mTOR Inhibitor PF-05212384

S196

International Journal of Radiation Oncology  Biology  Physics

Isobologram analysis showed that SAHA and GDC-0449 synergistically suppressed HNC cellular proliferation in vitro with concomitant G0/G1 cell cycle arrest associated with p53-independent up-regulation of p21waf. The combination of sub-clinical concentrations of SAHA, GDC-0449, and 2 Gy showed a synergistic reduction in colony formation corresponding with up-regulation of phosphorylated g-H2Ax. Conclusions: We have identified a novel interaction between HDAC, HHsignaling, and ionizing radiation in HNC that may be exploited to synergistically induce lethality in a p53-independent fashion. Ongoing mouse tumor xenograft studies in vivo will assess the potential to translate our findings into a phase I clinical trial for HNC as well as the mechanistic basis for this interaction. Author Disclosure: S.G. Chun: None. L.S. Li: None. H. Park: None. J.S. Yordy: None.

1094

1093 A Circulating Tumor Cell Assay for Tracking Treatment Response in Glioma M. Ju,1 G.D. Kao,2 D. Steinmetz,3 D. Patsch,2 M. Alonso-Basanta,2 S. Hahn,2 R.A. Lustig,2 and J.F. Dorsey2; 1Perelman School of Medicine, Philadelphia, PA, 2University of Pennsylvania Health System, Philadelphia, PA, 3University of Pennsylvania, Philadelphia, PA Purpose/Objective(s): Monitoring radiation therapy (RT) response in glioma patients is at times difficult, with post-RT effects and tumor progression often sharing similar appearances on magnetic resonance imaging. Circulating tumor cell (CTC) analysis provides biologically meaningful information that could contribute to the serial monitoring of treatment response with minimal risk to patients. We have previously established the effectiveness of a telomerase-based CTC assay for glioma. We present herein interim results of a prospective trial in patients with high grade glioma utilizing serial CTC assays throughout the RT course and during follow-up. Materials/Methods: Patients receiving RT and temozolomide for WHO grade 3 or 4 glioma were enrolled in an IRB-approved study in which CTC analysis was performed at time points before, during, and after RT. The assay incorporates a vector probe based on the human telomerase (hTERT) promoter, which amplifies the expression of green fluorescent protein (GFP) in cancer cells but not normal cells. The assay therefore identifies cancer cells with specificity, and is not affected by changes in or the absence of glioma cell surface markers. Results: The assay effectiveness in patients was not impeded by MGMT promoter methylation status, EGFR amplification, EGFR VIII expression, or IDH mutation status of tumors. Ten patients have now been followed for over six months, with a median age of 61 and 80% with grade 4 glioma. For the entire cohort, the mean pre-RT CTC count was 57.1 CTCs/mL (range 0-253) and the mean post-RT count was 3.9 CTCs/mL (range 1-9), with the overall decrease in CTC levels trending toward significance (p Z 0.17). Median pre- and post-RT counts were 19.3 and 2.0 CTCs/mL, respectively. The patient with the highest detected pre-RT CTC level (253 CTCs/mL) derived a good symptomatic and radiographic response to RT, with post-RT analysis showing 1.3 CTCs/mL. In contrast, the two patients with > 45% increase in CTC levels between pre-RT and RT developed rapidly progressive disease requiring re-resection within 6 months of initial resection; one died of disease progression, but the other’s CTC levels subsequently decreased from 67.1 to 9.4 CTCs/mL after re-resection of the tumor. Conclusions: These results support that CTCs may be detected in patients with high-grade glioma and that trends in CTC levels may reflect the tumor response to RT or resection. CTC assays may thus usefully complement conventional follow-up and imaging for tracking treatment response. Further updated results and implications for standard practice will be discussed. Author Disclosure: M. Ju: None. G.D. Kao: None. D. Steinmetz: None. D. Patsch: None. M. Alonso-Basanta: None. S. Hahn: None. R.A. Lustig: None. J.F. Dorsey: None.

