ROS1 Gene Fusions Research Articles

Detecting NTRK, ROS1, and ALK gene fusions in gastrointestinal tumor patients.

619 Background: The STARTRK-2 trial is a potentially registration-enabling Phase 2 global basket trial of the tyrosine kinase inhibitor entrectinib in patients with solid tumors harboring NTRK1, NTRK2, NTRK3, ROS1, or ALK gene fusions. Phase 1 studies of entrectinib reported a 79% ORR across multiple histologies in patients with gene fusions of these targets who were naïve to inhibitors of these targets, received an effective dose, and had extracranial disease. Patients harboring these fusions are typically rare in the cancer population ( < 3%); however, they have been seen across a wide variety of GI tumors. In this presentation we report on the occurrence of these fusions in GI tumors, based on Ignyta’s internal and partner testing. We discuss the development of an assay for GI samples and immunohistochemistry (IHC) screening efficiency rates in a clinical cohort of 157 GI samples. Methods: The occurrence of NTRK, ROS1, and ALK gene fusions in GI tumors was studied from Ignyta internal and partner collaborations. We developed a 2-step diagnostic test to identify fusions in FFPE clinical specimens. IHC screening using a pan-receptor tyrosine kinase cocktail of antibodies precedes an RNA-based anchored multiplex PCR next generation sequencing (NGS) assay. Results: IHC positivity rates ranged from 0 to 24 percent depending on histology and averaging 8% positivity rate across 15 tumor locations. The screen is particularly effective in cholangiocarcinoma, colon, esophagus, gastric, peritoneum, rectum and small bowel. It was highest in pancreas (24%). Out of 157 GI samples, no instances were detected where the IHC was screened negative and gene fusions were observed by NGS. The IHC cocktail enriches detection of the patient population for gene fusions over 11-fold and has a 100% negative predictive value. Conclusions: This 2-step assay is an efficient method for detection of gene rearrangements in both high-volume clinical testing and studies of archival formalin-fixed paraffin-embedded specimens. Depending on how it is implemented in clinical trials, the staged approach can substantially decrease the costs and tissue expended for NGS. Screening efficiency may be improved even further as additional diagnostic antibodies become available. Clinical trial information: NCT02568267.

Gene Expression and Clinical Characteristics of Molecular Targeted Therapy in Non-small Cell Lung Cancer Patients in Shandong

背景与目的分子生物学靶向治疗已逐渐成为非小细胞肺癌（non-small cell lung cancer, NSCLC）的一个重要治疗手段，本研究通过分析山东地区NSCLC多种驱动基因表达情况及临床病理特征，为筛选分子靶向治疗目标人群提供理论依据。方法采用荧光探针PCR法检测表皮生长因子受体（epidermal growth factor receptor, EGFR）、棘皮动物微管相关蛋白4-间变性淋巴瘤激酶（echinoderm microtubule-associated protein-like 4-anaplastic lymphoma kinase, EML4-ALK）、肉瘤致癌因子-受体酪氨酸激酶（ROS proto-oncogene 1, receptor tyrosine kinase, ROS1）、鼠类肉瘤病毒癌基因（Kirsten rat sarcoma viral oncgene, KRAS）基因表达情况，回顾性分析阳性病例的临床病理特征。结果EGFR基因突变阳性率为36.70%，主要为19、21外显子突变，突变人群主要为女性、腺癌、不吸烟患者，组间差异有统计学意义。EML4-ALK融合基因重排阳性率为9.37%。人群特征主要为60岁以下不吸烟人群，组间差异有统计学意义，基因突变与病理类型和性别间无明显差异。ROS1融合基因重排阳性率为3.67%，均为60岁以下患者，组间差异有统计学意义。23份病例标本开展KRAS基因检测，阳性标本数2例，阳性率为8.70%。2份阳性标本均为60岁以上病例，男女各占1例，病理类型均为腺癌，均无吸烟史。此外，未发现有两种基因同时突变的病例。结论EGFR、EML4-ALK、ROS1、KARS基因在NSCLC患者中存在较高的突变率，且具有不同的人群特征，在选择靶向治疗人群中具有重要意义。

Driver genes and targeted drugs in lung cancer

With the development of molecular biology of cancer, molecular targeted therapy which targeted the driver genes in lung cancer has become an inevitable part of the treatment of advanced lung cancer. The most successful examples of targeted therapy are targeting epidermal growth factor receptor (EGFR) and anaplastic lymphoma kinase (ALK). More and more driver genes have been discovered, including ROS1 gene fusion, fibroblast growth factor receptor 1 amplification, KRAS, BRAF, PIK3CA gene mutation and so on. It is meaningful for guiding the treatment of lung cancer that defines the mutation incidence and clinical significance of driver genes in lung cancer. Key words: Lung neoplasms; Driver genes; Targeted therapy

