with cisplatin plus irinotecan was initiated, and partial response was achieved. Seven months after initial diagnosis, the primary tumor enlarged again, and a second biopsy from the enlarged lesion detected only adenocarcinoma with the L861Q mutation. Erlotinib was started, but multiple brain metastases and enlarged mediastinal lymph nodes subsequently appeared. Whole-brain radiation therapy was performed, and endobronchial ultrasonography-guided transbronchial biopsy from the lymph node revealed reverse transformation to small-cell lung cancer with the L861Q mutation. Amrubicin therapy achieved partial response after two cycles, with the shrinkage lasting for eight months. Serum sialyl Lewis X antigen level increased when the adenocarcinoma component was dominant, whereas plasma pro-gastrin-releasing peptide level increased when the small-cell lung cancer component became dominant.ConclusionsTransformation of the tumor correlates with the difference between small-cell lung cancer and adenocarcinoma in sensitivity to therapies, so repeated biopsies are beneficial for choosing appropriate treatments. Noninvasively obtainable parameters such as tumor markers can support the need for biopsy.

Abstract C113: ERBB3 independent activation of the PI3K pathway in EGFR mutant lung adenocarcinomas.

Abstract Epidermal Growth Factor Receptor (EGFR) mutant lung adenocarcinomas are sensitive to the tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib. EGFR is one member of a family of four receptor tyrosine kinases (EGFR, ERBB2, ERBB3 and ERBB4) that can homo- and/or hetero-dimerize leading to activation of the RAS-MAPK and phosphoinositide 3-kinase (PI3K) pathways. In EGFR mutant lung cancer cell lines, ERBB3 has been implicated in activation of the PI3K pathway. However, the requirement for ERBB3 in the initiation, progression and therapeutic response of EGFR mutant tumors remains undetermined. Using genetically engineered mouse models we investigated the contribution of Erbb3 to the development of EGFR-induced lung adenocarcinomas and their response to TKIs. We found that upon conditional deletion of Erbb3 in the lungs of mice that express mutant EGFR tumorigenesis and erlotinib sensitivity were unaffected. In the absence of Erbb3, tumors retained activation of the Pi3k pathway via the adapter proteins Gab1 and Gab2. Acute loss of ERBB3 in human lung adenocarcinoma cell lines with sensitizing EGFR mutations had a modest effect on phosphorylation of AKT and cell viability that varied among cell lines. These data demonstrate that activation of the Pi3k pathway in EGFR mutant tumors is independent of Erbb3 and has implications for the potential of ERBB3 as a therapeutic target in lung cancer. Knowledge of the role EGFR heterodimerization partners play in mutant EGFR-driven lung cancer can help guide the selection of which EGFR family members to target in the treatment of this disease. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):C113. Citation Format: Xiaoling Song, Pang-Dian Fan, Udayan Guha, David Threadgill, Harold Varmus, Katerina Politi. ERBB3 independent activation of the PI3K pathway in EGFR mutant lung adenocarcinomas. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr C113.

Mutation of TP53 and Alteration of p14arf Expression in EGFR- and KRAS-Mutated Lung Adenocarcinomas

BackgroundIn lung adenocarcinoma, inactivation of the tumor suppressor p53 may abrogate a safeguard mechanism preventing the development of tumors with activating mutations in EGFR or KRAS. To assess this hypothesis, we analyzed TP53 mutations and downregulation of p14arf, a negative regulator of p53 activated by oncogenic signals, in a retrospective series of 96 patients with primary adenocarcinoma of the lung. Patients and MethodsMutations in TP53 (exons 4-9), KRAS (exon 1), and EGFR (exons 18-21) were identified by direct sequencing of DNA from formalin-fixed, paraffin-embedded resected tumors. Expression of p14arf was semiquantitatively evaluated by immunohistochemical analysis. ResultsTP53, KRAS, and EGFR mutations were detected in 42 of 93 (45.2%), 15 of 95 (15.8%), and 31 of 90 (34.4%) cases, respectively. Low p14arf expression was observed in 19 of 91 cases (20.9%). Disruption of the p53/p14arf pathway (defined as TP53 mutation or decreased p14arf expression, or both) was observed in 18 of 31 EGFR-mutated (58.1%) tumors and in 9 of 13 KRAS-mutated (69.2%) tumors. ConclusionInactivation of the p53/p14arf pathway is common but not systematic in EGFR- or KRAS-mutated lung adenocarcinomas. Our work highlights the need to better investigate the association between EGFR and KRAS mutations and alterations in tumor suppressor pathways.

