EGFR-mutant Tumors Research Articles

P1.06-005 An International Cohort of Patients with Small Cell Lung Cancer after a Non-Small Cell Lung Carcinoma Oncogene or Non-Oncogene Addicted

Phenotypic transformation from Non-small cell lung cancer (NSCLC) to small cell lung cancer (SCLC) is a resistance mechanism in tyrosine kinase inhibitors (TKIs) treated EGFR mutant tumors. SCLC is also however less frequently diagnosed in patients without EGFR mutations treated with chemotherapy. These transformations are rare and little is known about the clinical and therapeutic characteristics of these patients. In this study we describe and compare the characteristics of SCLC arising from mutant or non-mutant NSCLC. We performed a multicentric retrospective collection (27 centers in France and Italy) of cases. Between 2005 and 2015, patients with stage III or IV NSCLC with a secondary transformation to SCLC with histological proof were included. Forty seven cases of SCLC transformation were collected, 34 in EGFR mutant and 13 in non. Most of the patients (n=37, 82%) were stage IV, (n=27, 57%) female and (n=26, 76%) had an exon 19 mutation. The last treatment before transformation was a TKI in 23 (68%) cases in the mutant group and in 3 (23%) patients (erlotinib) in the non-mutant. Median time to SCLC transformation was 17 [IQR, 11-29] months in the mutant group and 26 [IQR 23-36] months in the other (p=0.03). Molecular analyses were not performed in the non-mutant group, 25 (74%) had molecular analyses in the EGFR mutant. A driver mutation was identified in 22/25 (88%) patients: in most of the cases the same as the initial, 1 case of ALK fusion and 1 of PI3K mutation. Thirty patients (88%) received at least one line of treatment after transformation in the mutant group, in all cases a platinum-etoposide (P-E) chemotherapy. Median survival from initial diagnosis in the EGFR mutant group was significantly worse 28 [17-41] months vs 49 [36-118] months in the non EGFR mutant group (p=0.01). After transformation, the same tendency was observed with a median survival of 8 [3-12] months for the EGFR mutated patients vs 13 [6-15] months for the non EGFR mutated patients (p=0.06). SCLC that occurs in EGFR mutant treated by TKIs is more aggressive than classic SCLC, and differs on epidemiological characteristics. These transformed SCLC are not fully explained and we need to define the molecular characteristics of this cohort, before and after transformation and if funded the whole genome sequencing of the tumors to understand this TKIs mechanism of resistant.

P1.02-059 Evaluation of Plasma DNA Extraction, Droplet PCR and Droplet next Generation Sequencing Methods for Liquid Biopsy Analysis

The ability to detect tumor mutations from blood and other bodily fluids promises many sample access, convenience, and monitoring benefits. However, the extremely rare levels at which mutations are present in these fluids obliges the use of optimal extraction and detection methods. Here, the performance of two extraction, two droplet PCR (dPCR) and a droplet next generation sequencing (dNGS) method for blood plasma analysis was systematically evaluated. Limits of detection were assessed using 15 blinded healthy donor blood samples spiked with equivolume mixtures of H1975 (containing EGFR L858R and T790M mutations) and H1650 (EGFR exon 19 deletion, e19del) cells at 10%, 1%, 0.1%, 0.01%, 0.001% H1650 cells in triplicate. A series of 32 blinded blood plasma samples from non-small cell lung cancer (NSCLC) patients with known tumor EGFR mutation status was also tested. Samples were processed for plasma, and 1ml plasma each underwent Qiagen Circulating Nucleic Acid and Promega Maxwell Circulation Cell Free DNA extraction processing. The plasma DNA samples were analyzed using the Biorad dPCR and Raindance Raindrop dPCR method for L858R and exon 19 deletion mutations, and the 50-gene Raindance Thunderbolts dNGS protocol. No significant difference in DNA yield and detection patterns was observed between the two extraction methods. L858R mutations were detected by both dPCR methods at 0.001% in 1/3 replicates and 0.01% in 3/3 replicates. Of 4 cases with L858R tumor mutations, mutations were detected in the same 3 plasma samples by both Biorad and Raindance dPCR (sensitivity 75%). “False positive” L858R mutations were identified in 2 (specificity 92%) and 7 (75%) cases respectively. For 8 tumor e19del mutations, the sensitivities were 38% and 25%, and specificities were 96% and 75% respectively. Of 16 clinical samples analyzed by dNGS, an average of 20 mutations per sample were identified after filtering for quality, non-synonymous, and non-germline variant status. The sensitivity and specificity for detecting 2 L858R tumor mutation was 100% and 50%, and 1 e19del tumor mutation was 100% and 55% respectively. The allele frequencies for the majority of “false positives” for dPCR and dNGS were less than 5%, although some “true positives” were also detected at that level. dPCR and dNGS methods can enable detection of tumor mutations in blood, albeit imperfectly. Future work to determine optimal detection thresholds will help to maximize sensitivity and specificity.