MTE11.01 The Clinical Impact of the 2015 WHO Classification of Lung Tumors

MTE11.01 The Clinical Impact of the 2015 WHO Classification of Lung Tumors

SC11.03 ROS1 as a Therapeutic Target in Advanced NSCLC

SC11.03 ROS1 as a Therapeutic Target in Advanced NSCLC

Correlation among Genetic Variations of c-MET in Chinese Patient with Non-Small Cell Lung Cancer

Aims: The aim of this study was to determine the relationship between amplification, protein expression and somatic mutation of c-MET in advanced Non-small cell lung cancer. The influence of c-MET abnormalities on clinical outcomes of patients undergoing Crizotinib therapy for treatment of Non-Small Cell Lung Cancer was also evaluated. Methods: c-MET protein expression, gene copy number (GCN) and somatic mutation for exon 14 were detected by Immunohistochemistry, fluorescent In Situ Hybridization and Denaturing High Performance Liquid Chromatography, respectively, in a large series of 196 NSCLC patients. The correlation of c-MET abnormalities and clinical outcome of targeted therapy was analyzed by McNemar’s test. Results: c-MET expression was observed in 28.6% (56/196) cases, and among those 13.8% (27/196) was shown to be FISH positive. Only 2.67% patients in this study carried the c-MET mutation. All cases that were c-MET FISH positive were also shown to express c-MET by IHC. However, only half of the cases that were positive for c- MET expression were found to be FISH positive. Among 31 patients with moderate c-MET IHC staining, 11 cases (35.5%) were FISH positive, while 16/25 cases with high IHC staining were also FISH positive. Six patients received Crizotinib as a first-line or second/third-line therapy. Among them, three cases showed ALK protein expression, two patients showed expression of the ROS1 fusion gene, and one was positive for c-MET expression by IHC. The response to Crizotinib in the three patients positive for ALK were all PR, while the c-MET positive patient showed SD and both cases with expression of the ROS1 gene showed PD. Conclusions: IHC could be a preliminary screening test to facilitate selection of patients with c-MET amplification for ALK inhibitor therapy.

P1.02-003 ROS1 (D4D6) is Reliable for Immunohisochemistry Detecting of ROS1 Fusion Lung Adenocarcinoma in Malignant Pleural Effusion

ROS1 fusion is of a low frequency genetic alteration in non-small cell lung cancer (NSCLC). The clinical trial showed that NSCLC patients harboring ROS1 fusion could benefit from crizotinib treatment. Several studies reported that immunohistochemistry (IHC) could be employed as a screening method for detecting ROS1 fusion in tumor tissue using Anti-ROS1(D4D6) antibody. Malignant pleural effusion (MPE) is the common sample type in advanced NSCLC, the reliability of ROS1(D4D6) for detecting ROS1 fusion in MPE cell blocks (CBs) should be explored. Anti-ROS1(D4D6) monoclonal antibody (Cell Signaling Technology, Danvers, USA) IHC testing was performed on 227 formalin fixed paraffin embedded (FFPE) MPE CBs from lung adenocarcinoma patients. RT-PCR using ROS1 fusion gene detection kit (AmoyDx) was performed to detected ROS1 gene fusion as a comfirming test. Some other lung cancer driver genes were also detected in both IHC/RT-PCR ROS1 positive samples, among which EGFR, KRAS, BRAF, PIK3CA and HER2 20 exon mutation was tested by amplification refractory mutation system kits(AmoyDx), ALK and RET fusion was tested by RT-PCR kits(AmoyDx). 4 of 227 MPE samples(1.76%) of lung adenocarcinoma were interpreted as ROS1 fusion-positive cases by IHC staining, and the cytoplasmic and membranous granular positive signals were displayed. Comparison with RT-PCR testing results, 3 of 4 IHC positive cases were verified by RT-PCR, the sensitivity was 100% and specificity of was 99.6%. The clinicopathologic features and other genes status of 3 both IHC/RT-PCR positive patients were showed Table 1. One ROS1 IHC/RT-PCR positive lung adenocarcinoma patient received crizotinib therapy and obtained partial response. ROS1 (D4D6) would be a reliable antibody for screening ROS1 fusion-positive lung adenocarcinoma on FFPE MPE CBs by IHC assay and shows high specificity in the FFPE MPE CBs samples.Table 1Clinicopathologic features and genes status of ROS1 IHC/RT-PCR positive patientsGenderAgeSmokingTTF1/ P63E/k/B/ P/HA/RTherapyEfficacyPFS/Follow-upp1Male55Smoker+/-WTWTcrizotinibPR10m+/alivep2Female60Never+/-WTWTNo1m/DeadP3Male62Never+/-WTWTPEM+CISPR9m+/aliveE/K/B/P/H: EGFR/KRAS/BRAF/PIK3CA/HER-2 20 exon mutation, A/R: ALK/RET fusion, WT: wild type, PEM+CIS: Pemetrexed plus Cisplatin, PR: partial response, m: months Open table in a new tab

