EGFR-mutant Tumors Research Articles

Crystal digital droplet PCR for detection and quantification of circulating EGFR sensitizing and resistance mutations in advanced non-small cell lung cancer.

Over the past years, targeted therapies using tyrosine kinase inhibitors (TKI) have led to an increase in progression-free survival and response rate for a subgroup of non-small cell lung cancer (NSCLC) patients harbouring specific gene abnormalities compared with chemotherapy. However long-lasting tumor regression is rarely achieved, due to the development of resistant tumoral subclones, which requires alternative therapeutic approaches. Molecular profile at progressive disease is a challenge for making adaptive treatment decisions. The aim of this study was to monitor EGFR-mutant tumors over time based on the quantity of mutant DNA circulating in plasma (ctDNA), comparing two different methods, Crystal™ Digital™ PCR and Massive Parallel Sequencing (MPS). In plasma circulating cell free DNA (cfDNA) of 61 advanced NSCLC patients we found an overall correlation of 78% between mutated allelic fraction measured by Crystal Digital PCR and MPS. 7 additional samples with sensitizing mutations and 4 additional samples with the resistance mutation were detected with Crystal Digital PCR, but not with MPS. Monitoring levels of both mutation types over time showed a correlation between levels and trends of mutated ctDNA detected and clinical assessment of disease for the 6 patients tested. In conclusion, Crystal Digital PCR exhibited good performance for monitoring mutational status in plasma cfDNA, and also appeared as better suited to the detection of known mutations than MPS in terms of features such as time to results.

Correlation between molecular analysis, diagnosis according to the 2015 WHO classification of unresected lung tumours and TTF1 expression in small biopsies and cytology specimens from 344 non-small cell lung carcinoma patients

We investigated correlations between diagnosis according to the 2015 World Health Organization (WHO) classification of unresected lung tumours, molecular analysis and TTF1 expression in small biopsy and cytology specimens from 344 non-small cell lung carcinoma (NSCLC) patients. One case failed testing for EGFR, KRAS and ALK abnormalities and six had insufficient tumour for ALK testing. Overall mutation rate in 343 cases was 48% for the genes tested, with 19% EGFR, 33% KRAS and 4% BRAF mutations, and 5% ALK rearrangements detected. More EGFR-mutant (78%) and ALK-rearranged (75%) tumours had morphologic adenocarcinoma than KRAS-mutant (56%) tumours. Despite no significant difference in the overall rate of any molecular abnormality between morphologic adenocarcinoma (52%) and NSCLC, favour adenocarcinoma (47%) (p=0.18), KRAS mutations were detected more frequently in the latter group. No significant difference in the overall rate of any molecular abnormality between TTF1 positive (49%) and TTF1 negative tumours (44%) (p=0.92) was detected, but more EGFR-mutant (97%) and ALK-rearranged tumours (92%) were TTF1 positive than KRAS-mutant tumours (68%). Rates of EGFR, KRAS and BRAF mutations and ALK rearrangements in this Australian NSCLC patient population are consistent with the published international literature. Our findings suggest that 2015 WHO classification of unresected tumours may assist in identifying molecular subsets of advanced NSCLC.

Selection of Non-small Cell Lung Cancer Patients for Intercalated Chemotherapy and Tyrosine Kinase Inhibitors.

BackgroundWhen treating patients with advanced non-small cell lung cancer (NSCLC) with tyrosine kinase inhibitors and chemotherapy, intercalated schedule with time separation between the two classes of drugs should avoid their mutual antagonism. In a survey of published trials, we focus on relation between eligibility criteria and effectiveness of intercalated treatment.MethodsPublished documents were identified using major medical databases, conference proceedings and references of published trials. Median progression-free survival (PFS) was taken as the basic parameter of treatment efficacy. Correlation between characteristics of patients and median PFS was assessed through the Pearson’s correlation coefficient and the coefficient of determination, separately for first-line and second-line setting.ResultsThe series includes 11 single-arm trials and 18 randomized phase II or phase III trials with a total of 2903 patients. Treatment-naive patients or those in progression after first-line treatment were included in 16 and 13 trials, respectively. In 14 trials, only patients with non-squamous histology were eligible. Proportion of patients with non-squamous carcinoma (in first-line setting), proportion of never-smokers (both in first- and second-line setting) and proportion of epidermal growth factor receptor (EGFR) mutant patients (both in first- and second-line setting) showed a moderate or strong correlation with median PFS. In six trials of intercalated treatment applied to treatment-naive EGFR-mutant patients, objective response was confirmed in 83.1% of cases and median PFS was 18.6 months.ConclusionsMost suitable candidates for intercalated treatment are treatment-naive patients with EGFR-mutant tumors, as determined from biopsy or liquid biopsy. For these patients, experience with intercalated treatment is most promising and randomized trials with comparison to the best standard treatment are warranted.