PUB121 Assessment of Prognostic Scores in Brain Metastases from Lung Adnocarcinoma with Status of EGFR Mutations

Our purpose was to analyze the prognostic factor and evaluate the value of four prognostic scores including RPA, DS-GPA BS-BM, GGS for the EGFR mutant BM patients from lung adenocarcinoma treated with EGFR-TKI. The data of NSCLC were retrospectively reviewed from August 2010 to June 2015 in medical database of Shanxi Provincial Cancer Hospital. Patients with BM from lung adenocarcinoma identified with EGFR mutations received treatment of EGFR-TKI or combination of EGFR-TKI plus WBRT were included. Potential prognostic factors were examined in uni=variable and multivariable analyses. To compare the predictive values of each scoring system, the C-index of each prognostic score were calculated. A total of 1063 BM patients with lung adenocarcinoma identified with status of EGFR mutations between August 2010 and June 2015 from lung cancer medical database was reviewed. Among them, 104 patients who diagnosed with BM confirmed with status of EGFR mutations in primary tumors. They received treatment of EGFR-TKI or EGFR –TKI plus WBRT to BM. The median survival was 15.83 months (95%CI 14.30-17.36). The 1 year survival rate was 60.3 %. The potential predictive factors in multivariable analysis included KPS (70 vs.70-80 vs.90-100), and number of brain metastatic lesions. In log-rank test, the indexes of RPA, DS-GPA BS-BM, GGS were all significant predictors of OS(p<0.001). The C-index value of each prognostic scores were 0.79,0.76,0.77,0.74 in DS-GPA,RPA,GGS,BS-BM respectively. The indexes of RPA, DS-GPA BS-BM, GGS were applicable for assessing survival stratification in brain metastases from lung adnocarcinoma with status of EGFR mutations in our independent population. The DS-GPA seems the best predictive value. However, all of the four indexes could not cover the exact independent prognostic factors in multivariable analysis. A new prognostic index specific for this group of patients was needed in the era of lung cancer target therapy.

P3.02c-087 The Relationship of TILs and PD-L1 Expression in NSCLC Adenocarcinoma in Little to Non-Smokers with Driver Mutations and Outcome Parameters: Topic: IT Biomarkers

Culminating evidence shows the importance of the immune response in NSCLC and other cancer types. TILs seem to be a marker of good prognosis in many different tumor types, including NSCLC. The prognostic importance of PD-L1 expression in NSCLC remains less clear. This study will contribute more information to this topic in NSCLC and will verify the influence of driver mutations on TILs levels and PD-L1 expression. In addition, the predictive role of TILs and PD-L1 expression in EGFR mutants, who received erlotinib, will be evaluated. Clinical data, genetic analysis and tumor biopsies of the FIELT-1 cohort (stage IIIb or IV NSCLC patients with little or non-smoking history) were retrospectively evaluated. PD-L1 expression was evaluated with a PD-1/PD-L1 IHC double staining. TILs were evaluated on H&E slides, using the method developed by an international working group under direction of R. Salgado. Measuring stromal TILs on H&E slides proved to be reproducible (ICC=0.74). The measurement of intratumor TILs (ICC=0.16) did not reach the cut-off ICC of 0.70. There was no difference in stromal TILs counts in KRAS (p=0.454) and EGFR (p=0.962) mutant tumors compared to their respective wild type tumors, nor was there any difference in sTILs counts between KRAS and EGFR mutants (p=0.605). The median OS in the general population was 49 weeks. There was a significant difference in median OS between the stromal TILs high tumors and the stromal TILs low tumors (68 weeks vs. 35 weeks respectively; p=0.003). A similar observation was made in the KRAS mutant tumors (95 weeks vs. 12 weeks; p=0.003). In the EGFR mutants no significant difference in median OS could be found according to the stromal TILs counts (p=0.65). There was no difference in the stromal TILs counts of EGFR mutants who responded (p=0.160) or showed clinical benefit (p=0.621) after receiving erlotinib, compared to those who did not. The analysis of the PD-1/PD-L1 double staining has been postponed. Results will be available by the end of August 2016. The results of the current study reinforce the prognostic role of TILs in NSCLC. Furthermore, this is the first study to confirm that the used scoring method on H&E slides is reproducible in NSCLC. This study is also the first to report about the relation between driver mutation and TILs, with results suggesting that the immune system plays a more crucial role in KRAS mutants than in EGFR mutants.