P2.06-002 Phase I Study of DS-6051b, a ROS1/NTRK Inhibitor, in Japanese Subjects with Advanced Solid Tumors Harboring Either a ROS1 or NTRK Fusion Gene: Topic: Phase I Trials

Oncogenic gene fusions of ROS1 or NTRK have been reported in various cancers. DS-6051b is an orally available small molecule receptor tyrosine kinase inhibitor with high affinity for the ROS1 and NTRK receptors. Non-clinical pharmacology studies demonstrated anticancer activity of DS-6051b against several types of human tumor harboring ROS1 or NTRK fusion gene in cultured cells and xenograft models. This is an ongoing phase 1 study in Japanese subjects with advanced solid tumors harboring either a ROS1 or NTRK fusion gene. Subjects receive doses of DS-6051b from 400mg to 800mg once daily (QD). Pharmacokinetics (PK) samples are collected from Day1 to Day22. Primary objective is to assess the safety profile and secondary objectives are to determine the maximum tolerated dose (MTD), the recommended phase 2 dose (RP2D), and to assess the PK profile. The efficacy of DS-6051b is an exploratory assessment performed by investigator judgment per RECIST v.1.1. As of June 27, 2016, a total of 9 subjects were enrolled. Median age was 51 (43-69) years, 56% were female, all 9 subjects were ROS1 fusion positive non-small cell lung cancer patients, and 3 subjects had prior crizotinib treatment. Subjects received DS-6051b at doses of 400mg QD (n=6) and 800mg QD (n=3). There were no DLTs in the 400mg QD cohort, and 2 out of 3 subjects in the 800mg QD cohort experienced DLT with grade 3 AST/ALT increased. To evaluate the MTD and RP2D more in detail, 600mg QD cohort is planned. Common adverse events were AST increased, ALT increased, diarrhea, and constipation. Among 7 patients who had target lesion, 4 subjects showed partial response, 3 subjects showed stable disease. PK data indicated the plasma drug concentration increases as the dose increases. This study is categorized as “Clinical Trial in Progress.” This study was initiated from February 2016 and estimated primary completion date will be September 2018.

P2.03b-080 A Comprehensive Test of Cancer Treatment-Related Genes for the Clinical Samples of Non-Small Cell Lung Cancer (NSCLC): Topic: Biomarkers

Molecular targeted therapies are one of the key drugs for NSCLC. For all advanced NCSLC patients to receive precise molecular targeted therapies, mutation analysis should be comprehensively analyzed from tissue and/or cytological samples with high sensitivity and cost-effectiveness. To satisfy these requirements, we established a system called MINtS(Mutation Investigation System using a Next-generation Sequencer). According to our basic data, by deep sequencing, MINtS enables us to detect genetic alterations from the clinical specimens of which cancer cell content is just over 1%. The lists of mutations are EGFR, KRAS, BRAF, HER2, and ALK/RET/ROS1 fusion genes. We report the data analyzed from the clinical cytological and tissue samples using MINtS. MINtS adapted amplicon sequencing strategy. For the EGFR, KRAS, BRAF and HER2 genes, the target regions are amplified by the multiplex PCR. For the ALK, RET and ROS1 fusion genes as well as OAZ1 housekeeping gene (internal control), the targets are amplified by the multiplex RT-PCR. The index sequences were then added for discriminating samples derived from different patients. Amplified fragments from multiple samples are combined, and run on a Next generation Sequencer. According to the index sequences, the sequencing reads obtained were de-multiplexed for each sample. Using MINtS system, we analyzed 65 tissue samples that were obtained surgically, and 76 cytological specimens that were obtained by bronchial brushing or pleural effusions from NSCLC patients. Our preliminary data indicated that mutations for EGFR, KRAS, BRAF, and HER2 were detected from DNA, and the test detected mutations from samples with cancer cell content > 1% with specificity and sensitivity >0.99. Fusion genes were detected from mRNA, and the test were considered to detect them from samples with cancer cell content >1%. Among 141samples, 140samples were successfully analyzed. EGFR mutations in 36 samples (25.5%), KRAS mutations in 10 samples (7%), BRAF mutations in 5 samples (3.5%), and HER2 mutations in 5 samples (3.5%). Regarding fusion genes, ALK fusion gene were 2 samples (1.4%), RET fusion gene were 2 samples (1.4%), and ROS fusion gene was 1 sample (0.7%). MINtS could analyze from both tissue and cytological samples. MINtS tests >100 samples in a single run. This fulfills clinical requirement of a high throughput testing at an affordable unit price; 30US dollars per sample. MINtS provides a protoytpe of mutation test applicable to cancers originating from organs other than lung, and may be also adaptable to liquid biopsy.

