EGFR Copy Number Research Articles

Clinical evaluation of a dedicated next generation sequencing panel for routine glioma diagnostics

Since 2013 next-generation sequencing (NGS) targeting genes mutated in diffuse gliomas is part of routine diagnostics in our institute. In the present report, we evaluate the use of this custom tailored NGS platform on 434 samples. The NGS panel assesses mutations in ATRX, CIC, EGFR, FUBP1, NOTCH1, PTEN; H3F3A, IDH1/2, PIK3CA, and BRAF, amplifications in EGFR or MDM2 and copy number alterations (CNA) of chromosome 1p, 7, 10 and 19q. TERT promoter mutations were assessed separately when indicated. Of the 433 samples of individual tumors with NGS data available, 176 cases were diagnosed as grade 2 or 3 glioma (40.6) and in 201 patients a glioblastoma (46.4%). Of the remaining 56 patients, 22 had inconclusive histology. In 378 cases (87.1%) a diagnosis solely based on glioma-targeted NGS could be established and resulted in a different diagnosis in ~ 1/4 of the cases. In 17 out of 22 cases without a conclusive histological diagnosis NGS resulted in a molecular diagnosis.The current study on a large cohort of patients confirms the diagnostic strength of the platform we developed, with a clear separation of glioma subgroups with different outcomes. It demonstrates the diagnostic value and the efficiency of glioma-targeted NGS for routine glioma diagnostics allowing with a single assay a glioma diagnosis in the large majority of cases. It allows in one run the molecular assessments required for the WHO classification of diffuse gliomas, including the recent recommendations to assess copy number alterations of chromosome 7 and 10, and of the TERT promoter region in IDHwt lower grade glioma.

Mouse avatar models of esophageal squamous cell carcinoma proved the potential for EGFR-TKI afatinib and uncovered Src family kinases involved in acquired resistance

BackgroundNo approved targeted agents are available for esophageal squamous cell carcinoma (ESCC). Informative genomic analysis and mouse patient-derived xenografts (PDX) also called mouse avatar can greatly expedite drug discovery.MethodsSix ESCC cell lines and 7 out of 25 PDX models derived from 188 biopsies with clear molecular features were employed to evaluate the sensitivity of several EGFR blockers in vitro and in vivo, as well as the underlying antitumor mechanisms of the most promising EGFR-TKI afatinib. Mechanisms involved in acquired resistance of afatinib were explored based on established resistant cell lines and PDX models followed by an attempt to reverse resistance.ResultsCompared with other EGFR blockers, the second-generation EGFR-TKI afatinib exerted superior antitumor effects in ESCC, and EGFR copy number gain (CNG) or overexpression was proposed to be predictive biomarkers. Afatinib played its antitumor effects by inhibiting EGFR downstream pathways, as well as inducing apoptosis and cell cycle arrest at G1. It was increased phosphorylation of Src family kinases (SFKs), rather than MET upregulation, that conferred to acquired resistance of afatinib. Dual blockade of EGFR and SFKs could overcome afatinib resistance and warrants validation in clinical practice.ConclusionBoth ESCC cell lines and PDXs with EGFR CNG or overexpression are potential candidates for afatinib, and concomitant EGFR/SFKs inhibition could reverse afatinib-acquired resistance caused by SFKs activation in ESCC.

Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining

BackgroundAlthough intratumoral heterogeneity is commonly observed in several cancers, few studies have shown its presence in EGFR-mutated lung cancer. We performed immunohistochemistry to analyze the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer and performed targeted sequencing in specific cases. We discuss the effects of intratumoral heterogeneity and acquired resistance to EGFR-TKI. MethodsTwenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody and the samples were scored by staining intensity (0–3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing was performed. Single nucleotide variations (SNVs) and copy number variations (CNVs) were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining. ResultsAmong 20 cases, 3 showed heterogeneous staining. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part compared to the negative part, and an increase in the copy number of CCNE1 was observed in the IHC-positive part compared to the negative part in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part compared to the negative part, and an increase in the copy number of MDM2 was observed in the IHC-positive part compared to the negative part. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes. ConclusionThese cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity.

Abstract 2643: Increased copy number of EGFR gene is an independent outcome predictor in resected non-small cell lung cancer patients treated by adjuvant chemotherapy