Genomic and Transcriptional Alterations in Lung Adenocarcinoma in Relation to EGFR and KRAS Mutation Status

IntroductionIn lung adenocarcinoma, the mutational spectrum is dominated by EGFR and KRAS mutations. Improved knowledge about genomic and transcriptional alterations in and between mutation-defined subgroups may identify genes involved in disease development or progression.MethodsGenomic profiles from 457 adenocarcinomas, including 113 EGFR-mutated, 134 KRAS-mutated and 210 EGFR and KRAS-wild type tumors (EGFRwt/KRASwt), and gene expression profiles from 914 adenocarcinomas, including 309 EGFR-mutated, 192 KRAS-mutated, and 413 EGFRwt/KRASwt tumors, were assembled from different repositories. Genomic and transcriptional differences between the three mutational groups were analyzed by both supervised and unsupervised methods.Results EGFR-mutated adenocarcinomas displayed a larger number of copy number alterations and recurrent amplifications, a higher fraction of total loss-of-heterozygosity, higher genomic complexity, and a more distinct expression pattern than EGFR-wild type adenocarcinomas. Several of these differences were also consistent when the three mutational groups were stratified by stage, gender and smoking status. Specific copy number alterations were associated with mutation status, predominantly including regions of gain with the highest frequency in EGFR-mutated tumors. Differential regions included both large and small regions of gain on 1p, 5q34-q35.3, 7p, 7q11.21, 12p12.1, 16p, and 21q, and losses on 6q16.3-q21, 8p, and 9p, with 20-40% frequency differences between the mutational groups. Supervised gene expression analyses identified 96 consistently differentially expressed genes between the mutational groups, and together with unsupervised analyses these analyses highlighted the difficulty in broadly resolving the three mutational groups into distinct transcriptional entities.ConclusionsWe provide a comprehensive overview of the genomic and transcriptional landscape in lung adenocarcinoma stratified by EGFR and KRAS mutations. Our analyses suggest that the overall genomic and transcriptional landscape of lung adenocarcinoma is affected, but only to a minor extent, by EGFR and KRAS mutation status.

EGFR Mutations Transform Tumors Inside and Out

EGFR mutant tumors are associated with an immunosuppressive tumor microenvironment and are susceptible to PD-1 blockade.

A Clinical Model for Identifying Radiosensitive Tumor Genotypes in Non–Small Cell Lung Cancer

Non-small cell lung cancer (NSCLC) includes a spectrum of radiosensitive and radioresistant tumors. However, little is known about the molecular determinants of cellular radiation responses. We examined clinical outcomes after gamma knife radiotherapy for NSCLC intracranial metastases to evaluate the use of this model for determining radiosensitive tumor genotypes. Between 2005 and 2012, 239 patients with NSCLC were enrolled in a prospective gamma knife data repository. Molecular pathology regarding EGF receptor (EGFR), ALK, and KRAS mutation status was available for 81 patients. Local and distant brain control was determined for 79 patients with 469 brain metastases. Modified Cox proportional hazards models were established to evaluate local control for treated lesions after serial gamma knife treatments. In total, 11% of patients developed in-field recurrence. No patients with metastases from tumors with EGFR mutations (0/164 lesions) or EML4-ALK translocations (0/61 lesions) recurred in-field. In contrast, 19% of patients without these mutations and 18% of patients with KRAS mutations recurred in-field (10/139 and 3/105 lesions, respectively). Rates of distant brain recurrence did not significantly differ across tumor genotypes. The predicted median in-field local control was significantly longer for EGFR-mutant and ALK-translocated tumors compared with other patients with NSCLC (P < 0.001), whereas distant brain recurrence time was equivalent (P = 0.97). On multivariate analysis, EGFR mutation, ALK translocation, and metastasis size were independent predictors for superior local control after gamma knife treatment. This study suggests that EGFR kinase domain mutations and EML4-ALK translocations are radiosensitive NSCLC genotypes, and proposes a novel model to identify radiosensitive subtypes of NSCLC.

