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Oncogenic ARAF as a New Driver in Lung Adenocarcinoma
Volume 90 Number 5S Supplement 2014 of Radiation Oncology, Fudan University Cancer Center, Shanghai Proton and Heavy Ion Center, Shanghai, China Purpose/Objective(s): The poor prognosis of esophageal squamous cell carcinoma (ESCC) is partially due to the lack of an effective means for early diagnosis. Moreover, the radioresistance of ESCC is a great obstacle to its treatment. Our study aimed to identify plasma microRNAs for early diagnosis of ESCC and to further investigate whether it can be used as predictors of radiation therapy outcomes. Materials/Methods: We selected 9 microRNA (miR-16, miR-21, miR-22, miR-126, miR-148b, miR-185, miR-221, miR-223 and miR-375), which were reported to be associated with carcinogenesis and radiobiology of ESCC, as the targets of our study. The peripheral blood from subjects was collected between August 2009 and June 2013. Plasma microRNA expression was investigated with 2 independent stages including 43 subjects (19 healthy, 24 pre-, after 1 week and postradiation therapy). Quantitative reverse-transcriptase-polymerase-chain-reaction (qRT-PCR) assay was applied to evaluate the expression of selected microRNAs. A logistic regression model was constructed based on the first cohort. Area under the receiver operating characteristic curve (AUC) was used to evaluate the diagnostic accuracy. Results: We found 4 microRNAs including miR-148b, miR-185, miR221 and miR-375 which had significantly higher expression levels in the ESCC group than those in the healthy group (fold change Z 2.9-19.3, P<.05). The analysis demonstrated that miR-375 had high diagnostic accuracy in ESCC from healthy (AUC: 0.924 with 95% CI: 0.799 to 0.983), the sensitivity and specificity were 91.67% and 77.78%, respectively. Next, we found that combination of the expression levels of miR375 and miR-148b produced the best model to predict ESCC. The predicated probability from the logit model based on the 2 significant predictors, logit (PZESCC) Z 7.8823+1.6537*miR-375-0.6523*miR148b, was used to construct the ROC curve (AUC: 0.965 with 95% CI: 0.857 to 0.998), the sensitivity and specificity were 91.67% and 94.44%, respectively. The expression profile of those nine individual microRNAs was further evaluated to evaluate the correlation between the microRNA level and the treatment outcomes. According to the imaging data taken at pree, after 1 week, and posteradiation therapy, the average expression level of miR-16 in the patients with complete response and partial response was significantly higher than that in the patients with stable disease and progressive disease after radiation therapy (fold change Z 3.2, P<.05). Conclusions: We find a plasma microRNA panel that has considerable clinical value in diagnosing ESCC at an early stage of ESCC development. Thus, patients who would have otherwise missed the curative treatment window can benefit from the optimal therapy. Moreover, miR-16 can be a potential biomarker for evaluation of the efficacy of ESCC radiation therapy. Author Disclosure: Q. Yu: None. B. Li: None. S. Fu: None.
269 Oncogenic ARAF as a New Driver in Lung Adenocarcinoma Translational Research L. Araujo,1 J. Amann,1 M. Imielinski,2 H. Greulich,2 M. Meyerson,2 and D. Carbone1; 1James Cancer Center - The Ohio State University, Columbus, OH, 2Broad Institute of Harvard and MIT, Dana*Farber Cancer Institute, and Brigham and Women’s Hospital, Boston, MA Purpose/Objective(s): We recently identified a novel somatic mutation in ARAF in a lung adenocarcinoma from a patient that demonstrated a remarkable response to sorafenib. The S214C lies in a negative regulatory domain of ARAF, distinct from the catalytic domain mutations commonly found in BRAF. The aim herein was to characterize the biochemical and functional aspects of ARAF S214C. Materials/Methods: ARAF constructs were generated and ectopically expressed in an immortalized bronchial epithelial cell line (BEAS-2B). We evaluated the acquisition of anchorage independence, MEK activation, and cell morphology. COS7 cells were used for co-immunoprecipitation (IP) and kinase assays.