P2.03b-008 The Impact of Brain Metastases and Their Treatment on Health Utility Scores in Molecular Subsets of Lung Cancer Patients: Topic: Brain Meta

New therapies, particularly in advanced patients with EGFR-mutated and ALK-rearranged tumors, result in prolonged survival. Brain metastases and/or their treatment, may have a negative impact on health-related quality of life. Technological assessment of the cost-effectiveness of various treatments for brain metastases will benefit from measurements of health-related qualify of life and health utility scores (HUS). This study evaluated the impact of brain metastases on HUS across multiple health states defined on the basis on disease stability, brain-specific therapies, and molecularly-defined subsets of NSCLC. A longitudinal cohort study at Princess Margaret Cancer Centre evaluated 1571 EQ5D-3L-derived HUS in 476 Stage IV lung cancer outpatients, from Dec, 2014 through May, 2016: EGFR+ (n=183), ALK+ (n=38), wild-type (WT) non-squamous (n=171), squamous (n=29), and small cell lung cancer (SCLC) (n=30). Patients were stratified according to presence or absence of brain metastases at the time of assessment; mean HUS (± standard error of the mean, SEM) by presence of brain metastases and various health states and disease subtypes were reported. For patients with repeated measures, only the earliest time point was analyzed. 172 patients had brain metastases, median age 62 (range 32-86) years and 304 patients did not have brain metastases, median age 66 (29-96) years. Overall HUS was related to disease subtype but not presence of brain metastases: EGFR/ALK+ patients with (0.78±0.02) or without brain metastases (0.79±0.01) versus WT/SCC/SCLC with (0.74±0.02) and without brain metastases (0.73±0.01) (p=0.01 by subtype; p>0.10 by presence of brain metastases). However, symptomatic CNS disease (0.69±0.04) had lower HUS (versus asymptomatic disease (0.77±0.02)) (p=0.03). Patients achieving intracranial stability or response to treatment had significantly higher HUS (0.81±0.05) than patients with progressive CNS metastases (0.72±0.02) (p=0.03). Extra-cranial control also correlated with higher HUS (0.81±0.02 versus 0.69±0.03, p<0.0001). When local treatment for brain metastases was delivered within 6 months, HUS was lower (0.71±0.02 versus 0.82±0.02, p=0.0005). CNS disease treated only with systemic therapy or on no active therapy had mean HUS of 0.81±0.03, while patients treated only with stereotactic radiosurgery (SRS) had values of 0.80±0.04; there was a trend for lower HUS with whole brain radiation (WBRT) only (0.72±0.03) or WBRT+SRS (0.74±0.03) (p=0.11). Brain metastasis stability has significant impact on HUS in lung cancer patients. Treatment modalities of brain metastases may also impact HUS. Data collection is ongoing; updated HUS data including longitudinal assessments and multivariable analyses will be presented.

Superior Treatment Response and In-field Tumor Control in Epidermal Growth Factor Receptor-mutant Genotype of Stage III Nonsquamous Non–Small cell Lung Cancer Undergoing Definitive Concurrent Chemoradiotherapy

BackgroundAlthough previous in vitro data have suggested a more radio-sensitive nature of epidermal growth factor receptor (EGFR)-mutant non–small cell lung cancer (NSCLC) cell lines, the clinical behavior according to the EGFR mutational status has not been well-established. In this study, we performed a comparative outcome analysis of EGFR-mutant and wild-type locally advanced NSCLC with chemoradiotherapy (CRT). Patients and MethodsA total of 102 patients with stage III nonsquamous NSCLC undergoing primary CRT were identified. Clinicopathologic characteristics, including the degree of glucose uptake, were evaluated. Failure patterns considering the radiation field and survival outcomes were compared according to the EGFR mutational status. ResultsPre- and post-CRT maximum standardized uptake values were significantly lower in EGFR-mutant tumors (P = .010 and .018, respectively). The overall response rate was higher in the EGFR-mutant group compared with the wild-type (89% vs. 64%, respectively; P = .023). The 3-year overall survival rate was better with the genetic alteration (68.0% vs. 47.4%, P = .046), but the statistical significance did not remain in multivariate analysis (hazard ratio, 0.68; 95% confidence interval, 0.30-1.55). Considering the tumor progression inside or outside the radiation field, the EGFR-mutant group showed longer in-field time to progression (P = .002), even after adjusting for other related baseline variables (hazard ratio, 0.27; 95% confidence interval, 0.11-0.71). ConclusionThe differential metabolic activity, failure patterns, and prognosis suggest the distinct nature of the EGFR-mutant tumors. EGFR mutational status needs to be considered for more precise curative-intent treatment strategies of locally advanced nonsquamous NSCLC.