Clinical utility of real-time fluorescent PCR for combined detection of anaplastic lymphoma kinase and c-ros oncogene 1 receptor tyrosine kinase in non-small cell lung cancer

Objective: To investigate the clinical application value of combined detection of ALK fusion gene and c-ros oncogene 1 receptor tyrosine kinase (ROS1) fusion gene in non-small cell lung cancer (NSCLC) using real-time fluorescent PCR. Methods: A kit for combined detection of ALK fusion gene and ROS1 fusion gene based on fluorescent PCR was used to simultaneously detect the two fusion genes in 302 cases of NSCLC specimens. The results were validated through Sanger sequencing. The consistency of the two detection methods was analyzed. Results: All 302 cases of NSCLC specimens were successfully analyzed through fluorescent PCR (302/302). 12 cases (4.0%) were found to contain ALK fusion gene, including 3 cases with ALK-M1, 3 with ALK-M2, 3 with ALK-M3, 1 with ALK-M4, and 2 with ALK-M6 fusion gene.12 cases (4.0%) were found to contain ROS1 fusion gene, including 1 case with ROS1-M7, 8 cases with ROS1-M8, 1 case with ROS1-M12, 1 case with ROS1-M14, and 1 case with double-positive ROS1-M3 and ROS1-M8 fusion genes. The total detection rate of ALK fusion gene and ROS1 fusion gene was 7.9% (24/302) and 278 cases showed to be negative for ALK fusion gene and ROS1 fusion gene. The successful detection rates for Sanger DNA sequencing were also 100%. The positive, negative and total coincidence rates obtained by real-time fluorescent PCR and by Sanger DNA sequencing were all 100%. Conclusions: The results of Sanger DNA sequencing demonstrate that the real-time fluorescent PCR assay is equally effective in detecting ALK and ROS1 fusion genes in NSCLC tissues. Furthermore, real-time fluorescent PCR assay can be used to detect trace ALK and ROS1 fusion gene simultaneously in tiny samples, and can save time and avoid repeated sampling. It is worthy of recommendation as a rapid and reliable detection technique.

Testing for ROS1 in non-small cell lung cancer: a review with recommendations.

Rearrangements of the ROS1 gene occur in 1–2 % of non-small cell lung cancers (NSCLCs). Crizotinib, a highly effective inhibitor of ROS1 kinase activity, is now FDA-approved for the treatment of patients with advanced ROS1-positive NSCLC. Consequently, focus on ROS1 testing is growing. Most laboratories currently rely on fluorescence in situ hybridisation (FISH) assays using a dual-colour break-apart probe to detect ROS1 rearrangements. Given the rarity of these rearrangements in NSCLC, detection of elevated ROS1 protein levels by immunohistochemistry may provide cost-effective screening prior to confirmatory FISH testing. Non-in situ testing approaches also hold potential as stand-alone methods or complementary tests, including multiplex real-time PCR assays and next-generation sequencing (NGS) platforms which include commercial test kits covering a range of fusion genes. In order to ensure high-quality biomarker testing, appropriate tissue handling, adequate control materials and participation in external quality assessment programmes are essential, irrespective of the testing technique employed. ROS1 testing is often only considered after negative tests for EGFR mutation and ALK gene rearrangement, based on the assumption that these oncogenic driver events tend to be exclusive. However, as the use of ROS1 inhibitors becomes routine, accurate and timely detection of ROS1 gene rearrangements will be critical for the optimal treatment of patients with NSCLC. As NGS techniques are introduced into routine diagnostic practice, ROS1 fusion gene testing will be provided as part of the initial testing package.