Abstract Background: Lung cancers are the leading cause of cancer-related deaths worldwide. Surgical resection alone remains preferred treatment for early stage patients with non-small cell lung cancer (NSCLC), whereas surgery plus platinum-based adjuvant chemotherapy is recommended for stages II - IIIa. Despite wide spectrum of known genetic alterations, there is a lack of biomarkers predicting response to adjuvant therapy. The aim of our study was to determine the predictive role of selected gene alterations. Methods and patients: Formalin-fixed paraffin embedded tumor samples were obtained from 209 NSCLC patients with stage I to IIIa. Of these, 59 patients were treated by surgical resection only, the follow-up adjuvant chemotherapy was indicated in remaining 150 cases. There were 146 males and 63 females (mean age 66; range 29-82 years), tumor histology revealed 79 adenocarcinomas, 105 squamous cell carcinomas, 21 large cell carcinomas, two adenosquamous carcinomas and two sarcomatoid carcinomas. Fluorescent in situ hybridization (FISH) was used for evaluation of EGFR, CMYC, MET and FGFR1 gene copy numbers and ALK and ROS1 copy numbers and rearrangements, respectively. EGFR, KRAS and BRAF mutations were screened using Cobas EGFR Mutation Test (Roche), TheraScreen: KRAS Mutation kit (Qiagen) and BRAF p.Val600Glu kit (IntellMed). Results: In the group of 150 resectable NSCLC patients treated with platinum-based adjuvant therapy, increased copy number of EGFR gene predicted poor overall survival (OS) (p = 0.00001, hazard ratio (HR) 1.66, 95% confidence interval (CI) 1.32-2.10) as well as disease-free survival (DFS) (p = 0.0001, HR 1.54, 95% CI 1.23-1.91) in univariate analysis. In multivariate analysis adjusted to age and stage, the EGFR copy number was independent predictor of OS (p = 0.001) as well as DFS (p = 0.0004). In the group of 59 patients treated by surgical therapy only, no correlation of tested markers with clinical-pathological data was identified. Conclusions: Increased copy number of EGFR gene was found to be an independent predictor of outcome in NSCLC patients treated by adjuvant platinum-based therapy. Acknowledgment: This work was financially supported by NPU LO1304, TE02000058 and EATRIS-CZ. Citation Format: Vladimira Koudelakova, Radek Trojanec, Jana Potockova, Jiri Drabek, Jana Stranska, Petra Smickova, Pavla Kourilova, Ivona Grygarkova, Vitezslav Kolek, Jozef Skarda, Marian Hajduch. Increased copy number of EGFR gene is an independent outcome predictor in resected non-small cell lung cancer patients treated by adjuvant chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2643.

Abstract 2200: Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining

Abstract Background: Acquired resistance to EGFR-TKI is an inevitable event for advanced lung cancer patients harboring the EGFR-activating mutation. Although intratumoral heterogeneity is commonly observed in several cancers, such as renal cell carcinoma, few studies have shown its presence in EGFR-mutated lung cancer. We analyzed the intratumoral heterogeneity in EGFR-mutated (L858R) lung cancer through immunohistochemistry (IHC) for the primary site and targeted sequencing for specific cases in order to clarify the mechanism of acquired resistance. Methods: Twenty resected primary lung cancers known to harbor EGFR L858R were analyzed. IHC was performed using an L858R mutant-specific rabbit monoclonal antibody (clone43B2; Japan) and the samples were scored by staining intensity (0-3) and proportion. For cases with heterogeneous L858R protein expression, the nucleic acids were extracted from each differently stained lesion, and targeted sequencing (Oncomine Cancer Research Panel) was performed. Single-nucleotide variations (SNVs) and copy number variations (CNVs) from each site were then analyzed. The cell proliferation and apoptosis were also evaluated by the ki-67 labeling index and TUNEL staining. Results: Among 20 cases, 3 (15%) showed heterogeneous staining, defined as having both an L858R IHC-positive part and an IHC-negative one. Genetic analyses for cases with heterogeneous staining revealed an increase in the copy number of EGFR in the IHC-positive part (CNV 19.54) compared to the negative part (CNV 3.03), and an increase in the copy number of CCNE1 was observed in the IHC-positive part (CNV 18.59) compared to the negative part (CNV 2.59) in one case (case 1). In another case (case 2), an increase in the copy number of EGFR was observed in the IHC-positive part (CNV 8.21) compared to the negative part (CNV 2.90), and an increase in the copy number of MDM2 was observed in the IHC-positive part (CNV 6.65) compared to the negative part (CNV 1.37). Case 3 did not show any CNV. In three cases, no SNV changes were observed. An increase in the ki-67 labeling index in the L858R-positive part in case 1 and increased apoptosis in the L858R-positive part in case 2 were observed, suggesting the functional significance of CNV changes. Conclusion: These cases exhibiting L858R IHC intratumoral heterogeneity suggest a heterogeneous effect on the cell activity due to CNV heterogeneity. Citation Format: Takafumi Hashimoto. Intratumoral heterogeneity of copy number variation in lung cancer harboring L858R via immunohistochemical heterogeneous staining [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2200.