Synonymous EGFR variant p.Q787Q is neither prognostic nor predictive in patients with lung adenocarcinoma

Patients with non-small cell lung cancer (NSCLC) harboring activating mutations in the Epidermal Growth Factor Receptor (EGFR) benefit from targeted therapies. A synonymous polymorphism (rs1050171, p.Q787Q) was shown to be associated with improved overall survival (OS) in colorectal cancer patients. As data in NSCLC are limited, we retrospectively analyzed associations of p.Q787Q with clinicopathological parameters including clinical response and outcome in patients with lung adenocarcinoma (ADC) who received tyrosine kinase inhibitor (TKI) therapy. Of 642 ADC patients whose tumors were profiled by next generation sequencing, 102 (15.9%) carried EGFR mutations targetable by TKIs (30.4% male patients, median age 65.1 y, 19.6% smokers with 12.8 median pack years). Seventy-nine patients (77.5%) received TKI therapy either as a first- or second-line therapy. Of the 102 EGFR-mutant tumors, 72 (70.6%) exhibited the p.Q787Q polymorphism and another 12 (11.8%) cases with p.Q787Q harbored an additional TKI insensitive mutation (p.T790M). The polymorphism was neither associated with classic clinicopathological parameters nor with overall survival (21.1 months vs. 20.1 months; P-value = 0.91) or clinical response (P-value = 0.122). The patients with p.T790M had worse survival compared to EGFR activating mutation carriers with and without p.Q787Q when analyzed as a separate group (27.5 months, P-value = 0.02). In conclusion, p.Q787Q is neither a suitable prognostic nor predictive biomarker for ADC patients receiving anti-EGFR therapy in first- or second-line of therapy. © 2016 Wiley Periodicals, Inc.

A Histopathological Feature of EGFR-Mutated Lung Adenocarcinomas with Highly Malignant Potential - An Implication of Micropapillary Element.

The purpose of this study was to define histological features determining the malignant potential of EGFR-mutated lung adenocarcinoma (LADC). Surgically resected tumors (EGFR-mutated LADCs with (21) and without (79) lymph node metastasis and EGFR wild-type LADCs with (26) and without (108) lymph node metastasis) and biopsy samples from inoperably advanced tumors (EGFR-mutated LADCs (78) and EGFR wild-type LADCs (99)) were examined. In surgically resected tumors, the EGFR-mutated LADCs with lymph node metastasis had the micropapillary element in a significantly greater proportion than others (Mann-Whitney tests P ≤0.026). The proportion of micropapillary element was higher in the EGFR-mutated LADC at the advanced stage (stage II, III, or IV) than in the tumor at the early stage (stage I) (Mann-Whitney test, P<0.0001). In the biopsy samples from inoperably advanced LADCs (177), EGFR-mutated tumors also had micropapillary element at a higher frequency than EGFR-wild type tumors (53/78 (68%), versus 30/99 (30%), Pearson x2 test, P<0.0001). In stage I EGFR-mutated LADCs (84), the tumors with the micropapillary element (34) exhibited a significantly higher recurrence rate than tumors without micropapillary element (50) (5-year Recurrence-free survival 64.4% versus 93.3%, log-rank test P = 0.028). The micropapillary element may be an exclusive determinant of malignant potential in EGFR-mutated LADC. It is suggested that EGFR-mutated LADC may develop through a distinct histogenesis, in which the micropapillary element is important for promoting progression.

Transcriptomic-metabolomic reprogramming in EGFR-mutant NSCLC early adaptive drug escape linking TGFβ2-bioenergetics-mitochondrial priming.

The impact of EGFR-mutant NSCLC precision therapy is limited by acquired resistance despite initial excellent response. Classic studies of EGFR-mutant clinical resistance to precision therapy were based on tumor rebiopsies late during clinical tumor progression on therapy. Here, we characterized a novel non-mutational early adaptive drug-escape in EGFR-mutant lung tumor cells only days after therapy initiation, that is MET-independent. The drug-escape cell states were analyzed by integrated transcriptomic and metabolomics profiling uncovering a central role for autocrine TGFβ2 in mediating cellular plasticity through profound cellular adaptive Omics reprogramming, with common mechanistic link to prosurvival mitochondrial priming. Cells undergoing early adaptive drug escape are in proliferative-metabolic quiescent, with enhanced EMT-ness and stem cell signaling, exhibiting global bioenergetics suppression including reverse Warburg, and are susceptible to glutamine deprivation and TGFβ2 inhibition. Our study further supports a preemptive therapeutic targeting of bioenergetics and mitochondrial priming to impact early drug-escape emergence using EGFR precision inhibitor combined with broad BH3-mimetic to interrupt BCL-2/BCL-xL together, but not BCL-2 alone.