The epidermal growth factor receptor-tyrosine kinase inhibitor era has changed the causes of death of patients with advanced non-small-cell lung cancer

BackgroundEpidermal growth factor receptor-tyrosine kinase inhibitors (EGFR-TKIs) are effective against tumor EGFR-mutated non-small cell lung cancer (NSCLC). Patients with the tumor EGFR-activating mutation (EGFRmu) had superior survival, compared to patients with EGFR wild-type tumors (EGFRwt). Many patients with the EGFRmu have had disease progression with EGFR-TKI treatment because of central nervous system (CNS) metastases. The objective of this retrospective study was to compare the causes of death in patients with a known tumor EGFR mutation status who had been treated with EGFR-TKIs. MethodsWe retrospectively reviewed the chart records of our patients with advanced NSCLC who had received diagnosis, treatment, and supportive and hospice care in our hospital between July 2005 and June 2010. The tumor EGFR mutation status was analyzed by using a DNA sequence method. All enrolled patients had a documented cause of death. ResultsNinety-four patients had documented tumor EGFR data, had received EGFR-TKI treatment (either erlotinib or gefitinib), and were with or without previous or salvage systemic chemotherapy. Of the 94 patients, 36 patients had EGFRwt and 58 patients had EGFRmu. The overall patient survival after starting EGFR-TKI treatment was significantly longer in the EGFRmu patients (median 17.2 months) than in the EGFRwt patients (median 11.6 months; p = 0.0058). Twenty-nine patients died of CNS metastases and 65 died of organ failure (other than the CNS). Patients who died of CNS metastases had undergone EGFR-TKI treatment significantly longer than patients who died of other organ failure (median, 8 months vs. 1.9 months; p = 0.0003) with a hazard ratio of 2.308 [95% confidence interval (C.I.), 1.452–3.668; p = 0.0004]. A significantly higher proportion of EGFRmu patients (26 of 58 patients; 44.8%) than EGFRwt patients (3 of 36 patients; 8.3%) (p < 0.001) died of CNS metastases. ConclusionThe EGFRmu NSCLC patients survived longer and had a significantly higher probability of mortality due to CNS metastases, compared to the EGFRwt patients. This change in the causes of death was noted after the era of EGFR-TKI treatment, and will have an important impact on the strategies and management of supportive and hospice care for patients.

Detection of EGFR mutations in circulating free DNA by PNA-mediated PCR clamping

BackgroundEpidermal growth factor receptor (EGFR)-activating mutations are major determinants in predicting the tumor response to EGFR tyrosine kinase inhibitors in non-small cell lung cancer (NSCLC). Noninvasive test for the detection of EGFR mutations is required, especially in NSCLC patients from whom tissue is not available. In this study, we assessed the feasibility of detection of EGFR mutations in free DNA circulating in plasma.MethodsPlasma samples of 60 patients with partial response to gefitinib were analyzed to detect EGFR-activating mutations in exons 19 and 21. Forty (66.7%) of patients had tumor EGFR mutation results. EGFR mutations in plasma were detected using the peptide nucleic acid (PNA)-mediated polymerase chain reaction (PCR) clamping method. All clinical data and plasma samples were obtained from 11 centers of the Korean Molecular Lung Cancer Group (KMLCG).ResultsOf the 60 patients, 39 were female and the median age was 62.5 years. Forty-three patients never smoked, 53 had adenocarcinomas, and seven had other histologic types. EGFR-activating mutation was detected in plasma of 10 cases (exon 19 deletion in seven and exon 21 L858R point mutation in three). It could not be found in plasma after treatment for 2 months. When only patients with confirmed EGFR mutation in tumor were analyzed, 17% (6 of 35) of them showed positive plasma EGFR mutation and the mutation type was completely matched with that in tumor. There was no statistically significant difference in clinical parameters between patients with EGFR mutations in plasma and those without EGFR mutations.ConclusionsThe detection rate of EGFR mutations from plasma was not so high despite highly sensitive EGFR mutation test suggesting that more advances in detection methods and further exploration of characteristics of circulating free DNA are required.

Impact of the new EGF receptor and ALK testing guideline on personalized lung cancer medicine.

