Somatic Epidermal Growth Factor Receptor Research Articles

AI-based pipeline for early screening of lung cancer: integrating radiology, clinical, and genomics data

The prognosis of lung carcinoma has changed since the discovery of molecular targets and their specific drugs. Somatic Epidermal Growth Factor Receptor (EGFR) mutations have been reported in lung carcinoma, and these mutant proteins act as substrates for targeted therapies. However, in a resource-constrained country like India, panel-based next-generation sequencing cannot be made available to the population at large. Additional challenges such as adequacy of tissue in case of lung core biopsies and locating suitable tumour tissues as a result of innate intratumoral heterogeneity indicate the necessity of an AI-based end-to-end pipeline capable of automatically detecting and learning more effective lung nodule features from CT images and predicting the probability of the EGFR-mutant. This will help the oncologists and patients in resource-limited settings to achieve near-optimal care and appropriate therapy. The EGFR gene sequencing and CT imaging data of 2277 patients with lung carcinoma were included from three cohorts in India and a White population cohort collected from TCIA. Another cohort LIDC-IDRI was used to train the AIPS-Nodule (AIPS-N) model for automatic detection and characterisation of lung nodules. We explored the value of combining the results of the AIPS-N with the clinical factors in the AIPS-Mutation (AIPS-M) model for predicting EGFR genotype, and it was evaluated by area under the curve (AUC). AIPS-N achieved an average AP50 of 70.19% in detecting the location of nodules within the lung region of interest during validation and predicted the score of five lung nodule properties. The AIPS-M machine learning (ML) and deep learning (DL) models achieved AUCs ranging from 0.587 to 0.910. The AIPS suggests that CT imaging combined with a fully automated lung-nodule analysis AI system can predict EGFR genotype and identify patients with an EGFR mutation in a cost-effective and non-invasive manner. This work was supported by a grant provided by Conquer Cancer Foundation of ASCO [2021IIG-5555960128] and Pfizer Products India Pvt. Ltd.

Advanced Non-small Cell Lung Cancer: EGFR Mutation Analysis Using Pyrosequencing and the Fully Automated qPCR-Based IdyllaTM System.

Epidermal growth factor receptor (EGFR) mutation status is of a major clinical significance in non-small cell lung cancer (NSCLC) management, as it guides therapeutic decision making to target patients for a better response to therapy. This implicates the introduction of EGFR mutation analysis as the standard of care for Moroccan NSCLC patients, which in itself entails the implementation of targeted methods for routine EGFR mutation analysis in our laboratories. In this study, we aimed to present 2 targeted methods for EGFR mutation identification and to determine the prevalence and spectrum of EGFR mutations in NSCLC Moroccan patients. A retrospective investigation of a cohort of 340 patients was undertaken to analyze somatic EGFR mutations in exons 18 to 21 using pyrosequencing and the IdyllaTM system. Of the enrolled patients, 70.9% were males and 29.1% were females. Predominately, 92% of cases had adenocarcinoma, and 53.7% of patients self-reported a history of smoking. Overall, 73 patients (21.7%) harbored an EGFR mutation, the most prevalent of which were the exon 19 deletions (53.4%) followed by exon 21 substitutions (31%). Exon 18 mutations and exon 20 alterations occurred in 8.1% and 6.7% of the positive EGFR mutation cases, respectively. Of the analyzed cases, all of the EGFR-mutated patients had adenocarcinoma. EGFR mutation prevalence was significantly higher in females (females vs males: 38.4% vs 14.5%, P < .001) and non-smokers (non-smokers vs non-smokers: 36% vs 10.3%, P < .001). The featured pyrosequencing and the IdyllaTM system are targeted methods endowed with high sensitivity and specificity as well as other compelling characteristics which make them great options for routine EGFR mutation testing for advanced NSCLC patients. These findings underline the imperious need for implementing quick and efficient targeted methods for routine EGFR mutation testing among NSCLC patients, which is particularly useful in determining patients who are more likely to benefit from targeted therapy.