AB036. Comparison of the effectiveness of gefitinib, erlotinib and afatinib in epidermal growth factor receptor mutated tumors of non-small cell lung cancer

AB036. Comparison of the effectiveness of gefitinib, erlotinib and afatinib in epidermal growth factor receptor mutated tumors of non-small cell lung cancer

Abstract A101: Role of TIM-3/Galectin-9 pathway in lung cancer

Abstract Purpose: T-cell inhibitory receptors and their ligands, and their roles in the tumor immune response have been extensively studied recently. Cytotoxic T-lymphocyte antigen 4 (CTLA-4), programmed cell death 1 (PD-1) or T cell immunoglobulin and mucin domain 3 (TIM-3) have been identified on the CD4 and CD8 T-cell surface, and it has been shown that the pathways of CTLA-4 and its ligand CD80 or CD86 (B7-2), PD-1 and its ligand PD-L1 or PD-L2 (B7-DC), and TIM-3 and its ligand Galectin-9 axes contributed to effector T-cell dysfunction in the tumor immune microenvironment. Those T-cell inhibitory receptors and their ligands also play important roles in the immune response in lung adenocarcinoma. In this study, we investigated inhibitory roles of TIM-3/Galectin-9 pathway in lung adenocarcinoma. Experimental Design: TILs or PBMCs were obtained from lung cancer patients. Expression of inhibitory molecules on those cells was analyzed by flow cytometry using FACS. PD-L1 and Galectin-9 expression in lung cancer was analyzed using tissue microarray by immunohistochemistry (IHC). To investigate soluble Galectin-9 released from the tumor cells, an EGFR-mutated tumor cell line, PC-9 and control EGFR wild type cell line, OU-LC-SK were treated with Afatinib at a concentration of 10 nM for 3 days. Then, the amount of Galectin-9 in culture supernatant was measured by ELISA. To investigate T-cell apoptosis induction by Galectin-9, established XAGE1-specifiic CD8 cloned T-cells were incubated with Galectin-9 protein for 8 hrs. Results: First, we examined expression of PD-1, TIM-3, BTLA and LAG-3 on CD4 and CD8 T-cells obtained from PBMCs and TILs in 11 lung cancer patients. Augmented expression in TILs compared to PBMCs was observed on PD-1 and TIM-3, but not on BTLA or LAG-3 in CD4 and CD8 T-cells. Next, we analyzed expression of the T-cell inhibitory receptor ligands PD-L1 and Galectin-9 in lung cancer by IHC. With adenocarcinoma, the frequencies of high PD-L1 and Galectin-9 expression in the cell membrane or cytoplasm were 49% and 31%, respectively. On the other hand, with squamous cell carcinoma, the frequencies were 32% and 16%, respectively. Furthermore, correlated expression of PD-L1, Galectin-9 and CD3 (T-cell infiltration) was observed at the periphery of the tumor nest. Those findings suggest the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer. To investigate soluble Galectin-9 released from the tumor cells, EGFR-mutated and -wild type tumor cell lines were treated with EGFR-TKI. Galectin-9 was detected in the medium of EGFR-mutated lung cancer cell lines following treatment with Afatinib. Moreover, apoptosis was induced in TIM-3-positive CD8 T-cell clones following interaction with Galectin-9 protein and this was inhibited by the addition of anti-Galectin-9 or an anti-TIM-3 antibody. The findings suggested that a significant amount of Galectin-9 could be released and induced T-cell apoptosis in tumor microenvironment. Conclusions: The results suggested the relevance of the PD-1/PD-L1 and TIM-3/Galectin-9 pathways in the tumor microenvironment in lung cancer and that release of soluble Galectin-9 from tumor cells could negatively regulate T-cell function. Citation Format: Yoshihiro Ohue, Koji Kurose, Yumi Nishio, Midori Isobe, Mikio Oka, Eiichi Nakayama. Role of TIM-3/Galectin-9 pathway in lung cancer [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr A101.

Predictive and prognostic value of preoperative serum tumor markers in resectable adenosqamous lung carcinoma.