Abstract 1838: TGFalpha (an EGFR ligand) promotes growth of EGFR-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model

Abstract Although an exogenous EGF is dispensable for EGFRL858R-mediated NIH3T3 cell transformation in vitro (Greulich et al., PLoS Med 2005), an exogenous EGF promotes growth of H1650 and H1975 lung adenocarcinoma cells that carry EGFR mutations in vitro (Sordella et al., Science 2004). However, it is unknown whether an exogenous EGFR ligand plays a role in growth of EGFR-mutant lung tumors in vivo. Here, using conditional transgenic mice expressing EGFRL858R or KRASG12D along with an exogenous TGFalpha (an EGFR ligand) in lung epithelium, we sought to determine the role of an EGFR ligand in EGFRL858R-lung tumors or wild type EGFR-lung tumors (KRASG12D-lung tumors) in vivo. As previously reported, conditional expression of TGFalpha (an EGFR ligand) in lung epithelium caused pulmonary fibrosis but not lung tumors (Korfhagen et al., J Clin Invest 1994; Hardie et al., AJP-LCMP 2004), indicating that the expression of an exogenous EGFR ligand is not sufficient to induce lung tumors. Conditional expression of EGFRL858R or KRASG12D in lung epithelium caused lung tumors as previously reported (Fisher et al., Genes Dev 2001; Politi et al., Gene Dev 2006). In our study using a new rtTA driver, Scgb1a1-rtTA line 2 (Perl et al., AJRCMB 2006), the majority of EGFRL858R-lung tumors occurred in alveolar regions while KRASG12D-lung tumors occurred in both alveolar and airway regions. Importantly, co-expression of EGFRL858R along with TGFalpha in lung epithelium induced the growth of lung tumors in airway regions but not in alveolar regions while the expression of EGFRL858R alone did not induce lung tumors in airway regions. Co-expression of EGFRL858R along with TGFalpha also shortened survival of the mice compared to the expression of either EGFRL858R or TGFalpha alone (median 32 days for EGFRL858R/TGFalpha vs 118 days for EGFRL858R alone or 248 days for TGFalpha alone). Co-expression of KRASG12D with TGFalpha did not influence tumor location or survival of the mice compared to the expression of either KRASG12D or TGFalpha alone. Consistent with the mouse study, TGFalpha expression in human EGFR-mutant lung cancer but not wild-type EGFR lung cancer was associated with worse overall survival (median 44 months for TGFalpha high vs 75 months for TGFalpha low; p<0.05) and recurrence free survival (median 21 months for TGFalpha high vs 61 months for TGFalpha low; p<0.05). Collectively, our results demonstrate that TGFalpha (an EGFR ligand) promotes not only the growth of EGFRL858R-lung tumors but also influences the regions where the EGFRL858R-lung tumors form. Citation Format: Koichi Tomoshige, Yutaka Maeda. TGFalpha (an EGFR ligand) promotes growth of EGFR-mutant lung tumors in airway regions but not in alveolar regions in a transgenic mouse model [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1838. doi:10.1158/1538-7445.AM2017-1838

Abstract 2078: CK-101 (RX518), a mutant-selective inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC

Abstract Patients with non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations initially respond to small molecule inhibitors of the EGFR tyrosine kinase such as gefitinib and erlotinib. However, secondary mutations in the kinase domain lead to acquired resistance to these inhibitors and limited clinical efficacy, with the EGFR T790M mutation being the most common mechanism of acquired resistance in more than half the patients that experience disease progression. CK-101 (also known as RX518) is a novel, irreversible, orally administered EGFR kinase inhibitor that specifically targets the mutant forms of EGFR, including T790M, while exhibiting minimal activity toward the wild-type (WT) EGFR. WT EGFR inhibition is believed to drive the commonly observed side effects of skin rash and diarrhea. The 50% inhibitory concentrations (IC50s) of CK-101 and 2 reference compounds, afatinib and AZD-9291, were determined in cell proliferation assays in human cancer cell lines in vitro after incubation for 72 hours. Xenograft studies were conducted in BALB/c nude mice or SCID/Beige mice using once daily oral administration of CK-101 or afatinib for 14 or 21 days. CK-101 selectively inhibited cell proliferation of cell lines expressing both the activating (e.g. HCC827, IC50 <0.015 µM) and resistance mutations (NCI-H1975, IC50 <0.005 µM), but was much less potent at inhibiting proliferation of cell lines expressing the WT form of the receptor (e.g. A431, IC50 >0.5 µM; i.e., CK-101 was over 100 fold less potent against A431 than against NCI-H1975). Single agent CK-101 significantly inhibited tumor growth in EGFR-mutated NSCLC tumor xenograft models, with no activity in a WT EGFR tumor xenograft model. In a xenograft study of PC-9 cells (exon 19 deletion) in SCID/Beige mice, CK-101 inhibited tumor growth by up to 90% (p <0.001). In a xenograft study of NCI-H1975 cells (L858R/T790M double mutant) in BALB/c nude mice, CK-101 inhibited tumor growth by up to 95% (p <0.001). The pre-clinical findings from this work strongly supported the clinical development of CK-101, and a first-in-human study of CK-101 was initiated in September 2016. Citation Format: Xiangping Qian, Yong-Liang Zhu, Edward Cullen, Robert M. Niecestro, James F. Oliviero. CK-101 (RX518), a mutant-selective inhibitor of EGFR that overcomes T790M-mediated resistance in NSCLC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2078. doi:10.1158/1538-7445.AM2017-2078

The impact of de novo liver metastasis on clinical outcome in patients with advanced non-small-cell lung cancer.