Perioperative chemotherapy with or without epidermal growth factor receptor blockade in unselected patients with locally advanced oesophagogastric adenocarcinoma: Randomized phase II study with advanced biomarker program of the German Cancer Society (AIO/CAO STO-0801)

BackgroundPerioperative chemotherapy significantly improves survival in patients with locally advanced oesophagogastric cancer (EGC). However, as approximately 60% of patients will die from their disease, new therapeutic agents such as molecular-targeted drugs are needed. Patients and methodsTo evaluate the role of panitumumab with perioperative chemotherapy, previously untreated patients with locally advanced EGC received, in an open-label randomised phase II study (NEOPECX), standard epirubicin, cisplatin, capecitabine (ECX) chemotherapy with or without panitumumab. The primary end-point was the histological response rate after neoadjuvant therapy. The expression status and gene copy number of EGFR, HER2, and MET were determined by immunohistochemistry and fluorescence in situ hybridization (FISH). Plasma samples were collected before the first cycle of neoadjuvant chemotherapy. ResultsOverall, 160 patients (80 versus 80) were eligible. The majority (82% versus 80%) showed lymph node involvement. Rate of R0-resection, percentage of patients with downstaging to ypT0-2 at pathohistological evaluation, and rate of major histological response was equal in both arms. Toxicity was increased by panitumumab with regard to thromboembolic events and skin toxicity. Patients with tumour EGFR, HER2 or MET expression had shorter progression-free and overall survival. FISH positivity for these markers was associated with shorter survival independent of therapy. High levels of soluble EGFR in particular predicted poor survival in the panitumumab arm. ConclusionThe addition of panitumumab to ECX did not improve downstaging of locally advanced EGC. Low plasma levels of pathway-associated proteins such as sEGFR may identify a group of patients that benefit from EGFR-directed therapy. ClinicalTrials.govNCT01234324.

Alterations in cancer stem-cell marker CD44 expression predict oncologic outcome in soft-tissue sarcomas

BackgroundCancer stem cells (CSCs) have been shown to resist chemotherapy and promote metastasis after cytotoxic therapies. We sought to determine if the expression of CSC markers (aldehyde dehydrogenase [ALDH], CD44, and epidermal growth factor receptor [EGFR]) predicted outcomes in soft-tissue sarcoma (STS) patients. MethodsWe queried an institutional database of 23 STS patients and evaluated immunohistochemical expression of CSC markers ALDH, CD44, and EGFR. The Cancer Genome Atlas (TCGA) was also queried for STS clinical and genomic data. Disease-specific (DSS) and overall survival (OS) were assessed by univariate and Kaplan–Meier analysis. ResultsOf the 23 institutional patients, the majority was female, had high-grade tumors and had extremity tumors. With a median follow-up of 27 months, nine patients (39%) experienced distant recurrence, and four (17%) died of disease. Mean H-scores at diagnosis (±standard error of the mean) for CD44, ALDH1, and EGFR were 169 ± 27, 77 ± 15, and 144 ± 23, respectively. On univariate analysis, there was a trend for increased CD44 score to predict both worse DSS and OS (hazard ratio = 1.01, 95% confidence interval 1-1.02, P = 0.056), whereas ALDH and EGFR scores did not. Analysis of 74 TCGA STS cases with complete clinical and genomic data revealed that CD44 copy number alterations predicted worse DSS (9.89 months versus 72.5 months, P = 0.007) and a trend for worse OS (14.03 months versus 38.6 months, P = 0.12), whereas ALDH1 and EGFR copy number alteration did not. Multivariate analysis of the combined data sets was consistent with worse DSS among patients with higher CD44 expression. ConclusionsInstitutional and national TCGA data show the association of elevated baseline CD44 expression with worse STS outcomes. Further study of CD44 as a possible novel STS biomarker appears indicated.

Cetuximab plus carboplatin and paclitaxel with or without bevacizumab versus carboplatin and paclitaxel with or without bevacizumab in advanced NSCLC (SWOG S0819): a randomised, phase 3 study

