IMRT and RapidArc commissioning of a TrueBeam linear accelerator using TG-119 protocol cases.

The purpose of this study is to evaluate the overall accuracy of intensity-modulated radiation therapy (IMRT) and RapidArc delivery using both flattening filter (FF) and flattening filter-free (FFF) modalities based on test cases developed by AAPM Task Group 119. Institutional confidence limits (CLs) were established as the baseline for patient specific treatment plan quality assurance (QA). The effects of gantry range, gantry speed, leaf speed, dose rate, as well as the capability to capture intentional errors, were evaluated by measuring a series of Picket Fence (PF) tests using the electronic portal imaging device (EPID) and EBT3 films. Both IMRT and RapidArc plans were created in a Solid Water phantom (30 × 30 × 15 cm3) for the TG-119 test cases representative of normal clinical treatment sites for all five photon energies (6X, 10X, 15X, 6X-FFF, 10X-FFF) and the Exact IGRT couch was included in the dose calculation. One high-dose point in the PTV and one low-dose point in the avoidance structure were measured with an ion chamber in each case for each energy. Similarly, two GAFCHROMIC EBT3 films were placed in the coronal planes to measure planar dose distributions in both high- and low-dose regions. The confidence limit was set to have 95% of the measured data fall within the tolerance. The mean of the absolute dose deviation for variable dose rate and gantry speed during RapidArc delivery was within 0.5% for all energies. The corresponding results for leaf speed tests were all within 0.4%. The combinations of dynamic leaf gap (DLG) and MLC transmission factor were optimized based on the ion chamber measurement results of RapidArc delivery for each energy. The average 95% CLs for the high-dose point in the PTV were 0.030 ± 0.007 (range, 0.022-0.038) for the IMRT plans and 0.029 ± 0.011 (range, 0.016-0.043) for the RapidArc plans. For low-point dose in the avoidance structures, the CLs were 0.029 ± 0.006 (range, 0.024-0.039) for the IMRT plans and 0.027 ± 0.013 (range, 0.017-0.047) for the RapidArc plans. The average 95% CLs using 3%/3 mm gamma criteria in the high-dose region were 5.9 ± 2.7 (range, 1.4-8.6) and 3.9 ± 2.9 (range, 1.5-8.8) for IMRT and RapidArc plans, respectively. The average 95% CLs in the low-dose region were 5.3 ± 2.6 (range, 1.2-7.4) and 3.7 ± 2.8 (range, 1.8-8.3) for IMRT and RapidArc plans, respectively. Based on ion chamber, as well as film measurements, we have established CLs values to ensure the high precision of IMRT and RapidArc delivery for both FF and FFF modalities.

A phase I clinical trial of oncolytic adenovirus mediated suicide and interleukin-12 gene therapy in patients with recurrent localized prostate adenocarcinoma.

In a phase I dose escalation and safety study (NCT02555397), a replication-competent oncolytic adenovirus expressing yCD, TK and hIL-12 (Ad5-yCD/mutTKSR39rep-hIL-12) was administered in 15 subjects with localized recurrent prostate cancer (T1c-T2) at increasing doses (1 × 1010, to 1 × 1012 viral particles) followed by 7-day treatment of 5-fluorocytosine (5-FC) and valganciclovir (vGCV). The primary endpoint was toxicity through day 30 while the secondary and exploratory endpoints were quantitation of IL-12, IFNγ, CXCL10 and peripheral blood mononuclear cells (PBMC). The study maximum tolerated dose (MTD) was not reached indicating 1012 viral particles was safe. Total 115 adverse events were observed, most of which (92%) were grade 1/2 that did not require any treatment. Adenoviral DNA was detected only in two patients. Increase in IL-12, IFNγ, and CXCL10 was observed in 57%, 93%, and 79% patients, respectively. Serum cytokines demonstrated viral dose dependency, especially apparent in the highest-dose cohorts. PBMC analysis revealed immune system activation after gene therapy in cohort 5. The PSA doubling time (PSADT) pre and post treatment has a median of 1.55 years vs 1.18 years. This trial confirmed that replication-competent Ad5-IL-12 adenovirus (Ad5-yCD/mutTKSR39rep-hIL-12) was well tolerated when administered locally to prostate tumors.