Abstract 75: Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™

Abstract Liquid biopsies are a minimally invasive sampling approach to overcome the heterogeneity of tumors and represent a valuable source of circulating tumor DNA (ctDNA) for oncological biomarker analysis. ctDNA measurements require a highly sensitive and reliable detection technology to quantify often low-level genetic aberrations within a high background of wild-type sequences. Digital PCR emerged as a powerful technology for the next-generation analysis of liquid biopsies. Stilla Technologies' 6-color naica® system is an ultrasensitive, easy-to-use digital PCR platform capable of simultaneously and precisely quantifying high numbers of biomarkers in a single reaction. Non-small cell lung cancer (NSCLC) is a leading cause of cancer mortality worldwide. Epidermal growth factor receptor (EGFR) is frequently mutated in NSCLC. A handful of anti-EGFR Tyrosine Kinase Inhibitor therapies are approved by the FDA; however, most patients develop resistance over time to these treatments. Both monitoring primary EGFR mutations to predict treatment response and precociously detecting resistance mutations to adapt treatments promptly through ctDNA analysis stand to improve the effectiveness of NSCLC patient management. Stilla has developed a highly multiplexed 6-color Crystal Digital PCR EGFR kit detecting more than 90% of EGFR mutations described in NSCLC from ctDNA. The assay detects 32 common and rare somatic EGFR mutations in exons 18, 19, 20, and 21, including both activating and resistant mutations. In this work, we evaluated the sensitivity, precision and specificity of the EGFR 6-color Crystal Digital PCR assay highly specific and sensitive for the detection of EGFR mutations, with a Limit of Detection in a high background of wild-type DNA ranging from 0.30 to 0.46 cp/μL (with observed MAFs ranging from 0.06 to 0.09%). Moreover, a high concordance was observed between those EGFR results obtained for NSCLC ctDNA samples using 6-color Crystal Digital PCR and other technologies. With a rapid time to results and straightforward ctDNA analysis workflow, Crystal Digital PCR on the naica system promises ultrasensitive, highly multiplexed 6-color mutation detection, maximizing the information obtained from precious samples. Citation Format: Cécile Jovelet, Myrtille Remy, Noémie Pata-Merci, Damien Vasseur, Ludovic Lacroix, Claudia Labrador-Rached, Allison C. Mallory. Highly multiplexed EGFR mutation detection from liquid biopsy samples using the 6-color Crystal Digital PCR™ [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 75.

Efficacy of liquid biopsy for disease monitoring and early prediction of tumor progression in EGFR mutation-positive non-small cell lung cancer.

15–40% of non-small cell lung cancer (NSCLC) patients harbor epidermal growth factor receptor (EGFR)-sensitizing mutations. Tyrosine kinase inhibitors (TKIs) provide significant clinical benefit in this population, yet all patients will ultimately progress. Liquid biopsy can reliably identify somatic tumor-associated EGFR mutations in plasma. This study aimed to assess the feasibility and value of the quantitative assessment of EGFR driver mutations in plasma in EGFR-mutated NSCLC patients treated with EGFR-TKIs as a tool to evaluate therapeutic response to TKIs and monitor for disease progression. The study included 136 patients with tissue biopsy-confirmed EGFR-sensitizing, mutation-positive lung adenocarcinoma with plasma collected prior to TKI treatment and at least two post-initiation TKI treatment/follow-up blood samples. Plasma samples were tested with the cobas® EGFR Mutation Test v2 (cobas EGFR Test), and semi-quantitative index (SQI) values for each identified mutation were reported by the assay software. The most common baseline EGFR mutations detected in tissue were L858R (53.7%) and exon 19 deletion (39.7%). Plasma cell-free DNA analysis detected EGFR mutations in 74% of the baseline samples. Objective response rate by RECIST 1.1 was achieved in 72% of patients, while 93% had a molecular response (defined as disappearance of the EGFR mutation from plasma). 83% of patients had molecular progression (MP; 1.5X SQI increase or new T790M mutation), and 82% who had a clinical response had clinical progression. On average, MP occurred 42 days prior to clinical progression. Patients who progressed while on first-line TKI showed MP of the original EGFR-sensitizing mutations prior to the emergence of a T790M mutation, which was detected in 27% of the EGFR plasma-positive patients. Longitudinal monitoring of EGFR mutational load in plasma is feasible and can predict both response and clinical progression in EGFR-mutated NSCLC patients treated with EGFR-TKIs, as well as detect treatment-emergent EGFR mutations.

Epidermal growth factor receptor mutations in adenocarcinoma lung: Comparison of techniques for mutation detection.

