Benjamin A. Hoff

Dynamic Imaging of Emerging Resistance during Cancer Therapy

One of the greatest challenges in developing therapeutic regimens is the inability to rapidly and objectively assess tumor response due to treatment. Moreover, tumor response to therapeutic intervention in many cases is transient, and progressive alterations within the tumor may mask the effectiveness of an initially successful therapy. The ability to detect these changes as they occur would allow timely initiation of alternative approaches, maximizing therapeutic outcome. We investigated the ability of diffusion magnetic resonance imaging (MRI) to provide a sensitive measure of tumor response throughout the course of treatment, possibly identifying changes in sensitivity to the therapy. Orthotopic 9L gliomas were subjected to two separate therapeutic regimens, with one group receiving a single 5-day cycle (1omega) of low-dose 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) and a second group receiving two cycles at the same dose, bisected with 2 days of rest (2omega). Apparent diffusion coefficient maps were acquired before and throughout treatment to observe changes in water mobility, and these observations were correlated to standard measures of therapeutic response and outcome. Our results showed that diffusion MRI was indeed able to detect the emergence of a drug-resistant tumor subpopulation subsequent to an initially successful cycle of BCNU therapy, leading to minimal gains from a second cycle. These diffusion MRI findings were highly correlated with tumor growth delay, animal survival, and ex vivo growth inhibition assays showing emerging resistance in excised tumors. Overall, this study highlights the ability of diffusion MRI to provide sensitive dynamic assessment of therapy-induced response, allowing early opportunities for optimization of therapeutic protocols.

The brain tumor window model: a combined cranial window and implanted glioma model for evaluating intraoperative contrast agents.

OBJECTIVEOptical contrast agents for brain tumor delineation have been previously evaluated in ex vivo specimens from animals with implanted gliomas and may not reflect the true visual parameters encountered during surgery. This study describes a novel model system designed to evaluate optical contrast agents for tumor delineation in vivo. METHODSBiparietal craniectomies were performed on 8-week-old Sprague-Dawley rats. 9L glioma cells were injected intraparenchymally. A cover slip was bonded to the cranial defect with cyanoacrylate glue. When the tumor radius reached 1 mm, Coomassie Blue was administered intravenously while the appearance of the cortical surface was recorded. Computerized image analysis of the red/green/blue color components was used to quantify visible differences between tumor and nonneoplastic tissue and to compare delineation in the brain tumor window (BTW) model with the conventional 9L glioma model. RESULTSThe tumor margin in the BTW model was poorly defined before contrast administration but readily apparent after contrast administration. Based on red component intensity, tumor delineation improved 4-fold at 50 minutes after contrast administration in the BTW model (P < .002). The conventional 9L glioma model overestimated the degree of delineation compared with the BTW model at the same dose of Coomassie Blue (P < .03). CONCLUSIONWindow placement overlying an implanted glioma is technically possible and well tolerated in the rat. The BTW model is a valid system for evaluating optical contrast agents designed to delineate brain tumor margins. To our knowledge, we have described the first in vivo model system for evaluating optical contrast agents for tumor delineation.

Assessment of Multiexponential Diffusion Features as MRI Cancer Therapy Response Metrics

The aim of this study was to empirically test the effect of chemotherapy‐induced tissue changes in a glioma model as measured by several diffusion indices calculated from nonmonoexponential formalisms over a wide range of b‐values. We also compared these results to the conventional two‐point apparent diffusion coefficient calculation using nominal b‐values. Diffusion‐weighted imaging was performed over an extended range of b‐values (120–4000 sec/mm2) on intracerebral rat 9L gliomas before and after a single dose of 1,3‐bis(2‐chloroethyl)‐1‐nitrosourea. Diffusion indices from three formalisms of diffusion‐weighted signal decay [(a) two‐point analytical calculation using either low or high b‐values, (b) a stretched exponential formalism, and (c) a biexponential fit] were tested for responsiveness to therapy‐induced differences between control and treated groups. Diffusion indices sensitive to “fast diffusion” produced the largest response to treatment, which resulted in significant differences between groups. These trends were not observed for “slow diffusion” indices. Although the highest rate of response was observed from the biexponential formalism, this was not found to be significantly different from the conventional monoexponential apparent diffusion coefficient method. In conclusion, parameters from the more complicated nonmonoexponential formalisms did not provide additional sensitivity to treatment response in this glioma model beyond that observed from the two‐point conventional monoexponential apparent diffusion coefficient method. Magn Reson Med, 2010.

Imaging proteolytic activity in live cells and animal models.