Feasibility of adenovirus-mediated hNIS gene transfer and 131I radioiodine therapy as a definitive treatment for localized prostate cancer.

We have developed a replication-competent adenovirus (Ad5-yCD/mutTK(SR39)rep-hNIS) armed with two suicide genes and the human sodium iodide symporter (hNIS) gene. In this context, hNIS can be used as a reporter gene in conjunction with nuclear imaging and as a potentially therapeutic gene when combined with (131)I radioiodine therapy. Here, we quantified the volume and magnitude of hNIS gene expression in the human prostate following injection of a high Ad5-yCD/mutTK(SR39)rep-hNIS dose using a standardized injection algorithm, and estimated the radiation dose that would be delivered to the prostate had men been administered (131)I with curative intent. Six men with clinically localized prostate cancer received an intraprostatic injection of Ad5-yCD/mutTK(SR39)rep-hNIS under transrectal ultrasound guidance. All men received 2 × 0.5 ml deposits (5 × 10(11) vp/deposit) in each of the four base and midgland sextants and 2 × 0.25 ml deposits (2.5 × 10(11) vp/deposit) in each of the two apex sextants for a total of 12 deposits (5 × 10(12) vp) in 5 ml. On multiple days after the adenovirus injection, men were administered sodium pertechnetate (Na(99m)TcO(4)) and hNIS gene expression in the prostate was quantified by single photon emission computed tomography (SPECT). hNIS gene expression was detected in the prostate of six of six (100%) men. On average, 45% (range 18-83%) of the prostate volume was covered with gene expression. Had men been administered 200 mCi (131)I, we estimate that the mean absorbed dose to the prostate would be 7.2 ± 4.8 Gy (range 2.1-13.3 Gy), well below that needed to sterilize the prostate. We discuss the obstacles that must be overcome before adenovirus-mediated hNIS gene transfer and (131)I radioiodine therapy can be used as a definitive treatment for localized prostate cancer.

Phase I trial of oncolytic adenovirus-mediated cytotoxic and interleukin-12 gene therapy for the treatment of metastatic pancreatic cancer.

The safety of oncolytic adenovirus-mediated suicide and interleukin-12 (IL 12) gene therapy was evaluated in metastatic pancreatic cancer patients. In this phase I study, a replication-competent adenovirus (Ad5-yCD/mutTKSR39 rep-hIL-12) expressing yCD/mutTKSR39 (yeast cytidine deaminase/mutant S39R HSV-1 thymidine kinase) and human IL-12 (IL 12) was injected into tumors of 12 subjects with metastatic pancreatic cancer (T2N0M1-T4N1M1) at escalating doses (1 × 1011, 3 × 1011, or 1 × 1012 viral particles). Subjects received 5-fluorocytosine (5-FC) therapy for 7 days followed by chemotherapy (FOLFIRINOX or gemcitabine/albumin-bound paclitaxel) starting 21 days after adenovirus injection. The study endpoint was toxicity through day 21. Experimental endpoints included measurements of serum IL 12, interferon gamma (IFNG), and CXCL10 to assess immune system activation. Peripheral blood mononuclear cells and proliferation markers were analyzed by flow cytometry. Twelve patients received Ad5-yCD/mutTKSR39 rep-hIL-12 and oral 5-FC. Approximately 94% of the 121 adverse events observed were grade 1/2 requiring no medical intervention. Ad5-yCD/mutTKSR39 rep-hIL-12 DNA was detected in the blood of two patients. Elevated serum IL 12, IFNG, and CXCL10 levels were detected in 42%, 75%, and 92% of subjects, respectively. Analysis of immune cell populations indicated activation after Ad5-yCD/mutTKSR39 rep-hIL-12 administration. The median survival of patients in the third cohort is 18.1 (range, 3.5-20.0) months. The study maximum tolerated dose (MTD) was not reached.

Inhibition of myostatin prevents microgravity-induced loss of skeletal muscle mass and strength.

