A Comprehensive Genome-Based Mutational Analysis by Next Generation Sequencing Technology in Patients With Malignant Pleural and Peritoneal Mesothelioma

A Comprehensive Genome-Based Mutational Analysis by Next Generation Sequencing Technology in Patients With Malignant Pleural and Peritoneal Mesothelioma

Volume 93  Number 3S  Supplement 2015 compared to wild-type. Mice with miR-21 loss demonstrated delay in spontaneous tumor formation, decreased grow...

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Volume 93  Number 3S  Supplement 2015 compared to wild-type. Mice with miR-21 loss demonstrated delay in spontaneous tumor formation, decreased growth rate, and reduced macroscopic lung metastases compared to MMTV-PyMT mice with intact miR-21. When challenged with orthotopic tumor implantation, miR-21+/and miR-21-/- mice had increased delay in allograft tumor formation compared to wild-type mice, suggesting stromal changes in the mammary microenvironment modulated by miR-21. Conclusion: Modulation of miR-21 expression in a murine tumor model appears to play an important role in breast cancer tumorigenesis and progression. Expression of miR-21 may be linked to treatment resistance and development of an aggressive tumor phenotype. As modulation of miR-21 appears to delay de novo tumorigenesis as well as tumor allograft formation, multiple mechanisms of tumorigenesis may be implicated. Targeted inhibition of the miR-21 pathway may be an attractive therapeutic option to both sensitize tumors to cytotoxic treatment while modulating the peritumoral stroma. Author Disclosure: T. Dan: Research Grant; ASTRO. A. Palagani: None. L. Jin: None. T. DeAngelis: None. E. Wickstrom: None. N.L. Simone: Research Grant; ASTRO.

1069 Generation of DNA Double Strand Breaks in Prostate Cancer Cells by Androgen Receptor Antagonists, Hydroxyflutamide and Bicalutamide J.B. Coulter,1 M.C. Haffner,1 O.Y. Mian,2 M. Hedayati,1 Y. Zhang,1 H. Zhou,1 W. Nelson,1 S. Yegnasubramanian,1 and T.L. DeWeese1; 1Johns Hopkins University, Baltimore, MD, 2Johns Hopkins University School of Medicine, Baltimore, MD Purpose/Objective(s): Androgen receptor (AR) signaling is central to the growth and survival of prostate cancers (PCa), and radiation therapy is a mainstay in PCa treatment. Our previous studies have shown that androgen-deprived PCa cells exposed to androgen display transient, topoisomerase 2-beta (TOP2B)-dependent, DNA double strand breaks (DSB) and exhibit enhanced radiosensitivity. Noting that various clinically-used AR antagonists have partial AR agonist activity with respect to nuclear translocation, the potential for hydroxyflutamide (FLU), bicalutamide (BIC), and enzalutamide (ENZ) to cause DSBs was tested. We anticipated that optimized delivery of AR antagonists, which promote AR nuclear translocation, may be a safe and effective strategy to enhance radiation sensitivity in PCa. Materials/Methods: Human PCa cell lines LNCaP, LAPC-4, and VCaP were androgen-deprived for 60 hours prior to 6 hour exposure to AR antagonists. The neutral comet assay was used to quantify DSBs and immunofluorescence microscopy was used to detect gH2AX as a marker of DNA damage; DNA content and EdU incorporation into DNA was measured using flow cytometry at 6, 24, and 48 hours of exposure. The potential roles of reactive oxygen and TOP2B in AR antagonist-induced DSBs were tested by co-incubation of AR antagonists with the antioxidant N-acetyl-L-cysteine (NAC) and siRNA-mediated knock-down of TOP2B, respectively. Cells exposed to AR antagonists for 6 hours were treated with 0 or 4 Gy ionizing radiation (IR) and viability was measured via resazurin oxidation 10 days later. Results: Six hour exposure of androgen-deprived PCa cells to FLU or BIC, but not ENZ, displayed increased DSBs and gH2AX foci formation comparable to the positive control, DHT. Changes in cell cycle or DNA synthesis in LNCaP PCa cells were not observed at this time-point, suggesting that the observed DSBs were not a result of recruitment of cells into cycle. Both co-incubation of LNCaP cells with NAC and knock-down of TOP2B abrogated DSB and gH2AX formation by FLU. Six hour FLU exposure prior to 4 Gy IR significantly decreased survival of LNCaP cells compared to vehicle-exposed control (P < .001). Conclusion: Here, we show that the AR antagonists FLU and BIC lead to widespread, transient DSBs and gH2AX formation in PCa cells. The ability of NAC co-incubation or TOP2B knock-down to completely abrogate FLUmediated DSBs suggests that reactive oxygen species and TOP2B are involved in widespread DSB formation; moreover, prostate cancer cells

