Epidermal Growth Factor Receptor Biomarkers Research Articles

Mini Review: Immunohistochemistry Using EGFR-Mutant Specific Antibodies in Non-Small Cell Lung Carcinoma: Accuracy and Reliability

Epidermal growth factor receptor (EGFR) is a predictive biomarker in many solid cancers including non-small cell lung carcinoma (NSCLC). Patients’ responsiveness to therapy is based on the prior determination of EGFR status. Many techniques were used to detect the potential predictive biomarker and considered as gold standards based on molecular genetic techniques as direct sequencing and polymerase chain reaction (PCR). Immunohistochemistry (IHC) using EGFR mutation-specific antibodies were generated as an alternative simple tool to identify EGFR status and its response to tyrosine kinase inhibitors (TKIs). EGFR gene copy number and wild-type EGFR are other parameters which determine the response to TKIs. The aim of this mini-review is to analyze the studies which investigated the IHC EGFR mutation-specific antibodies, in lung adenocarcinoma (ADC), to determine its accuracy and reliability as a pre-selection tool for candidate patients for TKIs therapy as well as the interplay with other EGFR biomarkers which are EGFR gene copy number and the wild-type EGFR in lung NSCLC. The later determine the response to TKIs and their detection methodology is standardized making them good candidates for comparison with the EGFR-mutation.

Genomic Profiling of Advanced Non–Small Cell Lung Cancer in Community Settings: Gaps and Opportunities

BackgroundNational guidelines have advocated broad molecular profiling as a part of the standard diagnostic evaluation for advanced non–small cell lung cancer (NSCLC), with the goal of identifying driver mutations for which effective therapies or clinical trials are available. However, adherence to genomic testing guidelines could present challenges to community oncologists. Patients and MethodsWe performed a retrospective review of genomic testing patterns in patients with nonsquamous NSCLC treated by 89 oncologists at 15 sites throughout New Jersey and Maryland from January 2013 to December 2015. ResultsA total of 814 patients (89% with stage IV; 11% with stage IIIB) were identified in the COTA Inc database. Of the 814 patients, 479 (59%) met the guideline recommendations for EGFR (epidermal growth factor receptor) and ALK (anaplastic lymphoma kinase) biomarker testing; 63 (8%) underwent comprehensive genomic profiling for all 4 major types of alterations (point mutations, indels, fusions, and copy number amplifications). Gender, age, race, site of care (referral vs. community center), and practice size did not influence comprehensive genomic profiling frequency. Active smokers and patients who died within 30 days were tested less frequently (P < .05). Among those not tested for EGFR and ALK, 52% received chemotherapy without documented reasons for no testing, 32% did not receive antineoplastic therapy, and 13% had insufficient tissue for genotyping. ConclusionGenomic testing presents multiple logistical challenges for the community-based oncologist, including coordination of sample handling, long turnaround times, test reimbursement, access to targeted therapies, insufficient tissue, and patient harm from the repeat biopsies necessary if the tissue sample is insufficient. Opportunities exist for improvement in guideline adherence, possibly through new technologies such as “liquid biopsies,” which obviates the need tissue biopsy samples in select settings.

P3.02b-008 Quantification and Monitoring of Treatment Response in EGFR Mutant NSCLC Patients by Digital-PCR in Plasma cftDNA: Topic: EGFR Biomarkers

The identification of activating epidermal growth factor receptor (EGFR) mutations is essential for deciding therapy of non-small cell lung cancer (NSCLC) patients. Circulating cell-free tumor DNA (cftDNA) holds promise as a non-invasive methodology for tumor monitoring in solid malignancies. Among advanced NSCLC patients with an acquired resistance to EGFR-tyrosine kinase inhibitors (TKIs), about 50% carry T790M mutation, but its frequency in EGFR-TKI-naıve patients and dynamic change during therapy remains unclear. We hypothesized that EGFRmutation analysis detection in cftDNA for NSCLC may be feasible for monitoring treatment response to EGFR-TKIs and also predict drug resistance. EGFR sensitive mutations and T790M were analyzed using digital PCR (d-PCR) (Quant studio 3D, life technologies) in longitudinally (at baseline, at 4, 8, 20, 60, 120, 180, 270, 360 days) collected plasma samples (n=50) from 8 tissue-confirmed EGFR-mutant NSCLC patients treated with an EGFR-TKI (Gefitinib N = 4; Erlotinib N = 1; Afatinib N = 3). DNA extracted from plasma of 8 healty blood donors were used to detect the specificity of EGFR mutant assay. Tumor assessment was performed according to RECIST criteria 1.1 every two months. The sensitivity of d-PCR in plasma versus tissue was 71.4%. No EGFR mutation was present in the 8 control cases (specificity of 100%). Of four patients who developed progression disease (PD), in the samples of progression, T790M was detected in 75% of cases. The frequency of T790M in pre-TKI plasma samples was of 37.5%. EGFR sensitive mutations decreased at PD while T790M mutation increased in 75% of patients. Patients with concomitant pre-TKI EGFR 19 deletion and T790M showed a PD before of 12 months compared to those with L858R. T790M was frequently detected when new lesions were developed. Four patients had T790M level decreased to undetectable level with longer PFS than those with detectable T790M in blood. Our results indicated that d-PCR was a highly sensitive and useful method for detecting the T790M mutation. Moreover, dynamically monitoring T790M change might help determining EGFR-TKI resistance. We thank Italian Association for Cancer Research (AIRC) for supporting the study.