EGFR antibodies have shown promise in patients with advanced non-small-cell lung cancer (NSCLC), particularly with squamous cell histology. We hypothesised that EGFR copy number by fluorescence in-situ hybridisation (FISH) can identify patients most likely to benefit from these drugs combined with chemotherapy and we aimed to explore the activity of cetuximab with chemotherapy in patients with advanced NSCLC who are EGFR FISH-positive. We did this open-label, phase 3 study (SWOG S0819) at 277 sites in the USA and Mexico. We randomly assigned (1:1) eligible patients with treatment-naive stage IV NSCLC to receive paclitaxel (200 mg/m2; every 21 days) plus carboplatin (area under the curve of 6 by modified Calvert formula; every 21 days) or carboplatin plus paclitaxel and bevacizumab (15 mg/kg; every 21 days), either with cetuximab (250 mg/m2 weekly after loading dose; cetuximab group) or without (control group), stratified by bevacizumab treatment, smoking status, and M-substage using a dynamic-balancing algorithm. Co-primary endpoints were progression-free survival in patients with EGFR FISH-positive cancer and overall survival in the entire study population. We analysed clinical outcomes with the intention-to-treat principle and analysis of safety outcomes included patients who received at least one dose of study drug. This study is registered with ClinicalTrials.gov (number NCT00946712). Between Aug 13, 2009, and May 30, 2014, we randomly assigned 1313 patients to the control group (n=657; 277 with bevacizumab and 380 without bevacizumab in the intention-to-treat population) or the cetuximab group (n=656; 283 with bevacizumab and 373 without bevacizumab in the intention-to-treat population). EGFR FISH was assessable in 976 patients and 400 patients (41%) were EGFR FISH-positive. The median follow-up for patients last known to be alive was 35·2 months (IQR 22·9-39·9). After 194 progression-free survival events in the cetuximab group and 198 in the control group in the EGFR FISH-positive subpopulation, progression-free survival did not differ between treatment groups (hazard ratio [HR] 0·92, 95% CI 0·75-1·12; p=0·40; median 5·4 months [95% CI 4·5-5·7] vs 4·8 months [3·9-5·5]). After 570 deaths in the cetuximab group and 593 in the control group, overall survival did not differ between the treatment groups in the entire study population (HR 0·93, 95% CI 0·83-1·04; p=0·22; median 10·9 months [95% CI 9·5-12·0] vs 9·2 months [8·7-10·3]). In the prespecified analysis of EGFR FISH-positive subpopulation with squamous cell histology, overall survival was significantly longer in the cetuximab group than in the control group (HR 0·58, 95% CI 0·36-0·86; p=0·0071), although progression-free survival did not differ between treatment groups in this subgroup (0·68, 0·46-1·01; p=0·055). Overall survival and progression-free survival did not differ among patients who were EGFR FISH non-positive with squamous cell histology (HR 1·04, 95% CI 0·78-1·40; p=0·77; and 1·02, 0·77-1·36; p=0·88 respectively) or patients with non-squamous histology regardless of EGFR FISH status (for EGFR FISH-positive 0·88, 0·68-1·14; p=0·34; and 0·99, 0·78-1·27; p=0·96; respectively; and for EGFR FISH non-positive 1·00, 0·85-1·17; p=0·97; and 1·03, 0·88-1·20; p=0·69; respectively). The most common grade 3-4 adverse events were decreased neutrophil count (210 [37%] in the cetuximab group vs 158 [25%] in the control group), decreased leucocyte count (103 [16%] vs 74 [20%]), fatigue (81 [13%] vs 74 [20%]), and acne or rash (52 [8%] vs one [<1%]). 59 (9%) patients in the cetuximab group and 31 (5%) patients in the control group had severe adverse events. Deaths related to treatment occurred in 32 (6%) patients in the cetuximab group and 13 (2%) patients in the control group. Although this study did not meet its primary endpoints, prespecified subgroup analyses of patients with EGFR FISH-positive squamous-cell carcinoma cancers are encouraging and support continued evaluation of anti-EGFR antibodies in this subpopulation. National Cancer Institute and Eli Lilly and Company.

EGFR Gene Copy Number by FISH May Predict Outcome of Necitumumab in Squamous Lung Carcinomas: Analysis from the SQUIRE Study.

Necitumumab is a monoclonal antibody targeting EGFR. In the SQUIRE trial, the addition of necitumumab to chemotherapy for squamous cell lung cancer significantly improved overall survival (OS) (hazard ratio [HR]= 0.84); in a post hoc analysis, EGFR copy number gain determined by fluorescence in situ hybridization (FISH) showed a trend toward improved OS (HR= 0.70) and progression-free survival (PFS) (HR= 0.71) with the addition of necitumumab. We present the analysis of granular EGFR FISH data from SQUIRE to examine the potential predictive role of high polysomy and gene amplification, as both were included in the FISH-positive category. Available specimens from SQUIRE underwent FISH analysis in a central laboratory, and each sample was evaluated by using the Colorado EGFR scoring criteria. Thecorrelation of granular FISH parameters with clinical outcomes was assessed. Samples were available for 557 of 1093 patients; 208 patients (37.3%) were FISH-positive, including 167 (30.0%) with high polysomy and 41 (7.4%) with gene amplification. In patients with high polysomy, the addition of necitumumab resulted in a statistically significant increase in PFS (6.08 versus 5.13 months [p= 0.044]) and nonstatistically significant increase in OS (12.6 versus 9.5 months [p= 0.133]); among patients with gene amplification, the addition of necitumumab did not significantly improve PFS (7.4 versus 5.6 months; [p= 0.334]) but did improve OS (14.8 versus 7.6 months; [p = 0.033]). EGFR copy number gain by FISH might have arole as a predictive biomarker for necitumumab in squamous cell lung cancer. In our opinion, these data encourage further studies to prospectively evaluate this potential biomarker.

Isocitrate Dehydrogenase Mutations are Better Prognostic Marker than O6-methylguanine-DNA Methyltransferase Promoter Methylation in Glioblastomas - a Retrospective, Single-centre Molecular Genetics Study of Gliomas.