Poster Presentations
S73
Results: Cells expressing ARAF S214C substantially increased soft agar colony formation relative to vector, wild-type, kinase-dead (D429A), and double-mutant (S214C+D429A) variants. Accordingly, ARAF S214C cells exhibited increased phospho-MEK levels, suggesting that the transforming potential is dependent on its kinase activity. We also demonstrated that cells expressing ARAF S214C with an additional RAS-binding domain mutation (R52L) lacked MEK activation and failed to form colonies, showing that RAS binding is essential for activity. Interestingly, ARAF S214C cells acquired an elongated, fibroblast-like shape, characteristic of MEK-active cells. Conversely, none of other variants presented this morphology. To determine if either BRAF or RAF1 were necessary for ARAF S214C activity, we performed BRAF and RAF1 knockdowns. ARAF S214C-induced MEK activation was not reverted by the knockdowns, suggesting that BRAF and RAF1 are not required. Subsequently, COS7 cells were co-transfected with tagged constructs of ARAF and either BRAF or RAF1, followed by co-IP. We showed that ARAF S214C does not heterodimerize, unless stimulated with sorafenib. Importantly, sorafenib-induced heterodimers lacked kinase activity, compatible with the clinical response reported. Conclusions: ARAF S214C demonstrates the in vitro features of a driver oncogene, and also a distinct mechanism of action. This oncogenic process can be successfully suppressed by RAF inhibitors like sorafenib, and could represent a new target for personalized therapy in advanced lung adenocarcinoma. Author Disclosure: L. Araujo: None. J. Amann: None. M. Imielinski: None. H. Greulich: None. M. Meyerson: None. D. Carbone: None.
270 A Comprehensive Genome-Based Mutational Analysis by Next Generation Sequencing in 22 Consecutive Patients With Thoracic Malignancies Translational Research B.W. Anderson,1 K. Chang,2 R.K. Funk,2 T.T. Sio,2 J.R. Molina,3 and R.C. Miller2; 1Department of Internal Medicine, Mayo Clinic, Rochester, MN, 2Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 3Department of Medical Oncology, Mayo Clinic, Rochester, MN Purpose/Objective(s): Exome-based next-generation sequencing (NGS) techniques may impact the management of molecular targeting and systemic therapies in patients (pts) with thoracic malignancies. To retrospectively review our institutional experience and determine the clinical utility of NGS in our practice and the molecular profiles exhibited in this cohort of pts. Materials/Methods: Twenty-two (22) consecutive pts with prior completion of NGS for thoracic malignancies were retrospectively identified between Aug 2012 and July 2013. NGS was performed for several histologic subtypes, which analyzed 236 somatic cancer-related genes involving 3230 exons and 47 introns from 19 genes at >900x mapping coverage by an outside vendor. Exploratory Kaplan-Meier analysis and univariate models were applied following genetic characterization. Three-year overall survival (OS) was compared between mutation subgroups; progression-free survival (PFS) and local control (LC) rates were reported for non-metastatic pts. Kaplan-Meier statistics were applied. Results: The median pt age was 59.4 years; 15 (68%) were female. Of the 22 pts, 17 (77%) had lung cancers (15 NSCLC, 2 SCLC) and 5 (23%) pts had other thoracic malignancies. Eight (36%) pts presented with metastatic disease at diagnosis. Selected pt characteristics and their mutations are summarized in Table. OS was 85% and 64% at 1 and 3 years, respectively; PFS was 75% and 36% at 1 and 2 years, respectively; LC rates were 75% and 22% at 1 and 3 years, respectively. Eleven (50%), 15 (86%), and 1 (5%) of genetic profiles harbored an oncogenic alteration (OA), tumor suppressor alteration, and chromosomal abnormality, respectively; 15 (86%) had additional mutations. Compared to other mutations, pts with an OA mutation had worse OS at 3 years (18% vs 63%, PZ.026). Of the mutations discovered by NGS, 19 (86%) yielded actionable results with existing FDA-approved therapies and open clinical trials; among these, 8 (42%) pts took the recommended therapies.
269 Oncogenic ARAF as a New Driver in Lung Adenocarcinoma Translational Research L. Araujo,1 J. Amann,1 M. Imielinski,2 H. Greulich,2 M. Meyerson,2 and D. Carbone1; 1James Cancer Center - The Ohio State University, Columbus, OH, 2Broad Institute of Harvard and MIT, Dana*Farber Cancer Institute, and Brigham and Women’s Hospital, Boston, MA Purpose/Objective(s): We recently identified a novel somatic mutation in ARAF in a lung adenocarcinoma from a patient that demonstrated a remarkable response to sorafenib. The S214C lies in a negative regulatory domain of ARAF, distinct from the catalytic domain mutations commonly found in BRAF. The aim herein was to characterize the biochemical and functional aspects of ARAF S214C. Materials/Methods: ARAF constructs were generated and ectopically expressed in an immortalized bronchial epithelial cell line (BEAS-2B). We evaluated the acquisition of anchorage independence, MEK activation, and cell morphology. COS7 cells were used for co-immunoprecipitation (IP) and kinase assays.
