Combinatorial effect of radium-223 and irreversible electroporation on prostate cancer bone metastasis in mice.

BACKGROUND: Metastatic prostate cancer in bone is difficult to treat as the tumor cells are relatively resistant to hormonal or chemotherapies when compared to primary prostate cancer. Irreversible electroporation (IRE) is a minimally invasive ablation procedure that has potential applications in the management of prostate cancer in bone. However, a common limitation of IRE is tumor recurrence, which arises from incomplete ablation that allows remaining cancer cells to proliferate. In this study, we combined IRE with radium-223 (Ra-223), a bone-seeking radionuclide that emits short track length alpha particles and thus is associated with reduced damage to the bone marrow and evaluated the impact of the combination treatment on bone-forming prostate cancer tumors. METHODS: The antitumor activity of IRE and Ra-223 as single agents and in combination was tested in vitro against three bone morphogenetic protein 4 (BMP4)-expressing prostate cancer cell lines (C4-2B-BMP4, Myc-CaP-BMP4, and TRAMP-C2-BMP4). Similar evaluation was performed in vivo using a bone-forming C4-2B-BMP4 tumor model in nude mice. RESULTS: IRE and Ra-223 as monotherapy inhibited prostate cancer cell proliferation in vitro, and their combination resulted in significant reduction in cell viability compared to monotherapy. In vivo evaluation revealed that IRE with single-dose administration of Ra-233, compared to IRE alone, reduced the rate of tumor recurrence by 40% following initial apparent complete ablation and decreased the rate of proliferation of incompletely ablated tumor as quantified in Ki-67 staining (53.58 ± 16.0% for IRE vs. 20.12 ± 1.63%; for IRE plus Ra-223; p = 0.004). Histological analysis qualitatively showed the enhanced killing of tumor cells adjacent to bone by Ra-223 compared to those treated with IRE alone. CONCLUSION: IRE in combination with Ra-223, which enhanced the destruction of cancer cells that are adjacent to bone, resulted in reduction of tumor recurrence through improved clearance of proliferative cells in the tumor region.

Molecular Imaging with 3'-deoxy-3'[(18)F]-Fluorothymidine (18F-FLT) PET/CT for Early Response to Targeted Therapies in Sarcomas: A Pilot Study.

Although 3'-deoxy-3'[(18)F]-fluorothymidine (FLT)- positron emission tomography (PET) has been utilized for tumor response assessment to neoadjuvant chemotherapy in soft tissue sarcomas, it has not been exploited for the assessment of early response to systematically targeted therapies. Herein, we investigated the 18F-FLT PET/CT kinetics in patients with sarcoma who received targeted therapies. Among 15 patients with sarcoma who underwent 18F-FLT PET/CT, 5 patients (33%) patients were imaged at three time points: At baseline and at 1-15 weeks (MDM2-inhibitor treatment), and 10 patients (67%) were imaged twice: At baseline and at 1-4 weeks (MDM2 inhibitor, n = 5 ;c-met inhibitor n = 5). The patients with sarcoma had a total of 18 identifiable tumors. Twelve of 15 patients (80%) demonstrated 18F-FLT concentrations changes early, i.e., at 1-4 weeks. Eight patients responded (53.3%), four patients progressed (26.7%) based on FLT change of more than 10% increase, and three patients (20%) demonstrated no change. 18F-FLT PET/CT may be used for early response imaging to molecularly targeted therapies in patients with sarcoma. Further larger studies in specific sarcoma sub-types are warranted.

Early Response Assessment to Targeted Therapy Using 3'-deoxy-3'[(18)F]-Fluorothymidine (18F-FLT) PET/CT in Lung Cancer.