Mutations in isocitrate dehydrogenase 1 and 2 (IDH1/2) are a promising prognostic biomarker of gliomas. The purpose of our study was to examine the clinical prognostic properties of IDH1/2 mutations in a glioma patient cohort from the Czech Republic using an improved platform for simple and reliable IDH genotyping. We retrospectively analyzed a group of 145 glioma patients by testing for the three most frequent IDH mutations, IDH1 R132H, IDH1 R132C, and IDH2 R172K, through the competitive amplification of differentially melting amplicons (CADMA) polymerase chain reaction (PCR). O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation, copy number of EGFR, p53, RB1, MDM2, CDKN2A genes, and deletions in 1p, 19q and 10p chromosomal regions were also analyzed and correlated with clinical characteristics. Of 145 gliomas, 36 harbored IDH1 R132H mutation and 1 IDH1 R132C mutation. We did not detect any IDH2 R172K mutation. IDH1 mutations were positively associated with MGMT methylation (OR 3.08, 95% CI 1.387-7.282; p = 0.007), 1p/19q co-loss (OR 8.85, 95% CI 2.367-42.786; p = 0.002) and negatively associated with epidermal growth factor receptor amplification (OR 0.12, 95% CI 0.019-0.437; p = 0.006) and 10p loss (OR 0.09, 95% CI 0.005-0.436; p = 0.019). The overall survival of IDH-mutant was 25 months, but only 9 months in IDH-wild type gliomas (p = 0.035); at the same time, survival associated with methylated vs. unmethylated MGMT promoter did not significantly differ (p = 0.166). Despite IDH1 mutations being closely associated with MGMT methylation in glioma patients, IDH1 mutations in glioblastoma patients are stronger marker of overall survival than MGMT methylation and should be the marker of choice, especially when using genotyping by CADMA PCR.Key words: isocitrate dehydrogenase - polymerase chain reaction - glioma - glioblastoma.

Genomic copy number gains of ErbB family members predict poor clinical outcomes in glioma patients.

The aim of this study was to investigate copy number of ErbB family members (including EGFR, HER2, HER3 and HER4) in a cohort of gliomas and benign meningiomas (control subjects), and explore the associations of their copy number with clinicopathological characteristics and clinical outcomes of glioma patients. Using real-time quantitative PCR assay, we demonstrated that copy number of EGFR, HER2, HER3 and HER4 in glioma patients was significantly increased compared to control subjects. Moreover, our data also showed that the risk of cancer-related death was positively associated with copy number gain (CNG) of EGFR, HER3 and HER4, but not HER2. CNG of EGFR and HER2 was positively related to radiotherapy, while CNG of HER3 and HER4 was negatively related to chemotherapy. Importantly, EGFR CNG significantly shortened median survival times of glioma patients regardless of gender, tumor grade and therapeutic regimens. Stratified analysis showed that CNG of HER2-4 almost did not influence the survival of male patients, patients with high-grade tumors and patients receiving chemotherapy, but dramatically shortened median survival times of female patients, those with low-grade tumors and those receiving radiotherapy. Collectively, our data not only demonstrate that the members of ErbB family are frequently amplified in gliomas, but also suggest that these common genetic events may be prognostic factors for poor clinical outcomes in glioma patients.

Amplification of the EGFR gene can be maintained and modulated by variation of EGF concentrations in in vitro models of glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common and lethal brain tumor in adults. It is known that amplification of the epidermal growth factor receptor gene (EGFR) occurs in approximately 40% of GBM, leading to enhanced activation of the EGFR signaling pathway and promoting tumor growth. Although GBM mutations are stably maintained in GBM in vitro models, rapid loss of EGFR gene amplification is a common observation during cell culture. To maintain EGFR amplification in vitro, heterotopic GBM xenografts with elevated EGFR copy number were cultured under varying serum conditions and EGF concentrations. EGFR copy numbers were assessed over several passages by quantitative PCR and chromogenic in situ hybridization. As expected, in control assays with 10% FCS, cells lost EGFR amplification with increasing passage numbers. However, cells cultured under serum free conditions stably maintained elevated copy numbers. Furthermore, EGFR protein expression positively correlated with genomic amplification levels. Although elevated EGFR copy numbers could be maintained over several passages in vitro, levels of EGFR amplification were variable and dependent on the EGF concentration in the medium. In vitro cultures of GBM cells with elevated EGFR copy number and corresponding EGFR protein expression should prove valuable preclinical tools to gain a better understanding of EGFR driven glioblastoma and assist in the development of new improved therapies.

551P - Comparative and multimodal analysis of the EGFR, HER2, c-MYC, and MET copy number alteration using in situ hybridization in Korean colorectal cancer patients with integration of array-based copy number data from The Cancer Genome Atlas

551P - Comparative and multimodal analysis of the EGFR, HER2, c-MYC, and MET copy number alteration using in situ hybridization in Korean colorectal cancer patients with integration of array-based copy number data from The Cancer Genome Atlas

EGFR copy number alterations in primary tumors, metastatic lymph nodes, and recurrent and multiple primary tumors in oral cavity squamous cell carcinoma