All advanced-stage lung adenocarcinoma patients should be tested for EGF receptor (EGFR) mutations and for EML4–ALK translocations as a guide for selecting them for tyrosine kinase inhibitor (TKI) therapy. The new EGFR and ALK guideline provides useful recommendations for appropriate testing [1]. Lung tumors are the leading cause of cancerrelated mortality worldwide [2]. Almost 80% of cases are non-small-cell lung cancer (NSCLC) and extensive disease is present at the time of diagnosis in the majority of patients. Systemic treatment with chemotherapy has reached a plateau of 10–20% response rates and 4–5 months of progression-free survival (PFS), and has not improved in more than a decade [3–5]. A first milestone in a new era of targeted therapy for this disease was reached with the discovery of a subset of tumors carrying activating mutations of EGFR, which can be selectively blocked with TKIs such as erlotinib and gefitinib. Numerous clinical studies have demonstrated the efficacy of these TKIs in the treatment of EGFR-mutated lung tumors, with 55–83% response rates and 9–13 month PFS [6,7]. A second milestone was the discovery, in 2007, of the EML4–ALK translocation in 6.7% of Japanese NSCLC patients [8]. The translocation was later found in 4–7% of Chinese [9], European [10] and US patients [11]. When treated with a novel ALK inhibitor, crizotinib, these patients showed response rates of 57% with a PFS greater than 6 months [12]. As a result of these new developments, many laboratories worldwide are currently testing lung tumor samples for EGFR mutations and EML4–ALK fusions. In an effort to standardize testing practices, the College of American Pathologists, the International Association for the Study of Lung Cancer and the Association for Molecular Pathology constituted writing and advisory panels, presided by three cochairs, to

Pulmonary Large Cell Carcinoma Lacking Squamous Differentiation Is Clinicopathologically Indistinguishable From Solid-Subtype Adenocarcinoma

Pulmonary large cell carcinoma (LCC) includes tumors not readily diagnosed as adenocarcinoma (ADC) or squamous cell carcinoma on morphologic grounds, without regard to immunophenotype, according to the World Health Organization (WHO). This ambiguous designation may cause confusion over selection of mutation testing and directed therapies. Several groups have proposed the use of immunohistochemistry (IHC) to recategorize LCC as ADC or squamous cell carcinoma; however, it remains unclear if strictly defined LCCs are a clinicopathologically distinct lung tumor subset. To compare the pathologic, molecular, and clinical features of 2 morphologically similar tumors: solid-subtype ADC and LCC. Tumors were included on the basis of solid growth pattern; tumors with squamous or neuroendocrine differentiation were excluded. Solid ADC (n = 42) and LCC (n = 57) were diagnosed by using WHO criteria (5 intracellular mucin droplets in ≥2 high-power fields for solid ADC) and tested for KRAS, EGFR, and ALK alterations. Both solid ADC and LCC groups were dominated by tumors with "undifferentiated"-type morphology and both had a high frequency of thyroid transcription factor 1 expression. KRAS was mutated in 38% of solid ADCs versus 43% of LCCs (P = .62). One ALK-rearranged and 1 EGFR-mutated tumor were detected in the solid ADC and LCC groups, respectively. There were no significant differences in clinical features or outcomes; the prevalence of smoking in both groups was greater than 95%. Other than a paucity of intracellular mucin, LCC lacking squamous or neuroendocrine differentiation is indistinguishable from solid-subtype ADC. We propose the reclassification of these tumors as mucin-poor solid adenocarcinomas.

Correlation of epidermal growth factor receptor (EGFR) mutation profile with computed tomography (CT) imaging features in lung adenocarcinomas.

7542 Background: Imaging studies provide essential clinical information for lung cancer diagnosis, treatment and management. Features such as ground glass opacity (GGO) or internal air bronchogram (IAB), while commonly seen in early stage lung cancer, have minimal clinical implication. By examining a cohort at a single institution, we explored the hypothesis that radiographic characteristics correlates with the molecular signatures. Methods: Since 2009, our institution has prospectively characterized the EGFR status of all non-squamous lung carcinomas. Our inclusion criteria included tumor size &lt; 3 cm and positive EGFR mutation. We randomly selected wild type (WT) patients matched by age and tumor size. The CT imaging of these patients was evaluated by a single radiologist (EO). Results: Imaging features were extracted from 20 WT and 19 EGFR-mutated tumors. Of the EGFR-mutated tumors, 7 had the exon 21 L858R point mutation and 12 had in-frame exon 19 deletion. There were 68% (13) solid nodules in EGFR-mutated and 90% (18) in WT tumors. While solid nodule was the primary feature seen in both WT and EGFR-mutated tumors, exon 21 mutation was associated with mixed ground glass/solid nodule (MGGS) (p = 0.005). This tendency, however, is not present in the exon 19 mutated tumors. No pure GGO was identified in either EGFR or WT group. IAB was present in 62% (24), pleural attachment (PA) in 87% (34), and spiculated (SP) as opposed to lobulated (LO) nodules in 90% (34) of the tumors, which do not differ significantly among groups. Conclusions: Early stage lung adenocarcinomas show a spectrum of imaging features that can be correlated with tumor’s genetic mutations. MGGS is a distinct imaging characteristic differentiating exon 19 and exon 21 EGFR-mutated tumors. [Table: see text]

