Proceedings of the 53rd Annual ASTRO Meeting S737

3101 Hedgehog Inhibitor and Radiotherapy in Non-small Cell Lung Cancer

J. Zeng, K. Aziz, B. Aftab, M. Armour, S. Chettair, R. Gajula, T. Salih, C. Rudin, P. T. Tran, R. Hales

Johns Hopkins University, Baltimore, MD

Purpose/Objective(s): Radiation concurrent with cytotoxic chemotherapy remains the standard of care for the management oflocally advanced NSCLC. Despite this treatment paradigm, local control remains a problem with local recurrence in 50 - 75% ofpatients, and may be an important contributor to metastatic spread. The hedgehog signaling pathway is aberrantly activated inadult malignancies, including NSCLC. We seek to investigate the effect of Hh pathway inhibition on radiation response in pre-clinical modals of NSCLC.

Materials/Methods: Utilizing in vitro and in vivo models, we studied hedgehog inhibitor (HhI) cytotoxicity and radiosensitiza-tion in NSCLC. We examined the effect of HhI with and without radiation on in vitro cell death, clonogenic survival, cell cycledistribution, and GLI1 expression. Using a hind-flank A549 xenograft model, we looked at tumor growth delay, proliferation, celldeath and hedgehog pathway gene expression changes following HhI treatment with or without radiation. In a transgenic mousemodel of Irasand Twist1-induced lung adenocarcinoma, tumor responses to lung radiation with or without HhI were assessed withserial micro-CT scans.

Results: HhI sensitizes A549 cells to ionizing radiation at high doses (5 mmol) as shown in colony formation assays with an en-hancement ratio of 1.4-1.8. Increased cell death is through apoptosis as determined by annexin-Vexpression. A549 has high GLI1expression via western, but did not show cell cycle changes upon treatment with HhI. Hind-flank xenograft experiments demon-strate the ability of HhI and radiation to delay tumor growth in vivo, p\0.001. Ki-67 immunofluorescence showed no change incellular proliferation, but cleaved caspase-3 expression increased in tumors treated with HhI. Using qPCR analysis, tumor cellsshow decreased GLI3 and Indian hedgehog (IHH) expression with HhI therapy. In transgenic mice with spontaneous lung car-cinomas, weekly micro-CT scans revealed improved tumor shrinkage with the addition of HhI to lung radiation. At 1-weekpost-radiotherapy, HhI plus radiation induced 45% tumor shrinkage, whereas radiation alone only induced 17% tumor shrinkage,and HhI alone showed 12% tumor growth, p\0.01.

Conclusions: Our data show that selectively targeting the hedgehog pathway increases the effectiveness of radiation in in-vitroand in-vivo systems of NSCLC. This data provides a framework for clinical translation of HhI with concurrent chemoradiother-apy in locally advanced NSCLC.

Author Disclosure: J. Zeng: None. K. Aziz: None. B. Aftab: None. M. Armour: None. S. Chettair: None. R. Gajula: None. T.Salih: None. C. Rudin: None. P.T. Tran: None. R. Hales: None.

3102 Oncolytic Vaccinia Virus Encoding an Anti-VEGFAntibody Improves the Efficacy of Fractionated

Radiation in Lung Tumor Xenografts

S. J. Advani1,2, L. Buckel3, A. Frentzen4, D. Scanderbeg1,2, N. Chen4, Q. Zhang4, Y. Yu4, A. J. Mundt1,2, A. Szalay4,1

1University of California San Diego, La Jolla, CA, 2UCSD Center for Advanced Radiotherapy Technologies, La Jolla, CA,3University of Wuerzburg, Wuerzburg, Germany, 4Genelux, San Diego, CA

Purpose/Objective(s):Dysregulated expression of vascular endothelial growth factors (VEGF) in tumors results in defective vas-culature. This has lead to the hypothesis that vasculature ‘‘renormalization’’ with anti-VEGF therapies may improve tumor ox-ygenation and efficacy of radiotherapy. Oncolytic viruses have been shown to interact with ionizing radiation (IR) to improvetumor regression. To further augment this treatment paradigm, we studied how oncolytic vaccinia virus that encodes for anti-VEGF affects the efficacy of IR on tumor regression in preclinical animal models.

Materials/Methods: A549 lung cancer xenografts were grown subcutaneously in athymic nude mice to. 200 mm3. Oncolyticvaccinia virus expressing anti-VEGF antibody (GLV-1h164) or the parental vaccinia virus (GLV-1h68) not encoding anti-VEGFantibody was injected systemically. Fractionated IR was delivered 4 Gy x 4. Tumor volumes of untreated controls, IR or virusalone and combination of viruses and IR treated xenografts were measured. Fractional tumor volumes (FTV) were calculatedby normalizing each mouse’s tumor volume to its initial tumor volume at the start of therapy, day 0.

Results: Injection of either GLV-1h68 or GLV-1h164 induced a tumor growth delay in A549 xenografts compared to untreatedcontrol tumors. At day 20, control tumors had a FTVof 6.4, while parental oncolytic vaccinia virus GLV-1h68 and anti-VEGFexpressing vaccinia virus had FTVof 4.5 and 3.1, respectively. IR alone also delayed tumor growth with a FTVof 3.4 at day 20.When GLV-1h68 was combined with fractionated IR, the FTV was 2.74. Interestingly, in mice treated with the combination of 4Gy x 4 and the anti-VEGF expressing GLV-1h164, the tumors had a FTVof 1.86. Tumors in this group were significantly smallerthan those of mice of all other groups including those treated with 4x 4Gy and the parental virus GLV-1h68 (p\0.05). Comparedto control tumors, GLV1h68 and IR resulted in a 57% reduction, while GLV-1h164 in combinationwith fractionated IR resulted ina 70% reduction in tumor volume by day 20 (p\0.05).

Conclusions: In addition to their inherent anti-tumor activity, oncolytic viruses can be constructed to express radiosensitizingproteins. Here, we show combining fractionated IR with an oncolytic vaccinia virus expressing an anti VEGF antibody furtherenhanced the efficacy of IR when compared with the combination of IR and parental virus not expressing an anti-VEGF antibody.These results indicate that in addition to the intrinsic interactions of oncolytic viruses with IR, further improvement of the ther-apeutic ratio of IR can be achieved using oncolytic viruses to locally express anti-angiogenic molecules and provide a basis forclinical development.

Author Disclosure: S.J. Advani: C. Other Research Support; 25,000. L. Buckel: None. A. Frentzen: A. Employment; Genelux.D. Scanderbeg: None. N. Chen: A. Employment; Genelux. Q. Zhang: A. Employment; Genelux. Y. Yu: A. Employment; Genelux.A.J. Mundt: None. A. Szalay: A. Employment; Genelux.