BackgroundThe EGFR and downstream signaling pathways play an important role in tumorigenesis in oral squamous cell carcinoma (OSCC). Gene copy number alteration is one mechanism for overexpressing the EGFR protein and was also demonstrated to be related to lymph node metastasis, tumor invasiveness and perineural invasion. Therefore, we hypothesized that EGFR gene copy number alteration in the primary tumor could predict amplification in recurrent tumors, lymph node metastatic foci or secondary primary tumors.MethodsWe recruited a group of newly diagnosed OSCC patients (n = 170) between Mar 1997 and Jul 2004. Metastatic lymph nodes were identified from neck dissection specimens (n = 57). During follow-up, recurrent lesions (n = 41) and secondary primary tumors (SPTs, n = 17) were identified and biopsied. The EGFR gene amplifications were evaluated by fluorescence in situ hybridization (FISH) assay in primary tumors, metastatic lymph nodes, recurrences and SPTs.ResultsOf the 170 primary OSCCs, FISH showed low EGFR amplification/polysomy in 19 (11.4%) patients and amplification in 33 (19.8%) patients. EGFR gene amplification was related to lymph node metastasis (χ2 trend test: p = 0.018). Of 57 metastatic lymph nodes, nine (15.8%) had EGFR polysomy and 14 (24.6%) had EGFR gene amplification. The concordance rate of EGFR gene copy number in primary tumors and lymph node metastasis was 68.4% (McNemar test: p = 0.389). Of 41 recurrent tumors, five (12.2%) had EGFR polysomy and five (12.2%) had gene amplification. The concordance rate of EGFR gene copy number between primary tumors and recurring tumors was 65.9% (McNemar test: p = 0.510). The concordance rate between primary tumors and SPTs was 70.6%. EGFR amplification in either primary tumors, metastatic lymph nodes or recurrent tumors had no influence on patient survival.ConclusionWe can predict two-thirds of the EGFR gene copy number alterations in lymph node metastasis or recurrent tumors from the analysis of primary tumors. For OSCC patients who are unable to provide lymph node or recurrent tumor samples for EGFR gene copy number analysis, examining primary tumors could provide EGFR clonal information in metastatic, recurrent or SPT lesions.

Abstract 4156: Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC)

Abstract Monoclonal antibody (mAb) therapy is limited by clinical resistance. For example, alterations in PI3K/Akt /Ras-MAPK signaling, over activation/expression of alternate receptor kinases (c-Met/ IGF-1R), or proteolysis of extracellular domains harboring target epitopes are postulated mechanisms of clinical resistance to trastuzumab. ADCC is a major mechanism of action for mAbs. Most studies have focused on cell models of resistance through in vitro selection, in presence of mAbs, in the absence of immune effector cells. We developed a unique model of immune selection, where target cells (HER2 +ve breast cancer cells SKBR3, BT474; and EGFR +ve colon cancer cells HT29, DLD1) were subjected to ADCC (&amp;gt;90% cell death) with saturating concentrations of trastuzumab or cetuximab (100 µg/ml), respectively, and human peripheral blood mononuclear cells (PBMCs). Selection (in triplicate) was continued for 10 consecutive passages and surviving cells were allowed to grow to confluence over a period of 8-10 weeks. Mock-treated parent cells, IgG1 isotype control, mAb-alone and PBMCs only were utilized as controls. Compared to parent controls, immune-selected DLD1 and SKBR3 cells demonstrated statistically significant increase in proliferation (WST viability assays, P&amp;lt;0.01), and significantly higher colony forming efficiency (soft agar assays, P&amp;lt;0.001); immune-selected HT29 and SKBR3 cells showed significantly higher motility (trans-well migration assays, P&amp;lt;0.05, and P&amp;lt;0.001 respectively). In vitro ADCC assays (calcein labeling) suggested significant reduction (max 20%, p&amp;lt;0.005) in cell lysis in all the immune-selected cell lines compared to parent. Immune-selected HT29 cells demonstrated a 5-fold reduction in gene copy number of EGFR by fluorescence in situ hybridization (FISH, p&amp;lt;0.0001), 2-fold reduction in mRNA levels (qPCR, p&amp;lt; 0.01) and 1.8-fold reduction in protein levels of EGFR (densitometry scans, western blot analyses, p&amp;lt;0.01). Transcriptome-wide next-generation RNA sequencing (Illumina NextSeq 500, 2 x 75 bp paired-end, with over 100 million paired-end reads/sample), coupled with bioinformatic analyses (Reactome pathway database) revealed gene expression changes in ADCC-selected cells. These involve changes in key immune signaling pathways such as class I/II MHC antigen processing, cell chemotaxis, inflammatory and immune regulatory pathways among others. Our data indicate immune-selection by effector cells contributes to ADCC resistance in vitro. Further investigation will determine whether mAb combination with an agonist antibody against CD137 (co-stimulatory NK cell receptor) or Fc-engineered MAbs (e.g. afucosylated) will sensitize the resistant cells. It is hoped that this investigation will better inform mAb therapy by elucidating potentially targetable pathways/ markers that emerge from immune-selection with therapeutic mAbs. Citation Format: Tanuka Biswas, Rebecca Fritzemeier, Adam Mark, Tobias Meißner, Brandon Young, Brian-Leyland Jones, Mark Pegram. Molecular and phenotypic characterization of breast and colorectal cancer cells selected for resistance to antibody-dependent cell-mediated cytotoxicity (ADCC) [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 4156. doi:10.1158/1538-7445.AM2017-4156

Targeted gene sequencing panel for identifying actionable genomic alterations in Taiwanese lung cancer patients.