Although 2-deoxy-2-[18F]-fluoro-D-glucose positron emission tomography/computed tomography (18F-FDG PET/CT) is a sensitive nuclear medicine modality, specificity for characterizing lung cancer is limited. Tumor proliferation and early response to molecularly targeted therapy could be visualized using 3'-deoxy-3'[(18)F]-fluorothymidine (18F-FLT) PET/CT. The superiority of 18F-FLT PET/CT over 18F-FDG PET/CT in early therapeutic monitoring has been well described in patients after chemotherapy, radiotherapy, and/or chemo/radiotherapy. In thispilot study, we explorethe use of 18F-FLT PET/CT as an early response evaluation modality in patients with lung cancerand provide specific case studies of patients with small cell lung cancer and non-small cell lung cancer who received novel targeted therapies. Early response for c-MET inhibitor was observed in four weeks and for MDM2 inhibitor in nine days.

Fully automated preparation of 68Ga-PSMA-11 at curie level quantity using cyclotron-produced 68Ga for clinical applications.

68Ga-PSMA-11 is currently one of the most investigated PET agents for imaging both recurrent prostate cancer and relevant metastases; however, the production and distribution of 68Ga-PSMA-11 is limited to a supply of only a few daily doses when using a commercially available 68Ge/68Ga generator. 68Ge/68Ga generators deliver only a modest amount of activity, up to 1850 MBq (50 mCi), when new, but it decreases with time. Additionally, the production of 68Ga/68Ge generators has not been able to meet the increasing demand of 68Ga radiotracers. In response to the need for a more economically viable alternative, the focus of this study was to provide a simple and efficient method for producing 68Ga-PSMA-11, using cyclotron-produced 68Ga that is ready for routine clinical practice.

Community Benefit Spending Among North Carolina's Tax-Exempt Hospitals After Performing Community Health Needs Assessments.

CONTEXT: As of March 23, 2012, the Internal Revenue Service (IRS) requires tax-exempt hospitals to conduct Community Health Needs Assessment (CHNA) every 3 years to incentivize hospitals to provide programs responsive to the health needs of their communities. OBJECTIVE: To examine the distribution and variation in community benefit spending among North Carolina's tax-exempt hospitals 2 years after completing their first IRS-mandated CHNA. DESIGN: Cross-sectional study using secondary analysis of published community benefit reports. Community benefit was categorized on the basis of North Carolina Hospital Association's community benefit reporting guidelines. Multiple regression analysis using generalized linear model was used to examine the variation in community benefit spending among study hospitals considering differences in hospital-level and community characteristics. SETTING: Fifty-three private, nonprofit hospitals across North Carolina. MAIN OUTCOME MEASURE: Dollar expenditures as a percentage of operating expenses of the 2 categories of community benefit spending: patient care financial assistance and community health programs. RESULTS: Study hospitals' aggregate community benefit spending was $2.6 billion, 85% of which was in the form of patient care financial assistance, with only 0.7% of total spending allocated to community-building activities such as affordable housing, economic development, and environmental improvements. On average, the study hospitals' community benefit spending was equivalent to 14.6% of operating expenses. Hospitals with 300 or more beds provided significantly higher investments in community health programs as a percentage of their operating expenses than hospitals with 101 to 299 beds (P = .03) or hospitals with 100 or fewer beds (P = .04). Access to care was not associated with patient care financial assistance (P = .81) or community health programs expenditures (P = .94). CONCLUSIONS: The study hospitals direct most of their community benefit expenditures to patient care financial assistance (individual welfare) rather than population health improvement initiatives, with virtually no investments in community-building activities that address socioeconomic determinants of health.

Did Performing Community Health Needs Assessments Increase Community Health Program Spending by North Carolina's Tax-Exempt Hospitals?

As of March 23, 2012, the Internal Revenue Service (IRS) requires tax-exempt hospitals to conduct a Community Health Needs Assessment (CHNA) every 3 years. This study assessed whether the IRS CHNA mandate incentivized North Carolina's tax-exempt hospitals to increase investments in community health programs. The authors gathered the 2012-2016 community benefit reports of 53 North Carolina private, nonprofit hospitals from the North Carolina Hospital Association. Community benefit spending data from the year of the first CHNA were compared to that 2 years later using paired t tests among matched subjects. No significant increases were found in hospitals' community health programs spending (P = 0.6920) or in providing patient care financial assistance (charity or discounted care) (P = 0.0934). In fact, aggregate community health programs spending effectively decreased by 4%, from $393.3 million to $377.5 million. Among all community benefit items, only the unreimbursed cost for treating Medicare patients increased significantly (P = 0.0297). The proportion of spending on community health programs relative to patient care financial assistance decreased significantly (P = 0.0338). Performing CHNAs did not incentivize North Carolina's tax-exempt hospitals to progressively invest in community health programs. The hospitals continue to spend heavily on patient care financial assistance and little on disease prevention and community health improvement activities. These findings suggest that tax-exempt hospitals continue to function as a safety net for the poor and the uninsured rather than as active partners in population health management initiatives. At present, performing CHNAs may be more a demonstration of compliance than a tool to improve population health.