P3.02b-002 Treatment Outcome Comparison between Exon 19 and 21 EGFR Mutations after Second-Line TKIs in Advanced NSCLC Patients: Topic: EGFR Biomarkers

Although superior clinical benefits of first-line epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in the treatment of advanced non-small-cell lung cancer (NSCLC) had been reported with different sensitivity. The sensitivity of second-line TKIs in NSCLC patients with different EFGR mutations was unknown. The purpose of this study is to investigate the clinical outcome of NSCLC patients with and without brain and/or bone metastases in different EGFR tumor genotypes receiving EGFR-TKIs as a second-line treatment. The treatment outcomes of 166 NSCLC patients harboring either the exon 19 deletion or the L858R point mutation of EGFR treated by second-line TKIs were retrospectively reviewed. The disease control rate (DCR) and objective response rate (ORR) of enrolled NSCLC patients were 77.7% and 11.4%, respectively. The exon 19 deletion group had a significantly longer median progression-free survival (PFS) (6.7 vs. 4.5 months, P=0.002) and overall survival (OS) (13.7 vs. 11.7 months, P=0.02) compared with the L858R mutation group for NSCLC patients, as well for patients with brain metastasis [PFS: (6.7 vs. 3.9 months, p<0.001), OS: (13.7 vs. 7.9 months, p=0.006)]. The median PFS and OS for NSCLC patients with bone metastasis were 4.8 months (95% Cl, 4.0-5.6 months) and 12.9 months (95% Cl, 8.7-17.1 months), respectively. No significant difference was observed for patients with bone metastasis for different mutations. EGFR genotype and ECOG PS were independent predictors of PFS for both NSCLC patients with and without brain metastasis. Never smoking (P=0.001), exon 19 deletion (P=0.03), EGOC PS (0-1) (P<0.001) and no brain metastasis (p=0.01) were correlated with longer OS for all NSCLC patients. For patients with brain metastasis, age at disease progression (p=0.009), genotype (p=0.02) and EGOC PS (p<0.001) were independent predictors of OS. For patients with bone metastasis, EGOC PS (p<0.001) was an independent predictor for both PFS and OS. Female (P=0.01), never smoking (P=0.005), number of bone metastases (P=0.03) and EGOC PS (0-1) (P=0.002) were related to a longer PFS. EGOC PS (0-1) (P<0.001) was associated with a longer OS. Rash, fatigue, and anorexia were the three most frequent side effects observed No significant side effects difference between exon 19 and 21 mutation groups were observed. NSCLC patients harboring exon 19 deletion achieved better PFS and OS than those with L858R mutation, indicating that EGFR mutations are significant prognostic factors for advanced NSCLC patients with and without brain metastasis receiving second-line EGFR-TKIs treatment.

P3.02b-040 A Comparison of ddPCR and ARMS for Detecting EGFR T790M Status from Advanced NSCLC Patients with Acquired EGFR-TKI Resistance: Topic: EGFR Biomarkers

To assess the ability of droplet digital PCR and ARMS technology to detect epidermal growth factor receptor (EGFR) T790M mutations from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance. A sensitive and convenient method for detecting T790M mutation would be desirable to direct patient sequential treatment strategy. To assess the ability of droplet digital PCR and ARMS technology to detect epidermal growth factor receptor (EGFR) T790M mutations from circulating tumor DNA (ctDNA) in advanced non-small cell lung cancer (NSCLC) patients with acquired EGFR-TKI resistance. A sensitive and convenient method for detecting T790M mutation would be desirable to direct patient sequential treatment strategy. A comparison of two platforms for detecting EGFR mutations in plasma ctDNA was undertaken. Plasma samples and tumor samples were collected from patients happening acquired EGFR-TKI resistance in Zhejiang cancer hospital from December 2014 to December 2015. Extracted ctDNA was analyzed using two platforms (Droplet Digital PCR and ARMS [dPCR]). And the associations between progression free survival (PFS) starting from initial TKI treatment and the T790M ctDNA status detected in plasma were analyzed. A total of 108 patients were enrolled in this study. 108 patient plasma samples were detected by ddPCR and 75 were detected by ARMS. And 16 patients experienced re-biopsy were detected T790M status by ARMS method. 43.7% (47/108) had acquired T790M mutation by ddPCR. In 75 patient plasma samples, comparing ddPCR with ARMS, the rates of T790M mutation were 46.7% (35/75) and 25.3% (19/75) by ddPCR and ARMS, respectively. Of all, 16 patients both had tumor and plasma samples, the T790M mutation rates were 56.3% (9/16) by ARMS in tissue and 50.5% (8/16) by ddPCR in plasma ctDNA. Among them, there were two ctDNA T790M mutations by ddPCR but T790M gene negative in tumor tissue by ARMS method. For all patients, the median PFS and OS were 12.3 months and 32.8 months, respectively. The patients with T790M-positive tumors had a longer time to disease progression after treatment with EGFR-TKIs (median, 13.1 months vs 10.8 months; P=0.010) and overall survival (median, 35.3 months vs 30.3 months; P=0.214) compared with those with T790M-negative patients. Our study demonstrates dPCR assay provide feasibility and sensitive method in detecting EGFR T790M status in plasma samples from NSCLC patients with acquired EGFR-TKI resistance. And T790M-positive patients have better clinical outcomes to EGFR-TKIs than patients with T790M negative.