ePoster Sessions S185 pretreated with FLU were significantly more sensitive to IR when radiation is delivered at the time of maximal FLU-induced DSB formation. Ongoing work is designed to determine the mechanisms through which AR antagonists produce these widespread DSBs and to develop a strategy to capitalize on them to sensitize cancer cells to radiation therapy. Author Disclosure: J.B. Coulter: None. M.C. Haffner: None. O.Y. Mian: None. M. Hedayati: None. Y. Zhang: None. H. Zhou: None. W. Nelson: None. S. Yegnasubramanian: None. T.L. DeWeese: None.

1070 A Comprehensive Genome-Based Mutational Analysis by Next Generation Sequencing Technology in Patients With Malignant Pleural and Peritoneal Mesothelioma G. Ugurluer,1,2 K. Chang,1 M. Mayeda,3 M.E. Gamez,4 A.L.H. Arnett,1 R. Jayakrishnan,1 B. Anderson,1 T.T.W. Sio,4 and R.C. Miller3; 1Mayo Clinic, Rochester, MN, 2Acibadem Adana Hospital, Adana, Turkey, 3Mayo Clinic, Jacksonville, FL, 4Mayo Clinic, Scottsdale, AZ Purpose/Objective(s): Malignant mesothelioma is a rare and aggressive malignancy primarily arising from the pleural or peritoneal cavity linings. Therapeutic options are very limited. Exome-based, next-generation sequencing (NGS) techniques may impact the management of molecular targeting and systemic therapies in patients (pts). We aimed to retrospectively review our institutional experience with NGS in clinical practice. The molecular profiles exhibited are reported. Materials/Methods: Eleven consecutive pts with prior completion of NGS were selected; NGS was performed on pt samples’ HE-stained slides. A total of 236 somatic cancer-related mutations involving 3 230 exons and 47 introns from 19 genes at >900x mapping coverage were analyzed by an outside vendor. Descriptive and Kaplan-Meier statistics were applied. Results: The median age was 65 years (range, 27-73 years); 36% was female. Of the 11 pts, 64% and 36% had pleural and peritoneal mesothelioma, respectively. Five pts presented with metastatic disease at diagnosis. Two pts had lymphoma. Detectable mutations were found in 55% of the pleural and 18% of the peritoneal mesothelioma pts (overall, 73%). The families of BAP1 (36%), CDKNA2A/B (27%) and NF2 (27%) represented the most frequently mutated genes (Table 1). One or more variants of unknown or additional genetic significance were also detected in 91% of pts. Although there was no U.S. Food and Drug Administration (FDA)-approved therapies specifically for the reported genomic alternations in these pts, mutations identified in 36% of pts did have existing FDA-approved clinical trials open for another type of malignancy or histology. One pt enrolled in a relevant experimental therapy (off-protocol), and survived for 14 months. The median overall survival for all pts was 20.8 months (95% CI, 15.7-25.9 months), with 1 and 2-year survival rates of 91% and 40%, respectively. Conclusion: Genetic mutations with therapeutic potential were found by NGS in a majority of pts. The detection of mutations, for which specific therapies are readily available, may provide clinical trial options; additionally, NGS-mediated identification of novel mutations can be applied to the development of new screening tools and systemically targeted therapies, which may have therapeutic and prognostic impact in the research and management of malignant mesothelioma in the future. ePoster Abstracts 1070; Table 1 Pt # 1 2 3 4

