POLG DNA testing as an emerging standard of care before instituting valproic acid therapy for pediatric seizure disorders.

PURPOSE: To review our clinical experience and determine if there are appropriate signs and symptoms to consider POLG sequencing prior to valproic acid (VPA) dosing in patients with seizures. METHODS: Four patients who developed VPA-induced hepatotoxicity were examined for POLG sequence variations. A subsequent chart review was used to describe clinical course prior to and after VPA dosing. RESULTS: Four patients of multiple different ethnicities, age 3-18 years, developed VPA-induced hepatotoxicity. All were given VPA due to intractable partial seizures. Three of the patients had developed epilepsia partialis continua. The time from VPA exposure to liver failure was between 2 and 3 months. Liver failure was reversible in one patient. Molecular studies revealed homozygous p.R597W or p.A467T mutations in two patients. The other two patients showed compound heterozygous mutations, p.A467T/p.Q68X and p.L83P/p.G888S. Clinical findings and POLG mutations were diagnostic of Alpers-Huttenlocher syndrome. CONCLUSION: Our cases underscore several important findings: POLG mutations have been observed in every ethnic group studied to date; early predominance of epileptiform discharges over the occipital region is common in POLG-induced epilepsy; the EEG and MRI findings varying between patients and stages of the disease; and VPA dosing at any stage of Alpers-Huttenlocher syndrome can precipitate liver failure. Our data support an emerging proposal that POLG gene testing should be considered in any child or adolescent who presents or develops intractable seizures with or without status epilepticus or epilepsia partialis continua, particularly when there is a history of psychomotor regression.

APPLICATIONS OF EPHB4 RECEPTOR SPECIFIC PEPTIDES IN TARGETED CANCER IMAGING AND THERAPY

EphB4 receptors, a member of the largest family of receptor tyrosine kinases, are found over-expressed in a variety of tumors cells including glioma cells as well as angiogenic blood vessels. Noninvasive imaging of EphB4 could potentially increase early detection rates, monitor response to therapy directed against EphB4, and improve patient outcomes. Targeted delivery of EphB4 receptor specific peptide conjugated hollow gold nanoshells (HAuNS) into tumors has great potential in cancer imaging and photothermal therapy. In this study, we developed an EphB4 specific peptide named TNYL-RAW and labeled with radioisotope 64Cu and Cy5.5 dye. We also conjugate this specific peptide with hollow gold nanoshells (HAuNS) to evaluate targeted photothermal therapy of cancers. In vitro, 64Cu-DOTA-TNYL- RAW specifically bind to CT26 and PC-3M cells but not to A549 cells. In vivo, Small-animal PET/CT clearly showed the significant uptake of 64Cu-DOTA-TNYL-RAW in CT26 and PC-3M tumors but not in A549 tumors. Furthermore, µPET/CT and near-infrared optical imaging clearly showed the uptake of the dual labeled TNYL-RAW peptide in both U251 and U87 tumors in the brains of nude mice. In U251 tumors, Cy5.5-labeled peptide can bind to EphB4-expressing tumor blood vessels and tumors cells. But in U87 models, dual labeled peptide only could bind to tumor associated blood vessels. Also, Irradiation of PC-3M and CT-26 cell treated with TNYL-PEG-HAuNS nanopatilces with near-infrared (NIR) laser resulted in selective destruction of these cells in vitro. EphB4 targeted TNYL-PEG-HAuNS showed more photothermal killing effect on CT26 tumor model than PEG-HAuNS did. In summary, tumors with overexpression of EphB4 receptors can be noninvasively visualized by micro PET/CT with 64Cu labeled or dual labeled TNYL-RAW peptide. Targeted delivery of TNYL-RAW conjugated HAuNS into tumors can greatly improve the treatment effect of photothermal therapy. The information acquired with this study should be advantageous in improving diagnostics and future applications in photothermal ablation therapy in clinical.

Circumvention of cellular defense responses to DNA -directed nucleoside analogues by the protein kinase inhibitor, UCN-01

DNA-directed nucleoside analogues, such as ara-C, fludarabine, and gemcitabine, are antimetabolites effective in the treatment of a variety of cancers. However, resistance to nucleoside analogue-based chemotherapy in treatments is still a major problem in therapy. Therefore, it is essential to develop rationales for optimizing the use of nucleoside analogues in combination with other anticancer drugs or modalities such as radiation. The present study focuses on establishing mechanism-based combination strategy to overcome resistance to nucleoside analogues. ^ I hypothesized that the cytostatic concentrations of nucleoside analogues may cause S-phase arrest by activating an S-phase checkpoint that consists of a series of kinases. This may allow cells to repair damaged DNA over time and spare cytotoxicity. Thus, the ability of cells to enact an S-phase arrest in response to incorporation of potentially lethal amounts of nucleoside analogue may serve as a mechanism of resistance to S-phase-specific agents. As a corollary, the addition of a kinase inhibitor, such as UCN-01, may dysregulate the checkpoint response and abrogate the survival of S-phase-arrested cells by suppression of the survival signaling pathways. Using gemcitabine as a model of S-phase-specific nucleoside analogues in human acute myelogenous leukemia ML-1 cells, I demonstrated that cells arrested in S-phase in response to cytostatic conditions. Proliferation continued after washing the cells into drug-free medium, suggesting S-phase arrest served as a resistance mechanism of cancer cells to spare cytotoxicity of nucleoside analogues. However, nontoxic concentrations of UCN-01 rapidly killed S-phase-arrested cells by apoptosis. Furthermore, the molecular mechanism for UCN-01-induced apoptosis in S-phase-arrested cells was through inhibition of survival pathways associated with these cells. In this regard, suppression of the PI 3-kinase-Akt-Bad survival pathway as well as the NF-κB signaling pathway were associated with induction of apoptosis in S-phase-arrested cells by UCN-01, whereas the Ras-Raf-MEK-ERK pathway appeared not involved. This study has provided the rationales and strategies for optimizing the design of effective combination therapies to overcome resistance to nucleoside analogues. In fact, a clinical trial of the combination of ara-C with UCN-01 to treat relapsed or refractory AML patients has been initiated at U.T.M.D. Anderson Cancer Center. ^