Targeted therapy using tyrosine kinase inhibitors in cases of non-small-cell lung carcinoma (NSCLC) that harbor epidermal growth factor receptor (EGFR) mutations has drastically improved the overall survival rate. The current study estimated the frequency of EGFR mutations in the Indian population by analyzing the diagnostic parameters of various techniques available for the detection of these mutations. A case series of 100 histologically diagnosed and immunohistochemically confirmed NSCLC with the adenocarcinoma phenotype comprises the study sample. EGFR mutations were detected using clone-specific immunohistochemistry (IHC), real-time polymerase chain reaction (PCR), and Sanger sequencing. EGFR mutations were identified in 48% cases with 72.78% mutations involving exon 19. Clone-specific IHC had a low sensitivity of 46.43%, and the specificity was 79.17%. Sanger sequencing yielded interpretable results in 16% cases only, which were in concordance with the results of real-time PCR. EGFR mutations are increasingly being explored for targeted therapy and personalized medicine. Real-time PCR was found to be the best and the most accurate method for the detection of somatic EGFR mutations in adenocarcinoma primarily in the lungs.

Mutation patterns of epidermal growth factor receptor gene in non-small cell lung cancer among Egyptian patients

ABSTRACT Epidermal growth factor receptor (EGFR) mutations have been reported to be associated with non–small cell lung cancer (NSCLC) and correlated to the responsiveness of tumors to EGFR tyrosine kinase inhibitors (TKIs). EGFR mutations in NSCLC Egyptian patients was investigated in formalin-fixed paraffin-embedded lung tumor tissues of 120 NSCLC patients and 20 control tissues from patients with benign lung tumor using ViennaLab StripAssay. Patients showed higher rates of females (87/120, 72.5%) (P=0.043) and never-smokers (82/120, 68.3%) (P=0.003), than the control group. EGFR mutations were significantly related to NSCLC adenocarcinoma, where 49 (40.8%) of patients were mutant (P=0.013) compared with the control group who have no mutations. EGFR mutations were most found in exons 1821, whereas the most mutated exons were exon 19 (55.1%) and exon 21 (26.5%). While exon 18 (10.2%) and exon 20 (8.2%) were the less mutated exons. Moreover, the most common mutations were; L858R (Leu858Arg) in exon 21 and L747-P753 in exon 19; representing 22.4% and 18.4% of all mutations; respectively. Our findings imply that, somatic EGFR mutations could be helpful for NSCLC diagnosis and can be used in combination with clinical factors to select the patients in Egypt who will respond more effectively to TKIs.

Profiling Oncogenic Germline Mutations in Unselected Chinese Lung Cancer Patients.

IntroductionEmerging evidence has suggested that inherited factors are also involved in lung cancer development. However, most studies focused on well-elucidated cancer predisposition genes, the majority of which are tumor suppressor genes. The profile of germline mutations in oncogenic driver genes remains unrevealed, which might also provide potential clinical implications for lung cancer management.MethodsSequencing data from 36,813 unselected lung cancer patients who underwent somatic mutation profiling were retrospectively reviewed. All recruited patients had matched white blood cell samples sequenced in parallel using a capture-based panel including eight key lung cancer driver genes (epidermal growth factor receptor (EGFR), anaplastic lymphoma kinase (ALK), MET proto-oncogene, receptor tyrosine kinase (MET), Kirsten rat sarcoma viral oncogene homolog (KRAS), Erb-B2 receptor tyrosine kinase 2(ERBB2), ROS proto-oncogene 1, receptor tyrosine kinase (ROS1), ret proto-oncogene (RET), and B-Raf proto-oncogene, serine/threonine kinase (BRAF)). Likely pathogenic/pathogenic (LP/P) variants were called according to the classification criteria of the American College of Medical Genetics and Genomics. Variants of uncertain significance (VUS) located in the kinase domains of driver genes and occurring recurrently (n ≥3) were also included for further analyses.ResultsSeven different LP/P variants in EGFR, MET, or RET were identified in 0.03% of lung cancer patients (n = 14) and 25 different VUS in the kinase domains of seven driver genes (except KRAS) were found with a prevalence of 0.3% (n = 117).Collectively, germline mutations were most frequently seen in ROS1 (n = 31, 0.084%), followed by MET (n = 23, 0.062%), EGFR (n = 22, 0.06%), ALK (n = 22, 0.06%) and RET (n = 17, 0.046%). LP/P variants and VUS fell the most commonly in EGFR (n = 10, 72%) and ROS1 (n = 31, 26%), respectively. Of the 10 patients with EGFR LP/P germline mutation, 70% also acquired somatic EGFR driver mutation exon21 p.L858R or exon19 deletion at baseline; while the three patients with pathogenic germline RET mutation displayed distinct baseline somatic profiles of rare EGFR mutation or KRAS exon2 p.G12C. We discovered 11 germline mutations that also occurred somatically, including four LP/P variants and seven VUS.ConclusionWe present the first study to systemically characterize the germline mutation in oncogenic driver genes in a large cohort of unselected patients with lung cancers.