Liver metastasis has been found to affect outcome in prostate cancer and colorectal cancer, but its role in lung cancer is unclear. The current study aimed to evaluate the impact of de novo liver metastasis (DLM) on stage IV non-small cell lung cancer (NSCLC) outcomes and to examine whether tyrosine kinase inhibitors (TKI) reverse poor prognosis in patients with DLM and epidermal growth factor receptor (EGFR)-mutant NSCLC. Among 1392 newly diagnosed NSCLC patients, 490 patients with stage IV disease treated between November 2010 and March 2014 at Kaohsiung Chang Gung Memorial Hospital were included. Patients were divided into two groups according to DLM status. There were 75 patients in the DLM group and 415 patients in the non-DLM group. The DLM group included more patients with bone metastasis, fewer patients with a lymphocyte-to-monocyte ratio (LMR) > 3.1, and fewer patients with pleural metastasis. In the DLM group, Eastern Cooperative Oncology Group performance status 3–4 and LMR ≦3.1 were associated with poor outcome. In patients without DLM, overall survival (OS) was longer in patients with EGFR-mutant NSCLC than in those without (20.2 vs. 7.3 months, p < 0.001). Among DLM patients, OS was similar between the EGFR-mutant and wild-type EGFR tumor subgroups (11.9 vs. 7.7 months, p = 0.155). We found that DLM was a significant poor prognostic factor in the EGFR-mutant patients treated with EGFR-TKIs, whereas DLM did not affect the prognosis of EGFR-wild-type patients.

SFK/FAK Signaling Attenuates Osimertinib Efficacy in Both Drug-Sensitive and Drug-Resistant Models of EGFR-Mutant Lung Cancer.

Mutant-selective EGFR tyrosine kinase inhibitors (TKI), such as osimertinib, are active agents for the treatment of EGFR-mutant lung cancer. Specifically, these agents can overcome the effects of the T790M mutation, which mediates resistance to first- and second-generation EGFR TKI, and recent clinical trials have documented their efficacy in patients with EGFR-mutant lung cancer. Despite promising results, therapeutic efficacy is limited by the development of acquired resistance. Here we report that Src family kinases (SFK) and focal adhesion kinase (FAK) sustain AKT and MAPK pathway signaling under continuous EGFR inhibition in osimertinib-sensitive cells. Inhibiting either the MAPK pathway or the AKT pathway enhanced the effects of osimertinib. Combined SFK/FAK inhibition exhibited the most potent effects on growth inhibition, induction of apoptosis, and delay of acquired resistance. SFK family member YES1 was amplified in osimertinib-resistant EGFR-mutant tumor cells, the effects of which were overcome by combined treatment with osimertinib and SFK inhibitors. In conclusion, our data suggest that the concomitant inhibition of both SFK/FAK and EGFR may be a promising therapeutic strategy for EGFR-mutant lung cancer. Cancer Res; 77(11); 2990-3000. ©2017 AACR.

The first- or second-line use of pemetrexed sequentially with EGFR-TKI did not differ in PFS for advanced NSCLC patients harboring EGFR-mutant tumors.

e20531 Background: Both EGFR-TKI and pemetrexed doublet chemotherapy (P) are two effective therapies in advanced NSCLC patients with EGFR mutant tumors. But which one should be used in first-line remains controversial. Methods: This was a retrospective study where patients prescribed with EGFR-TKI (gefitinib or icotinib) from Jan 2013 to Jan 2016 were screened. Patients must have metastastic diseases harboring TKI-sensitizing EGFR mutation. They must be older than 18 years, and have evaluable target lesions. Whether TKI or P was used in the first line, it must be switched to the other in the second line. PFS in both first line (PFS1) and second line (PFS2) was collected. Results: We screened totally 550 patients (gefitinib n = 455, icotinib n = 95) to enroll a cohort of 36 patients. They were all adenocarcinoma except 1 adenosquamous carcinoma. Gender (M or F), PS (0 or 1), mutation type (Exon19Ddel or other) were equally distributed. The median age was 50.5 years. For the whole cohort, the total PFS (PFS1+PFS2) was 18.1 m (95%CI: 15.2-21.1 m). For those (n = 24) receiving first-line TKI, PFS1, PFS2, and total PFS were 10.3 m, 6.6 m, and 19.2 m. And for those receiving first-line P (n = 12), they were were 3.4 m, 11.5 m, and 16.5m. The total PFS in both groups did not differ significantly (p = 0.548). Conclusions: Our results argued the sequential usage of TKI and P achieve a comparable PFS irrespective of the sequence.