Targeted Therapy in Advanced Thyroid Cancer to Resensitize Tumors to Radioactive Iodine.

Context: Many differentiated thyroid cancers (DTC) dedifferentiate and become radioactive iodine (RAI)-refractory (RAIR) with worse outcomes. Targeted therapy (TTx) may downregulate MAPK signaling and sensitize tumors to RAI. Objective: We describe patients with RAIR DTC receiving TTx with demonstrated RAI uptake allowing for iodine-131 (I131) administration. Design: Charts of patients with metastatic, progressive, RAIR DTC in whom TTx increased RAI uptake on a diagnostic whole-body scan (WBS), were reviewed. Results of WBS, I131 administration, thyroglobulin (TG) panels, and cross-sectional studies were recorded. Setting: Thirteen patients [median age (range), 56 (45 to 75) years; seven men] were included; 11 (85%) had DTC, two (15%) had poorly DTC. Nine (69%) had BRAF mutations, three (23%) had RAS mutations, and one (8%) was wild type. Selective BRAF or an MEK inhibitor TTx was continued for a median (range) of 14.3 (1 to 76.4) months before diagnostic WBS. Results: Nine (69%) patients were treated with I131 [median (range) activity, 204.4 (150 to 253) mCi], after which TTx was discontinued. Median (range) follow-up was 8.3 (0 to 17.4) months after I131 therapy. All nine patients had durable disease control (three had partial response, six had stable disease). TG and TG antibody levels increased in patients who demonstrated uptake before TTx, and declined in eight of the nine patients after I131 treatment. Adverse events included pneumonitis and sialadenitis. Conclusion: TTx in BRAF-/RAS-mutated RAIR DTC resensitizes tumors to iodine. Subsequent I131 administration results in meaningful responses. Patient selection, adverse events, response duration, and survival impact require additional study.

The unintended consequences of The Centers for Medicare and Medicaid Services pay-for-performance structures on safety-net hospitals and the low-income, medically vulnerable population.

Safety-net hospitals are hospitals with patient mix that is substantially composed of the uninsured, underinsured, and low-income, medically vulnerable patient populations. They are the hospitals of last resort for poor patients. This article examined the impact of The Centers for Medicare and Medicaid Services pay-for-performance reimbursement policies on the financial viability of safety-net hospitals. Studies showed that these policies, which are based on the principle of reward and punishment, might have unintentionally placed safety-net hospitals on financial disadvantage compared to other hospital organizations. Several studies implied that these payment structures might have resulted in a situation where safety-net hospitals that are serving poor patient populations become more susceptible to penalties than hospitals that are serving affluent patients.

Characterization of a novel bidirectional distraction spinal cord injury animal model.

Scoliosis corrective surgery requires the application of significant multidirectional stress forces, including distraction, for correction of the curved spine deformity and the application of fixation rods. If excessive, spine distraction may result in the development of new neurological deficits, some as severe as permanent paralysis. Current animal models of spinal cord injury, however, are limited to contusion, transection, or unidirectional distraction injuries, which fail to replicate the multidirectional forces that occur during spine corrective surgery. To address such limitation, we designed a novel device that relies on intervertebral grip fixation and linear actuators to induce controllable bidirectional distraction injuries to the spine. The device was tested in three (i.e., 3, 5, and 7 mm) distention paradigms of the rat T9-T11 vertebra, and the resulting injuries were evaluated through electrophysiological, behavioral, and histological analysis. As expected, 3mm bilateral spine distractions showed no neurological deficit. In contrast, those with 5 and 7 mm showed partial and complete paralysis, respectively. The relationship between the severity of the spine distraction and injury to the spinal cord tissue was determined using glial fibrillary acidic protein immunocytochemistry for visualization of reactive astrocytes and labeling of ED1-positive activated macrophages/microglia. Our results demonstrate that this device can produce bidirectional spine distraction injuries with high precision and control and, thus, may be valuable in contributing to the testing of neuroprotective strategies aimed at preventing unintended new neurological damage during corrective spine surgery.