Cell Free Tumoral DNA Versus Paraffin Block Epidermal Growth Factor Receptor Mutation Detection in Patients with Non-Small Cell Lung Cancer.

Introduction:Increasing knowledge about the molecular profile of tumors has led to personalized treatment for achieving better outcomes in patients with nonsmall cell lung cancer (NSCLC). Currently, finding exact somatic genomic changes of tumor has gained great importance. On the other hand, crescendoing needs to actual tumor tissue at different time points during cancer treatment may produce major discomfort for NSCLC patients. Tumor genomes can be reconstructed by information obtained from circulating cell-free deoxyribonucleic acid (cfDNA) of peripheral blood. cfDNA may be represented as a suitable alternative test for epidermal growth factor receptor (EGFR) mutation detection in these patients. This study aimed to assess validity of cfDNA in somatic EGFR mutation identification in Iranian NSCLC cases. Methods:Somatic mutation of EGFR gene was studied in both tissue specimens and plasma. Then, mutations were detected by polymerase chain reaction(PCR) and sequencing. Results:We observed a high concordance (90%) between tissue samples and cfDNA for EGFR gene mutation. The sensitivity, accuracy, and positive precision value were 90%, 90% and 100%, respectively. A false negative rate of 10% was also demonstrated in this study. Conclusion:We established sensitive methods for detecting EGFR gene mutation which may be very useful in clinical practice.

Abstract 1412: High EGFR somatic mutation frequency targeting the TK domain in colorectal cancer patients with microsatellite instability (MSI)

Abstract CRCs arise through genetic changes that impact various driver genes and in some tumors increased mutation rate in microsatellite unstable tumors. The hypermutable phenotype associated with microsatellite instability (MSI) results from loss of the mismatch repair system (MMR) activity. MSI is detected in 15% of all CRCs, and such tumors have a better prognosis and different chemotherapeutic outcome patterns, including high clinical benefit from immune checkpoint therapy or relative resistance to 5-FU as compared to microsatellite stable (MSS) tumors. The epidermal growth factor receptor (EGFR) signaling pathway plays an essential role in carcinogenesis of CRCs and is known to be overexpressed in MSI CRCs while highly mutated in Lung cancer. We analyzed the mutation frequency of deregulated pathways including EGFR and its downstream genes KRAS and BRAF in CRCs. We found a significantly elevated EGFR mutation frequency in CRC MSI-H subtype (45.5% vs. 6.5% in MSS CRCs, p&amp;lt;0.0000001). Although KRAS and NRAS are mutated with high frequency in both MSI-H and MSS groups, BRAF corresponding to RTK-RAS pathways is more altered in MSI-H than MSS CRCs (32.67% vs 13.10%; p=0.001), consistent with the known association between MSI-H CRCs and BRAF mutations. We hypothesized that there might be a mutation pattern in EGFR in CRC subtypes that provide a rationale for EGFR-targeted therapy for a subtype of CRC. Of 1104 profiled CRCs in the COSMIC v73 database, somatic EGFR mutations were mapped for 101 MSI-High versus 916 MSS CRCs. EGFR mutations mapped on the protein structure revealed that mutations are mainly targeting tyrosine kinase (TK) domain while the extracellular domain remained mostly wild-type with potential for targeting by anti-EGFR monoclonal antibodies. It is known that downstream genes such as KRAS as well as expression levels of EGFR ligands can affect the sensitivity of CRCs to anti-EGFR therapy. We didn’t detect any difference in mRNA expression level between MSI-H and MSS group but our analysis is indicative of the presence of G12D mutations in a high proportion of KRAS-mutated MSS CRCs, therefore resistance to anti-EGFR antibodies such as cetuximab would be expected. However, EGFR tyrosine kinase inhibitors such as erlotinib and gefitinib could be considered for further testing in patients with MSI-H tumors that have a lower frequency of G12D KRAS mutations and may have mutant EGFR. Citation Format: Safoora Deihimi, Michael Slifker, Eric A. Ross, Wafik S. El-Deiry. High EGFR somatic mutation frequency targeting the TK domain in colorectal cancer patients with microsatellite instability (MSI) [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 1412. doi:10.1158/1538-7445.AM2017-1412

An improved CTC isolation scheme for pairing with downstream genomics: Demonstrating clinical utility in metastatic prostate, lung and pancreatic cancer