Abstract CT024: First-in-human phase 1 dose-escalation study of DS-6051b, an oral ROS1 and NTRK inhibitor, in subjects with advanced solid tumors

Abstract Background: DS-6051b is an orally available small molecule receptor tyrosine kinase inhibitor with high affinity for the ROS1 and NTRK receptors. Non-clinical pharmacology studies demonstrated activity of DS-6051b against tumors harboring ROS1 or NTRK fusion genes in cell culture and xenograft models. The objectives of this first in human (FIH) study are to assess safety and tolerability of DS-6051b in cancer subjects and identify the maximum tolerated dose (MTD) and the tentative RP2D. Pharmacokinetics (PK), QT prolongation, and tumor responses are also assessed in this study (Clinical Trial Information: NCT02279433). Methods: Dose escalation initially followed an accelerated titration design enrolling a single subject at each dose level, and then adopted the modified continuous reassessment method using a Bayesian logistic regression model and escalation with overdose control. Adult subjects with advanced solid tumors harboring ROS1 or NTRK1-3 fusion genes; or with neuroendocrine carcinoma; or with advanced solid tumors with tumor-induced pain are eligible for the study. DS-6051b is orally administered once-daily in 21-day cycles. Tumor assessments are performed every 3 cycles using RECIST 1.1 criteria. Plasma PK samples and time-matched ECG data are collected at multiple time points. Results: A total of 22 subjects have been enrolled at 50, 100, 200, 400, 800, and 1200 mg once daily (QD) dose levels. Enrolled population included 11 subjects with neuroendocrine carcinomas, 9 subjects with advanced solid tumors with tumor-induced pain, 1 subject with ROS1 fusion-positive NSCLC liver metastases, and 1 subject with leiomyosarcoma with a ROS1 point mutation. Two dose-limiting toxicities occurred in subjects who received 1200 mg QD, consisting of grade 3 transaminase elevations. The MTD is 800 mg QD. The most frequent (?20%) TEAEs were diarrhea, nausea, vomiting, dehydration, dizziness, dysgeusia, and fatigue, which were predominantly grade ?2. Cmax and AUC increased with the dose. Plasma terminal half-life was 11-24 hours. Steady state AUC was approximately 3 to 4-fold higher than that of Cycle 1 Day 1. Two subjects showed partial response: One with neuroendocrine carcinoma, who received 800 mg QD (33% decrease in tumor size), and one with ROS1 fusion-positive NSCLC liver metastases, who received 1200 mg QD (44% decrease in tumor size). Notably, the latter subject had progressed on crizotinib (best objective response stable disease) and ceritinib (progressive disease). Conclusions: DS-6051b has been well tolerated up to 800 mg QD in subjects with advanced solid tumors. The observed efficacy signals in two subjects with partial response warrant further evaluation. This trial is now selectively accruing subjects with ROS1 fusion-positive tumors. Citation Format: Kyriakos P. Papadopoulos, Erkut Borazanci, Daniel Von Hoff, Leena Gandhi, Amita Patnaik, Masaya Tachibana, Hamim Zahir, Roohi Gajee, Terri Goldberg, Giorgio Senaldi, Sai-Hong Ou. First-in-human phase 1 dose-escalation study of DS-6051b, an oral ROS1 and NTRK inhibitor, in subjects with advanced solid tumors. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr CT024.