Preclinical antitumor activity of oral silibinin-meglumine in combination with erlotinib or gefitinib in the treatment of erlotinib- and gefitinib-resistant EGFR-mutant NSCLC mouse xenografts.

e19003 Background: Silibinin (SLB), a bioactive flavonolignan isolated from the dried fruits of milk thistle (Silybum marianum), suppresses the resistance to erlotinib (ERL) caused by the EGFR T790M mutation in NSCLC xenograft models. Poor water solubility and low bioavailability might severely limit the efficacy of SLB. The ability of SLB to overcome ERL resistance attributed to mechanisms other than the second-site EGFR mutations remains completely unexplored. Methods: Using PC-9 cells harboring the ERL-sensitizing EGFR exon 19-mutation ΔE746-A750, we developed ERL-refractory derivatives (PC-9 ERL-R) that lacked EGFR T790M mutation. An oral formulation of milk thistle extract enriched (30%) with a water-soluble form of SLB complexed with the excipient amino-sugar meglumine (SLB-m) was explored for its ability to inhibit the growth of PC-9 ERL-R xenografts. Results: In the absence of second-site EGFR mutations or the alternative activation of MET or AXL, acquired cross-resistance to ERL and GEF correlated with enhanced epithelial-to-mesenchymal transition (EMT) features. Single-agent SLB-m significantly decreased the overall tumor volumes of PC-9 ERL-R xenografts, which exhibited enhanced engraftment and faster growth compared with the ERL-/GEF-sensitive PC-9 parental xenografts. Combination treatment with ERL + SLB-m or GEF + SLB-m significantly suppressed tumor growth (&gt; 90%) compared with single-agent treatment. The SLB-m treatment fully inhibited the development of an EMT phenotype in the ERL-/GEF-refractory cells in vitro. Systemic SLB-m treatment significantly affected the expression of EMT-related transcription factors in the ERL-/GEF-refractory xenografts. Conclusions: The oral administration of SLB-m delays tumor progression in ERL-/GEF-refractory EGFR-mutant NSCLC mouse xenografts. Given that the EMT phenomenon is required by a multiplicity of mechanisms of acquired resistance mechanisms to ERL, our data might facilitate the design of clinical trials to test the administration of SLB-m in combination with ERL or GEF in patients with EGFR-mutated tumors that progress to these drugs.

Impact of body surface area (BSA) on efficacy of gefitinib in patients with non-small cell lung cancer (NSCLC).

e19167 Background: Drug dosing of chemotherapeutic agents has traditionally been based on an estimated BSA to reduce the pharmacokinetic inter-patient variability. However, the approved dose of gefitinib was fixed at 250 mg/body/day. Our preliminary retrospective study showed a potential association between BSA and progression-free survival (PFS) in the gefitinib monotherapy in advanced NSCLC [ASCO 2012, #2607]. Here, we investigated the influence of BSA on efficacy of gefitinib, using data from a prospective cohort of NSCLC patients receiving gefitinib. Methods: The subjects were 154 consecutive, EGFR-tyrosine kinase inhibitor (TKI)-naïve NSCLC patients who were to receive gefitinib monotherapy between 2007 and 2012. Baseline BSA was estimated by height and weight in each patient. The association between BSA and survival time was assessed using log-rank test and stepwise multivariate Cox proportional hazard model. Results: Median BSA of the subjects was 1.42 m2 (range: 0.98-1.95). At the cutoff level in BSA of 1.50 m2, overall survival (OS) and PFS in the large BSA group were not different from those in the small BSA group in the univariate analysis, which was also shown in multivariate analysis (hazard ratio: 1.12, 95% confidence interval: 0.66-1.92 and 1.67, 0.86-3.44, respectively). Among subjects with EGFR-mutant tumors (n=125; 81%), BSA had no association with OS, but patients with larger BSA had significantly worse PFS in the multivariate analysis (1.59, 1.01-2.51). The association of BSA with PFS was highly sensitive to increase or decrease in the cutoff level of BSA: BSA ≥ 1.45 m2 vs. &lt;1.45 m2, [1.18, 0.80-1.75]; BSA ≥ 1.55 m2 vs. &lt;1.55 m2, [1.75, 1.11-2.78]; BSA ≥ 1.60 m2 vs. &lt;1.60 m2, [1.32, 0.82-2.11]. Conclusions: In this cohort, PFS was significantly associated with BSA in EGFR-mutant NSCLC when BSA level was cut off by around the median score. Our findings might suggest the need for further investigation of dose finding PK/PD study stratified by BSA level in gefitinib therapy in EGFR-mutant NSCLC.