Improvements in technologies to yield purer circulating tumor cells (CTCs) will enable a broader range of clinical applications. We have previously demonstrated the use of a commercially available cell-adhesion matrix (CAM) assay to capture invasive CTCs (iCTCs). To improve the purity of the isolated iCTCs, here we used fluorescence-activated cell sorting (FACS) in combination with the CAM assay (CAM + FACS). Our results showed an increase of median purity from the CAM assay to CAM + FACS for the spiked-in cell lines and patient samples analyzed from three different metastatic cancer types: castration resistant prostate cancer (mCRPC), non-small cell lung cancer (mNSCLC) and pancreatic ductal adenocarcinoma cancer (mPDAC). Copy number profiles for spiked-in mCRPC cell line and mCRPC patient iCTCs were similar to expected mCRPC profiles and a matched biopsy. A somatic epidermal growth factor receptor (EGFR) mutation specific to mNSCLC was observed in the iCTCs recovered from EGFR+ mNSCLC cell lines and patient samples. Next-generation sequencing (NGS) of spiked-in pancreatic cancer cell line and mPDAC patient iCTCs showed mPDAC common mutations. CAM + FACS iCTC enrichment enables multiple downstream genomic characterizations across different tumor types.

Enhanced gefitinib-induced repression of the epidermal growth factor receptor pathway by ataxia telangiectasia-mutated kinase inhibition in non-small-cell lung cancer cells.

The epidermal growth factor receptor (EGFR) tyrosine kinase signaling pathways regulate cellular activities. The EGFR tyrosine kinase inhibitors (EGFR‐TKIs) repress the EGFR pathway constitutively activated by somatic EGFR gene mutations and have drastically improved the prognosis of non‐small‐cell lung cancer (NSCLC) patients. However, some problems, including resistance, remain to be solved. Recently, combination therapy with EGFR‐TKIs and cytotoxic agents has been shown to improve the prognosis of NSCLC patients. To enhance the anticancer effects of EGFR‐TKIs, we examined the cross‐talk of the EGFR pathways with ataxia telangiectasia‐mutated (ATM) signaling pathways. ATM is a key protein kinase in the DNA damage response and is known to phosphorylate Akt, an EGFR downstream factor. We found that the combination of an ATM inhibitor, KU55933, and an EGFR‐TKI, gefitinib, resulted in synergistic cell growth inhibition and induction of apoptosis in NSCLC cell lines carrying the sensitive EGFR mutation. We also found that KU55933 enhanced the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors. ATM inhibition may facilitate the gefitinib‐dependent repression of the phosphorylation of EGFR and/or its downstream factors, to exert anticancer effects against NSCLC cells with the sensitive EGFR mutation.

Genotyping of EGFR Mutations from Bronchial Cytological Specimens in Slovakian Lung Cancer Patients.

Non-small cell lung cancer (NSCLC) is a histologically and molecularly heterogeneous disease predominating in Slovakia among newly diagnosed oncological disorders and leading in the number of associated deaths. NSCLC diagnostics has advanced especially in molecular typing of epidermal growth factor receptor (EGFR) and subsequent targeted molecular therapy using tyrosine-kinase inhibitor(s) (TKI). The selection of patients for targeted therapy, we describe in this study, is mostly guided through bronchial smears rather than more invasive biopsies. We identified 32 adenocarcinomas, 40 squamous-cell carcinomas, 12 large-cell carcinomas, along with two unspecified carcinomas, in the NSCLC group who had bronchial smears taken. The assessment of tumor cell number, and genomic DNA allowed for screening of clinically relevant somatic EGFR mutations in 86 patients. Using quantitative PCR, 12 patients (14 %) were recommended for EGFR-TKI therapy. The most prevalent EGFR HIT-a in the somatosome, terms introduced and defined in this study, were exon 19 deletions, which were found in combination with the TKI-resistant p.T790M mutation in exon 20 in one patient. The study describes a method that is minimally invasive, reliable, and meets all criteria of routine molecular diagnostics. A multidisciplinary approach of EGFR genotyping from bronchial smears implemented in the study allows expanding targeted molecular therapy in NSCLC patients.

Heterogeneity in resistance mechanisms causes shorter duration of epidermal growth factor receptor kinase inhibitor treatment in lung cancer.

Epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs) are used as a first line therapy for metastatic lung cancer harboring somatic EGFR mutation. However, acquisition of resistance to these drugs is almost inevitable. T790M (threonine to methionine substitution at codon 790 of the EGFR gene) and MET amplification are well-known resistance mechanisms, and we previously demonstrated that three of six autopsied patients showed inter-tumor heterogeneity in resistance mechanisms by analyzing T790M and MET gene copy number (Suda et al., 2010). To further elucidate the role of heterogeneity in acquired resistance, here we performed further analyses including additional five patients. We analyzed somatic mutations in 50 cancer-related genes for 26 EGFR-TKI refractory lesions from four autopsied patients using target sequencing. MET and ERBB2 copy numbers were analyzed by real-time PCR. Data for additional one patient was obtained from our recent study (Suda et al., 2015). Relationship between heterogeneity in resistance mechanism(s) and time to treatment failure (TTF) of EGFR-TKI and post-progression survival (PPS) were analyzed. We observed heterogeneity of resistance mechanisms in two of four patients analyzed (T790M+MET gene copy number gain, and mutant EGFR loss+unknown). We also identified quantitative heterogeneity in EGFR T790M mutation ratio among EGFR-TKI refractory lesions. In analyzing patient outcomes, we found that patients who developed multiple resistance mechanisms had shorter TTF compared with those who developed single resistance mechanism (p=0.022). PPS after EGFR-TKI treatment failure was compatible between these two groups (p=0.42). These findings further our understanding of acquired resistance mechanisms to EGFR-TKIs, and may lead to better treatment strategies after acquisition of resistance to first generation EGFR-TKIs in lung cancer patients with EGFR mutations.

Abstract 121: The EGFR gatekeeper mutation T790M is present in selected patients with early breast cancer

Abstract Introduction. The epidermal growth factor receptor (EGFR) is one of the major oncogenes in a variety of human cancers including breast cancer (BC). While EGFR overexpression and/or amplification has been shown to occur frequently in human breast cancer, EGFR-activating mutations are suggested to be rare if not absent. Thus, the aim of our study was to examine the presence of somatic EGFR gene mutations in a group of norwegian patients diagnosed with early breast cancer. Patients. We investigated tumor biopsies obtained from 132 patients with early breast cancer treated at the Akershus University Hospital (University of Oslo) in 2007/2008. The majority of patients (n = 82) belonged to the luminal- A/B subtypes of BC. In addition, 33 patients were classified as HER-2 positive patients (IHC 3+ or amplification verified by FISH) and 17 as triple-negative breast cancer (TNBC) patients. Methods. Briefly, DNA was extracted from formalin-fixed, paraffin-embedded (FFPE) tumor tissue using QIAsymphony DSP DNA Mini Kit (Qiagen Inc.). The extraction of DNA was performed by fully automated purification using the QIAsymphony SP/AS system. EGFR-mutations were detected using a Therascreen EGFR RGQ PCR kit according to the manufacturer´s protocol. We tested for point mutations in exon 18 (G719A/C/S), in-frame deletions in exon 19 (ΔE746-A750), in-frame insertions as well as point mutations in exon 20 (S768I, T790M) and a point mutation in exon 21 (L858R). Results. In the present study we detected three individuals with an EGFR-T790M mutation. Two of the patients were diagnosed with TNBC, while the disease of the third patient was classified as a luminal-A type breast cancer. Interestingly, the T790M-mutation was present in the biopsies obtained during surgery before adjuvant therapy was initiated. No anti-cancer therapy has been given prior to surgery. According to our best knowledge, the T790M-”TKI-resistance-mutation” has not been detected in early breast cancer previously. This finding contrasts the observations made in lung cancer patients where the T790M mutation in general is a classical second mutation causing drug resistance during ongoing TKI-therapy. No other EGFR-mutations were detected in our patients. Conclusion: To our best knowledge we provide first evidence for the presence of the EGFR-T790M-mutation in human early breast cancer. Thus, treatment with novel EGFR-targeting cancer drugs (like “pan-HER-inhibitors”) affecting also T790M-mutated-EGFR may be worthwhile to be tested in highly selected breast cancer patients. Citation Format: Vahid Bemanian, Torill Sauer, Joel Touma, Bjørn A. Lindstedt, Ying Chen, Hilde P. Ødegård, Ida R. Bukholm, Jürgen Geisler. The EGFR gatekeeper mutation T790M is present in selected patients with early breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 121. doi:10.1158/1538-7445.AM2015-121

JAK2 inhibitor TG101348 overcomes erlotinib-resistance in non-small cell lung carcinoma cells with mutated EGF receptor