Increased CD73 and reduced IFNG signature expression in relation to response rates to anti-PD-1(L1) therapies in EGFR-mutant NSCLC.

11505 Background: Anti-PD-1(L1) therapies appear to be less efficacious in NSCLC patients whose tumors have EGFR activating mutations, but the underlying mechanism is poorly understood. We investigated the relationship between Methods: Flow cytometry and/or quantitative PCR were used to evaluate genes and proteins in five NSCLC EGFR mt cell lines and 6 wt lines. Anti-EGFR TKIs gefitinib and osimertinib were used at concentrations ranging from 0.001-100uM; EGF was used at 50 ng/mL. CP1108/NCT01693562 was a nonrandomized phase 1/2 trial evaluating durvalumab (10 mg/kg Q2W) in advanced NSCLC. As of 24OCT16, 304 previously treated patients in CP1108 were enrolled. RNA sequencing was conducted on available tumor specimens from 97 patients in CP1108. CP1108 and TCGA were separated by EGFR status for genomic comparisons. Results: Median CD73 expression was increased 10-fold in EGFR mt NSCLC cell lines (n = 5) compared to wt cell lines (n = 6). EGF induced CD73 protein levels 5-40-fold in 3/6 EGFR wt lines. There was dose-dependent inhibition of CD73 expression (45-70 fold maximum) following treatment with gefitinib or osimertinib in 3/5 mt cell lines and 4/6 wt lines, suggesting a causal relationship between the EGFR pathway and CD73 expression. Consistent with these observations, EGFR mutant tumors had ≥2 fold increased expression of CD73 compared to wt (p &lt; 0.05) in TCGA and CP1108 NSCLC adenocarcinoma patients. These EGFR mutants had significantly lower levels of IFNg signature, previously reported to be associated with enhanced benefit from durvalumab. Conclusions: Our findings identify a novel relationship in NSCLC between EGFR pathway activation, expression of the immunosuppressive molecule CD73 and reduced expression of IFNg mRNA signature. These results prompt the hypothesis that over-expression of CD73 in EGFR-mt NSCLC may explain, at least in part, the reduced benefit from anti-PD-1(L1) in this subset of NSCLC, and suggest evaluating anti-CD73 in combination with EGFR TKIs or anti-PD-L1 in EGFR-mt NSCLC.

Phase I study of ABBV-399, a c-Met antibody-drug conjugate (ADC), as monotherapy and in combination with erlotinib in patients (pts) with non-small cell lung cancer (NSCLC).

2509 Background: The c-Met receptor is overexpressed in ~50% of pts with NSCLC. ABBV-399 is a first-in-class ADC composed of ABT-700, an anti–c-Met antibody, conjugated to monomethyl auristatin E (a microtubule inhibitor). Preclinical data demonstrate that ABBV-399 can deliver a potent cytotoxin directly to c-Met+ tumor cells. Methods: ABBV-399 was administered at doses ranging from 2.4 to 3.0 mg/kg (dose expansion and combination cohorts at 2.7 mg/kg) once every 21 days to 29 pts with advanced c-Met+ (immunohistochemistry [IHC] H-score ≥150) NSCLC both as monotherapy (ABBV-399/monoT; 16 pts) and in combination with oral erlotinib 150 mg daily (ABBV-399/ERL; 13 pts) (NCT02099058). c-Met overexpression was assessed by IHC utilizing the SP44 antibody (Ventana; Tucson, AZ, USA). Results: As of January 9, 2017, 16 pts with c-Met+ NSCLC received ≥1 dose of ABBV-399/monoT. Monotherapy treatment-related adverse events (TRAEs) occurring in ≥10% of pts (all dose levels and all grades) were fatigue (43.8%), nausea (37.5%), neuropathy (25.0%), vomiting (18.8%), and anemia, constipation, and diarrhea (12.5% each). Three of 16 (19%) ABBV-399–treated c-Met+ NSCLC pts had a confirmed partial response (PR) with duration of response (DOR) 3, 4.5, and 10+ months. At week 12, 6 of 16 pts (37.5%) had disease control. TRAEs in ABBV-399/ERL occurring in ≥10% of pts (all grades) were neuropathy (30.8%), and acneiform rash, diarrhea, fatigue, nausea, and dry skin (15.4% each). Four of 13 (31%) evaluable ABBV-399/ERL–treated c-Met+ pts had a PR (3 confirmed, 1 unconfirmed) with DOR 1+, 2.7, 5.3+, and 11+ months. Three of the 4 pts with PR had EGFR-mutated tumor and recently progressed on TKI. At week 12, 8 of 13 pts (61.5%) had disease control. There were no treatment-related deaths as monotherapy or in combination with erlotinib. Responses were seen in both squamous and non-squamous histology. Conclusions: ABBV-399 is well tolerated at 2.7 mg/kg once every 21 days and has demonstrated antitumor activity in pts with c-Met+ NSCLC both as monotherapy and in combination with erlotinib. Updated efficacy/safety data and MET gene status will be presented. Clinical trial information: NCT02099058.