The microgravity conditions of prolonged spaceflight are known to result in skeletal muscle atrophy that leads to diminished functional performance. To assess if inhibition of the growth factor myostatin has potential to reverse these effects, mice were treated with a myostatin antibody while housed on the International Space Station. Grip strength of ground control mice increased 3.1% compared to baseline values over the 6 weeks of the study, whereas grip strength measured for the first time in space showed flight animals to be -7.8% decreased in strength compared to baseline values. Control mice in space exhibited, compared to ground-based controls, a smaller increase in DEXA-measured muscle mass (+3.9% vs +5.6% respectively) although the difference was not significant. All individual flight limb muscles analyzed (except for the EDL) weighed significantly less than their ground counterparts at the study end (range -4.4% to -28.4%). Treatment with myostatin antibody YN41 was able to prevent many of these space-induced muscle changes. YN41 was able to block the reduction in muscle grip strength caused by spaceflight and was able to significantly increase the weight of all muscles of flight mice (apart from the EDL). Muscles of YN41-treated flight mice weighed as much as muscles from Ground IgG mice, with the exception of the soleus, demonstrating the ability to prevent spaceflight-induced atrophy. Muscle gene expression analysis demonstrated significant effects of microgravity and myostatin inhibition on many genes. Gamt and Actc1 gene expression was modulated by microgravity and YN41 in opposing directions. Myostatin inhibition did not overcome the significant reduction of microgravity on femoral BMD nor did it increase femoral or vertebral BMD in ground control mice. In summary, myostatin inhibition may be an effective countermeasure to detrimental consequences of skeletal muscle under microgravity conditions.

Does hyperthermia increase adenoviral transgene expression or dissemination in tumors?

PURPOSE: Viral vectors used for cancer gene therapy are usually delivered by direct intratumoral administration. We studied the role of hyperthermia (HT) in vitro and in vivo in an attempt to achieve higher transfection rates (especially, larger volume of spread). MATERIALS AND METHODS: Replication-deficient adenoviruses containing either the human sodium-iodide symporter (Ad5-CMV-hNIS) or green fluorescent protein (Ad5-CMV-eGFP) as reporter genes were used. For in vitro studies, human lung cancer A549 cells were transfected with the virus and assayed for hNIS expression by radioactive pertechnetate uptake or green fluorescence activity using a gamma-counter or fluoroscopy respectively in the presence and absence of HT. For in vivo studies, A549 tumors were established intramuscularly in CD1 athymic mice. The adenoviral constructs (10(10) viral particles/tumor) were injected intratumorally when the tumors reached 10-11 mm in diameter. Different timing sequences of HT were examined and viral spread was assessed using technetium-autoradiography or GFP-fluorescence microscopy. RESULTS: In the in vitro studies, A549 cells infected with the adenoviral construct did not show any difference in gene expression level in the presence or absence of HT. In vivo, the effect of HT on the volume of gene expression in A549 tumors was highly variable with some groups of mice showing better spread in the presence of HT and others showing reduced spread with HT. CONCLUSION: Improvements in intratumoral adenoviral spread in response to hyperthermia were not consistently observed in a mouse tumor model using two quantitative endpoints of gene expression.

Phase I study of noninvasive imaging of adenovirus-mediated gene expression in the human prostate.

To monitor noninvasively potentially therapeutic adenoviruses for cancer, we have developed a methodology based on the sodium iodide symporter (NIS). Men with clinically localized prostate cancer were administered an intraprostatic injection of a replication-competent adenovirus, Ad5-yCD/utTK(SR39)rep-hNIS, armed with two suicide genes and the NIS gene. NIS gene expression (GE) was imaged noninvasively by uptake of Na(99 m)TcO(4) in infected cells using single photon emission-computed tomography (SPECT). The investigational therapy was safe with 98% of the adverse events being grade 1 or 2. GE was detected in the prostate in seven of nine (78%) patients at 1 x 10(12) virus particles (vp) but not at 1 x 10(11) vp. Volume and total amount of GE was quantified by SPECT. Following injection of 1 x 10(12) vp in 1 cm(3), GE volume (GEV) increased to a mean of 6.6 cm(3), representing, on average, 18% of the total prostate volume. GEV and intensity peaked 1-2 days after the adenovirus injection and was detectable in the prostate up to 7 days. Whole-body imaging demonstrated intraprostatic gene expression, and there was no evidence of extraprostatic dissemination of the adenovirus by SPECT imaging. The results demonstrate that noninvasive imaging of adenovirus-mediated gene therapy in humans is feasible and safe.