Concurrent visualization of trafficking, expansion, and activation of T lymphocytes and T-cell precursors in vivo.

We have developed a dual bioluminescent reporter system allowing noninvasive, concomitant imaging of T-cell trafficking, expansion, and activation of nuclear factor of activated T cells (NFAT) in vivo. NFAT activation plays an important role in T-cell activation and T-cell development. Therefore we used this system to determine spatial-temporal activation patterns of (1) proliferating T lymphocytes during graft-versus-host disease (GVHD) and (2) T-cell precursors during T-cell development after allogeneic hematopoietic stem cell transplantation (HSCT). In the first days after HSCT, donor T cells migrated to the peripheral lymph nodes and the intestines, whereas the NFAT activation was dominant in the intestines, suggesting an important role for the intestines in the early stages of alloactivation during development of GVHD. After adoptive transfer of in vitro-derived T-cell receptor (TCR) H-Y transgenic T-cell precursors into B6 (H-2(b)) hosts of both sexes, NFAT signaling and development into CD4(+) or CD8(+) single-positive cells could only be detected in the thymus of female recipients indicating either absence of positive selection or prompt depletion of double-positive thymocytes in the male recipients. Because NFAT plays an important role in a wide range of cell types, our system could provide new insights into a variety of biologic processes.

Sensitive in vivo imaging of T cells using a membrane-bound Gaussia princeps luciferase.

We developed a new approach to bioluminescent T cell imaging using a membrane-anchored form of the Gaussia luciferase (GLuc) enzyme, termed extGLuc, which we could stably express in both mouse and human primary T cells. In vitro, extGLuc+ cells emitted significantly higher bioluminescent signal when compared to cells expressing GLuc, Renilla luciferase (RLuc) or membrane-anchored RLuc (extRLuc). In vivo, mouse extGLuc+ T cells showed higher bioluminescent signal when compared to GLuc+ and RLuc+ T cells. Application of this imaging approach to human T cells genetically modified to express tumor-specific chimeric antigen receptors (CARs) enabled us to show in vivo CAR-mediated T cell accumulation in tumor, T cell persistence over time and concomitant imaging of T cells and tumor cells modified to express firefly luciferase. This sensitive imaging technology has application to many in vivo cell-based studies in a wide array of mouse models.

Synthesis and biological evaluation of a fluorine-18 derivative of dasatinib.

Tyrosine kinases often play pivotal roles in the pathogenesis of cancer and are good candidates for therapeutic intervention and targeted molecular imaging. The precursor synthesis, radiosynthesis, and biological characterization of a fluorine-18 analog of dasatinib, a multitargeted kinase inhibitor, are reported. Compound 5 potently inhibits Abl, Src, and Kit kinases and inhibits K562 and M07e/p210bcr-abl human leukemic cell growth. Using positron emission tomography, we visualized K562 tumor xenografts in mice with [18F]-5.

Pretargeted radioimmunotherapy with a single-chain antibody/streptavidin construct and radiolabeled DOTA-biotin: strategies for reduction of the renal dose.