Achieving patient health outcomes via CME: A case-study in educating oncologists in the treatment of non-small cell lung cancer.

e19104 Background: The Validation Model is an educational series built on the concept of strengthening education by incorporating the patient perspective. After conducting two iterations of the Validation Model to educate oncologists in the treatment of NSCLC, Quintiles Medical Education was able to demonstrate that CME can lead to improved patient health outcomes by changing physician behaviors in practice. Educational measurement tools, including chart audits, revealed an increase in tumor histology and EGFR mutation testing. Histologic and molecular marker testing leads to optimization of treatment based on tumor pathology and genotype, respectively, as per NCCN guidelines. Personalization of treatment according to guidelines results in improved health outcomes. Methods: The Validation Model consisted of 6 educational activities for which efficacy was measured by conducting pre- and post-tests of knowledge and clinical competence among participants. Participants were then asked to complete a follow-up survey which was designed to determine if specific practices had been implemented since participation. The self-self reported behavioral data was validated by the distribution of patient surveys, designed to measure patient satisfaction and expectation with care, and chart audits, designed to determine the actual practice habits of the participating practitioners. Results: More than 500 participants have completed all 6 of the educational activities. The activities have demonstrated improved knowledge and competence in the educational setting and changes to clinical practice and, as a result, patient health outcomes. Conclusions: CME can be used as an effective tool to improve patient health outcomes in the treatment of NSCLC.

Evaluation of the prognostic/predictive role of tumor EGFR and KRAS mutations in Greek metastatic non-small cell lung cancer patients treated with first-line chemotherapy.

e19048 Background: KRAS mutations are reported in 20-25% of non-small cell lung cancer (NSCLC). Little is known about the prognostic/predictive role of KRAS in advanced NSCLC, with conflicting results among small studies. Recent evidence showed that KRAS mutations could predict for worse outcome in patients treated with platinum-based adjuvant chemotherapy. Methods: KRAS and EGFR genotypes were evaluated in 414 NSCLC patients with available clinical data, diagnosed from March 2000 to December 2012 (tissue blocks from the HeCOG tumor repository). KRAS and EGFR mutations were associated with clinicopathological parameters (mutated vs. wild-type). Outcome comparisons were performed in 214 metastatic patients with treatment data available, following 1st-line chemotherapy without tyrosine kinase inhibitors. Results: The majority of the patients were male (80%), current smokers (53%), with adenocarcinoma (AC) histology (65%). EGFR mutations were found in 10% and KRAS mutations in 17% of all histological types, while in AC they were 14% and 21%, respectively. Most EGFR mutations were classical (79%), while most common KRAS mutations were p.G12C (39%), p.G12D (30%) and p.G12V (15%). Two tumors had concurrent EGFR and KRAS mutations. EGFR mutations were significantly associated with female gender, AC histology and non-smoking status, as previously described. KRAS mutations were associated with AC histology and younger age (&lt;60). At a median follow-up of 31 months, EGFR status did not show any significant associations with OS or PFS, while KRAS mutations were prognostic for worse OS compared to KRAS and EGFR wild-type tumors (HR=1.67, CI=1.08-2.59, p=0.021). This effect was also significant (p=0.022) in the presence of treatment type, with platinum-based therapy being a positive prognostic factor for OS (HR=0.62, CI=0.4-0.92, p=0.019). The association of KRAS mutations with PFS was not significant. Conclusions: EGFR and KRAS genotype incidences are presented for the first time for Greek metastatic NSCLC patients. In this setting, the presence of KRAS mutations significantly increases and platinum 1st-line treatment decreases the risk of death.