P3.02b-031 T790M: A Favorable Mutation?: Topic: EGFR Biomarkers

Patients with lung adenocarcinoma harboring somatic activating mutations in the epidermal growth factor receptor (EGFR) gene have benefited significantly from EGFR tyrosine kinase inhibitors (TKIs). However, a vast majority would eventually develop resistance to such TKIs, resulting in disease progression. T790M, a secondary mutation in exon 20 of the EGFR gene, has been identified as the major mechanism responsible for acquired EGFR-TKI resistance, accounting for roughly 50% of resistance. However, the association of T790M status and clinical outcome remains elusive. Here, we conducted a retrospective study examining the association between T790M status and prognosis on clinical samples obtained from 43patietns with EGFR-mutant lung adenocarcinoma and later acquired resistance to EGFR-TKIs. We performed capture-based targeted ultra-deep sequencing on either tumor biopsies or blood samples of 43 lung adenocarcinoma patients, who harbored EGFR mutations, and subsequently developed resistance to EGFR TKIs. We used BurningRock Biotech’s panels, consisting of critical exons and introns, covering multiple classes of somatic mutations, including single nucleotide variation (SNVs), rearrangements, copy number variations (CNVs) and insertions and deletions (INDELs) to detect genetic alterations both qualitatively and quantitatively. We investigated the mutation spectrum after the development of resistance to EGFR-TKIs. Our analysis revealed 64% (18/28) of patients, harboring exon 19 deletions at baseline, acquired T790M at the time of progression. In contrast, only 33% (5/15) of patients, harboring exon 21 L858R substitutions at baseline, acquired T790M, indicating T790M mutation is more commonly found in patients with exon 19 deletions (p=0.052). Next, we associated T790M status with cumulative EGFR-TKI exposure time, and found patients had longer TKI exposure are more likely to acquire T790M mutation. The median TKI exposure time for patients who eventually acquired T790M was 19.20m ±1.86. In contrast, the median exposure time for patients who never acquired T790M 12.20m±1.20m (P =0.017). Furthermore, we investigated the presence of T790M mutation with progression free survival (PFS). Our data revealed that 68% (15/22) of patients with PFS≥12 months acquired T790M mutation after TKI exposure. In contrast, among patients with PFS< 12 months, only 38% of them acquired T790M mutation (p=0.048). Patients who eventually acquired T790M mutation have longer cumulative TKI exposure and are associated with longer PFS before the emergence of drug resistance. In addition, we also discovered that patients harboring exon 19 deletions are more likely to develop T790M mutation. Therefore, T790M status can be used as a potential biomarker for prognosis.

P3.02b-019 TTF-1 Expression as a Predictor of Response to EGFR-TKIs in Patients with Lung Adenocarcinoma: Topic: EGFR Biomarkers

Several studies have shown that overexpression of thyroid transcription factor (TTF-1) may be associated with the presence of epidermal growth factor receptor (EGFR) gene mutations and predict survival in patients with non-small cell lung cancer (NSCLC). Nevertheless, the potential significance of TTF-1 immunostaining as a predictor of response to EGFR tyrosine kinase inhibitors (TKIs) has received limited research attention thus far. The aim of this study was to further explore the potential association between TTF-1 immunohistochemical expression and response to EGFR TKIs in patients with lung adenocarcinoma. The medical records of 129 patients with stage IV lung adenocarcinoma, treated at the Oncology Unit of “Sotiria” Athens General Hospital between January 2011 and December 2014, were retrospectively reviewed. All patients had received treatment with EGFR TKIs (erlotinib or gefitinib) and had a known TTF-1 immunohistochemical expression status in formalin-fixed, paraffin-embedded tumor tissue. Demographic and clinicopathological features (age, gender, smoking status and performance status), TTF-1 immunohistochemistry and EGFR mutation status results were correlated to each other as well as with the response rate (RR) to EGFR TKIs, using univariate and multivariate regression analysis. Median age of our study population was 68 years (range 26-88 years), while the majority were male (79/129 cases, 61.2%). Patients with EGFR mutant tumors had significantly higher response rates to EGFR TKIs compared to patients with wild-type EGFR tumors (p=0.001). TTF-1 positive staining was weakly associated with the presence of EGFR mutations, without reaching the level of statistical significance (p=0.053). Most importantly, TTF-1 positive staining was not significantly correlated with RR to EGFR TKIs, when gender, age, smoking status, EGFR mutation status and PS were included in the multivariate model. The present results failed to demonstrate any independent association between TTF-1 overexpression and the RR to EGFR TKIs in patients with advanced-stage lung adenocarcinoma. Prospective data from larger cohorts of patients are needed to clarify the exact predictive value of TTF-1 immunostaining in this setting.

P3.02b-041 Genome-Wide Screen of DNA Methylation Changes Reveals GABBR2 as a Novel Potential Target for EGFR 19 Deletion Adenocarcinoma with Erlotinib: Topic: EGFR Biomarkers