UNLABELLED: Multistep immune targeting holds great promise for radioimmunodiagnosis and therapy of cancer. Pretargeting of the tetrameric single-chain, variable-fragment streptavidin construct of the tetrameric monoclonal antibody CC49 with subsequent administration of radiolabeled 1,4,7,10-tetraazacyclododecane-N,N',N'',N'''-tetraacetic acid (DOTA)-biotin has yielded promising results in TAG-72-expressing tumor xenograft models. A potential limitation of this approach, however, has been high and prolonged renal uptake of radioactivity. The objective of the current study, therefore, was to evaluate the reduction of kidney uptake of radiolabeled DOTA-biotin achieved by each of 4 different methods. METHODS: A human pancreatic adenocarcinoma xenograft model (HPAC) in nude mice was used. The animals were intravenously injected with the antibody-streptavidin construct and a synthetic clearing agent (biotinylated N-acetyl-galactosamine) 24 and 4 h, respectively, before the administration of (67)Ga-DOTA-biotin. For reduction of the renal uptake, different groups of mice were treated with streptavidin saturated with biotin, with several administrations of lysine or colchicine or with a succinylated antibody-streptavidin construct (resulting in a decreased electrical charge). All animals were sacrificed 24 h after injection of the (67)Ga-DOTA-biotin for biodistribution and quantitative autoradiography (QAR) studies and selected animals underwent microSPECT/microCT studies. RESULTS: There was marked targeting of the radiolabeled DOTA-biotin to tumor in all groups except in negative-control animals. Only succinylation of the scFv-CC49-streptavidin fusion protein significantly reduced ( approximately 30%) kidney uptake without affecting tumor activity. QAR corroborated these results and demonstrated that radiolabeled DOTA-biotin localized selectively in the renal cortex. Among the other experimental groups, there was no change in kidney uptake of the radiolabeled biotin. CONCLUSION: In contrast to directly labeled antibodies and antibody fragments, administration of the negatively charged amino acid lysine was largely ineffective in pretargeting strategies with a single-chain-immuno-streptavidin fusion protein. Succinylation of the scFv-CC49-streptavidin construct, on the other hand, reduces kidney uptake of subsequently administered radiolabeled biotin, presumably by inhibiting reuptake of the fusion protein in the proximal renal tubules, and, therefore, could significantly reduce renal doses and improve therapeutic indices associated with multistep immune targeting approaches to radioimmunotherapy.

Protection of beagle dogs from mucosal challenge with canine oral papillomavirus by immunization with recombinant adenoviruses expressing codon-optimized early genes.

Replication-deficient adenoviral (rAd5) vaccines containing codon-optimized E1, E2, E4, and E7 genes of canine oral papillomavirus (COPV) were tested singly or in combination to determine which vaccines could protect against mucosal challenge with COPV. In three studies, groups of 4-6 beagle dogs were immunized subcutaneously (s.c.) with 10(11) rAd5 at 8-10 weeks and 4-6 weeks prior to challenge with infectious COPV particles at multiple oral mucosal sites. Control dogs were immunized with equivalent doses of rAd5 expressing human papillomavirus (HPV) type 16 L1 (rAd5-HPV-16 L1). In the first study, complete protection from COPV-induced papillomas was achieved by immunization with rAd5 vaccine combinations expressing either E1 + E2 or E1 + E2 + E4 + E7; whereas two of six dogs immunized with rAd5-E4 + rAd5-E7 and six of six rAd5-HPV16-L1-immunized control dogs developed oral papillomas. In two subsequent studies, rAd5-E1 and rAd5-E2 vaccines were tested singly or in combination to assess levels of protective immunity to COPV challenge. Subcutaneous immunization with either one or two doses of rAd5 expressing the COPV E1 and E2 genes could protect > 90% of challenged dogs from wart formation. In contrast, all eight dogs immunized with rAd5-HPV-16 L1 developed papillomas at multiple sites. Protection was accompanied by significant IFN-gamma responses to COPV E1 and E2 peptides. Partial protection was conferred by two immunizations with either rAd5-E1 (6 of 9 protected) or rAd5-E2 (8 of 9 protected). These data indicate that rAd5 expressing papillomavirus E1 and E2 proteins can induce strong protective responses even in outbred populations under practical immunization conditions.

Intraepithelial DNA immunisation with a plasmid encoding a codon optimised COPV E1 gene sequence, but not the wild-type gene sequence completely protects against mucosal challenge with infectious COPV in beagles.