Non-small cell lung cancer (NSCLC) patients with epidermal growth factor receptor (EGFR) mutations are responsive to EGFR-tyrosine kinase inhibitor (EGFR-TKI). However, NSCLC patients with secondary somatic EGFR mutations are resistant to EGFR-TKI treatment. In this study, we investigated the effect of TG101348 (a JAK2 inhibitor) on the tumor growth of erlotinib-resistant NSCLC cells. Cell proliferation, apoptosis, gene expression and tumor growth were evaluated by diphenyltetrazolium bromide (MTT) assay, flow cytometry, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling (TUNEL) staining, Western Blot and a xenograft mouse model, respectively. Results showed that erlotinib had a stronger impact on the induction of apoptosis in erlotinib-sensitive PC-9 cells but had a weaker effect on erlotinib-resistant H1975 and H1650 cells than TG101348. TG101348 significantly enhanced the cytotoxicity of erlotinib to erlotinib-resistant NSCLC cells, stimulated erlotinib-induced apoptosis and downregulated the expressions of EGFR, p-EGFR, p-STAT3, Bcl-xL and survivin in erlotinib-resistant NSCLC cells. Moreover, the combined treatment of TG101348 and erlotinib induced apoptosis, inhibited the activation of p-EGFR and p-STAT3, and inhibited tumor growth of erlotinib-resistant NSCLC cells in vivo. Our results indicate that TG101348 is a potential adjuvant for NSCLC patients during erlotinib treatment.

Epidermal growth factor receptor (EGFR) mutations in lung cancer: preclinical and clinical data

Lung cancer leads cancer-related mortality worldwide. Non-small-cell lung cancer (NSCLC), the most prevalent subtype of this recalcitrant cancer, is usually diagnosed at advanced stages, and available systemic therapies are mostly palliative. The probing of the NSCLC kinome has identified numerous nonoverlapping driver genomic events, including epidermal growth factor receptor (EGFR) gene mutations. This review provides a synopsis of preclinical and clinical data on EGFR mutated NSCLC and EGFR tyrosine kinase inhibitors (TKIs). Classic somatic EGFR kinase domain mutations (such as L858R and exon 19 deletions) make tumors addicted to their signaling cascades and generate a therapeutic window for the use of ATP-mimetic EGFR TKIs. The latter inhibit these kinases and their downstream effectors, and induce apoptosis in preclinical models. The aforementioned EGFR mutations are stout predictors of response and augmentation of progression-free survival when gefitinib, erlotinib, and afatinib are used for patients with advanced NSCLC. The benefits associated with these EGFR TKIs are limited by the mechanisms of tumor resistance, such as the gatekeeper EGFR-T790M mutation, and bypass activation of signaling cascades. Ongoing preclinical efforts for treating resistance have started to translate into patient care (including clinical trials of the covalent EGFR-T790M TKIs AZD9291 and CO-1686) and hold promise to further boost the median survival of patients with EGFR mutated NSCLC.

Egfr Ca Repeat Polymorphism Predict Clinical Outcome in Egfr Mutation Positive Nsclc Patients Treated with Erlotinib

Aim: Somatic mutations in the epidermal growth factor receptor (EGFR) are predictors of efficacy for treatment with the EGFR tyrosine kinase inhibitor erlotinib in non-small cell lung cancer (NSCLC). However, not all patients with EGFR mutations respond to erlotinib and the duration of the response varies markedly between patients. ACA repeat polymorphism in intron 1 of the EGFR gene influences the transcription of the EGFR gene. Here the association between the CA repeat polymorphism and outcome in NSCLC patients treated with erlotinib is evaluated. Methods: The number of CA repeats in the EGFR gene was evaluated in 432 advanced NSCLC patients treated with erlotinib irrespective of EGFR mutation status. Patients were dichotomized into harboring short allele (CA ≤16 in any allele) or long alleles (CA > 16 in both alleles). Number of repeats was correlated with clinical characteristic and outcome. A subgroup analysis was performed based on the somatic EGFR mutation status. Results: We demonstrate a significant better median progression free survival (HR= 0.39 (0.22-0.70); p = 0.002) and overall survival (HR= 0.43, (0.23-0.78); p = 0.006) in EGFR mutation positive patients (N = 62) harboring a short allele versus a long allele (median follow-up time of 52.2 months). The result remained highlysignificant in a multivariate Cox proportional hazards model. This correlation was not seen in EGFR mutation negative patients. Further, there were an association between having a short allele and developing the L585R mutation in exon 21 and between long allele and exon 20 insertions. Conclusions: We demonstrate that in EGFR mutation positive NSCLC patients treated with erlotinib a low number of CA repeats in intron 1 of the EGFR gene is a predictor for longer progression free survival and overall survival. Disclosure: All authors have declared no conflicts of interest.