Lung cancer is the leading cause of cancer related death around the world. The last decade has witnessed the rapid development of epidermal growth factor receptor (EGFR) directly targeted therapies that have significantly changed the treatment of non-small-cell lung cancer (NSCLC). However, the challenge of acquired resistance to EGFR tyrosine kinase inhibitors (TKIs) has been an issue when talking with activating EGFR mutations, which makes it crucial to elucidate the mechanism of EGFR-TKI targeted drug resistance. Methyl-sensitive cut counting sequencing (MSCC), one of the most commonly used whole-genome DNA methylation sequencing technology, was applied to investigate the changes of paired tissue DNA methylation before and after TKI (erlotinib) treatment lasting two cycles with partial response (PR) for stage IIIa (N2) lung adenocarcinoma patients (N=2) with activating EGFR 19 deletion. Sequenom EpiTYPER assay method was further analyzed to double confirm the changed methylated candidate genes in these two patients, through comparing the methylated changes in paired tissues before and after TKI treatment. Western blotting, cell cycle and apoptosis analysis by the up/down regulation of a candidate gene (GABBR2) were performed in three lung adenocarcinoma cell lines, A549 (EGFR wild type), HCC4006 (EGFR 19 deletion, DelL747-E749) and HCC827 (EGFR 19 deletion, DelE746-A750), to elucidate the mechanism of GABBR2 gene in the regulation of EGFR-TKI treatment. Sixty aberrant methylated genes were firstly discovered using MSCC method in these two patients harboring EGFR 19 del mutation treated with erlotinib. Two aberrant methylated genes, CBFA2T3 and GABBR2, were clearly validated by the Sequenom EpiTYPER assay subsequently. GABBR2 was significantly down regulated in HCC4006 and HCC827 cells harboring EGFR 19 mutations but remained conservative in A549 cells with EGFR wild-type after erlotinib treatment by Western blotting. The phenomenon was in line with the obvious apoptosis of HCC4006 and HCC827 cell lines after erlotinib treatment, but not in A549 cells. GABBR2 was further induced down regulation after erlotinib exposure through apoptosis method silenced by siRNA using RNAi technology. Meanwhile, GABBR2 gene was demonstrated up regulation rescued the apoptosis significantly, when overexpressing GABBR2 in HCC827 cell lines along with erlotinib treatment. Genome-wide screen of DNA methylation changes demonstrated that GABBR2 gene might be as a novel potential treatment target for stage IIIa (N2) EGFR 19 deletion adenocarcinoma with erlotinib treatment. Our research provides a new theoretical basis for the epigenetic study of EGFR mutated lung adenocarcinoma treatment.

P3.02b-015 Impact of Metastatic Status on the Prognosis of Patients Treated with First Generation EGFR-TKIs: Topic: EGFR Biomarkers

First generation epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) such as gefitinib (GEF) or erlotinib (ERL) are effective for patients harboring EGFR mutations positive non-small cell lung cancer (NSCLC). To know the impact of metastatic status on patients treated with EGFR-TKIs is important. The objective of this study is to know the impact of metastatic status including brain metastasis, bone metastasis, liver metastasis and pleural effusion on the prognosis of patients treated with first generation EGFR-TKIs. The pathology files of National Hospital Organization Kinki-chuo Chest Medical Center from 2009 to 2014 were retrospectively reviewed and 533 patients were EGFR mutation positive NSCLC. Patients with stage IA to IIIA and small cell lung cancer were excluded. From 299 stage IIIB, IV and relapsed NSCLC patients, 178 patients treated with GEF or ERL as first line treatment were enrolled for the study. Metastatic status, progression free survival (PFS), overall survival (OS) and response rate (RR) were investigated. We also evaluated the relationship between the number of metastatic organs and prognosis. Fifty-one patients were male and median age at the time of first line treatment was 72 years (range 39-91). Number of patients with adenocarcinoma was 168 and 156 patients were treated with GEF. In log-rank test, PFS and OS were significantly worse in patients with brain metastasis (8.0m vs 13.2m, p= 0.008; 20.2m vs 38.0m, p< 0.001 respectively), bone metastasis (8.8m vs 15.4m, p< 0.001; 24.0m vs 32.1m, p= 0.020 respectively), liver metastasis (6.7m vs 12.5m, p< 0.001; 13.4m vs 32.1m, p< 0.001 respectively) and pleural effusion (10.8m vs 12.2m, p= 0.033; 21.9m vs 34.9m, p= 0.004 respectively) at the time of first line treatment compared with patients without each metastasis. In cox proportional hazards models multivariate analysis, bone metastasis had correlated with worse PFS (HR 2.110, 95% CI 1.437-3.093, p< 0.001) and moreover, brain metastasis had correlated with worse OS (HR 2.414, 95%CI 1.464-3.951, p< 0.001). There were no relationships between metastatic status and RR. Furthermore, the much number of metastasized organs (no metastasis, 1 metastasis and 2 or more metastasis) was significantly related to worse PFS (19.3m vs 12.5m vs 6.6m, p< 0.001) and OS (46.1m vs 29.9m vs 15.1m, p< 0.001). Bone metastasis had correlated with worse PFS and brain metastasis had correlated with worse OS in patients treated with first generation EGFR-TKIs. Moreover, the much number of metastasized organs was related to worse PFS and OS.