Prospective cohort study of serum sialylated glycoprotein (KL-6) as a prognostic marker in patients (pts) with non-small cell lung cancer (NSCLC) receiving gefitinib monotherapy: Okayama Lung Cancer Study Group 0703.

e19023 Background: Gefitinib is now crucial in the treatment of EGFR-mutant NSCLC. Its anti-tumor activity could be modulated by MUC1 mucins [Cancer Res 2006], similar to KL-6 in the biochemical property, currently used as an indicator for interstitial lung diseases (ILDs). Our preliminary retrospective study showed the elevated serum KL-6 level is a potential negative prognostic value in pts with advanced NSCLC receiving gefitinib therapy [Lung Cancer 2008]. We conducted an observational study to evaluate this issue prospectively. Methods: Consecutive, EGFR-TKI-naïve NSCLC pts who were to receive gefitinib were enrolled and stratified by pretreatment KL-6 levels (&lt; 500 U/ml: normal group [NG] and ≥ 500 U/ml: abnormally elevated group [EG]). The primary endpoint was overall survival (OS). The planned accrual number of pts was 150, assuming an estimated HR of 2.0, α=0.05, ß=0.10). We also evaluated ILD events during the gefitinib treatment. Results: Between 2007 and 2012, 154 pts were registered (NG: 89 [58%] and EG: 65 [42%] with median KL-6 score of 305 and 1,050 IU/mL, respectively). Those with pretreatment pulmonary fibrosis were similarly distributed between the groups. Median follow-up time was 13.7 months. The EG had significantly worse OS in the univariate analysis (log-rank p=0.0011), which was retained in the multivariate analysis (hazard ratio [HR]: 1.95, 95% confidence interval [95% CI]: 1.34-2.81). Exploratory analyses showed an interaction between EGFR-mutation status and KL-6; HR of KL-6 for OS: 1.96, [1.30-2.97], and HR: 4.80, [1.05-21.95] in pts with EGFR-mutant and wild-typed tumors, respectively (interaction p=0.07). As for ILD event it occurred similarly during the gefitinib therapy (HR: 2.76, [0.62-12.33]). Conclusions: The elevated KL-6 level could predict a poor prognosis in NSCLC pts with gefitinib therapy, and this association seemed modulated by EGFR-mutation status. Further studies are needed to clarify the biological behavior of KL-6 expression on tumor progression especially in the EGFR pathway.

Optimisation of EGFR TKI efficiency in the therapeutic scheme of EGFR wild-type lung cancer.

e18532 Background: EGFR tyrosine kinase inhibitors (EGFR TKI) have improved the therapeutic care of lung cancer patients but only a small sub-population of patients, namely those harboring EGFR-mutated tumors, benefit from this therapy. The observation that EGFR TKI enhance prognosis and quality of life in all patients when used as second line or maintenance treatment impelled us into the search of potential markers of the optimal introduction kinetics of EGFR TKI in the therapeutic scheme. Methods: We used lung cancer cell lines harboring either wild-type or mutated EGFR (NCI-H1650, NCI-H1975) to study the consequences of cisplatin treatment in vitro on the consecutive sensitivity to erlotinib. Results: Sub-lethal cisplatin pretreatment (3µM) enhances erlotinib toxicity in EGFR wild-type, but not EGFR mutated cells (A549 IC50 drops from 28 to 15µM for short-term or 12µM for long-term exposure). This correlates with EGFR activation following short-term or prolonged cisplatin treatment through the secretion of EGFR ligands. This activation of EGFR is concomitant to the decrease in other receptor tyrosine kinases phosphorylation including Met. Conclusions: The sensitivity of EGFR wild-type lung cancer cells to erlotinib in vitro is enhanced by cisplatin pretreatment. We identified potential markers of this sensitization, namely EGFR ligands, which serum level might be predicitive of the optimal efficiency of EGFR TKI. In vivo validation of these markers is under investigation. The concomitant decrease in other receptor tyrosine kinases phosphorylation suggests that the targeting of other receptor tyrosine kinases might potentiate EGFR TKI efficiency.

Abstract 1106: Mechanism for activation of mutated EGF receptors in lung cancer.

Abstract Previous studies have demonstrated that wild-type (WT) EGFRs are activated by an allosteric mechanism in which a ‘donor’ tyrosine kinase domain (TKD) monomer contacts an ‘acceptor’ TKD monomer, leading to its activation. Here, we present biochemical reconstitution data demonstrating that receptor activation by the most frequently occurring EGFR mutations found in lung cancer does not adhere to this mechanism. Mutated EGFRs hyperphosphorylate their WT counterparts and appear to adopt a conformation that allows them to function as acceptors but not as donors to WT EGFR. As acceptors, the mutated EGFRs also show enhanced association with WT EGFR. We suggest the term “superacceptor activity” to describe this directionally-limited mutant-specific mechanism of EGFR transactivation. Mutated EGFRs also hyperphosphorylate the related ErbB family member, ErbB-2, in a similar manner. Collectively, these data provide insights into a mechanism for activation of lung cancer-associated mutant EGFRs. Since WT EGFRs are usually present in EGFR mutant tumor cells, our studies additionally argue that WT EGFRs contribute to tumorigenesis and may alter sensitivity to EGFR-targeted therapy. Citation Format: Monica L. Red Brewer, Darson Lai, Mark A. Lemmon, William Pao. Mechanism for activation of mutated EGF receptors in lung cancer. [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 1106. doi:10.1158/1538-7445.AM2013-1106