The Effect of Ionizing Radiation on cFMS-Mediated Immune Polarization in High-Grade Astrocytomas L. Gerngross, M.P. Achary, and T. Fischer-Smith; Temple University School of Medicine, Philadelphia, PA Purpose/Objective(s): Immune polarization has been suggested to contribute to tumor progression in high-grade astrocytomas (HGA). The cFMS receptor tyrosine kinase signaling in tumor-associated MFs (TAMs) is believed to upregulate markers associated with type II macrophages (MF-2). Although MF-colony stimulating factor (M-CSF), one of two known cFMS ligands, is acutely increased in irradiated HGA cell lines, the effect of ionizing radiation (IR) on cFMS-mediated immune polarization has yet to be explored. Here, we test the hypothesis that IR, through a cFMS-dependent mechanism, supports immune polarization in HGA. Materials/Methods: U87 cells were irradiated with a single dose of 2 or 4 Gy and cultured for 24 hrs. U87 supernatants were collected and used in peripheral blood mononuclear cell (PBMC) culture media, with and without GW2580, a cFMS inhibitor. To assess IR-mediated changes in phenotype and activation status, PBMC (MF-1: IL-12 and TNFa; MF-2: CD163 and IL-10; CD16), and U87s (cFMS, IL-34, and M-CSF) were stained with antibodies against select markers and analyzed by flow cytometry. The mean percent frequency (%fx) and mean fluorescence intensity of each marker were compared between treatment groups by oneand two-way ANOVA or Student t-test. P-values < 0.05 were considered statistically significant. To begin to understand the implications of our in vitro findings in vivo, we performed immunohistochemistry (IHC) studies on formalin-fixed paraffin-embedded resected astrocytoma tissue using antibodies against CD163, CD16, and IL-34. Results: Compared to sham irradiated cells, IR did not affect the number of U87 cells or the %fx of cells expressing M-CSF. It did, however, increase the %fx of IL-34-expressing U87s (4 Gy, p Z 0.026). Culturing PBMC with supernatant from irradiated U87 cells did not alter expression of our selected makers by CD14+ monocytes. The %fx of CD163+ (2 Gy, p < 0.0001; 4 Gy, p < 0.0001) and CD16+ (2 Gy p < 0.0001; 4 Gy, p < 0.0001) CD14+ monocytes, however, decreased when cultured in supernatant from either irradiated and non-irradiated U87 cells with the addition of the cFMS specific inhibitor, GW2580. IHC of resected astrocytoma tissue revealed grade-dependent increases in the number of cells expressing IL-34 and CD16 in the astrocytoma tumor microenvironment. Conclusions: Here, we show for the first time that IR increases expression of the cFMS ligand, IL-34, by U87 cells, but does not alter expression of its other ligand, M-CSF, at 24hrs post-IR. Additionally, cFMS-mediated immune polarization does not appear to be affected by IR. While modulation of the immune system via cFMS inhibition may be a valuable adjunctive therapy in individuals with HGA, further studies exploring the role of IR-induced IL-34 expression are warranted. Author Disclosure: L. Gerngross: None. M.P. Achary: None. T. FischerSmith: None.

1095 Radiation Enhancement of Head and Neck Squamous Cell Carcinoma by the Dual PI3K/mTOR Inhibitor PF-05212384 A. Leiker,1 W. DeGraff,1 R. Choudhuri,1 A.L. Sowers,1 A. Thetford,1 J.A. Cook,1 C. Van Waes,2 and J.B. Mitchell1; 1National Cancer Institute, Bethesda, MD, 2National Institute on Deafness and Other Communications Disorders, Bethesda, MD Purpose/Objective(s): Oncogenic aberrations in the PI3k/mTOR signaling in head and neck squamous cell carcinoma (HNSCC) point toward dual specificity PI3K and mTOR inhibitors as molecular targeted therapies. Dual PI3k/mTOR inhibitors have displayed radiation modulatory effects in preclinical studies in other human cancer cell lines. We investigated the dual PI3K/mTOR inhibitor, PF-05212384, as a potential radiation modifier of human HNSCC tumor cells.