e20522 Background: Lung cancer boasts an arsenal of targeted therapies directed at various molecular aberrations such as EGFR mutations and fusion genes. Simultaneous assessment for genetic alterations provides biomarkers to assist clinicians in their treatment selections. Methods: A total of 43 FFPE (formalin-fixed, paraffin-embedded) samples obtained from lung cancer patients in Taiwan were subjected to next-generation sequencing (NGS), using a compact panel encompassing 17 potentially actionable, lung cancer-associated genes. NGS was performed on the Ion Torrent PGM or Proton System with a targeted average depth of &gt; 800x. Genomic alterations detected were categorized as single nucleotide variants (SNV) or chromosomal copy number alterations (CNA). Results: A total of 74 putatively actionable genomic alterations were detected across 38 patients (88.4%), which may predict sensitivity or resistance to established and/or therapies that are still in clinical development. 19 patients (44.2%) had tumors harboring activating EGFR mutations in the tyrosine kinase (TK) domain – L858R substitution or exon 19 deletion, which may suggest clinical benefit to first- and second-generation EGFR TK inhibitors (TKIs). Three patients (7.0%) whose tumor had acquired the EGFR T790M mutation were advised to switch to third-generation EGFR TKI. Possible resistance mechanisms to EGFR TKIs via increased copy number of EGFR (14.0%, n = 6) and MET (11.6%, n = 5) were observed. Deletion or loss of CDKN2A gene were detected in 14 patients (32.6%), which may confer sensitivity to CDK4/6 inhibitors. Inhibition of downstream effectors in the RAS/RAF/MEK/ERK and PI3K/AKT/mTOR pathways were suggested for patients whose tumor harbored PIK3CA and/or KRAS oncogenic mutations. Conclusions: Next-generation sequencing using a compact panel of genes may be sufficient to identify biomarkers for targeted therapies selection for most lung cancer patients in Taiwan.

Phase I study (BLOOM) of AZD3759, a BBB penetrable EGFR inhibitor, in EGFRm NSCLC patients with leptomeningeal metastasis (LM) who progressed after other anti-cancer therapy.

2069 Background: AZD3759 is the first EGFR inhibitor primarily designed to effectively cross the blood brain barrier (BBB) to treat central nervous system (CNS) metastases. A phase I study is ongoing to assess AZD3759 in EGFRm NSCLC patients with leptomeningeal metastasis (LM) who progressed after other treatment (NCT02228369). Methods: The primary objective is safety and tolerability, and secondary objectives include anti-tumor efficacy. Two dose levels of AZD3759, 200 mg and 300 mg BID, were assessed. Results: As of24 September, 2016,38 patients were recruited into the expansion cohorts of this phase I study. Of those, 18 were TKI pre-treated EGFRm NSCLC patients with LM. 10 and 8 patients were in the 300 and 200 mg BID cohorts, respectively. No DLTs were observed at either dose, while AZD3759 was better-tolerated at 200 than 300 mg BID during &gt; 2 month treatment. The longest duration on treatment was &gt; 9 months. Most common AEs included skin rash and diarrhea. 67% (12 out of 18) patients had drug-related dose interruption and/or reduction, however, there were no drug-related discontinuations.The Ctrough free plasma and CSF exposure were above pEGFR IC90at the two doses. 40% of patients (4 out of 10) had &gt; 50% inhibition of pEGFR in CSF tumor cells at C1D8, and 70% of patients (7 out of 10) had reduced EGFR copy numbers in CSF at C3D1. 53% of evaluable patients (9 out of 17) had confirmed improved/stable LM MRI imaging. 2 out of 3 patients with concomitant measurable BM lesions achieved confirmed/unconfirmed partial CNS response. Among 18 patients with measurable extracranial lesions, 6 (33%) had confirmed stable extracranial disease as best response. 3 patients achieved clearance of tumor cells in CSF by two consecutive assessments. After 6 months follow-up, 29% patients (5 out of 17 evaluable) were still surviving, plus 5 surviving patients with less than 6 months follow-up. Conclusions: AZD3759 was well-tolerated, achieved sufficient CNS exposure for target inhibition and demonstrated promising anti-tumor activity in patients with LM who progressed after multiple lines of treatment. Updated clinical data will be shared at the meeting. Clinical trial information: NCT02228369.

P1.02-058 EGFR Amplification and Sensitizing Mutations Correlates with Survival from Erlotinib in Lung Adenocarcinoma Patients (MutP-CLICAP)