Age at Diagnosis (Years)/Sex 69/M 65/M 70/M 73/M

5 27/M 6 73/F 7 29/M 8 71/F

All Detected Genomic Alternations CDKN2A/B (loss) BAP1(N142fs*12); SF3B1(Y623C) CDKN2A/B (loss) NF2 (E427); PTCH1(deletion, exons 6-14); BAP1 (S460); MYD88 (L220P); SETD2 (E1756fs*33) MAP2K1 (E203K); NF2 (V24fs*25); TP53 (S314F) STK11 (T24fs*138); BAP1 (splice site 659+1G>T); CDKN2A/B (loss) NF2 (splice site 1341-6_1347del13) BAP1( loss ); KDM6A (L338fs*26); ASXL1 (L764fs*8); BRIP1 (K998fs*5); SETD2 (D653fs*5)

S186

International Journal of Radiation Oncology  Biology  Physics

Author Disclosure: G. Ugurluer: None. K. Chang: None. M. Mayeda: None. M.E. Gamez: None. A.L. Arnett: None. R. Jayakrishnan: None. B. Anderson: None. T.T. Sio: None. R.C. Miller: Stock; Tekcapital Europe. Board member; Rare Cancer Network. Board of Trustees; Mayo Clinic Health System Albert Lea Austin.

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1071 Next Generation Sequencing of Sarcoma Cells With HIF-1a Deletion Reveals Context-Dependent Regulation of HIF-1 Transcriptional Targets Induced by Radiation Versus Hypoxia T.J. Robinson, M. Zhang, and D.G. Kirsch; Duke University Medical Center, Durham, NC Purpose/Objective(s): Radiation resistance caused by hypoxia and its master regulator, HIF-1a, remains a promising therapeutic target; however, the consequences of HIF-1a activation via non-hypoxic stimuli remain poorly understood. In this study, we set out to clarify how hypoxia versus radiation impacts the HIF1a-dependent tumor cell transcriptome. Materials/Methods: We used RNA-seq to perform comprehensive transcriptional analyses in cell lines from a genetically engineered mouse model of soft tissue sarcoma with intact versus deleted HIF-1a. We exposed cells with or without intact HIF-1a to 6 Gy of ionizing radiation and analyzed transcription at 8H, 24H, 48H, and 72H following irradiation, and separately to normoxia or hypoxia (1% O2 for 24 hours). We employed multivariable regression analyses using DESeq2 to identify radiation-induced versus hypoxia-induced genes, whose expression was dependent on HIF-1a. Results: A total of 533 genes were induced by hypoxia and 247 by radiation in a HIF-dependent manner; HIF-dependent, radiation-induced genes (N Z 114) were enriched for metabolism (N Z 41) and tRNA modification (Aars, Gars, Qtrt1, Sars1); HIF-dependent radiation-inhibition (N Z 133) was enriched for roles in steroid biosynthesis (N Z 9), WNT-receptor signaling (Fzd2, Fzd8, Ldb1, Tcf7l2, Tle2), and included 24 snRNAs and 3 snoRNAs. We identified 15 HIF-dependent genes induced by both hypoxia and radiation, including known HIF-1 a targets and 3 non-coding RNAs, including the lncRNA Hotair. The remaining upregulated genes were radiation-specific, with 12 demonstrating an opposite effect in response to hypoxia-induced HIF. Of these, we examined OSGIN2 by RT-qPCR and confirmed HIF-dependent, radiation-specific upregulation. All of the genes inhibited by radiation in a HIF-dependent manner were radiation-specific, 7 of which were upregulated by HIF in hypoxia (Table 1; all P  .05). Conclusion: HIF-1a demonstrates context-dependent transcriptional inhibition or activation depending on whether HIF-1a is activated by hypoxia or radiation. This marks the first study to use RNA-seq technology in a model of hypoxia and radiation therapy, allowing detection of both coding and non-coding RNA genes to elucidate novel mechanistic insights and potential targets in radioresistance. ePoster Abstracts 1071; Table 1

HIF-1 Dependent Targets

UP-regulation by BOTH XRT and hypoxia Protein Coding Pfkl Mthfd1l Wsb1 Pgk1 Vegfa Pdk1 Cgref1 Bhlhe40 Ero1l Higd1a Vldlr Ak4

lncRNA Gm25687 Gm22567 Hotair

UP-regulation by XRT and DOWN-regulation in hypoxia Protein Coding Osgin2 Dnajc2 Rae1 Gtpbp4 Rrp12 Fam195a Ccdc86 Prps1 Trmt61a Ankrd13b Pcgf6 Odc1 DOWN-regulation by XRT and UP-regulation in hypoxia Protein Coding lncRNA Nr2f1 Ikbip Fzd2 Gm11816 Hoxa10 Arhgef19 Fut11

Author Disclosure: T.J. Robinson: None. M. Zhang: None. D.G. Kirsch: None.