Clinical mutational profiling of 1006 lung cancers by next generation sequencing.

Analysis of lung adenocarcinomas for actionable mutations has become standard of care. Here, we report our experience using next generation sequencing (NGS) to examine AKT1, BRAF, EGFR, ERBB2, KRAS, NRAS, and PIK3CA genes in 1006 non-small cell lung cancers in a clinical diagnostic setting. NGS demonstrated high sensitivity. Among 760 mutations detected, the variant allele frequency (VAF) was 2–5% in 33 (4.3%) mutations and 2–10% in 101 (13%) mutations. A single bioinformatics pipeline using Torrent Variant Caller, however, missed a variety of EGFR mutations. Mutations were detected in KRAS (36% of tumors), EGFR (19%) including 8 (0.8%) within the extracellular domain (4 at codons 108 and 4 at codon 289), BRAF (6.3%), and PIK3CA (3.7%). With a broader reportable range, exon 19 deletion and p.L858R accounted for only 36% and 26% of EGFR mutations and p.V600E accounted for only 24% of BRAF mutations. NGS provided accurate sequencing of complex mutations seen in 19% of EGFR exon 19 deletion mutations. Doublet (compound) EGFR mutations were observed in 29 (16%) of 187 EGFR-mutated tumors, including 69% with two non-p.L858R missense mutations and 24% with p.L858 and non-p.L858R missense mutations. Concordant VAFs suggests doublet EGFR mutations were present in a dominant clone and cooperated in oncogenesis. Mutants with predicted impaired kinase, observed in 25% of BRAF-mutated tumors, were associated with a higher incidence of concomitant activating KRAS mutations. NGS demonstrates high analytic sensitivity, broad reportable range, quantitative VAF measurement, single molecule sequencing to resolve complex deletion mutations, and simultaneous detection of concomitant mutations.

Distinct prognostic roles and heterogeneity of TTF1 copy number and TTF1 protein expression in non-small cell lung cancer.

Thyroid transcription factor 1 (TTF1) located on chromosome band 14q13.3 is an oncogene and a suppressor gene in non-small cell lung cancer (NSCLC). The prognostic relevance of TTF1 copy number alterations (CNAs) and their association with TTF1 protein expression are poorly understood. Here, we assessed TTF1 CNAs and protein expression using microarrays in a cohort of 636 NSCLC, including 423 adenocarcinoma (ADC) and 171 squamous cell carcinoma (SCC). In addition, fluorescent in situ hybridization and immunohistochemistry were performed. TTF1 CNAs were detected in 23% of NSCLC (23% of ADC and 20% of SCC). Specifically, TTF1 amplification and polysomy were observed in 5% and 18% of NSCLC, and in 7% and 16% of ADC, respectively. TTF1 expression was detected in 85% of ADC. TTF1 CNAs were significantly associated with advanced tumor stage, EGFR mutations, and TTF1 expression. A multivariate Cox hazards model analysis of overall survival and recurrence-free survival demonstrated that both TTF1 amplification and polysomy were independent indicators of an unfavorable prognosis in patients with NSCLC. Survival was inversely correlated with TTF1 copy number. In contrast, TTF1 protein expression was an independent favorable prognostic factor. Intratumoral and intertumoral heterogeneities of TTF1 CNAs and TTF1 protein expression were assessed using primary cores from 138 pairs of primary tumors and corresponding nodal metastases. The concordance rate for TTF1 CNAs and TTF1 protein expression was high within tumors and between primary and metastatic tumors. Altogether, these results suggest that TTF1 CNAs are correlated with TTF1 protein expression, but have opposing effects on survival.

109P - Sequential administration of EGFR-TKI and pemetrexed achieved a long period of response in advanced NSCLC patients with EGFR-mutant tumors

109P - Sequential administration of EGFR-TKI and pemetrexed achieved a long period of response in advanced NSCLC patients with EGFR-mutant tumors

MA05.10 Cell-Free DNA Testing for EGFR Mutations in Clinical Practice - Facts and Figures from an Austrian Lung Cancer Center