DNA plasmids encoding the open reading frames of canine oral papillomavirus (COPV) nonstructural early genes E1, E2, or E7 protein were delivered into both oral mucosal and cutaneous epithelial sites in beagle dogs using particle-mediated immunotherapeutic delivery (PMID) technology. Control dogs were vaccinated with plasmid encoding either hepatitis B virus surface antigen (HBVs) or COPV L1. Using a prophylactic immunisation protocol, a priming dose of plasmid DNA was followed by a booster dose 6 weeks later. Four weeks after boost, all dogs were challenged with infectious COPV particles. Following viral challenge, as shown previously (M. A. Stanley et al., 2001, Vaccine 19, 2783-2792), mucosal papillomas developed in the negative-control HBVs vaccinated dogs, but all animals in the COPV L1 group were fully protected from disease development. In the early gene-vaccinated groups five of six in the E1-vaccinated dogs, two of six in E2-vaccinated dogs, and three of six in the E7-vaccinated beagles developed oral papillomas. Compared to the HBVs negative-control group the oral papillomas that did develop in the early-gene vaccinated beagles were significantly smaller, shorter in duration, and fewer in number. Taken together the disease burden was markedly reduced and this was statistically significant. In a second experiment one group of animals was vaccinated with plasmid encoding the wild-type COPV E1 gene, and a separate group was vaccinated with plasmid encoding a synthetic codon-optimised COPV E1 gene sequence. None of the codon-optimised E1-vaccinated animals developed papillomas at any challenge site. However, all animals vaccinated with wild-type E1 had papillomas. These data suggest that immunisation by PMID with papillomavirus early genes can significantly impact upon subsequent disease development and that full protection can be achieved using improved vectors encoding codon-optimised gene sequences perhaps emphasizing the importance of antigen load in the generation of protective responses to papillomavirus proteins.

Perfusion method for thoracoabdominal aneurysm repair using the open distal technique.

Challenges related to perfusion support of thoracoabdominal aneurysm repair include maintenance of distal aortic perfusion, rapidity of fluid resuscitation, and avoidance of both hypothermia and excessive hemodilution. Using available technology, we have devised a circuit and protocol that addresses these issues. To accomplish such support a bypass circuit consisting of 3/8 inch tubing connected to a centrifugal pump and low-prime heat exchanger was constructed. The circuit was primed via 1/4 inch spiked connectors attached to a 3-liter bag of normal saline. After initial de-airing, the solution was recirculated through this bag. Patients were anticoagulated with 1 mg/kg of heparin prior to initiation of support. Left atrial-descending aorta bypass was used primarily. A cell salvage device was used for autotransfusion. All blood products were delivered via a rapid infusion device. During partial exsanguination, shed blood was not processed, but directed to the rapid infusor for immediate retransfusion. Any packed cells given were washed prior to transfusion. Citrate dextrose solution was used as an anticoagulant for the cell scavenger. This configuration was used successfully in 50 procedures during an 18-month period. Use of this low-prime, custom circuit reduced both hemodilution and cost. A connection off the cell salvage pump offers fast retransfusion of shed blood during partial exsanguination. Minimal heparinization and citrate anticoagulation appears to reduce coagulopathy.

Liquid crystal thermography in the evaluation of chronic back pain: a comparative study.

This research involves the relative value of liquid crystal contact thermography (LCT) as compared to the physical examination, myelography, electromyography and CT scanning in the examination of 155 chronic low back pain patients. Thermograms were interpreted by two independent examiners as positive for nerve root compromise. Results demonstrate excellent interobserver reliability when used for this specific purpose and respectable correlations with the physical examination and EMG studies were obtained. Somewhat less correlation was found between LCT and the diagnostic procedures of CT scanning and myelography, especially in the postoperated patient. Liquid crystal contact thermography shows some promise as an adjunctive diagnostic tool in the assessment of chronic back pain patients with radicular symptoms, especially where further surgery is contemplated and the more structural tests of CT scanning and myelography may be falsely positive as a result of previous back surgery.