Abstract 1889: AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene

Abstract Background: The multi-tyrosine kinase inhibitor (TKI) crizotinib elicits a dramatic response in patients with non-small cell lung cancer (NSCLC) harboring the c-ros oncogene 1 (ROS1) fusion gene (Shaw et al. NEJM 2014); however, these patients inevitably develop resistance to crizotinib within a year, which limits the efficacy of crizotinib. Although reported molecular changes include ROS1 tyrosine kinase mutations, epidermal growth factor receptor (EGFR) activation, and epithelial-to mesenchymal transition, the detailed mechanism of crizotinib resistance has not been elucidated. Methods: To explore the molecular mechanisms of acquired crizotinib resistance in detail, we used a cell line model. HCC78 cells harboring the SLC34A2-ROS1 fusion gene, which are sensitive to crizotinib, were exposed continuously to increasing concentrations of crizotinib in a step-wise manner. A crizotinib-resistant cell line designated HCC78R was established and assessed by MTT assay, Western blotting, a receptor tyrosine kinase (RTK) array, quantitative PCR (RT-PCR), ELISA, and an RNA kinome targeted kinome panel (612 genes) with next-generation sequencing. Results: Crizotinib-resistant HCC78R (50% inhibitory concentration [IC50] 4085.9 nmol/L) was 47-fold more resistant to crizotinib than was parental HCC78 (IC50 85.8 nmol/L) (p = 0.0145) according to MTT assay. The RTK array revealed that EGFR phosphorylation was upregulated in HCC78R cells. Western blotting confirmed the activation of EGFR and its downstream signaling pathways. RT-PCR screening of EGFR ligand family member expression showed increased heparin-binding EGF-like growth factor (HB-EGF) in HCC78R compared with HCC78 cells. Consistently, addition of the HB-EGF or conditioned medium from HCC78R cells rendered the HCC78 parental cells moderately resistant to crizotinib. EGFR overexpression or activating mutations were not detected. Furthermore, RNA kinome sequencing revealed 16-fold higher AXL mRNA expression and 1.68-fold lower ROS1 fusion gene expression in HCC78R compared with HCC78. Overexpression of the AXL protein and downregulation of the ROS1 protein were confirmed by Western blotting. Monotherapy with gefitinib (EGFR-TKI) or R428 (AXL inhibitor) or cabozantinib (AXL inhibitor) moderately inhibited the growth of HCC78R cells, while combined therapy suppressed proliferation more significantly compared with each monotherapy. Conclusions: AXL and EGFR signaling mediate resistance to crizotinib, and combination treatment of an EGFR-TKI and AXL inhibitor may be an alternative strategy in ROS1 fusion gene-driven lung cancer cells. Citation Format: Yuka Kato, Kadoaki Ohashi, Eiki Ichihara, Shuuta Tomida, Hiroe Kayatani, Kenichiro Kudo, Daisuke Minami, Takashi Ninomiya, Toshio Kubo, Toshiyuki Kozuki, Katsuyuki Hotta, Nagio Takigawa, Mitsune Tanimoto, Katsuyuki Kiura. AXL and EGFR signaling mediate resistance to Crizotinib in non-small cell lung cancer cells harboring the ROS1 fusion gene. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1889.

Abstract 5227: Molecular profiling in African American NSCLC patients to identify novel potential driver mutations

Abstract African Americans continue to have poorer 5 year survival after a lung cancer diagnosis than whites for reasons that remain to be fully characterized. This disparity remains even with advances in treatments targeting specific driver mutations that have improved outcomes for some patients. Although the relative frequency of these genetic alterations varies in subsets of individuals defined by sex, histologic subtype, smoking history and race, little is known about the occurrence of these mutations in African Americans. In this study, we characterize the spectrum of known driver mutations in 200 African American NSCLC patients and seek to identify novel somatic mutations in this population. Initially, the population was screened using the Sequenom LungCarta panel of 216 mutations in 24 genes known to contain alterations associated with lung cancer, RET and ROS1 fusion gene expression, and amplification of FGFR1. Whole-exome sequencing is being performed on tumors from those patients with no known somatic mutations. Paired normal and tumor DNA/RNA samples are being genotyped to distinguish germline from somatic mutations in both the screening and sequencing phases. Initial screening has been completed on 130 patients, with the remaining 70 underway. The mean age of the patients enrolled is 61.7 years, 58.5% are female, and 8.5% are never smokers. In profiling the first 130 African American patients, we find that only 28% of patients (N = 36) carry a known somatic mutation that is not present in the germline, far lower than the 41% reported in white patients in our previous studies. Of the identified mutations, approximately 23.8% are KRAS and 23.8% are PT53 alterations. The next most frequent somatic alterations are in EGFR (16.7%). The remaining alterations occurred in only 1 or 2 tumors and include EPHA3, ERBB2, FGFR1, MET, NOTCH1, NRAS, NTRK2, PIK3CA, PTEN, RET and STK11. Five percent of patients have tumors with two driver mutations. Half of the tumors carrying 2 driver mutations had concurrent alterations in KRAS and TP53. Patients with known genetic alterations were more likely to be female than patients with no known genetic alterations (p = 0.42), but did not differ in age, smoking status, pack-years, family history of lung cancer, history of COPD, stage at diagnosis or histology. Mutations in EGFR were responsible for the differences by sex. Exome sequencing is complete for the first 48 of the 92 patients with no known somatic mutations. Sequences are being aligned using Novoalign and GATK will be used for variant calling; results will be available soon. Genetic profiling of NSCLC in African Americans has the potential to both identify novel mutations, expanding the list of potential targets for tailored treatments, and aid clinical decision making in African American patients leading to improvements in outcomes. Citation Format: Ann G. Schwartz, Angela S. Wenzlaff, Chrissy M. Lusk, Greg Dyson, Aliccia Bollig-Fischer, Susan Land, Sneh Lata, Michele L. Cote, Gerold Bepler, Shirish M. Gadgeel. Molecular profiling in African American NSCLC patients to identify novel potential driver mutations. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5227.