Detection of EGFR mutations using cell-free DNA in plasma has recently emerged as a novel diagnostic approach to lung carcinoma. This “liquid biopsy” (LB) may also be useful for monitoring disease activity in EGFR-mutated tumors as well as in the management of EGFR-tyrosine kinase inhibitor (TKI) therapy. We present data collected in one year of use and report on applicability and diagnostic value in daily clinical practice. EGFR Mutations were analyzed using the semi-quantitative Roche cobas® EGFR Mutation Test v2, comprising 42 mutations. Cell free DNA was extracted from EDTA-Blood with the Qiagen QIAsymphony circulating DNA Kit. LB was initially used for follow-up of known EGFR-mutated tumors and/or in patients under TKI-therapy. Subsequently, we introduced routine LB testing in the primary diagnostic workup of lung cancer patients. From July 2015 to June 2016 we performed a total of 92 liquid biopsies in 77 patients (60% male, mean age 65y) in whom EGFR mutations could be detected in 10 cases (13%). EGFR status from histological samples was available in 40 patients, in 14 (18%) of them mutations were reported. Compared to histological EGFR status, LB reached a sensitivity and specificity of 0.57 and 0.96, respectively. A total of 9 patients had multiple LB testing during follow-up. Three of them initially had detectable mutations by LB, which turned undetectable upon tumor-specific treatment. Two patients remained EGFR-positive during follow-up despite of therapy, whereas four patients remained negative throughout follow-up and therapy. Resistance mutations under TKI-therapy were not observed. Primary LB (before initiation of any tumor-specific therapy) was obtained from 47 patients (72% male, mean age 66y). EGFR mutations by LB were detected in 2 patients (4%; 1 Ins. Exon-20, 1 L858R), while histology revealed EGFR mutations in 2 out of 22 patients (9%; both L858R). Comparison yielded a sensitivity of 0.5 and a specificity of 0.95 for LB. Testing for EGFR mutations using cell-free DNA has been established as a new powerful tool in the field of pulmonary oncology. Apart from sole detection of EGFR mutations, especially the application of LB in following patients over time will provide valuable new opportunities in clinical routine and decision making.

P1.02-073 Characterizing the Genomes of Lung Adenocarcinomas from Never Smokers Reveals SHPRH as a Novel Candidate Tumor Suppressor Gene

Approximately 15 to 25% of lung adenocarcinomas (LAC) arise in never smokers. They develop through mechanisms distinct from those that affect smokers and are associated with unique histological and molecular characteristics. A significant fraction of LACs in never smokers do not have mutations in known oncogenic driver genes such as EGFR/ALK/KRAS. Furthermore, mutations in oncogenic driver genes appear to be insufficient for tumorigenesis, suggesting that additional alterations are required. To address these issues, we used whole-exome sequencing to comprehensively study 15 LACs from never smokers - seven “triple negative” tumors (with normal EGFR/ALK/KRAS) and eight EGFR-mutant tumors - with the goal of identifying novel mutant genes in these subsets. To identify mutated genes that confer a selective advantage a multistep approach was used to filter variants based on gene expression level, background mutation rate and gene size. Targeted sequencing of 180 genes in the original 15 and an extended panel of 85 tumor/normal pairs validated these alterations and indicated their prevalence in LAC. Sequence data was integrated with copy number and gene expression levels to determine mechanisms, and consequences, of gene disruption. Animal and cell models were used to functionally validate identified genes of interest and explore their role in LAC biology. 32 unique genes demonstrated significant evidence of conferring a selective advantage including known oncogenes (EGFR/ERBB2/MET) and tumor suppressor genes (p53/RB1/ATM). In addition, RNA-seq revealed fusions involving RET or ROS1 in one tumor each. The variations in MET consisted of truncating and splice-site mutations that we are currently investigating in transgenic mouse models. Pathway analysis indicated frequent mutation in genes implicated in PI3-kinase signaling, RNA splicing and histone modification. Importantly, we identified the hemizygous and homozygous loss of multiple genes from chromosome arm 6q - a genetic locus associated with familial lung cancer susceptibility - including a novel candidate tumor suppressor gene, SHPRH, based on its high frequency of biallelic disruption. SHPRH is an evolutionarily conserved E3-ligase that mediates crucial processes related to DNA repair. We found that SHPRH silencing increased transformation of normal lung cells, increased DNA damage and induced cell cycle changes while SHPRH inhibition sensitized LAC cells to topoisomerase II and PARP inhibitors. SHPRH inactivation may induce genetic alterations that cooperate with mutations in driver oncogenes to promote LAC development. Together, this work will expand our understanding of LAC initiation and progression in never smokers and may offer new biomarkers for response to therapy.

P1.02-020 The Effect of EGF-Pathway Targeted Immunization (EGF PTI) on STAT3 and Cancer Stem Cells in EGFR Mutant NSCLC Cells