Five-year follow-up of trial of replication-competent adenovirus-mediated suicide gene therapy for treatment of prostate cancer.

Replication-competent adenovirus-mediated suicide gene therapy is an investigational cancer treatment that combines the oncolytic actions of human adenoviruses with the cytotoxic effects of chemo-radiosensitizing genes. Previously, we reported the short-term effects of this therapy in men with local recurrence of prostate cancer after definitive radiotherapy. With a median prostate-specific antigen (PSA) follow-up of 5 years, we report here the effect of the gene therapy on prostate-specific antigen doubling time (PSADT), a surrogate end point with significant prognostic power. When considering all evaluable subjects, the PSADT increased following the gene therapy from a mean of 17 to 31 months (median 16 to 22 months) (P=0.014). Assuming that salvage androgen suppression therapy androgen suppression therapy (AST) was uniformly initiated at a PSA of 15 ng/mL, the gene therapy would have delayed the projected onset of salvage therapy by an average of 2 years. The results indicate that replication-competent adenovirus-mediated suicide gene therapy may provide a potential long-term benefit to patients, as shown by a lengthening of the PSADT, and delay in when salvage therapy is indicated. Given the high morbidity associated with AST, we believe this approach could provide an attractive treatment option for selection of patients experiencing PSA relapse following definitive therapy.

Design considerations for incorporating sodium iodide symporter reporter gene imaging into prostate cancer gene therapy trials.

This study was done to aid in the design of a phase I gene therapy trial in patients with prostate cancer. We determined the dosimetric characteristics of our reporter gene system when coupled with intravenous administration of radioactive sodium pertechnetate (Na(99m) TcO(4)) and determined the feasibility of using human sodium iodide symporter (hNIS) as a reporter gene to study the dynamics of adenoviral transgene expression in a large animal tumor. A replication-competent Ad5-yCD/mutTK(SR39) rep-hNIS adenovirus was injected into the prostate gland of dogs for dosimetry purposes, and into a canine soft tissue sarcoma (STS) for imaging purposes. After resection of the prostate, the amount of (99m)TcO(4)() sequestered in the prostate was determined, the radiation dose absorbed by the prostate and nontarget critical organs was calculated, and hNIS reporter gene expression was imaged in the STS by single-photon emission computed tomography (SPECT). On the basis of the findings from 25 dogs, the amount of (99m)TcO (4)() sequestered in the prostate ranged from 13 to 276 muCi. Using the highest value observed, absorbed radiation dose to critical organs was calculated and found to be below U.S. Food and Drug Administration limits for diagnostic imaging. Also, (99m)TcO (4)() uptake was readily detected by SPECT and found to persist in vivo for at least 4 days. On the basis of our dosimetry calculations, up to five imaging procedures can be safely performed in humans after intraprostatic injection of the Ad5-yCD/mutTK(SR39)rep-hNIS adenovirus and the hNIS reporter gene system can be used to study the dynamics of adenoviral gene therapy vectors in large animal tumors.

A model for optimizing adenoviral delivery in human cancer gene therapy trials.