Stereotactic Ablative Radiation Therapy for Treatment of Adrenal Gland Metastasis: Toxicity, Patterns of Failure, and Factors Impacting Outcomes W.W. Chance, Q. Nguyen, R.J. Mehran, J.W. Welsh, D.R. Gomez, P. Balter, R.U. Komaki, Z. Liao, and J.Y. Chang; MD Anderson Cancer Center, Houston, TX Purpose/Objective(s): To evaluate the toxicity, patterns of failure, and factors impacting outcomes in patients undergoing stereotactic ablative radiation therapy (SABR) for metastasis to the adrenal gland. Materials/Methods: From 2009-2014, 47 adrenal metastasis were treated with SABR at our institution. Thirty-five patients received treatment to a single adrenal gland, and 6 patients received treatment to the bilateral adrenal glands. Primary sites included lung (n Z 36), ovary (n Z 2), bladder (n Z 1), esophagus (n Z 1), and skin (n Z 1). The prescription dose was 60 Gy in 10 fractions (n Z 28), 50 Gy in 10 fractions (n Z 7), 50 Gy in 4 fractions (n Z 5), or other (n Z 7). Toxicity was evaluated with Common Terminology Criteria for Adverse Events version 4.0. Failure within the high-dose region was considered local failure. New or progressive disease outside of the treatment field was considered distant failure. Survival, local failure, and distant failure were estimated by a Kaplan-Meier method. Prognostic factors were evaluated using a Cox proportional hazards model. Results: Median follow-up from diagnosis was 30 months (range, 6-155 months). Median follow-up after SABR was 11 months (range, 1-37 months). Eight patients developed grade 1-2 gastrointestinal toxicity. Two patients treated to the bilateral adrenal glands developed grade 2 adrenal insufficiency at 4 and 7 months after therapy. No grade 3 or 4 toxicity was observed. Median overall survival (OS) from diagnosis was 50 months. Factors associated with worse OS from diagnosis included a lung primary (P Z .04), synchronous adrenal metastasis (P Z .02), and bilateral adrenal metastasis (P Z .03). The median OS from SABR to the first adrenal metastasis was 20 months. The 1 and 2-year OS rates after treatment were 67% and 39%, respectively. Factors associated with worse OS after treatment included bilateral adrenal metastasis (P Z .02) and right-sided adrenal metastasis (P Z .04). Median progression-free survival (PFS) after SABR was 6 months. The 1-year PFS was 25%. Progression-free survival was better in patients with a solitary adrenal metastasis who received definitive treatment to the primary site compared to all others (median PFS 9 months vs 4 months, P Z .03). Median time to distant progression was 6 months. One-year freedom from distant progression was 33%. Median time to local failure was not reached. The 1-year freedom from local failure was 87%. Total dose, dose per fractions, tumor size, and laterality did not significantly impact local control. There were 6 in-field local failures, and 1 marginal failure observed. Conclusion: In patients with adrenal metastasis, SABR is well tolerated and results in excellent local control. Bilateral adrenal gland treatment is associated with a risk of adrenal insufficiency. In patients with solitary adrenal metastasis who received definitive treatment to the primary site of disease, PFS was promising. Author Disclosure: W.W. Chance: None. Q. Nguyen: None. R.J. Mehran: None. J.W. Welsh: None. D.R. Gomez: None. P. Balter: None. R.U. Komaki: None. Z. Liao: None. J.Y. Chang: None.

1073 Higher Facility Case Volume of Stereotactic Body Radiation Therapy Is Associated With Improved Overall Survival in Clinical Stage I Non-Small Cell Lung Cancer H.S.M. Park, E.H. Wang, C.D. Corso, C.E. Rutter, L.D. Wilson, A.W. Kim, C.P. Gross, J.B. Yu, and R.H. Decker; Yale School of Medicine, New Haven, CT Purpose/Objective(s): Increased provider experience has been associated with beneficial clinical outcomes for facilities performing lobectomy for stage I non-small cell lung cancer (NSCLC) and definitive chemoradiation therapy for stage III NSCLC; however, it is unknown if a similar benefit