P3.02b-026 Association of EGFR Exon 19 Deletion and EGFR-TKI treatment duration with Frequency of T790M Mutation in EGFR-Mutant Lung Cancer Patients: Topic: EGFR Biomarkers

The most common event responsible for resistance of epidermal growth factor receptor (EGFR)-tyrosine kinase Inhibitor (TKI) is acquisition of the T790M mutation, which occurs in approximately 50% of patients who initially respond to EGFR-TKI. Recently, third-generation EGFR-TKIs have been shown to exert a remarkable effect against T790M resistance mutation-positive non-small cell lung cancer (NSCLC). T790M is an important predictive marker for third-generation EGFR-TKIs; therefore, determining the clinicopathologic characteristics of T790M-harboring NSCLC showing relapse after EGFR-TKI therapy is of high clinical relevance. we evaluated whether T790M mutation is related to clinicopathologic or prognostic factors in patients with relapse of EGFR-mutant NSCLC after treatment with EGFR-TKIs. We retrospectively reviewed T790M status and clinical characteristics of advanced-stage or recurrent NSCLC patients who had received EGFR-TKI treatment and rebiopsy at Kurume University Hospital between March 2005 and December 2015. we identified 193 patients with advanced or recurrent EGFR-mutant NSCLC who developed resistance to initial EGFR-TKI treatment. Of these patients, 105 (54%) underwent re-biopsy. Adequate histological specimens were available for 73 of these patients, who were enrolled in the study; 38 (52%) of them had T790M mutation and 35 (48%) did not. T790M mutation was present more frequently in patients with exon 19 deletion mutation (63%, 26 of 41) than in those with L858R mutation (38%, 12 of 32) (p=0.035) and in patients who received EGFR-TKI treatment for at least 10 months in total (71%, 32 of 45) than in those who did not (21%, 6 of 28) (p<0.001). The median PFS after initial EGFR-TKI treatment was longer in the T790M mutation-positive group (13.6 months, 95% CI: 9.2-15.8) than in the negative group (7 months, 95% CI: 3.7-8.5) (p=0.037). The median total duration of EGFR-TKI treatment in patients with T790M mutation was 15.3 months, which was significantly longer than that (8.1 months) in patients without T790M mutation (p<0.001). Multivariate analyze revealed that The type of EGFR mutation (exon 19 deletion mutation versus L858R point mutation, p=0.011, OR=0.21, 95% CI=0.05-0.71) and total duration of EGFR-TKI treatment (≥10 months versus <10 months, p<0.001, OR=0.09, 95% CI=0.02-0.28) were significantly associated with the presence of T790M mutation. The patients with EGFR exon 19 deletion mutation and long-term treatment with EGFR-TKI exposure demonstrated a high prevalence of T790M mutation. These data are potentially important for clinical decision making in the NSCLC patients with EGFR mutation.

P3.02b-011 Comparison of Four Leading Technologies for Detecting EGFR Mutations in Circulating Tumor DNA from Patients with Non-Small Cell Lung Carcinoma: Topic: EGFR Biomarkers

This study aimed to assess the ability of different technology platforms to detect epidermal growth factor receptor (EGFR) mutations including L858R, E19-dels, T790M, and G719X from circulating tumor DNA (ctDNA) in patients with non-small cell lung cancer (NSCLC). Plasma samples were collected from 20 patients with NSCLC including detailed clinical information along with data regarding treatment response. ctDNA was extracted from 10 mL plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen). Extracted ctDNA was analyzed using two real time-amplification refractory mutation system-quantitative PCR platforms (cobas® EGFR Mutation Test: cobas; and AmoyDx® EGFR 29 Mutations Detection Kit: ADx), one digital platform (Droplet DigitalTM PCR, ddPCR: Bio-rad), and one next-generation sequencing platform (firefly NGS: Accuragen). If a positive result was obtained from any one of the four platforms, the sample was categorized as positive. We identified 15 EGFR mutations in 20 patients with NSCLC using the four platforms, for which 7, 11, 10, and 12 mutations were detected by ADx, cobas, ddPCR, and firefly NGS, respectively. Among the 15 EGFR mutations, six and seven EGFR alterations demonstrated an allele frequency of more or less than 1% (group A or B, respectively), and two exhibited unknown allele frequency. In group A, 5, 5, 5, and 6 EGFR mutations were detected by ADx ,cobas, ddPCR, and firefly NGS, respectively. The positive coincidence rate of any two platforms ranged from 66.7% to 100% and the kappa value varied from 0.787 to 1.000 in group A. In group B, 1, 5, 5, and 6 EGFR mutations were detected and the positive coincidence rate of any two platforms ranged from 16.7% to 100% and the kappa value varied from 0.270 to 1.000. The output of cobas, ddPCR, and firefly NGS were highly correlated, whereas ADx displayed weak concordance with these three platforms in group B. In addition, we identified 75 wild-type loci when EGFR alleles identified as negative by one or more platforms were considered as negative. ADx, cobas, ddPCR, and firefly NGS uncovered 73, 69, 70, and 68 EGFR wild-type loci, respectively. The concordance and negative coincidence rates between any two platforms were over 90%. The detection rate and concordance were probably affected by the abundance of EGFR mutations and the sensitivity of different platforms. Three platforms, including cobas, ddPCR, and firefly NGS, exhibited higher positive coincidence and detection rates when the allele frequency was lower than 1%.

P3.02b-022 Early Experience of Detecting EGFR Activating Mutations in Cell Free DNA Using Droplet Digital PCR: Topic: EGFR Biomarkers

Early detection of resistance conferring epidermal growth factor receptor (EGFR) T790M mutation is of clinical significance in previously EGFR positive NSCLC patients. Plasma from 40 advanced NSCLC patients with known L858R or Del 19 745_750 Mutations were tested for cell free DNA on Droplet Digital PCR. This included biopsy confirmed treatment naive cases as well as those who developed clinical resistance to tyrosine kinase inhibitors. The later cases were examined for previous known and new T790M mutations using highly sensitive Droplet Digital PCR. Where ever feasible the results were compared to the secondary biopsy findings, duration of development of new T790M mutation and its serial plasma quantification results. 20 Treatment naive biopsy positive DEL19/L858R cases were tested for cell free DNA. All cases were positive with values ranging from .04% to 24%. 19 cases were checked for primary and secondary mutations on progression of disease. 12 cases showed secondary mutation along with primary mutation. The values of T790M mutation ranged from 0.04% to 4.5%. We also had 3 cases with biopsy and cell free correlation of secondary mutation. None of them correlated. Droplet digital PCR is a robust platform to detect the primary driver EGFR mutations and can be used as a substitute / addendum to biopsy for initiating early treatment. It also appears to be too sensitive for detection of secondary T790M mutations. However response to treatment in patients with minimal cell free T790M values in plasma may need to be further investigated for clinical utilization of this platform.