Volume 90  Number 1S  Supplement 2014 Digital Poster Abstract 1096; Table

Digital Poster Discussion Abstracts S197

GNE-317 and GDC-0980 drug concentrations in brain, unirradiated vs irradiated GNE-317 drug level

Time Points*

RT Schema

Control 16 hours 64 hours 112 hours 160 hours 2 hours 64 hours

No RT 40 Gy x1 40 Gy x1 40 Gy x1 40 Gy x1 4 Gy x10 4 Gy x10

Number of Mice 3 3 3 2** 3 4** 5

Unirradiated Brain, Mean (S.D.), microgram/gram 1.2 1.8 2.5 1.2 1.8 1.6 2.3

(1.3) (1.2) (1.7) (-) (1.2) (1.1) (1.3)

Irradiated Brain, Mean (S.D.), microgram/gram 2.2 2.8 3.8 1.8 2.7 2.1 3.0

(1.4) (0.3) (0.4) (-) (0.3) (0.5) (0.5)

GDC-0980 drug level

P-value

Unirradiated Brain, Mean (S.D.), microgram/gram

.55 .06 .14 N/A .33 .19 .12

0.08 0.10 0.10 0.09 0.14 0.04 0.03

(0.13) (0.06) (0.07) (-) (0.11) (0.03) (0.02)

Irradiated Brain, Mean (S.D.), microgram/gram 0.09 0.14 0.15 0.15 0.20 0.04 0.04

(0.08) (0.03) (0.02) (-) (0.05) (0.02) (0.02)

P-value .26 .32 .07 N/A .53 .95 .22

* Brain levels for 2-week and 6-week harvests after 4 Gy x 10 will be reported at the meeting. ** One mouse per group was not evaluable due to technical difficulties; not included as part of analysis.

Materials/Methods: Survival in vitro of two HNSCC cell lines (UMSCC1- wtP53, UMSCC46-mtP53) and human fibroblast (AG1522) was assessed by clonogenic assay; cell lines were pre-treated 24 hours with PF-05212384 (10mM) prior to irradiation or 24 hours post-irradiation in separate survival studies. Dose modifying factors (DMF) were determined from radiation survival curves as the ratio of control (DMSO) radiation dose divided by drug treatment radiation dose required for 10% survival in each group. Cell cycle analysis by flow cytometry and Western blotting were performed. In vivo efficacy was assessed by UMSCC1 xenograft tumor regrowth delay with 10 mg/kg PF-05212384 (i.v.) administered twice weekly and daily 3 Gy fractionated radiation. Results: Radiation DMFs for UMSCC1 and UMSCC46 pre-treated with PF-05212384 was 1.9  0.1 (Mean  STD) and 1.0, respectively; whereas, post-treatment with PF-05212384 demonstrated significant DMFs for both cell lines of 3.6  0.2 and 1.8  0.3, respectively. PF-05212384 exposure alone displayed minimal normal cell (AG1522) toxicity (Surviving Fraction Z 0.94), and a DMF of 1.4  0.2 for post-treatment drug exposure and 1.0 for pre-treatment drug exposure. PF-05212384 exposure resulted in an evident G1/S phase block in p53 competent HNSCC cells and persistent g-H2AX foci at 24 hours with post-irradiation drug treatment (P < 0.05, ttest). UMSCC1 xenograft tumor regrowth delay (time for tumor to grow to 3x the tumor volume at the initiation of treatment) was significantly delayed in the PF-05212384 plus radiation treatment arm versus radiation alone (21  4 vs 16  5 days, P < 0.05, t-test); control and PF-05212384 treatment alone tumor growth delay were 15  1 and 17  3 days, respectively. Conclusions: The dual PI3K/mTOR inhibitor PF-05212384 significantly enhanced the radiosensitivity of HNSCC both in vitro and in vivo, with considerably less enhancement to normal fibroblasts. Our data suggest PF05212384 radiation modulation is at least in part secondary to DNA-DSB repair inhibition with further experimental investigation warranted. Author Disclosure: A. Leiker: None. W. DeGraff: None. R. Choudhuri: None. A.L. Sowers: None. A. Thetford: None. J.A. Cook: None. C. Van Waes: None. J.B. Mitchell: None.