Correlation of Mutation Status and Survival with Predominant Histologic Subtype According to the New IASLC/ATS/ERS Lung Adenocarcinoma Classification in Stage III (N2) Patients

We investigated the relationship between predominant subtype, according to the International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Lung Adenocarcinoma Classification; mutation status; and patient outcome in stage III (N2) lung adenocarcinoma. We identified 69 patients with stage III (N2) lung adenocarcinoma operated on with curative intent between 1993 and 2011 who had adequate tumor tissue for molecular analysis and adequate follow-up time for survival analysis. DNA was isolated and tested for mutations using Sequenom's OncoCarta Panel (v1.0; Sequenom, San Diego, CA). The majority of tumors were acinar (26 of 69 tumors; 38%), solid (24 of 69 tumors; 35%), and micropapillary predominant (13 of 69 tumors; 19%) subtypes. EGFR and KRAS mutations were identified in 17 of 59 tumors (29%) and 13 of 59 tumors (22%), respectively. EGFR mutations occurred most often in acinar (11 of 25 tumors; 44%) and micropapillary predominant tumors (five of 13 tumors; 38%) (p = 0.009), whereas KRAS mutations occurred most often in solid predominant tumors (nine of 21 tumors; 43%) (p = 0.016). Patients with acinar predominant tumors had significantly improved overall survival compared with those with non-acinar predominant tumors (hazard ratio: 0.45; 95% confidence interval: 0.22-0.91; p = 0.026), which remained significant after adjustment for EGFR status, T-stage, sex, and age. Patients with EGFR-mutant micropapillary predominant tumors had similar survival to those with EGFR-mutant acinar predominant tumors. The predominant subtype in the primary tumor was most often seen in the N2 node in micropapillary and solid predominant tumors but not in acinar predominant tumors. The predominant subtype in the primary tumor was associated with overall survival in resected stage III (N2) lung adenocarcinoma and was independent of mutation status. Histologic subtyping provides important prognostic information and potentially molecular correlates.

Erlotinib versus Pemetrexed for Pretreated Non-Squamous Non-Small Cell Lung Cancer Patients in Clinical Practice

Background/Aim: Erlotinib and chemotherapy have shown similar efficacy for pretreated non-small cell lung cancer (NSCLC) patients, but none of the large studies have selected patients based on histology. We present a retrospective single-center series of advanced non-squamous NSCLC patients treated with erlotinib or pemetrexed as second-line therapy. Our aim was to compare the efficacy and safety data under clinical practice conditions and to identify subgroups of patients who could benefit more from these therapies. Methods: A total of 88 patients were included. Squamous histology was our main exclusion criterion. EGFR mutation status was known for 54.5% of the patients; 6 patients treated with erlotinib and 2 with pemetrexed had EGFR-mutated tumors. Smoking history was analyzed as possible predictive factor of efficacy. Results: No significant differences in progression-free survival (PFS; 3 vs. 2.5 months, p = 0.06) or overall survival (OS; 4.9 vs. 7.4 months, p = 0.733) between the erlotinib and pemetrexed groups were found in the overall population. EGFR wild-type patients had a similar median PFS with erlotinib compared to pemetrexed (2.7 vs. 2.3 months, p = 0.42), with no statistical differences in OS. Statistically significant differences in OS in favor of pemetrexed for current smokers (3 vs. 7.1 months, p = 0.017) were found, while erlotinib achieved significantly better PFS in never-smokers compared to former smokers (3.5 vs. 2.7 months, p = 0.005). Serious adverse events were uncommon but more frequent with pemetrexed, and were mainly related to hematologic toxicity. Conclusions: Erlotinib should be considered as another equal option in second-line treatment for EGFR wild-type patients as well as for subpopulations with unknown mutational status. Smoking history could be a useful clinical marker to choose a second-line treatment.