A. Rehemtulla

Fusion of the HSV-1 tegument protein vp22 to cytosine deaminase confers enhanced bystander effect and increased therapeutic benefit

A major limitation in cancer gene therapy, specifically gene-dependent enzyme prodrug therapy (GDEPT), is inefficient gene delivery and expression. The suicide gene cytosine deaminase (CD) and its substrate, 5-fluorocytosine (5-FC), have been extensively explored due to the inherent ‘bystander’ effect achieved through diffusion of the toxic metabolite 5-fluorouracil (5-FU). In this study, we aimed to enhance this ‘bystander’ effect by fusing the Saccharomyces cerevisiae CD to the HSV-1 tegument protein vp22, a novel translocating protein. Two constructs were created: one with vp22 fused to CD (vp22CD) and a second wherein a truncated vp22, lacking the necessary residues for trafficking, fused to CD (delvp22CD). The generated 9L stable lines exhibited similar growth rates, enzyme expression, CD activity, and sensitivity to 5-FC and 5-FU. However, mixed population colony formation assays demonstrated greater bystander effect with the vp22CD fusion as compared to delvp22CD. This enhancement was maintained in vivo where 9L tumors expressing 20 or 50% vp22CD exhibited increased growth delay compared to the respective delvp22CD tumors. Moreover, adenoviral transduction of established wild-type 9L tumors showed increased growth delay with vp22CD (Ad-EF_vp22CD) as compared to equivalent CD (Ad-EF_CD) transduced tumors. Finally, confirming the increased efficacy, 19F magnetic resonance spectroscopy (MRS) of vp22CD-expressing tumors demonstrated increased 5-FU levels as compared to tumors expressing the nontranslocating CD. These results together demonstrated that fusion of vp22 to CD resulted in CD translocation, which in turn amplified conversion of 5-FC to 5-FU in vivo and enhanced the therapeutic benefit of this GDEPT strategy.

Abstract 4299: Evaluation of a new multimodality voxel-based imaging biomarker for therapeutic response assessment in GBM

The purpose of this study was to evaluate the diagnostic performance of MRI based T1 and relative cerebral blood volume (rCBV) maps analyzed a multi-modality voxel-based analysis referred to as multi-parametric response mapping (mPRM) for early chemoradiation response prediction in patients diagnosed with high-grade gliomas. Patients (n=23) with Grade III/IV glioma were recruited in a prospective imaging trial. Patients underwent MRI before RT, 1 and 3 weeks after RT. The MRI protocol included fluid-attenuated inversion recovery imaging (FLAIR), quantitative T1 mapping, dynamic contrast-susceptibility T2*-weighted imaging (rCBV) and contrast-enhanced T1-weighted imaging (CE-T1w). All images were co-registered to CE-T1w images acquired before RT. Following co-registration, the tumor VOIs were manually contoured either on the CE-T1w or on the FLAIR images and applied to the rCBV and T1maps. For each patient, mid-treatment time point and VOI the percentage change, PRM and mPRM techniques were used to analyze data. Briefly, PRM was performed by calculating the difference in the rCBV values of each voxel within the tumor at mid-treatment values with respective pre-treatment values. A threshold was then applied to the absolute difference of the rCBV in a voxel and all like voxels were summed to obtain tumor volume fractions that showed significantly increasing (PRMrCBV+), decreasing (PRMrCBV-), and unchanged (PRMrCBV0) rCBV values following therapy. We used the same procedure for determining the PRM of T1 maps. mPRMs maps were computed by combining both PRMs (T1 and rCBV) in a single color map. Receiver operator characteristic analysis (ROC), assessed for 12 month survival, was used to determine the optimal cutoff for each parameter. The patient population was then stratified based on the optimal cutoffs obtained from the ROC analysis. Overall survival for each parameter was determined using Kaplan-Meier curves and the log-rank test. Standard PRM, using T1or rCBV maps (specifically PRMT1+ and PRMrCBV- metrics) and percentage change methods were found to be predictive of survival only for specific time of acquisition and VOI used. It was determined that mPRMT1+/rCBV- significantly identified patients resistant to therapy irrespective of tumor volume delineation on CE-T1w or FLAIR images and the time point mid-treatment used. Our results show that mPRM improves the sensitivity of quantitative T1 and rCBV maps to predict overall patient survival. This study introduces a new approach for analyzing and combining multi-parametric MR images into a single multi-parametric response map. The mPRM metric showed promise as an early and robust imaging biomarker of treatment response in patients diagnosed with high grade gliomas. This novel approach is a very sensitive tool for analysis of multiparametric and/or multimodal data with enhanced sensitivity for early detection of therapy response. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4299. doi:1538-7445.AM2012-4299

Abstract 2355: Assessment of glioma recurrence following radiotherapy using the parametric response map: A preclinical study