Mouse models for ROS1-fusion-positive lung cancers and their application to the analysis of multikinase inhibitor efficiency.

ROS1-fusion genes, resulting from chromosomal rearrangement, have been reported in 1-2% of human non-small cell lung cancer cases. More than 10 distinct ROS1-fusion genes, including break-point variants, have been identified to date. In this study, to investigate the in vivo oncogenic activities of one of the most frequently detected fusions, CD74-ROS1, as well as another SDC4-ROS1 fusion that has also been reported in several studies, we generated transgenic (TG) mouse strains that express either of the two ROS1-fusion genes specifically in lung alveolar type II cells. Mice in all TG lines developed tumorigenic nodules in the lung, and a few strains of both TG mouse lines demonstrated early-onset nodule development (multiple tumor lesions present in the lung at 2-4 weeks after birth); therefore, these two strains were selected for further investigation. Tumors developed progressively in the untreated TG mice of both lines, whereas those receiving oral administration of an ALK/MET/ROS1 inhibitor, crizotinib, and an ALK/ROS1 inhibitor, ASP3026, showed marked reduction in the tumor burden. Collectively, these data suggest that each of these two ROS1-fusion genes acts as a driver for the pathogenesis of lung adenocarcinoma in vivo The TG mice developed in this study are expected to serve as valuable tools for exploring novel therapeutic agents against ROS1-fusion-positive lung cancer.

Bronchoscopic Re-biopsy for Mutational Analysis of Non-small Cell Lung Cancer.

Currently, several acquired resistance mechanisms and rare driver oncogenes are identified in non-small cell lung cancer (NSCLC) relapses. Re-biopsy increases valuable information to guide treatment strategies, but the utility and feasibility of bronchoscopic re-biopsy has not been investigated. We studied 70 patients who underwent bronchoscopic for re-biopsy of NSCLC that was resistant to at least one regimen of chemotherapy or molecular-targeted therapy between January 2013 and December 2014. We assessed clinical data, technical success rate, and mutational analysis. Procedures performed were transbronchial biopsy (n=52) and endobronchial ultrasound-guided transbronchial needle aspiration (EBUS-TBNA) (n=18). Overall detection rate of re-biopsy for malignant cells was 87% (83% for TBB and 100% for EBUS-TBNA). Mutational analysis was possible in almost all technically successful cases; likewise, acquired-resistant mutations (55% of EGFR mutants) and small cell lung cancer transformation were identified from the bronchoscopy specimens. Other driver mutations were seen in four cases, including ALK fusion gene (n=2) and ROS1 fusion gene (n=2). There were no associated severe complications. This study shows that bronchoscopic re-biopsy for NSCLC is feasible and provides adequate samples that enable identification of resistance mutations and rare driver oncogenes.

Smoking, hormonal factors and molecular markers in female lung cancer.

There has been substantial argumentation about differences in lung cancer occurrence and characteristics between men and women. Lung cancer incidence suggests that gender-related factors may influence lung cancer risk. The carcinogenic effect of tobacco smoke and the use of hormone replacement therapy could result in susceptibility to lung cancer among women. Mutations in EGFR and HER-2/neu genes, and ROS1 gene fusions may also play a role in gender-based survival rate differences. This review summarizes the latest data of disease markers and its usefulness in female lung cancer.