1096 The Impact of Hemi-brain Irradiation on Accumulation of PI3K/ mTOR Inhibitors With Limited (GDC-0980) and Robust (GNE-317) BloodeBrain Barrier Penetration T.T. Sio,1 R.K. Oberoi,2 M.P. Grams,1 K.M. Furutani,1 S.K. Gupta,1 Z.C. Wilson,1 J.L. Pokorny,1 K.K. Bakken,1 M.A. Schroeder,1 B.L. Carlson,1 K. Chang,1 W.F. Elmquist,2 and J.N. Sarkaria1; 1 Department of Radiation Oncology, Mayo Clinic, Rochester, MN, 2 Department of Pharmaceutics, University of Minnesota, Minneapolis, MN Purpose/Objective(s): Previous studies have demonstrated the physical and biochemical effects of radiation on the blood-brain barrier (BBB), which has led to a commonly held belief that radiation therapy (RT) can improve the penetration of small molecules into the brain and associated brain tumors. However, the impact of brain RT on therapeutic drug delivery

has not been previously published. Here, we used hemi-brain irradiation to compare accumulation of two structurally similar PI3K/mTOR inhibitors, GNE-317 (BBB-penetrant), and GDC-0980 (BBB-impenetrant), at various time points following RT with 40 Gy in 1 or 10 fractions. Materials/Methods: C57BL/6 mice were treated with hemi-brain irradiation using a tungsten collimated high-dose-rate 192Ir beam. One group of mice was irradiated with 40 Gy in a single fraction and euthanized up to 160 hours later. In a second experiment, mice were treated with 4 Gy x10 fractions, and euthanized up to 6 weeks after the end of RT. All mice were orally dosed with GNE-317 (30 mg/kg) and GDC-0980 (7.5 mg/kg) concomitantly one hour prior to euthanasia, and irradiated and unirradiated brain hemispheres and plasma were collected. Drug concentrations in both hemispheres and plasma were assessed by mass spectrometry. A paired Student t-test was used to compare drug levels in unirradiated vs irradiated brain. Results: Consistent with previous data, the mean  SD brain-to-plasma ratio for GNE-317 and GDC-0980 in unirradiated brain for all animals was 0.91  0.23 and 0.048  0.018, respectively. For drug levels in the irradiated brains, the brain-to-plasma ratio for the 2 drugs was 1.01  0.27 and 0.054  0.020. To readily compare the effects of radiation on drug accumulation at each time-point, the drug levels in unirradiated vs irradiated brain were compared by a paired t-test. As seen in the Table, there was no significant difference in accumulation of GNE-317 or GDC-0980 associated with irradiation at any of the time-points investigated. Conclusions: Neither fractionated nor single-dose RT had significant impact on the accumulation of either GNE-317 or GDC-0980 in the brain, and importantly, did not significantly increase the penetration of the brain-impenetrant GDC-0980 compound. Further experiments investigating the effect of RT on the blood-brain-tumor-barrier are currently ongoing. Author Disclosure: T.T. Sio: None. R.K. Oberoi: None. M.P. Grams: None. K.M. Furutani: None. S.K. Gupta: None. Z.C. Wilson: None. J.L. Pokorny: None. K.K. Bakken: None. M.A. Schroeder: None. B.L. Carlson: None. K. Chang: None. W.F. Elmquist: None. J.N. Sarkaria: None.

1097 Shining Light on the Implementation of Cherenkov Emission for Image Guidance and Intensity Modulation in Radiation Therapy Y. Zlateva,1 N. Quitoriano,1 S. Davis,2 and I. El Naqa1; 1McGill University, Montreal, QC, Canada, 2McGill University Health Centre, Montreal, QC, Canada Purpose/Objective(s): X-ray imaging, commonly used to localize a lesion in radiation therapy (RT), increases patient exposure. Yet, Cherenkov emission (CE) by charged particles traveling faster than the phase speed of light in a medium is inherent to all high-energy RT beams and consists of (nonionizing) optical photons. However, its clinical application is limited by tissue penetrability and its detectability. We hypothesize that CE is correlated with radiation dose and its detection can be maximized by a spectral shift