Optimization of adenoviral delivery to the target volume is required for adenovirus-mediated cancer gene therapy to reach its maximal potential. The purpose of these studies was to develop a model of gene expression to improve adenovirus-mediated cancer gene therapy in the clinic. We measured the distribution of gene expression after a single deposit of a replication-competent adenovirus carrying the human sodium iodide symporter (hNIS) reporter gene was delivered to naive canine prostate and to human tumor xenografts. We generated hypothetical treatment plans for two prospective prostate cancer patients, using standard brachytherapy algorithms. In both models, the gene expression distribution from a single adenoviral deposit could be accurately described by a Gaussian function. In the naive canine prostate, a 0.1-ml deposit of 3 x 10(11) viral particles (VP) resulted in a gene expression volume of 1.14 +/- 0.70 cm(3), indicating that a minimum of 40 adenoviral deposits would be required to cover a 40-cm(3) prostate with therapeutic gene expression. On a viral particle basis, the gene expression volume obtained in human tumor xenografts (7 x 10(-12) cm(3)/VP) was twice that (3.5 x 10(-12) cm(3)/VP) measured in the naive canine prostate. Hypothetical treatment plans for two prostates indicated that 26 and 57 0.1-ml adenoviral deposits would be required to cover, respectively, 24- and 49-cm(3) prostates with gene expression. Although our studies focused on prostate, we believe the methodology to model gene expression presented here has much broader application to optimize treatment plans in other solid tumor sites; this assertion should be confirmed experimentally.

Immunotherapeutic intervention with oncolytic adenovirus in mouse mammary tumors.

The goal is to elucidate the immune modulating activity of an adenovirus (Adv) vector which showed therapeutic activity in human clinical trials. The oncolytic adenovirus (Adv/CD-TK) expressing two suicide genes was tested in two HER2/neu positive BALB/c mouse mammary tumor systems: rat neu-induced TUBO and human HER2-transfected D2F2/E2. Intra-tumoral (i.t.) Adv/CD-TK injection of TUBO tumor plus systemic prodrug therapy showed limited antitumor activity, not exceeding that by the virus itself. Antibody (Ab) to the virus was induced in Adv-/Luc-treated mice, to coincide with the loss of transgene expression. Low replication activity of adenoviruses in rodent cells may limit viral persistence. Host immunity against Adv or Adv-infected cells further mutes suicide gene activity. Treatment of TUBO tumors with Adv/CD-TK alone, however, induced neu-specific Ab responses. Treatment with Adv/CD-TK/GM (Adv/GM) that also expressed mouse granulocyte macrophage colony stimulating factor (GM-CSF), but without prodrug treatment, delayed tumor growth, enhanced anti-neu Ab production and conferred complete protection against secondary tumor challenge. D2F2/E2 tumor-bearing mice showed decreased tumor growth following i.t. Adv/GM treatment and they generated greater HER2-specific T-cell responses. These data suggest that i.t. injection of Adv itself induces immune reactivity to tumor-associated antigens and the encoded cytokine, GM-CSF, amplifies that immune response, resulting in tumor growth inhibition. Incorporation of suicide gene therapy did not improve the efficacy of Adv therapy in this mouse mammary tumor system. Oncolytic adenoviral therapy may be streamlined and improved by substituting the suicide genes with immune modulating genes to exploit tumor immunity for therapeutic benefit.

Gene therapy strategies to enhance the effectiveness of cancer radiotherapy.

Altering the genetic makeup of a cancer cell by gene transfer is a potentially powerful strategy for treating human cancer. However, a low efficiency of gene delivery in vivo and poor tumor specificity has prevented the widespread implementation of this technology in the clinic. Despite these formidable obstacles, the first successful application of gene therapy in the treatment of cancer could occur when it is combined with local modalities such as radiation therapy. A small number of gene therapy strategies have been evaluated in clinical trials in combination with external-beam radiation therapy. Combined therapy has been well tolerated and has not exacerbated the side effects of radiation therapy. Gene transfer and tumor cell destruction have been demonstrated in vivo. Although the results await confirmation in larger, prospective phase III trials, there is suggestive evidence that combined therapies may be demonstrating better than expected antitumor activity. The vast knowledge of the molecular defects that drive the cancer process, coupled with expanding the understanding of the genes responsible for tumor cell radioresistance, have led to the development of rational, targeted gene therapies designed to increase tumor cell radiosensitivity. Results of the clinical trials conducted to date are reviewed, followed by a description of new approaches currently under development.

Vision 20/20: the role of Raman spectroscopy in early stage cancer detection and feasibility for application in radiation therapy response assessment.