489P A reverse transcription PCR based screening for oncogenic mutations in non-small cell lung cancer in Xuanwei population of China

Aim/Background: Air pollution-related lung cancer has been considered as an exacerbating public health problem worldwide. For instance, Xuanwei and Fuyuan County in Yunnan Province in China are heavily air-polluted regions with exceptionally high lung cancer rates. Hence, these regions can be used as very good models to study air pollution-related lung cancer.In cancer, changes in driver genes are crucial to carcinogenesis. Clinically, patients with specific genetic alterations exhibit higher sensitivity to targeted therapy than to other therapeutic strategies. This study mainly aimed to define the status of clinically significant oncogenes in air pollution-related lung cancers and to establish a simple and sensitive test to screen patients with particular genetic changes. Methods: 82 tissue samples of lung cancer and adjacent nonmalignant lung tissues were obtained from patients in Xuanwei and Fuyuan. These tissue samples and 13 cell lines were analyzed through the combined reverse transcription PCR and DNA sequencing to determine the expression and gene mutation of HER2 and BRAF. These tissue samples and cell lines were also examined through RT-PCR to identify the incidence of two fusion genes, namely EML4-ALK and CD74-ROS1. Results: Of the 82 patients with non-small cell lung cancer, 20.7% (17/82) exhibited HER2 upregulation, 1.21% (1/82) harbored HER2 insertion mutation at exon 20 and 6.1% (5/82) showed ALK gene rearrangements. Of the 5 cases with the gene rearrangements, two belonged to EML4-E2 + ALK-E20 and three were EML4-E13 + ALK-E20. Of the 82 patients, 3.6% (3/82) carried the ROS1 fusion gene with CD74 (CD74-E6 + ROS1-E34). Conclusions: The overexpression and mutation of HER2, EML4-ALK and CD74-ROS1 were observed in patients living in highly air-polluted regions such as Xuanwei and Fuyuan. These genetic alterations may participate in air pollution-induced lung carcinogenesis. The HER2 overexpression and the EML4-ALK fusion are molecular targets of personalized therapy. To guide treatments, we should screen these genetic alterations in air pollution-related lung cancers through various methods such as an RT-PCR-based test. This test is indeed a useful tool to screen these genetic alterations in clinical applications. Disclosure: All authors have declared no conflicts of interest.

Abstract A1-43: Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations

Abstract Background: Although precision medicine approaches have revolutionized oncology, widespread adoption requires robust, inexpensive approaches enabling the targeted assessment of all relevant alteration classes from routine tissue samples. Methods: Here we interrogated >7,000 cancer exomes and transcriptomes, along with >30,000 array based cancer genomes to identify recurrent somatic alterations (mutations, copy number alterations [CNAs] and gene fusions) across solid tumors. From this analysis, we developed and validated an integrated multiplexed PCR based Ion Torrent next generation sequencing panel (Oncomine Cancer Research Panel [OCP]) targeting the actionable somatic cancer genome optimized for 20ng of formalin-fixed, paraffin-embedded (FFPE) tissue isolated DNA/RNA. Results: We validated the OCP using FFPE cell line mixtures, as well as a prospective cohort of 104 FFPE tumor specimens sent for concurrent clinical molecular testing, with >97% sensitivity and specificity for the presence/absence of KRAS, EGFR, BRAF and ALK point mutations, indels or gene fusions in this molecular testing cohort. We also applied the OCP to 100 lung cancers, identifying known and novel alterations, including ALK and ROS1 gene fusions. Lastly, applying the OCP to 116 prostate cancers, including 50 previously treated samples, we recapitulated known molecular subtypes, observed distinct profiles according to previous treatment and obtained 100% concordance for isoform specific TMPRSS2:ERG gene fusion detection compared to qPCR. Additionally, OCP profiling supports a novel molecular subtype of prostate cancer defined by IDH1 R132 hotspot mutations and informed on resistance mechanisms in a pre- and post-treatment sample pair. Importantly, 44%, 35% and 9% of patients in the molecular testing, lung and prostate cancer cohorts, respectively, harbored additional alterations (beyond routine molecular testing) associated with FDA approved or NCCN guideline referenced therapies. Conclusions: Through analysis of both DNA and RNA to assess the actionable somatic cancer genome, the validated OCP panel may have utility in both clinical and research settings. Citation Format: Daniel H. Hovelson, Andrew S. McDaniel, Bryan Johnson, Andi K. Cani, Kate Rhodes, Paul D. Williams, Chia-Jen Liu, Santhoshi Bandla, Catherine S. Grasso, Michael J. Quist, Seth Sadis, Daniel R. Rhodes, Scott A. Tomlins. Targeted amplicon-based next-generation sequencing of routine solid tumor specimens to detect clinically relevant somatic alterations. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-43.

Cell Line Usage as Positive Controls in ThinPrep Specimens for Detecting ROS1 Gene Fusions in Non-Small Cell Lung Carcinoma

Cell Line Usage as Positive Controls in ThinPrep Specimens for Detecting ROS1 Gene Fusions in Non-Small Cell Lung Carcinoma