In addition to their degradative role in protein turnover, proteases play a key role as positive or negative regulators of signal transduction pathways and therefore their dysregulation contributes to many disease states. Regulatory roles of proteases include their hormone-like role in triggering G protein-coupled signaling (Protease-Activated-Receptors); their role in shedding of ligands such as EGF, Notch and Fas; and their role in signaling events that lead to apoptotic cell death. Dysregulated activation of apoptosis by the caspase family of proteases has been linked to diseases such as cancer, autoimmunity and inflammation. In an effort to better understand the role of proteases in health and disease, a luciferase biosensor is described which can quantitatively report proteolytic activity in live cells and mouse models. The biosensor, hereafter referred to as GloSensor Caspase 3/7 has a robust signal to noise (50–100 fold) and dynamic range such that it can be used to screen for pharmacologically active compounds in high throughput campaigns as well as to study cell signaling in rare cell populations such as isolated cancer stem cells. The biosensor can also be used in the context of genetically engineered mouse models of human disease wherein conditional expression using the Cre/loxP technology can be implemented to investigate the role of a specific protease in living subjects. While the regulation of apoptosis by caspases was used as an example in these studies, biosensors to study additional proteases involved in the regulation of normal and pathological cellular processes can be designed using the concepts presented herein.

DCE and DW-MRI monitoring of vascular disruption following VEGF-Trap treatment of a rat glioma model

Vascular‐targeted therapies have shown promise as adjuvant cancer treatment. As these agents undergo clinical evaluation, sensitive imaging biomarkers are needed to assess drug target interaction and treatment response. In this study, dynamic contrast enhanced MRI (DCE‐MRI) and diffusion‐weighted MRI (DW‐MRI) were evaluated for detecting response of intracerebral 9 L gliosarcomas to the antivascular agent VEGF‐Trap, a fusion protein designed to bind all forms of Vascular Endothelial Growth Factor‐A (VEGF‐A) and Placental Growth Factor (PGF). Rats with 9 L tumors were treated twice weekly for two weeks with vehicle or VEGF‐Trap. DCE‐ and DW‐MRI were performed one day prior to treatment initiation and one day following each administered dose. Kinetic parameters (Ktrans, volume transfer constant; kep, efflux rate constant from extravascular/extracellular space to plasma; and vp, blood plasma volume fraction) and the apparent diffusion coefficient (ADC) over the tumor volumes were compared between groups. A significant decrease in kinetic parameters was observed 24 hours following the first dose of VEGF‐Trap in treated versus control animals (p < 0.05) and was accompanied by a decline in ADC values. In addition to the significant hemodynamic effect, VEGF‐Trap treated animals exhibited significantly longer tumor doubling times (p < 0.05) compared to the controls. Histological findings were found to support imaging response metrics. In conclusion, kinetic MRI parameters and change in ADC have been found to serve as sensitive and early biomarkers of VEGF‐Trap anti‐vascular targeted therapy. Copyright

Phosphorylation of FADD by the kinase CK1α promotes KRASG12D-induced lung cancer

Mutant KRAS promotes mitosis by stimulating phosphorylation of the adaptor protein FADD. Conversion of a death adaptor to a proliferation mediator Activating mutations in the protein RAS drive cell proliferation and tumor growth. Although best known for mediating cell death signaling through its death domain, when phosphorylated, the adaptor protein FADD promotes cell survival and proliferation. Bowman et al. found that, compared with KRAS mutant mice, KRAS mutant mice engineered to lack FADD or its upstream kinase CK1α developed fewer lung tumors. Lung tissue and cells from KRAS mutant mice had increased abundance of CK1α, phosphorylated FADD, and proliferative markers. In lung tumor samples from patients, expression of FADD was greater in tumors that had mutant KRAS. A CK1α inhibitor prevented FADD from physically interacting with mitotic kinases and suppressed cell proliferation in culture. Thus, blocking the phosphorylation of FADD may be a new strategy for patients with KRAS mutant lung tumors. Genomic amplification of the gene encoding and phosphorylation of the protein FADD (Fas-associated death domain) is associated with poor clinical outcome in lung cancer and in head and neck cancer. Activating mutations in the guanosine triphosphatase RAS promotes cell proliferation in various cancers. Increased abundance of phosphorylated FADD in patient-derived tumor samples predicts poor clinical outcome. Using immunohistochemistry analysis and in vivo imaging of conditional mouse models of KRASG12D-driven lung cancer, we found that the deletion of the gene encoding FADD suppressed tumor growth, reduced the proliferative index of cells, and decreased the activation of downstream effectors of the RAS–MAPK (mitogen-activated protein kinase) pathway that promote the cell cycle, including retinoblastoma (RB) and cyclin D1. In mouse embryonic fibroblasts, the induction of mitosis upon activation of KRAS required FADD and the phosphorylation of FADD by CK1α (casein kinase 1α). Deleting the gene encoding CK1α in KRAS mutant mice abrogated the phosphorylation of FADD and suppressed lung cancer development. Phosphorylated FADD was most abundant during the G2/M phase of the cell cycle, and mass spectrometry revealed that phosphorylated FADD interacted with kinases that mediate the G2/M transition, including PLK1 (Polo-like kinase 1), AURKA (Aurora kinase A), and BUB1 (budding uninhibited by benzimidazoles 1). This interaction was decreased in cells treated with a CKI-7, a CK1α inhibitor. Therefore, as the kinase that phosphorylates FADD downstream of RAS, CK1α may be a therapeutic target for KRAS-driven lung cancer.