Human Papillomavirus and Epidermal Growth Factor Receptor in Oral Cavity and Oropharyngeal Squamous Cell Carcinoma: Correlation With Dynamic Contrast-Enhanced MRI Parameters.

The objective of this study was to investigate differences in dynamic contrast-enhanced MRI (DCE-MRI) parameters on the basis of the status of human papillomavirus (HPV) and epidermal growth factor receptor (EGFR) biomarkers in patients with squamous cell carcinoma (SCC) of the oral cavity and oropharynx by use of histogram analysis. A total of 22 consecutive patients with oral cavity and oropharyngeal SCC underwent DCE-MRI before receiving treatment. DCE parameter maps of the volume transfer constant (K(trans)), the flux rate constant (kep), and the extravascular extracellular volume fraction (ve) were obtained. The histogram parameters were calculated using the entire enhancing tumor volume and were compared between the patient subgroups on the basis of HPV and EGFR biomarker statuses. The cumulative histogram parameters of K(trans) and kep showed lower values in the HPV-negative and EFGR-overexpression group than in the HPV-positive EGFR-negative group. These differences were statistically significant for the mean (p = 0.009), 25th, 50th, and 75th percentile values of K(trans) and for the 25th percentile value of kep when correlated with HPV status in addition to the mean K(trans) value (p = 0.047) and kep value (p = 0.004) when correlated with EGFR status. No statistically significant difference in ve was found on the basis of HPV and EGFR status. DCE-MRI is useful for the assessment of the tumor microenvironment associated with HPV and EGFR biomarkers before treatment of patients with oral cavity and oropharyngeal SCC.

ET-19 * A PHASE 1 STUDY EVALUATING ABT-414 WITH TEMOZOLOMIDE (TMZ) OR CONCURRENT RADIOTHERAPY (RT) AND TMZ IN GLIOBLASTOMA (GBM)

BACKGROUND: Standard therapies have little effect on the poor survival rates of GBM. Abnormal epidermal growth factor receptor (EGFR) expression and signaling are common in GBM. ABT-414 is a unique antibody-drug conjugate, with a toxic payload (MMAF) targeted to active EGFR or mutant EGFRvIII, that has demonstrated high antitumor activity in preclinical GBM tumor models. METHODS: Objectives were to evaluate the safety, pharmacokinetics (PK), and the maximum tolerated dose (MTD) of ABT-414 when administered with TMZ (recurrent or unresectable GBM, Arm B) or concurrent RT and TMZ (newly diagnosed GBM, Arm A). Adverse events (AEs), PK parameters, objective response (RANO), and tumor tissue EGFR biomarkers were assessed. Dose escalation was determined by the continual reassessment method (CRM). RESULTS: As of April, 2014, 22/21 pts were treated in Arm A/B. Common treatment-emergent AEs included fatigue(n= 11/ 6, Arm A/B); blurred vision(n = 10/9); AST increase(n = 9, Arm A); and nausea(n = 7/7) Grade 3/4 AEs (≥ 2 pts) included lymphopenia(n = 3); eye toxicity(n = 3); brain edema, ALT, AST(n = 2 each) in Arm A and keratitis(n = 2) in Arm B. Doses from 0.5-3.2mg/kg have been explored. Dose limiting toxicities primarily affecting the eye (keratitis) and liver occurred at multiple doses. The CRM has predicted 2.4mg/kg as the MTD in Arm A and 1.25mg/kg in Arm B. Patient samples are being evaluated for EGFR expression, amplification and EGFRvIII status. Efficacy endpoints are not yet mature in Arm A, but 4 patients in Arm B have best responses of 3 PRs and 1 CR. CONCLUSIONS: PK and safety data support doses of 2.4 and 1.25mg/kg as the predicted MTD in Arms A and B. Preliminary safety data demonstrate liver and eye toxicities in Arm A and primarily eye toxicities in Arm B. Updated biomarker results as they correlate with efficacy will be reported. Phase 2 studies assessing ABT-414 in GBM are planned.