Toward precision medicine with next-generation EGFR inhibitors in non-small-cell lung cancer.

The use of genomics to discover novel targets and biomarkers has placed the field of oncology at the forefront of precision medicine. First-generation epidermal growth factor receptor (EGFR) inhibitors have transformed the therapeutic landscape of EGFR mutant non-small-cell lung carcinoma through the genetic stratification of tumors from patients with this disease. Somatic EGFR mutations in lung adenocarcinoma are now well established as predictive biomarkers of response and resistance to small-molecule EGFR inhibitors. Despite early patient benefit, primary resistance and subsequent tumor progression to first-generation EGFR inhibitors are seen in 10%–30% of patients with EGFR mutant non-small-cell lung carcinoma. Acquired drug resistance is also inevitable, with patients developing disease progression after only 10–13 months of antitumor therapy. This review details strategies pursued in circumventing T790M-mediated drug resistance to EGFR inhibitors, which is the most common mechanism of acquired resistance, and focuses on the clinical development of second-generation EGFR inhibitors, exemplified by afatinib (BIBW2992). We discuss the rationale, mechanism of action, clinical efficacy, and toxicity profile of afatinib, including the LUX-Lung studies. We also discuss the emergence of third-generation irreversible mutant-selective inhibitors of EGFR and envision the future management of EGFR mutant lung adenocarcinoma.

Assessing standardization of molecular testing for non-small-cell lung cancer: results of a worldwide external quality assessment (EQA) scheme for EGFR mutation testing.

Background:The external quality assurance (EQA) process aims at establishing laboratory performance levels. Leading European groups in the fields of EQA, Pathology, and Medical and Thoracic Oncology collaborated in a pilot EQA scheme for somatic epidermal growth factor receptor (EGFR) gene mutational analysis in non-small-cell lung cancer (NSCLC).Methods:EQA samples generated from cell lines mimicking clinical samples were provided to participating laboratories, each with a mock clinical case. Participating laboratories performed the analysis using their usual method(s). Anonymous results were assessed and made available to all participants. Two subsequent EQA rounds followed the pilot scheme.Results:One hundred and seventeen labs from 30 countries registered and 91 returned results. Sanger sequencing and a commercial kit were the main methodologies used. The standard of genotyping was suboptimal, with a significant number of genotyping errors made. Only 72 out of 91 (72%) participants passed the EQA. False-negative and -positive results were the main sources of error. The quality of reports submitted was acceptable; most were clear, concise and easy to read. However, some participants reported the genotyping result in the absence of any interpretation and many obscured the interpretation required for clinical care.Conclusions:Even in clinical laboratories, the technical performance of genotyping in EGFR mutation testing for NSCLC can be improved, evident from a high level of diagnostic errors. Robust EQA can contribute to global optimisation of EGFR testing for NSCLC patients.

EGFR CA repeat polymorphism predict clinical outcome in EGFR mutation positive NSCLC patients treated with erlotinib

ObjectivesSomatic mutations in the epidermal growth factor receptor (EGFR) are predictors of efficacy for treatment with the EGFR tyrosine kinase inhibitor erlotinib in non-small cell lung cancer (NSCLC). A CA repeat polymorphism in intron 1 of the EGFR gene influences the transcription of the EGFR gene. This study evaluates the association between the CA repeat polymorphism and outcome in NSCLC patients treated with erlotinib. Materials and methodsNumber of CA repeats in the EGFR gene was evaluated with PCR-fragment length analysis by capillary electrophoresis in 432 advanced NSCLC patients treated with erlotinib irrespective of EGFR mutation status. Patients were dichotomized into harboring short allele (CA≤16 in any allele) or long alleles (CA>16 in both alleles). Number of repeats was correlated with clinical characteristic and outcome. A subgroup analysis was performed based on the somatic EGFR mutation status. ResultsIn EGFR mutation positive patients (N=62) we demonstrate a significantly higher median progression free survival (HR=0.39 (0.22–0.70); p=0.002) and overall survival (HR=0.43 (0.23–0.78); p=0.006) in patients also harboring a short CA repeat length vs. a long (median follow-up time of 52.2 months). The result remained highly significant in a multivariate Cox proportional hazards model. This correlation was not seen in EGFR mutation negative patients. ConclusionOur study demonstrate that in EGFR mutation positive NSCLC patients treated with erlotinib a low number of CA repeats in intron 1 of the EGFR gene is a predictor for both longer progression free survival and overall survival.