Parametric response mapping of CT images provides early detection of local bone loss in a rat model of osteoporosis

Loss of bone mass due to disease, such as osteoporosis and metastatic cancer to the bone, is a leading cause of orthopedic complications and hospitalization. Onset of bone loss resulting from disease increases the risk of incurring fractures and subsequent pain, increasing medical expenses while reducing quality of life. Although current standard CT-based protocols provide adequate prognostic information for assessing bone loss, many of the techniques for evaluating CT scans rely on measures based on whole-bone summary statistics. This reduces the sensitivity at identifying local regions of bone resorption, as well as formation. In this study, we evaluate the effectiveness of a voxel-based image post-processing technique, called the Parametric Response Map (PRM), for identifying local changes in bone mass in weight-bearing bones on CT scans using an established animal model of osteoporosis. Serial CT scans were evaluated weekly using PRM subsequent to ovariectomy or sham surgeries over the period of one month. For comparison, bone volume fraction and mineral density measurements were acquired and found to significantly differ between groups starting 3 weeks post-surgery. High resolution ex vivo measurements acquired four weeks post-surgery validated the extent of bone loss in the surgical groups. In contrast to standard methodologies for assessing bone loss, PRM results were capable of identifying local decreases in bone mineral by week 2, which were found to be significant between groups. This study concludes that PRM is able to detect changes in bone mineral with higher sensitivity and spatial differentiation than conventional techniques for evaluating CT scans, which may aid in clinical decision making for patients suffering from bone loss.

Diffusion MRI in early cancer therapeutic response assessment.

Imaging biomarkers for the predictive assessment of treatment response in patients with cancer earlier than standard tumor volumetric metrics would provide new opportunities to individualize therapy. Diffusion‐weighted MRI (DW‐MRI), highly sensitive to microenvironmental alterations at the cellular level, has been evaluated extensively as a technique for the generation of quantitative and early imaging biomarkers of therapeutic response and clinical outcome. First demonstrated in a rodent tumor model, subsequent studies have shown that DW‐MRI can be applied to many different solid tumors for the detection of changes in cellularity as measured indirectly by an increase in the apparent diffusion coefficient (ADC) of water molecules within the lesion. The introduction of quantitative DW‐MRI into the treatment management of patients with cancer may aid physicians to individualize therapy, thereby minimizing unnecessary systemic toxicity associated with ineffective therapies, saving valuable time, reducing patient care costs and ultimately improving clinical outcome. This review covers the theoretical basis behind the application of DW‐MRI to monitor therapeutic response in cancer, the analytical techniques used and the results obtained from various clinical studies that have demonstrated the efficacy of DW‐MRI for the prediction of cancer treatment response. Copyright

Apparent diffusion coefficient is highly reproducible on preclinical imaging systems: Evidence from a seven-center multivendor study

To evaluate between‐site agreement of apparent diffusion coefficient (ADC) measurements in preclinical magnetic resonance imaging (MRI) systems.

The Impact of Sources of Variability on Parametric Response Mapping of Lung CT Scans.

Parametric response mapping (PRM) of inspiration and expiration computed tomography (CT) images improves the radiological phenotyping of chronic obstructive pulmonary disease (COPD). PRM classifies individual voxels of lung parenchyma as normal, emphysematous, or nonemphysematous air trapping. In this study, bias and noise characteristics of the PRM methodology to CT and clinical procedures were evaluated to determine best practices for this quantitative technique. Twenty patients of varying COPD status with paired volumetric inspiration and expiration CT scans of the lungs were identified from the baseline COPDGene cohort. The impact of CT scanner manufacturer and reconstruction kernels were evaluated as potential sources of variability in PRM measurements along with simulations to quantify the impact of inspiration/expiration lung volume levels, misregistration, and image spacing on PRM measurements. Negligible variation in PRM metrics was observed when CT scanner type and reconstruction were consistent and inspiration/expiration lung volume levels were near target volumes. CT scanner Hounsfield unit drift occurred but remained difficult to ameliorate. Increasing levels of image misregistration and CT slice spacing were found to have a minor effect on PRM measurements. PRM-derived values were found to be most sensitive to lung volume levels and mismatched reconstruction kernels. As with other quantitative imaging techniques, reliable PRM measurements are attainable when consistent clinical and CT protocols are implemented.