416O - A Phase 1 Study Evaluating Abt-414 with Concurrent Radiotherapy (Rt) and Temozolomide (Tmz) in Newly Diagnosed Glioblastoma (Gbm)

ABSTRACT Aim: Despite standard therapy for GBM, median survival is 1-2 years. Abnormal epidermal growth factor receptor (EGFR) expression and signaling are common in GBM. ABT-414 is a unique antibody-drug conjugate, with a toxic payload (monomethylauristatin F) targeted to active EGFR or mutant EGFRvIII, that has demonstrated high antitumor activity in preclinical GBM tumor models. Methods: Objectives were to evaluate the safety, pharmacokinetics (PK), and the maximum tolerated dose (MTD) of ABT-414 when administered every 14 days with concurrent RT and TMZ in newly diagnosed GBM. Adverse events, PK parameters, objective response (RANO), and tumor tissue EGFR biomarkers were assessed. Dose escalation was determined by the exposure-adjusted continual reassessment method (EACRM). Results: As of April 9, 2014, 22 pts were treated (13/9 Male/Female, median age 58 years, range 34-79). Common treatment-emergent adverse events (TEAEs, ≥ 5 pts) included fatigue (n= 11); blurred vision (n = 10); thrombocytopenia (n = 8); AST increase (n = 9); ALT increase (n = 8); nausea (n = 7); eye pain (n = 6); lacrimation increase (n = 6); dry eye, headache, and constipation (n = 5 each). Grade 3/4 TEAEs (≥ 2 pts) included lymphopenia (n = 3); eye toxicity (n = 3); brain edema, ALT, AST (n = 2 each). Doses from 0.5 - 3.2 mg/kg have been explored. Dose limiting toxicities primarily affecting the eye (keratitis) and liver occurred at the 2, 2.6, 3.0, and 3.2 mg/kg doses. The EACRM has predicted 2.4 mg/kg as the MTD. Confirmatory safety data are being collected. PK data from 9 subjects in the dose range of 0.5 - 3 mg/kg indicate that exposure (Cmax and AUC) of ABT‐414 appeared to be dose proportional with a half‐life of approximately 11 days. Efficacy endpoints are not yet mature. Patient samples are being evaluated for EGFR expression, amplification and EGFRvIII status to determine which marker best associates with clinical benefit. Conclusions: PK and safety data support a dose of 2.4 mg/kg as the predicted MTD. Preliminary safety data demonstrate increased liver and eye toxicities in addition to common RT + TMZ toxicities. Further follow-up may demonstrate whether ABT-414 improves outcome. Disclosure: H.K. Gan: employee of Ludwig Institute for Cancer Research, which has licensed ABT-806; A. Lassman: Consultancy: Amgen, Celgene, Genentech, Sigma-Tau, Agenus, Roche, Stemline, Synapse, RadMD, Venture Inflections, Novartis, Kyowa Hakko Kirin, Abbott, Scientia Advisors, MSD, GSK, Able Assoc, Defined Health, Easton Assoc.; M.J. van den Bent: Consultancy: Roche, ABBVIE, Actelion, Celldex, AMGEN. Research support: Roche, ABBVIE. Speakers bureau: MSD; A. Scott: employee of Ludwig Institute for Cancer Research, which has licensed ABT-806; Consultancy and stock ownership - Life Science Pharmaceuticals; M. Pedersen, E. Gomez, J. Fischer, W. Ames, H. Xiong, M. Dudley, L. Roberts-Rapp, P.J. Ansell and K. Holen: is an AbbVie employee and may own stock; D.A. Reardon: Speaker's Bureau – Merck/Schering and Genentech/Roche; Advisory Board – Novartis; Amgen; Roche/Genentech; EMD Serono; Stemline Therapeutics; Momenta Pharmaceuticals. All other authors have declared no conflicts of interest.

Human Papillomavirus, p16, and Epidermal Growth Factor Receptor Biomarkers and CT Perfusion Values in Head and Neck Squamous Cell Carcinoma

Head and neck squamous cell carcinoma tumors positive for laboratory biomarkers hrHPV and p16 and negative for EGFR often respond better to nonsurgical organ-preservation therapy than hrHPV-negative, p16-negative, and EGFR overexpressing tumors. CTP has been shown to distinguish which locally advanced head and neck squamous cell carcinomas will respond to induction chemotherapy or chemoradiation. Our purpose was to determine whether a relationship exists between CTP measures and the expression of these laboratory biomarkers, because both appear to separate head and neck squamous cell carcinoma tumors into similar groups. We conducted an institutional review board-approved, Health Insurance Portability and Accountability Act-compliant retrospective review of head and neck CTP in 25 patients with locally advanced head and neck squamous cell carcinoma who had signed informed consent. Eight women and 17 men, 41-80 years of age, constituted a pretreatment group of 18 patients and a palliative group of 7 patients. Tumor biopsy samples were analyzed for overexpression of hrHPV, p16, and EGFR. The hrHPV, p16, and EGFR status of the tumors was correlated with CTP parameters (MTT, BV, BF, CP) by using the Wilcoxon evaluation and Fischer exact test. There were significantly lower CP values in pretreatment tumors overexpressing EGFR (P = .04). CP values ≤17.23 were significantly correlated with EGFR overexpression (P = .015). A trend toward higher CP values was present in hrHPV-positive and p16-overexpressing pretreatment tumors (P = .14). A significant correlation exists between CTP measures and EGFR overexpression in head and neck squamous cell carcinomas, suggesting an association between certain imaging findings and molecular biomarkers. These results may be related to a tumor cell survival mechanism linking perfusion and biomarker expression.