BackgroundAdenosquamous carcinoma is a rare and aggressive form of lung cancer. The prognostic and predictive value of preoperative serum tumor markers and frequency of EGFR mutations in adenosquamous lung carcinoma are unclear.MethodsWe retrospectively analyzed data and samples collected from 106 radically resected adenosquamous lung carcinoma patients with pathological stage I-IIIA between 2008 and 2013. Correlations between serum tumor marker levels and EGFR mutations as well as survival parameters were analyzed and prognostic factors were identified.ResultsOf the 106 adenosquamous lung carcinoma patients, 29 (27.4%) harbored EGFR mutations. By univariate analysis, advanced clinical stage (P = 0.009 for disease-free survival [DFS]; P = 0.046 for overall survival [OS]), larger tumor size (P = 0.001 for DFS; P = 0.002 for OS), regional lymph node metastasis (P = 0.024 for DFS; P = 0.030 for OS), higher NSE level (P = 0.002 for DFS; P < 0.001 for OS), and higher TMI (tumor marker index) (P = 0.009 for OS) were significantly correlated with a worse prognosis. By multivariate analysis, NSE (P = 0.014) was confirmed as independent predictor for DFS, while NSE (P = 0.001) and TMI (P = 0.038) were independent prognostic factors for OS.ConclusionAdenosquamous lung carcinoma is an aggressive malignancy with relatively high EGFR mutation frequency. Elevated preoperative NSE level and TMI are adverse predictive and prognostic indicators.

A Randomized Phase 2 Study Comparing the Combination of Ficlatuzumab and Gefitinib with Gefitinib Alone in Asian Patients with Advanced Stage Pulmonary Adenocarcinoma

IntroductionA randomized phase 2 study was designed to compare the combination of ficlatuzumab (AV-299), a humanized hepatocyte growth factor–neutralizing monoclonal antibody, plus gefitinib versus gefitinib monotherapy in a pulmonary adenocarcinoma population clinically enriched for EFGR tyrosine kinase inhibitor–sensitizing mutations. MethodsA total of 188 patients were randomized 1:1 to receive either gefitinib or ficlatuzumab plus gefitinib treatment. Patients who demonstrated disease control in the single-agent gefitinib arm were allowed to cross over to ficlatuzumab plus gefitinib treatment upon disease progression. Molecular analyses included tumor EGFR mutation status and retrospective proteomic testing using VeriStrat, a multivariate test based on mass spectrometry. ResultsThe addition of ficlatuzumab to gefitinib did not provide significant improvement over gefitinib monotherapy for the primary end point of overall response rate or the secondary end points of progression-free survival and overall survival. In the subgroup classified as VeriStrat poor, the addition of ficlatuzumab to gefitinib showed significant improvement in both progression-free survival and overall survival in both the intent-to-treat population and the subgroup with EGFR tyrosine kinase inhibitor–sensitizing mutations. For all patients, the most frequent adverse events were diarrhea, dermatitis acneiform, and paronychia. ConclusionsAlthough the trial showed no significant benefit from the addition of ficlatuzumab to gefitinib in the overall population of Asian patients with advanced-stage pulmonary adenocarcinoma, the biomarker data suggest that patients classified as VeriStrat poor may benefit from ficlatuzumab combination therapy.

Advanced non small cell lung cancer: response to microwave ablation and EGFR Status.

To verify the association between EGFR status and clinical response to microwave ablation (MWA) and survival. NSCLC patients with known EGFR status and treated with MWA in combination with chemotherapy were retrospectively enrolled in the study. A total of 61 patients were recruited. EGFR mutations were found in 28 patients (39.4%), and were more common in women (67.7%) and nonsmokers (74.1%). Complete ablation was achieved in 69.7% of patients with EGFR mutant tumours and in 82.1% of patients with EGFR wild-type tumours (p = 0.216). The median progression-free survival (PFS) and overall survival (OS) were 8.3months and 27.2months in patients with an EGFR mutant tumour. The corresponding values were 5.4months (p = 0.162) and 17.8months (p = 0.209) in patients with an EGFR wild-type tumour. Patients with complete ablation had longer PFS (7.8months vs. 4.2months, p = 0.024) and OS (28.1months vs. 12.6months, p = 0.001) than those with incomplete ablation. Multivariate analyses also showed that response to MWA was an independent prognostic factor for OS, but EGFR status was not, and that neither response to MWA nor EGFR status was a prognostic factor for PFS. The EGFR status was not related to response to MWA, and response to MWA was a predictor of survival. • EGFR mutations were commonly seen in women and in nonsmokers • EGFR status had no correlation with the response to MWA, PFS and OS. • The response to MWA could predict PFS and OS.