Raman spectroscopy is an optical technique capable of identifying chemical constituents of a sample by their unique set of molecular vibrations. Research on the applicability of Raman spectroscopy in the differentiation of cancerous versus normal tissues has been ongoing for many years, and has yielded successful results in the context of prostate, breast, brain, skin, and head and neck cancers as well as pediatric tumors. Recently, much effort has been invested on developing noninvasive "Raman" probes to provide real-time diagnosis of potentially cancerous tumors. In this regard, it is feasible that the Raman technique might one day be used to provide rapid, minimally invasive real-time diagnosis of tumors in patients. Raman spectroscopy is relatively new to the field of radiation therapy. Recent work involving cell lines has shown that the Raman technique is able to identify proteins and other markers affected by radiation therapy. Although this work is preliminary, one could ask whether or not the Raman technique might be used to identify molecular markers that predict radiation response. This paper provides a brief review of Raman spectroscopic investigations in cancer detection, benefits and limitations of this method, advances in instrument development, and also preliminary studies related to the application of this technology in radiation therapy response assessment.

Prospective randomized phase 2 trial of intensity modulated radiation therapy with or without oncolytic adenovirus-mediated cytotoxic gene therapy in intermediate-risk prostate cancer.

PURPOSE: To assess the safety and efficacy of combining oncolytic adenovirus-mediated cytotoxic gene therapy (OAMCGT) with intensity modulated radiation therapy (IMRT) in intermediate-risk prostate cancer. METHODS AND MATERIALS: Forty-four men with intermediate-risk prostate cancer were randomly assigned to receive either OAMCGT plus IMRT (arm 1; n=21) or IMRT only (arm 2; n=23). The primary phase 2 endpoint was acute (≤90 days) toxicity. Secondary endpoints included quality of life (QOL), prostate biopsy (12-core) positivity at 2 years, freedom from biochemical/clinical failure (FFF), freedom from metastases, and survival. RESULTS: Men in arm 1 exhibited a greater incidence of low-grade influenza-like symptoms, transaminitis, neutropenia, and thrombocytopenia than men in arm 2. There were no significant differences in gastrointestinal or genitourinary events or QOL between the 2 arms. Two-year prostate biopsies were obtained from 37 men (84%). Thirty-three percent of men in arm 1 were biopsy-positive versus 58% in arm 2, representing a 42% relative reduction in biopsy positivity in the investigational arm (P=.13). There was a 60% relative reduction in biopsy positivity in the investigational arm in men with <50% positive biopsy cores at baseline (P=.07). To date, 1 patient in each arm exhibited biochemical failure (arm 1, 4.8%; arm 2, 4.3%). No patient developed hormone-refractory or metastatic disease, and none has died from prostate cancer. CONCLUSIONS: Combining OAMCGT with IMRT does not exacerbate the most common side effects of prostate radiation therapy and suggests a clinically meaningful reduction in positive biopsy results at 2 years in men with intermediate-risk prostate cancer.

The potential of iodine for improving breast cancer diagnosis and treatment.

Early detection through modalities such as mammography remains pivotal in the fight against breast cancer. The detectability of breast cancer through mammography is rooted in the differential X-ray attenuation properties of cancerous and normal breast tissue. An unexplored component of the X-ray contrast between fibrous breast tissue and similarly composed tumor tissue is the presence of naturally localized iodine in the cancer but not healthy breast tissue. It is hypothesized that differing amounts of iodine are present in tumor versus normal breast tissue that leads to more easily detectable cancer due to an increased Z value of the tumor tissue relative to the healthy tissue, which results in enhanced differences in X-ray attenuation properties between the two tissues and thus greater radiographic contrast. The hypothesis is supported by experimental observations explaining how iodine could localize in the tumor tissue but not surrounding healthy tissue. Breast cancer cells express the sodium-iodide symporter (NIS), an ion pump which sequesters iodine in tumor cells. Healthy non-lactating breast tissue, in contrast, does not express NIS. Further evidence for the differential expression of NIS resulting in X-ray contrast enhancement in breast cancer is the established correlation between expression of insulin growth factor (IGF) and enhanced X-ray contrast, and the evidence that IGF is a promoter for NIS. Ultimately, if the expression of iodine can be shown to be a component of radiographic contrast between healthy and tumor breast tissue, this could be used to drive the development of new technology and techniques for use in the detection and treatment of breast cancer. The proof of this hypothesis could thus have a substantial impact in the fight against breast cancer.