Phase III, randomized, open‐label, first‐line study in Asia of gefitinib versus carboplatin/paclitaxel in clinically selected patients with advanced non‐small‐cell lung cancer: evaluation of patients recruited from mainland China

In the IRESSA Pan-Asia Study (IPASS), 1217 patients in East Asia with pulmonary adenocarcinoma who were never-smokers or ex/light-smokers received first-line gefitinib (250 mg/day) or carboplatin/paclitaxel (area under the curve 5/6; 200 mg/m(2) ). Efficacy analyses were pre-planned in patients in China. In China, 372 patients (30.6% of the overall group) were randomized. The primary end-point was progression-free survival (PFS). Secondary end-points were overall survival (OS), objective response rate (ORR), health-related quality of life (HRQoL), symptom improvement, safety and tolerability. For patients in China, PFS did not significantly differ from the overall IPASS population (interaction test P= 0.427). PFS was numerically longer (hazard ratio [HR] 0.79; 95% CI 0.62-1.01; P= 0.065; median PFS 6.8 months for both treatments) and ORR significantly higher (ORR 44.6 vs 29.8%; odds ratio 1.88; 95% CI 1.22-2.89; P= 0.004) for gefitinib than carboplatin/paclitaxel. OS (mature data) was similar for both treatments (HR 0.92; 95% CI 0.73-1.17; P= 0.511; median OS gefitinib 18.1 months vs 18.3 months carboplatin/paclitaxel). HRQoL improvement rates favored gefitinib; symptom improvement rates were similar for both treatments. Gefitinib had a more favorable tolerability profile than carboplatin/paclitaxel. Efficacy by epidermal growth factor receptor biomarker status (exploratory analyses) was difficult to interpret due to low patient numbers with known biomarker status. For the Chinese subgroup of IPASS, gefitinib demonstrated improved PFS and ORR, similar OS, higher HRQoL, similar symptom improvement rates and a more favorable tolerability profile than carboplatin/paclitaxel, generally consistent with the overall IPASS population.

Labeling approaches for the GE11 peptide, an epidermal growth factor receptor biomarker

The epidermal growth factor receptor (EGFR) is involved in the proliferation and differentiation of normal and malignant cells and is a major therapeutic target for a variety of human cancers. The peptide GE11 was reported to bind efficiently to the EGFR. Labeling GE11 with radionuclides may aid in the quantification of EGFR expression in tumors via noninvasive imaging. To this end, a GGGK linker was attached to the peptide (GE11′), which was conjugated with prosthetic‐labeled groups such as [18F]N‐succinimidyl 4‐fluorobenzoate ([18F]SFB), [18F](2‐{2‐[2‐(2‐fluoro‐ethoxy)‐ethoxy]‐ethoxy}‐ethoxy)‐propyne ([18F]F‐PEG4‐propyne), [124I]N‐succinimidyl 4‐iodobenzoate, and the NOTA‐Bn‐NCS chelator for 111In labeling. All labeled analogs were successfully prepared with radiochemical purity &gt;95% and were identified by HPLC based on their non‐labeled standards. [18F]SFB and [18F]F‐PEG4‐propyne were obtained with a decay‐corrected yield (DCY) of 26% and 30% and a specific activity (SA) of 844 and 2580 Ci/mmol, respectively. [124I]N‐Succinimidyl 4‐iodobenzoate was obtained with a DCY of 65% and an SA of 3600 Ci/mmol. The DCYs for [18F]4‐fluoro‐benzoate‐GE11′, [18F]F‐PEG4‐1,3‐triazole‐GE11′, and [124I]4‐iodo‐benzoate‐GE11′ were 8%, 10%, and 30%, and the SAs were 108, 500, and 300 Ci/mmol, respectively. [111In]‐NOTA‐GE11′ was prepared with 60% radiochemical yield (RCY) and a SA of 0.35 Ci/mmol. GE11 was also labeled directly with 124I, using chloramine‐T, yielding [124I]GE11 with 47% RCY and a SA of 1030 Ci/mmol. Copyright © 2011 John Wiley &amp; Sons, Ltd.

Nano-Bio-Chip Sensor Platform for Examination of Oral Exfoliative Cytology

Oral cancer is a deadly and disfiguring disease that could greatly benefit from new diagnostic approaches enabling early detection. In this pilot study, we describe a nano-bio-chip (NBC) sensor technique for analysis of oral cancer biomarkers in exfoliative cytology specimens, targeting both biochemical and morphologic changes associated with early oral tumorigenesis. Here, oral lesions from 41 dental patients, along with normal epithelium from 11 healthy volunteers, were sampled using a noninvasive brush biopsy technique. Specimens were enriched, immunolabeled, and imaged in the NBC sensor according to previously established assays for the epidermal growth factor receptor (EGFR) biomarker and cytomorphometry. A total of 51 measurement parameters were extracted using custom image analysis macros, including EGFR labeling intensity, cell and nuclear size, and the nuclear-to-cytoplasmic ratio. Four key parameters were significantly elevated in both dysplastic and malignant lesions relative to healthy oral epithelium, including the nuclear area and diameter (P < 0.0001), the nuclear-to-cytoplasmic ratio (P < 0.0001), and EGFR biomarker expression (P < 0.03). Further examination using logistic regression and receiver operating characteristic curve analyses identified morphologic features as the best predictors of disease (area under the curve < or =0.93) individually, whereas a combination of all features further enhanced discrimination of oral cancer and precancerous conditions (area under the curve, 0.94) with high sensitivity and specificity. Further clinical trials are necessary to validate the regression model and evaluate other potential biomarkers, but this pilot study supports the NBC sensor technique as a promising new diagnostic tool for early detection of oral cancer, which could enhance patient care and survival.

Epidermal Growth Factor Receptor Biomarkers in Non–Small-Cell Lung Cancer: A Riddle, Wrapped in a Mystery, Inside an Enigma

Epidermal Growth Factor Receptor Biomarkers in Non–Small-Cell Lung Cancer: A Riddle, Wrapped in a Mystery, Inside an Enigma