Poster Presentations
S73
Results: Cells expressing ARAF S214C substantially increased soft agar colony formation relative to vector, wild-type, kinase-dead (D429A), and double-mutant (S214C+D429A) variants. Accordingly, ARAF S214C cells exhibited increased phospho-MEK levels, suggesting that the transforming potential is dependent on its kinase activity. We also demonstrated that cells expressing ARAF S214C with an additional RAS-binding domain mutation (R52L) lacked MEK activation and failed to form colonies, showing that RAS binding is essential for activity. Interestingly, ARAF S214C cells acquired an elongated, fibroblast-like shape, characteristic of MEK-active cells. Conversely, none of other variants presented this morphology. To determine if either BRAF or RAF1 were necessary for ARAF S214C activity, we performed BRAF and RAF1 knockdowns. ARAF S214C-induced MEK activation was not reverted by the knockdowns, suggesting that BRAF and RAF1 are not required. Subsequently, COS7 cells were co-transfected with tagged constructs of ARAF and either BRAF or RAF1, followed by co-IP. We showed that ARAF S214C does not heterodimerize, unless stimulated with sorafenib. Importantly, sorafenib-induced heterodimers lacked kinase activity, compatible with the clinical response reported. Conclusions: ARAF S214C demonstrates the in vitro features of a driver oncogene, and also a distinct mechanism of action. This oncogenic process can be successfully suppressed by RAF inhibitors like sorafenib, and could represent a new target for personalized therapy in advanced lung adenocarcinoma. Author Disclosure: L. Araujo: None. J. Amann: None. M. Imielinski: None. H. Greulich: None. M. Meyerson: None. D. Carbone: None.
270 A Comprehensive Genome-Based Mutational Analysis by Next Generation Sequencing in 22 Consecutive Patients With Thoracic Malignancies Translational Research B.W. Anderson,1 K. Chang,2 R.K. Funk,2 T.T. Sio,2 J.R. Molina,3 and R.C. Miller2; 1Department of Internal Medicine, Mayo Clinic, Rochester, MN, 2Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 3Department of Medical Oncology, Mayo Clinic, Rochester, MN Purpose/Objective(s): Exome-based next-generation sequencing (NGS) techniques may impact the management of molecular targeting and systemic therapies in patients (pts) with thoracic malignancies. To retrospectively review our institutional experience and determine the clinical utility of NGS in our practice and the molecular profiles exhibited in this cohort of pts. Materials/Methods: Twenty-two (22) consecutive pts with prior completion of NGS for thoracic malignancies were retrospectively identified between Aug 2012 and July 2013. NGS was performed for several histologic subtypes, which analyzed 236 somatic cancer-related genes involving 3230 exons and 47 introns from 19 genes at >900x mapping coverage by an outside vendor. Exploratory Kaplan-Meier analysis and univariate models were applied following genetic characterization. Three-year overall survival (OS) was compared between mutation subgroups; progression-free survival (PFS) and local control (LC) rates were reported for non-metastatic pts. Kaplan-Meier statistics were applied. Results: The median pt age was 59.4 years; 15 (68%) were female. Of the 22 pts, 17 (77%) had lung cancers (15 NSCLC, 2 SCLC) and 5 (23%) pts had other thoracic malignancies. Eight (36%) pts presented with metastatic disease at diagnosis. Selected pt characteristics and their mutations are summarized in Table. OS was 85% and 64% at 1 and 3 years, respectively; PFS was 75% and 36% at 1 and 2 years, respectively; LC rates were 75% and 22% at 1 and 3 years, respectively. Eleven (50%), 15 (86%), and 1 (5%) of genetic profiles harbored an oncogenic alteration (OA), tumor suppressor alteration, and chromosomal abnormality, respectively; 15 (86%) had additional mutations. Compared to other mutations, pts with an OA mutation had worse OS at 3 years (18% vs 63%, PZ.026). Of the mutations discovered by NGS, 19 (86%) yielded actionable results with existing FDA-approved therapies and open clinical trials; among these, 8 (42%) pts took the recommended therapies.