Tumor heterogeneity, which causes different EGFR mutation abundance, is believed to be responsible for varied progression-free survival (PFS) in lung adenocarcinoma (ADC) patients receiving EGFR-TKI treatment. Frequent EGFR amplification and its common affection in EGFR mutant allele promote the hypothesis that EGFR mutant abundance might be determined by EGFR copy number variation and therefore examination of EGFR amplification status in EGFR mutant patients could predict the efficacy of EGFR-TKI treatment. 72 lung ADC patients who harbored EGFR activating mutations and received erlotinib as first line treatment, were examined for EGFR amplification by FISH. EGFR mutational and copy number status were compared with response, overall-survival (OS), and progression-free-survival (PFS). Median age was 62-yo (r, 20-87 years), 53 patients were females (73%), and 89% have common mutations. Twenty-two (30.6%) samples with EGFR activating mutations were identified with EGFR amplification. EGFR amplification was more frequent in patients with exon 19 deletion (p=0.05) and in those with better performance status (p=0.01). Patients with EGFR gene amplification had a significantly longer PFS than those without [(25.2 months, 95%CI 22.0-38.5) vs. (12.4 months, 95%CI 5.3-19.5); p=0.002] as well as better OS [(EGFR amplified 37.8 months, 95%CI 30.9-44.7) vs. (EGFR non-amplified 27.1 months, 95%CI 12.8-41.3); p=0.009]. EGFR amplification significantly influenced the response to erlotinib (p=0.0001). EGFR amplification occurs in one third of patients with lung ADC harboring EGFR activating mutations, and could serve as an indicator for better response and survival from EGFR-TKI treatment.

MA11.01 Molecular Profiling of Large Cell Neuroendocrine Carcinoma Using Capture-Based Targeted Sequencing

Conventionally, the classification of lung cancer and many other malignancies is determined by the histology of a tumor. Large cell neuroendocrine carcinoma (LCNEC) is traditionally classified as a histological variant of large cell carcinoma (LCC), which is a subtype of non-small cell lung cancer (NSCLC). However, LCNEC exhibits differential cytological, morphological, clinical and biological features than those of classic LCC, thus rendering controversies regarding its classification. In 2015, with the integration of immunohistochemical analyses, the World Health Organization (WHO) has re-classified LCNEC under neuroendocrine tumors. Due to the rareness of LCNEC, few studies have been conducted on the molecular genetic profiling of LCNEC. In this study, we characterized molecular signature associated with a cohort of LCNEC, SCLC and LCC using capture-based targeted sequencing. We performed capture-based targeted sequencing on 30 surgically resected samples from patients with lung cancer using BurningRock Biotech’s OncoScreen Panel. This panel, consisting of all exons and critical introns of 295 genes, covering multiple classes of somatic mutations, including single nucleotide variation (SNVs), rearrangements, copy number variations (CNVs) and insertions and deletions (INDELs), can be used to detect genetic alterations both qualitatively and quantitatively. Among the 30 patients, 15 of them were diagnosed with LCNEC, 5 with LCC and 10 with small cell lung cancer (SCLC). While no statistically significant difference was observed in total number of mutations among the three subtypes, LCC carries the most number of somatic mutations followed by LCNEC then SCLC. Overall, we identified 331 mutations with TP53 being the most frequently mutated gene in all three subtypes. Genes with recurrent somatic mutations detected in LCNEC, but not in LCC or SCLC include RUNX1, ERBB4, BRCA1, and EPHA3. Copy number analysis revealed a higher prevalence of CNV in LCNEC, with 60% of cases harboring such alteration. There is no common CNVs shared among all three subtypes. NFкBIA amplification is the only common CNV found in both LCNEC and LCC; and AKT2 amplification is shared by LCNEC and SCLC. Most CNVs are subtype-specific. Interestingly, one RET-fusion was discovered in one LCC sample and one EGFR exon 19 deletion accompanied by EGFR copy number amplification was discovered in one LCNEC sample. Targeted deep sequencing reveals distinct genetic profile for LCNEC compared to LCC and SCLC. LCNEC harbors more CNV and contains a panel of genes, including RUNX1, ERBB4, BRCA1 and EPHA3 that are more frequently mutated comparing to LCC and SCLC.

EGFR Gene Amplification and KRAS Mutation Predict Response to Combination Targeted Therapy in Metastatic Colorectal Cancer.

Genetic variability in KRAS and EGFR predicts response to cetuximab in irinotecan refractory colorectal cancer. Whether these markers or others remain predictive in combination biologic therapies including bevacizumab is unknown. We identified predictive biomarkers from patients with irinotecan refractory metastatic colorectal cancer treated with cetuximab plus bevacizumab. Patients who received cetuximab plus bevacizumab for irinotecan refractory colorectal cancer in either of two Phase II trials conducted were identified. Tumor tissue was available for 33 patients. Genomic DNA was extracted and used for mutational analysis of KRAS, BRAF, and p53 genes. Fluorescence in situ hybridization was performed to assess EGFR copy number. The status of single genes and various combinations were tested for association with response. Seven of 33 patients responded to treatment. KRAS mutations were found in 14/33 cases, and 0 responded to treatment (p=0.01). EGFR gene amplification was seen in 3/33 of tumors and in every case was associated with response to treatment (p<0.001). TP53 and BRAF mutations were found in 18/33 and 0/33 tumors, respectively, and there were no associations with response to either gene. EGFR gene amplification and KRAS mutations are predictive markers for patients receiving combination biologic therapy of cetuximab plus bevacizumab for metastatic colorectal cancer. One marker or the other is present in the tumor of half of all patients allowing treatment response to be predicted with a high degree of certainty. The role for molecular markers in combination biologic therapy seems promising.