Monitoring adenoviral based gene delivery in rat glioma by molecular imaging.

AIM: To determine whether endothelial progenitor cells (EPCs) can be used as delivery vehicle for adenoviral vectors and imaging probes for gene therapy in glioblastoma. METHODS: To use cord blood derived EPCs as delivery vehicle for adenoviral vectors and imaging probes for glioma gene therapy, a rat model of human glioma was made by implanting U251 cells orthotopically. EPCs were transfected with an adenovirus (AD5/carrying hNIS gene) and labeled with iron oxide and inoculated them directly into the tumor 14 d following implantation of U251 cells. Magnetic resonance imaging (MRI) was used to in vivo track the migration of EPCs in the tumor. The expression of gene products was determined by in vivo Tc-99m single photon emission computed tomography (SPECT). The findings were validated with immunohistochemistry (IHC). RESULTS: EPCs were successfully transfected with the adenoviral vectors carrying hNIS which was proved by significantly (P < 0.05) higher uptake of Tc-99m in transfected cells. Viability of EPCs following transfection and iron labeling was not altered. In vivo imaging showed the presence of iron positive cells and the expression of transgene (hNIS) product on MRI and SPECT, respectively, all over the tumors following administration of transfected and iron labeled EPCs in the tumors. IHC confirmed the distribution of EPC around the tumor away from the injection site and also showed transgene expression in the tumor. The results indicated the EPCs' ability to deliver adenoviral vectors into the glioma upon intratumor injection. CONCLUSION: EPCs can be used as vehicle to deliver adenoviral vector to glioma and also act as imaging probe at the same time.

Endothelial progenitor cells (EPCs) as gene carrier system for rat model of human glioma.

BACKGROUND: Due to their unique property to migrate to pathological lesions, stem cells are used as a delivery vehicle for therapeutic genes to tumors, especially for glioma. It is critically important to track the movement, localization, engraftment efficiency and functional capability or expression of transgenes of selected cell populations following transplantation. The purposes of this study were to investigate whether 1) intravenously administered, genetically transformed cord blood derived EPCs can carry human sodium iodide symporter (hNIS) to the sites of tumors in rat orthotopic model of human glioma and express transgene products, and 2) whether accumulation of these administered EPCs can be tracked by different in vivo imaging modalities. METHODS AND RESULTS: Collected EPCs were cultured and transduced to carry hNIS. Cellular viability, differential capacity and Tc-99m uptake were determined. Five to ten million EPCs were intravenously administered and Tc-99-SPECT images were acquired on day 8, to determine the accumulation of EPCs and expression of transgenes (increase activity of Tc-99m) in the tumors. Immunohistochemistry was performed to determine endothelial cell markers and hNIS positive cells in the tumors. Transduced EPCs were also magnetically labeled and accumulation of cells was confirmed by MRI and histochemistry. SPECT analysis showed increased activity of Tc-99m in the tumors that received transduced EPCs, indicative of the expression of transgene (hNIS). Activity of Tc-99m in the tumors was also dependent on the number of administered transduced EPCs. MRI showed the accumulation of magnetically labeled EPCs. Immunohistochemical analysis showed iron and hNIS positive and, human CD31 and vWF positive cells in the tumors. CONCLUSION: EPC was able to carry and express hNIS in glioma following IV administration. SPECT detected migration of EPCs and expression of the hNIS gene. EPCs can be used as gene carrier/delivery system for glioma therapy as well as imaging probes.

Adaptive seamless design for an efficacy trial of replication-competent adenovirus-mediated suicide gene therapy and radiation in newly-diagnosed prostate cancer (ReCAP Trial).