Abstract 3192: Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples

Abstract Introduction: Approximately 30% of patients with an adenocarcinoma of the lung have an actionable driver mutation. Further understanding the molecular mechanisms of acquired resistance to targeted therapies provides key information for determining subsequent treatment options. Access to tumor tissue to perform either the initial molecular profile or at the point of acquired resistance, however, is often limited. Circulating tumor DNA (ctDNA) can be used as a minimally invasive method for the detection and quantification of molecular abnormalities. We performed a prospective study to assess molecular alterations in the ctDNA of NSCLC patients in whom the initial molecular profile or profile at acquired resistance was unknown due to lack of tumor tissue biopsy or insufficient cellularity in the biopsy. Methods: Plasma samples were collected from 52 pre-treated advanced NSCLC patients at the Gustave Roussy. DNA was extracted from &amp;lt; 5 ml of plasma and analysed using Inivata's enhanced TAm-SeqTM assay covering regions from 35 cancer-related genes. Sequences were generated using Illumina sequencing. We also analysed plasma taken following treatments prescribed after the original molecular profile detected using plasma ctDNA. Results: From July 2015 to October 2015, 52 patients were included (63% female, 37% never-smoker, 95% diagnosed with an adenocarcinoma subtype, 95% with stage IV disease, and 54% had EGFR mutant tumors of which 68% had mutations in exon 19 and 32% had mutations in exon 21). ctDNA profiling was successfully performed for all patients, and mutations were detected in 38 of 52 patients. The median number of mutations detected in plasma samples was 1. Within the EGFR mutant subpopulation, T790M mutations were identified including 8 acquired cases (with a concomitant C797S mutation in 1 case) and 1 primary T790M mutation. Of these patients, 5 started personalised treatment with AZD9291 based on the results of ctDNA analysis. In the other 18 patients with EGFR mutant tumors, no acquired mutations associated with resistance were detected. Other results encompassed: 2 plasma samples with EGFR mutation exon 18 (G719A, G719C) leading to initiation of afatinib in one case, 1 case with EGFR mutation exon 21 (L861Q), 1 patients with ERBB2 exon 20 insertion, 3 KRAS mutant detected in plasma (G12C, G12S, G12F), 2 STK11 mutant samples, and 1 patient with a MET mutation (exon 14) who subsequently started crizotinib. Conclusions: ctDNA analysis with Inivata's enhanced TAm-Seq™ provides an alternative method of ‘liquid biopsy’ for obtaining molecular profile of mutations present in NSCLC patients in the absence of an invasive tissue biopsy. Liquid biopsy identified cancer mutations in 73% of the study population, and 18% of those patients subsequently received treatment tailored to the plasma ctDNA detected mutations. An update on the analysis of 75 patients will be presented during the conference. Citation Format: Jordi Remon, Jean Charles Soria, David Planchard, Cecile Jovelet, Chloe Pannet, Ludovic Lacroix, Annas Gazzah, Andrew Lawson, Sarah Smalley, Kenth Howarth, David Gale, Emma Green, Vincent Plagnol, Nitzan Rosenfeld, Ken Oulassen, Nathalie Chaput, Benjamin Besse. Liquid biopsies for molecular profiling of mutations in non-small cell lung cancer patients lacking tissue samples. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3192.

Systematic evaluation of pembrolizumab dosing in patients with advanced non-small-cell lung cancer

In the phase I KEYNOTE-001 study, pembrolizumab demonstrated durable antitumor activity in patients with advanced non-small-cell lung cancer (NSCLC). We sought to characterize the relationship between pembrolizumab dose, exposure, and response to define an effective dose for these patients. Patients received pembrolizumab 2 mg/kg every 3 weeks (Q3W) (n = 55), 10 mg/kg Q3W (n = 238), or 10 mg/kg Q2W (n = 156). Response (RECIST v1.1) was assessed every 9 weeks. The relationship between the estimated pembrolizumab area under the concentration-time curve at steady state over 6 weeks (AUCss-6weeks) and the longitudinal change in tumor size (sum of longest diameters) was analyzed by regression and non-linear mixed effects modeling. This model was simultaneously fit to all tumor size data, then used to simulate response rates, normalizing the trial data across dose for prognostic covariates (tumor PD-L1 expression and EGFR mutation status). The exposure-safety relationship was assessed by logistic regression of pembrolizumab AUCss-6weeks versus occurrence of adverse events (AEs) of interest based on their immune etiology. Overall response rates were 15% [95% confidence interval (CI) 7%-28%] at 2 mg/kg Q3W, 25% (18%-33%) at 10 mg/kg Q3W, and 21% (95% CI 14%-30%) at 10 mg/kg Q2W. Regression analyses of percentage change from baseline in tumor size versus AUCss-6weeks indicated a flat relationship (regression slope P > 0.05). Simulations showed the exposure-response relationship to be similarly flat, thus indicating that the lowest evaluated dose of 2 mg/kg Q3W to likely be at or near the efficacy plateau. Exposure-safety analysis showed the AE incidence to be similar among the clinically tested doses. No significant exposure dependency on efficacy or safety was identified for pembrolizumab across doses of 2-10 mg/kg. These results support the use of a 2 mg/kg Q3W dosage in patients with previously treated, advanced NSCLC. NCT01295827.