The vast majority of advanced non-small cell lung cancer (NSCLC) patients with EGFR mutant tumors will develop disease progression following successful treatment with an EGFR tyrosine kinase inhibitor (TKI). Resistance to EGFR-TKIs is due to various mechanisms, such as the secondary mutation (T790M) or the activation of alternative pathways (MET, AXL). What has not been fully appreciated is that EGFR blockade induces an imbalance in favor of survival, increases activity of STAT3 and enriches lung CSCs through Notch3-dependent signaling. EGF-PTI was designed to elicit an antibody response against EGF, in order to reduce EGF receptor signaling and limit tumor growth. We have explored whether EGF-PTI alone or in combination with EGFR TKIs may efficiently inhibit STAT3 and target CSCs. EGF PTI was provided by Bioven (Europe) Ltd. Gefitinib, erlotinib and the recently FDA-approved third-generation EGFR TKI, AZD9291 (osimertinib) were purchased from Selleck chemicals. Western blotting was used to assess the effect of the drugs on ERK, AKT and STAT3 phosphorylation and on Notch and PARP cleavage in EGFR (del19) mutant NSCLC PC9 cells and gefitinib-resistant PC9-GR4 cells. PC9-GR4 cells have been established in our lab and harbor the resistant T790M mutation (T790M+). The protein expression of AXL and CSCs markers such as HES1 (downstream effector of Notch) and Bmi1 was also examined. Gefitinib, erlotinib or AZD9291 suppressed EGFR, ERK1/2 and AKT phosphorylation in PC9 cells but increased STAT3 phosphorylation on the tyrosine residue 705 in both PC9 and PC9-GR4 cells. EGF-PTI suppressed STAT3, EGFR and ERK1/2 and the combination of each of the three EGFR TKIs with EGF-PTI lead to more potent inhibition of STAT3, EGFR and ERK1/2. The EGF-PTI induced AKT phosphorylation was reversed when EGF PTI was combined with EGFR TKIs. Interestingly, EGF-PTI blocked Notch cleavage and decreased the expression of HES1. The expression of Bmi1 and AXL were also attenuated with EGF PTI and apoptosis was enhanced through the induction of PARP cleavage. EGF PTI may reverse mechanisms of resistance to single EGFR inhibition and the combination of EGF PTI with EGFR TKIs efficiently inhibits downstream signaling pathways in T790M+ cells. Based on these results, the design of a proof-of-concept trial with the combination of EGF PTI with gefitinib for the first line treatment of EGFR mutant NSCLC patients is in progress.

MA09.09 First-In-Human Phase 1 Study of ABBV-399, an Antibody-Drug Conjugate (ADC) Targeting C-Met, in Patients with Non-Small Cell Lung Cancer (NSCLC)

The c-Met receptor is overexpressed in ∼50% of patients with NSCLC. ABBV-399 is a first-in-class ADC composed of ABT-700, an anti–c-Met antibody, conjugated to monomethyl auristatin E (a microtubule inhibitor). Preclinical data support ABBV-399 as a unique strategy to deliver a potent cytotoxin directly to c-Met+ tumor cells. In a 3+3 dose-escalation design, ABBV-399 was administered at doses ranging from 0.15 to 3.3 mg/kg once every 21 days to patients with advanced metastatic solid tumors (NCT02099058). ABBV-399 was then studied in a dose-expansion cohort in 16 patients with advanced c-Met+ (immunohistochemistry [IHC] H-score ≥150) NSCLC that had progressed on ≥2 prior lines of therapy. ABBV-399 was also studied in combination with erlotinib in 10 patients with NSCLC, 8 of whom were c-Met+. Overexpression of c-Met was assessed by an IHC assay utilizing the SP44 antibody (Ventana; Tucson, AZ, USA). As of June 27, 2016, 48 patients with solid tumors received ≥1 dose of ABBV-399. The dose-limiting toxicity (DLT) for ABBV-399 was febrile neutropenia, which occurred in 2 patients (1 each at 3 and 3.3 mg/kg). There were no treatment-related deaths. Monotherapy treatment-related adverse events (AEs) occurring in ≥10% of patients (including all dose levels and all grades) were fatigue (25.0%), nausea (22.9%), neuropathy (14.6%), decreased appetite (12.5%), vomiting (12.5%), and hypoalbuminemia (10.4%). Based primarily on safety and tolerability, a 2.7-mg/kg dose was chosen for dose expansion in patients with c-Met+ advanced NSCLC. Three of 16 (19%) ABBV-399–treated c-Met+ NSCLC patients had a confirmed partial response (PR) with duration of response (DOR) 3+, 3, and 4.5 months. At week 12, 6 of 16 patients treated (37.5%) had disease control. Ten patients received ABBV-399 in combination with erlotinib. No DLTs were observed and AEs related to ABBV-399 occurring in ≥2 patients were acneiform rash (40.0%), fatigue (30.0%), and dry skin (20.0%). Three of 8 (37.5%) evaluable ABBV-399 + erlotinib-treated c-Met+ patients had a confirmed PR with DOR 2+, 4+, and 5+ months. Two of the 3 patients with PR had EGFR-mutated tumor, and previous TKI- and platinum-based chemotherapy had failed. ABBV-399 is well tolerated at a dose of 2.7 mg/kg every 21 days and has demonstrated antitumor activity in patients with c-Met+ NSCLC both as monotherapy and in combination with erlotinib. Updated data of antitumor activity and safety of ABBV-399 as monotherapy and in combination with erlotinib in c-Met+ NSCLC patients will be presented.

SC22.01 How Do I Define Optimal Candidates for Immunotherapy in My Practice?

SC22.01 How Do I Define Optimal Candidates for Immunotherapy in My Practice?