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Heterogeneity of the glioblastoma multiforme (GBM) response to standard therapies can be due to spatially varying phenotypes within an individual tumor due to genomic instability. The goal of this study was to evaluate the parametric response map PRM, a voxel-based analytical approach, as a biomarker of tumor recurrence on an animal-by-animal basis in a radiation dose escalation protocol. Twenty-four genetically engineered murine GBM model [Ink4a/Arf-/- PtenloxP/loxp/ Ntv-a RCAS/PDGF(+)/Cre(+)] were imaged by MRI (contrast-enhanced (CE-T1w) and diffusion-weighted MRI) daily for a week and then every two days thereafter. Tumor bearing animals were introduced to the study (D0) when the tumors reached a volume of ∼20 mm3. Mice were randomized in 4 groups (n=6 per group): 0G and daily doses of 1G, 2G and 4G delivered daily for 5 days. Tumor volumes of interest (VOI) were manually contoured on the CE-T1w images and reported on the diffusion map. The relative ADC (rADC) maps were computed by normalized values using a VOI defined within the contralateral striatum. PRM was performed by calculating the difference in mid-treatment from pre-treatment rADC values for each voxel in the tumor. Voxels were classified, based on a user-defined threshold set at ±337 rADC units, as significantly increasing (PRMrADC+), significantly decreasing (PRMrADC-), and unchanged (PRMrADC0) rADC values following therapy. The volume fraction of each classification was calculated by summing the class specific voxels and normalizing by the tumor volume. Finally, for each animal the maximum PRMrADC+ value (maxPRMrADC+) was determined within the first 5 days following therapy initiation. The maxPRMrADC+ was correlated to time to recurrence using a linear regression (TTR; was defined as the time for the tumor to triple its initial volume at baseline (D0)). We observed a large inter- and intra-group variability of TTR (intra-group variability in day: [min-max] = [3-5], [3-9], [13-21] and [19-25] for the 0G, 1G, 2G and 4G groups; respectively). The day of the maxPRMrADC+ value measured animal by animal was somewhat variable over the week of treatment administration. We observed a linear increase of maxPRMrADC+ value which correlated with the dose of radiation received (from 4.5±2.7 % to 33.9±11.3 %; for control and 4G groups; respectively). We also found excellent correlation between TTR and the maxPRMrADC+ metric value of individual animals during the week of treatment (R2 = 0.85). This study demonstrates the efficacy of PRM as an imaging biomarker of tumor recurrence following radiotherapy. The maxPRMrADC+ values measured during radiation therapy were correlated strongly with TTR. PRMrADC technique may serve as a biomarker of tumor recurrence that is insensitive to the response heterogeneity typically observed in GBM. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2355. doi:1538-7445.AM2012-2355

Abstract 452: Novel MRI-based biomarker for early assessment of glioma recurrence

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Gliomas continue to be the most common form of brain malignancy in adult patients. Even with advancements in the clinical management of these patients, assessment of tumor recurrence continues to be based on late or serial changes in tumor volume as measured by CT or MRI [1; 2]. The purpose of this study was to develop and evaluate a new voxel-based method, referred as parametric response mapping (PRM), for early detection of brain tumor progression using standard MR images (FLAIR and contrast-enhanced T1-weighted). PRM results were evaluated relative to standard MRI-based criteria of clinical progression assessment in patients with Grade III/IV gliomas. Fourteen patients with grades III/IV glioma underwent MRI before and during treatment (every 2 months). MRI scans, acquired on a 1.5T or a 3T clinical scanner, consisted of fluid-attenuated inversion recovery imaging (FLAIR) and contrast-enhanced T1-weighted (CE-T1w) images. All images were spatially registered to CE-T1w pre-treatment scans and tumors were manually contoured on the T1w-Gd images. Subsequent to PRM analysis, FLAIR images were normalized to the signal using the white matter (rFLAIR). PRM, applied to the whole brain volume on rFLAIR, was performed by first generating voxel-based difference maps at each longitudinal follow-up scan using the baseline as the subtrahend. Baseline was defined as either the pre-treatment or subsequent rFLAIR image. Individual voxels were classified based on the extent of change observed in the rFLAIR difference maps. Three classifications were used: voxels with a significant increase in rFLAIR values (PRMrFLAIR+), significant decrease (PRMrFLAIR-) or statistically unchanged (PRMrFLAIR0). Disease recurrence was defined by PRM as the sum of red voxels that exceed 20% of the tumor volume as delineated on T1w-Gd. PRMrFLAIR detected tumor recurrence on average 18±3 weeks earlier than the MacDonald criteria. It was also observed that PRMrFLAIR+ found outside the tumor CE-T1w regions revealed the spatial location at which the tumor would progress on subsequent scans based on current standard progression criteria. These results show that the PRM approach provides for the early detection and spatial depiction of brain tumor progression prior to detection by currently available conventional MRI-based criteria. While further validation of PRM for detection of tumor recurrence is currently underway, the ability to detect tumor progression significantly earlier than currently possible is anticipated to have a major impact on patient care. [1] Macdonald, D. R., et al. (1990). J Clin Oncol 8(7): 1277-1280. [2] Wen, P. Y., et al. (2010). J Clin Oncol 28(11): 1963-1972. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 452. doi:1538-7445.AM2012-452

Abstract LB-334: Imaging caspase dependent cell death as a surrogate for efficacy of cancer therapeutics

Induction of cell death in malignant lesions still represents the major goal in the treatment of cancer patients. Imaging of apoptosis would enhance the development of optimal dosing, schedule and combination therapies. Here we used a luciferase biosensor (Promega) and developed a bioluminescent assay system with optimized signal to noise for the detection of cell death in vitro and in living animals. Using D54-MG cells stably expressing the reporter, we demonstrate that TRAIL treatment resulted in a 100–200 fold induction of bioluminescence activity which correlated with cell death as demonstrated by increased cleavage of Caspase-3. Furthermore inhibition of apoptosis using the pan caspase inhibitor Z-VAD-FMK resulted in inhibition of reporter activity. The utility of this cell death reporter was further demonstrated in human glioma xenografted tumors wherein induction of apoptosis was correlated with reporter activation. The high signal to noise and dynamic range of reporter activity provides for a sensitive and quantitative surrogate for evaluation of experimental therapeutics. In addition, the ability to image repeatedly provides a unique opportunity to understand the dynamics of cell death in response to specific drugs or combination therapies. We are also investigating the application of this technology in a high throughput screening of compound and targeted siRNA libraries in diverse cells and in pre-clinical cancer mouse models. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr LB-334. doi:10.1158/1538-7445.AM2011-LB-334