Unravelling signal escape through maintained EGFR activation in advanced non-small cell lung cancer (NSCLC): new treatment options

The discovery of activating epidermal growth factor receptor (EGFR) mutations has opened up a new era in the development of more effective treatments for patients with non-small cell lung cancer (NSCLC). However, patients with EGFR-activating mutated NSCLC treated with EGFR tyrosine kinase inhibitors (TKIs) ultimately develop acquired resistance (AR). Among known cases of patients with AR, 70% of the mechanisms involved in the development of AR to EGFR TKI have been identified and may be categorised as either secondary EGFR mutations such as the T790M mutation, activation of bypass track signalling pathways such as MET amplification, or histologic transformation. EGFR-mutant NSCLC tumours maintain oncogenic addiction to the EGFR pathway beyond progression with EGFR TKI. Clinical strategies that can be implemented in daily clinical practice to potentially overcome this resistance and prolong the outcome in this subgroup of patients are presented.

IL-8 signaling is involved in resistance of lung carcinoma cells to erlotinib.

A signaling pathway that is frequently deregulated in human carcinomas and has been explored as a therapeutic target involves the activation of the epidermal growth factor receptor (EGFR). Inhibition of EGFR via the small molecule inhibitors erlotinib and gefitinib commonly results in tumor resistance, even in patients with EGFR-mutant tumors that initially show substantial clinical responses. This study was designed to broaden our understanding of the molecular mechanisms of acquired resistance to erlotinib in lung cancer cells bearing wild type or mutated EGFR. We report here that generation of erlotinib-resistant lung cancer cells in vitro resulted in a phenotypic alteration reminiscent of an epithelial-mesenchymal transition (EMT) concomitant with a robust upregulation of the IL-8/IL-8R axis. Our results also demonstrate that upregulation of p38 MAPK signaling is responsible for the enhanced IL-8 secretion in the erlotinib-resistant tumor cells. Blockade of IL-8 signaling effectively reduced mesenchymal features of the resistant cells and also markedly enhanced their susceptibility to erlotinib. These results provide a rationale for the development of new therapeutic approaches involving blockade of IL-8 signaling for the management of acquired resistance to EGFR inhibition in patients with lung cancer.

Transformation to Small Cell Lung Cancer of Pulmonary Adenocarcinoma: Clinicopathologic Analysis of Six Cases.

Background:Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) are considered the first line treatment for a subset of EGFR-mutated non-small cell lung cancer (NSCLC) patients. Although transformation to small cell lung cancer (SCLC) is one of the known mechanisms of resistance to EGFR TKIs, it is not certain whether transformation to SCLC is exclusively found as a mechanism of TKI resistance in EGFR-mutant tumors.Methods:We identified six patients with primary lung adenocarcinoma that showed transformation to SCLC on second biopsy (n = 401) during a 6-year period. Clinicopathologic information was analyzed and EGFR mutation results were compared between initial and second biopsy samples.Results:Six patients showed transformation from adenocarcinoma to SCLC, of which four were pure SCLCs and two were combined adenocarcinoma and SCLCs. Clinically, four cases were EGFR-mutant tumors from non-smoking females who underwent TKI treatment, and the EGFR mutation was retained in the transformed SCLC tumors. The remaining two adenocarcinomas were EGFR wild-type, and one of these patients received EGFR TKI treatment.Conclusions:NSCLC can acquire a neuroendocrine phenotype with or without EGFR TKI treatment.

Epithelial NF-κB signaling promotes EGFR-driven lung carcinogenesis via macrophage recruitment

ABSTRACTSeveral studies have demonstrated that NF-κB activation is common in lung cancer; however, the mechanistic links between NF-κB signaling and tumorigenesis remain to be fully elucidated. We investigated the function of NF-κB signaling in epidermal growth factor receptor (EGFR)-mutant lung tumors using a transgenic mouse model with doxycycline (dox)-inducible expression of oncogenic EGFR in the lung epithelium with or without a dominant inhibitor of NF-κB signaling. NF-κB inhibition resulted in a significant reduction in tumor burden in both EGFR tyrosine kinase inhibitor (TKI)-sensitive and resistant tumors. However, NF-κB inhibition did not alter epithelial cell survival in vitro or in vivo, and no changes were detected in activation of EGFR downstream signaling pathways. Instead, we observed an influx of inflammatory cells (macrophages and neutrophils) in the lungs of mice with oncogenic EGFR expression that was blocked in the setting of NF-κB inhibition. To investigate whether inflammatory cells play a role in promoting EGFR-mutant lung tumors, we depleted macrophages and neutrophils during tumorigenesis and found that neutrophil depletion had no effect on tumor formation, but macrophage depletion caused a significant reduction in tumor burden. Together, these data suggest that epithelial NF-κB signaling supports carcinogenesis in a non-cell autonomous manner in EGFR-mutant tumors through recruitment of pro-tumorigenic macrophages.