PURPOSE: Cumulative evidence has suggested investigation of the efficacy of Replication-Competent Adenovirus-mediated Suicide Gene Therapy in newly-diagnosed Prostate Cancer (ReCAP). There is a challenge in designing an efficacy trial for newly-diagnosed prostate cancer given its long natural history. The regulatory agency recommended a Phase II trial for safety before conducting the efficacy trial. EXPERIMENTAL DESIGN: The ReCAP trial is an adaptive seamless, multi-site open-label, randomized Phase II/III trial. Two hundred eighty men will be randomized to receive either replication-competent adenovirus-mediated suicide gene therapy followed by radiation (Arm 1) or radiation alone (Arm 2). Phase II trial component will include the first 21 patients in Arm 1 with complete toxicity through day 90 for safety evaluation. The primary efficacy endpoint is the time free from biochemical and/or clinical failure (FFF). The secondary efficacy endpoints are 2-year prostate biopsies and overall survival. Unequal spaced interim looks are proposed with the adaptive sample-size re-estimation. RESULTS: This trial has been approved by the FDA for the study therapy investigation and is currently recruiting patients. CONCLUSIONS: Challenges remain in designing newly-diagnosed prostate cancer trials. Adaptive seamless design is time-saving and a cost-effective design in the development of novel medical therapies, but requires a specified statistical plan in the decision process involved.

CDDO-Me: A Novel Synthetic Triterpenoid for the Treatment of Pancreatic Cancer.

Pancreatic ductal adenocarcinoma (PDA) is one of the most lethal human malignancy with dismal prognosis and few effective therapeutic options. Novel agents that are safe and effective are urgently needed. Oleanolic acid-derived synthetic triterpenoids are potent antitumorigenic agents, but their efficacy or the mechanism of action for pancreatic cancer has not been adequately investigated. In this study, we evaluated the antitumor activity and the mechanism of action of methyl-2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oate (CDDO-Me), a oleanane-derived synthetic triterpenoid for human pancreatic cancer cell lines. CDDO-Me inhibited the growth of both K-ras mutated (MiaPaca2, Panc1 and Capan2) and wild-type K-ras (BxPC3) pancreatic cancer cells at very low concentrations. The growth inhibitory activity of CDDO-Me was attributed to the induction of apoptosis characterized by increased annexin-V-FITC binding and cleavage of PARP-1 and procaspases-3, -8 and-9. In addition, CDDO-Me induced the loss of mitochondrial membrane potential and release of cytochrome C. The antitumor activity of CDDO-Me was associated with the inhibition of prosurvival p-Akt, NF-κB and mammalian target of rapamycin (mTOR) signaling proteins and the downstream targets of Akt and mTOR, such as p-Foxo3a (Akt) and p-S6K1, p-eIF-4E and p-4E-BP1 (mTOR). Silencing of Akt or mTOR with gene specific-siRNA sensitized the pancreatic cancer cells to CDDO-Me, demonstrating Akt and mTOR as molecular targets of CDDO-Me for its growth inhibitory and apoptosis-inducing activity.

A novel method of boron delivery using sodium iodide symporter for boron neutron capture therapy.

Boron Neutron Capture Therapy (BNCT) effectiveness depends on the preferential sequestration of boron in cancer cells relative to normal tissue cells. We present a novel strategy for sequestering boron using an adenovirus expressing the sodium iodide symporter (NIS). Human glioma grown subcutaneously in athymic mice and orthotopic rat brain tumors were transfected with NIS using a direct tumor injection of adenovirus. Boron bound as sodium tetrafluoroborate (NaBF(4)) was administered systemically several days after transfection. Tumors were excised hours later and assessed for boron concentration using inductively coupled plasma atomic emission spectroscopy. In the human glioma transfected with NIS, boron concentration was more than 10 fold higher with 100 mg/kg of NaBF(4), compared to tumor not transfected. In the orthotopic tumor model, the presence of NIS conferred almost 4 times the boron concentration in rat tumors transfected with human virus compared with contralateral normal brain not transfected. We conclude that adenovirus expressing NIS has the potential to be used as a novel boron delivery agent and should be explored for future clinical applications.