P1.06-001 Sluga R, Roepman P, Kummer A, vd Bosch W, vd Borne BEEM, Schramel FMNH

Tyrosine kinase inhibitors (TKIs) for treatment of advanced EGFR mutated adenocarcinoma were shown in many studies to be superior to chemotherapy in terms of progression-free survival. Since most data is derived from studies on Asian populations, there is a lack of data from other ethnicities. In the Netherlands the guidelines recommend that EGFR-mutation analysis should be performed in all patients with stage IIIb and IV adenocarcinoma and a non-small cell lung cancer-not otherwise specified (NSCLC- NOS). The aim of this study was to investigate the compliance to the guidelines in terms of determination of EGFR mutations, prevalence of EGFR mutations and the outcomes of treatment with the TKIs in a cohort of European patients with advanced NSCLC harboring an EGFR mutation. Data was obtained by retrospective analysis of the medical records of patients with a stage IIIb and IV non-small cell lung cancer between 2009 and 2014 in the two top-clinical hospitals in the Netherlands. The total number of patients included in the study was 1022. Molecular diagnostic tests were performed in 57.8% of patients with advanced adenocarcinoma or NSCLC-NOS. The prevalence of performing molecular diagnostic tests improved significantly between 2009 and 2014 (25,2% to 74,4% respectively). Positive EGFR mutation was found in 43 patients (in 9,1% of all molecular diagnostic tests performed). 72,1% of patients harboring an EGFR mutation were treated with TKIs.A significant overall survival benefit with a mean survival of 763 days was seen in EGFR positive patients treated with TKIs (with or without prior/subsequent chemotherapy) versus 435 days in patients treated with conventional chemotherapy (hazard ratio (HR): 0.719. 95% confidence interval (CI): 0.587-0.881). A large fraction of the patients with EGFR-mutated tumors were either initially or after progression treated with chemotherapy. EGFR positive patients who were prior to TKI treatment treated with chemotherapy had significantly longer survival in comparison to patients treated only with chemotherapy (1201 days vs 435 days respectively (HR: 3.289, 95% CI: 1.551-6.997). 10 patients were not treated with TKIs, either because of poor performance status (40%), patient’s refusal (30%), rapid disease progression (20%) or T790M mutation (10%). This study showed that incidence of molecular testing improved significantly over the course of the last years, leading to more effective targeted therapy. Overall survival was significantly prolonged in patients harboring an EGFR mutation treated with chemotherapy prior to TKIs, compared to patients without EGFR mutation treated only with conventional chemotherapy

P1.02-054 Genomic Complexity in KRAS Mutant Non-Small Cell Lung Cancer (NSCLC) by Smoking Status with Comparison to EGFR Mutant NSCLC

KRAS is the most frequently mutated oncogene in NSCLC and lacks an effective targeted therapy. Notably, KRAS mutations occur in both never/light-smokers and smokers. However, the relationship between smoking status and a KRAS+ tumor’s genomic complexity (mutation burden, copy number changes, concurrent mutations in key oncogenic pathways) is unclear. Similarly, the relationship between genomic complexity in tumors from never/light smokers but with a KRAS v EGFR activating mutation is also unknown. Targeted next-generation sequencing (NGS) data at our institution from 7/13-1/16 was reviewed to identify KRAS+ NSCLC tumors. All patients with a <10 pack-year (py) smoking history (NS) and a subset of heavy smokers (HS) (>40 py) were identified, with clinical and genomic analysis. A comparison cohort of 48 patients with EGFR+ NSCLC was also identified. Fisher’s exact test was used to compare frequency of gene mutations. 41 NS and 104 HS KRAS patients were evaluated. NS patients were more likely to be female (34/41 v 66/104, p<.01) and diagnosed with Stage I disease (14/41 v 13/104, p<.01). Compared to KRAS NS patients, tumors from KRAS HS patients were also genomically more complex, with increased total nucleotide variants (median=10 v 7, p<.001) and total copy number variations (median=22.5 v 5, p<.01). Intriguingly, in the cohort of EGFR tumors, total nucleotide variants resembled the KRAS NS cohort (median=6.5) but the total copy number variants were more similar to the KRAS HS cohort (median=25). Compared to KRAS NS tumors, KRAS HS tumors were also more likely to have: a) concurrent mutation in TP53 (43/104 v 8/41, p=.012) and b) concurrent mutations/2-copy deletions in >1 tumor suppressor (TS)/tumor (panel of TP53, STK11, APC, CDKN2A/B, RB) (26/104 v 4/41, p=.042). Although the total number of nucleotide variants in the EGFR cohort was most similar to the KRAS NS cohort, TS distribution in these EGFR tumors was closer to the KRAS HS cohort (TP53 variants in 31/48 and multiple TS variants in 14/48). Finally, median OS for KRAS HS patients with Stage IV disease was 9.7m v 28.7m in KRAS NS patients (HR=0.56). The genomic landscape of KRAS+ NSCLC from HS patients is distinct from NS patients and includes increased total mutations and frequency of TS loss. EGFR mutant tumors share some similarities with KRAS tumors from both NS and HS patients. Overall, NS KRAS+ tumors may be a genetically distinct cohort within the broader context of KRAS+ NSCLC.