Drug penetration in pediatric brain tumors: Challenges and opportunities.

Larger clinical trial enrollments and a greater understanding of biological heterogeneity have led to improved survival rates for children diagnosed with brain tumors in the last 50 years. However, reducing long-term morbidities and improving survival rates of high-risk tumors remain major challenges. Chemotherapy can reduce tumor burden, but effective drug penetration at the tumor site is limited by barriers in the route of drug administration and within the tumor microenvironment. Bioavailability of drugs is impeded by the blood-brain barrier, plasma protein binding, and structural components by the tumor including the matrix and vasculature contributing to increased interstitial fluid pressure, hypoxia, and acidity. Designing drug delivery systems to circumvent these barriers could lead to improved drug penetration at the tumor site and reduce adverse systemic side effects. In this review, we expand on how systemic and local barriers limit drug penetration and present potential methods to enhance drug penetration in pediatric brain tumors.

Comprehensive analysis of MYB/MYBL1-altered pediatric-type diffuse low-grade glioma.

BACKGROUND: Pediatric-type diffuse low-grade gliomas (pLGG) harboring recurrent genetic alterations involving MYB or MYBL1 are closely related tumors. Detailed treatment and outcome data of large cohorts are still limited. This study aimed to comprehensively evaluate pLGG with these alterations to define optimal therapeutic strategies. METHODS: We retrospectively reviewed details of pLGG with MYB or MYBL1 alterations from patients treated or referred for pathologic review at St. Jude Children's Research Hospital. Tumor specimens were centrally reviewed, and clinical data were collated. RESULTS: Thirty-three patients (18 male; median age, 5 years) were identified. Two tumors had MYBL1 alterations; 31 had MYB alterations, MYB::QKI fusion being the most common (n = 10, 30%). Most tumors were in the cerebral hemispheres (n = 22, 67%). Two patients (6%) had metastasis at diagnosis. The median follow-up was 6.1 years. The 5-year event-free survival (EFS) rate was 81.3% ±â€…8.3%; the 5-year overall survival (OS) rate was 96.4% ±â€…4.1%. Patients receiving a near-total or gross-total resection had a 5-year EFS of 100%; those receiving a biopsy or subtotal resection had a 5-year EFS rate of 56.6% ±â€…15.2% (P < .01). No difference in EFS was observed based on location, histology, or molecular alterations. However, the tumors that progressed or metastasized may have distinct methylation profiles with evidence of activation of the MAPK and PI3K/AKT/mTOR pathways. CONCLUSIONS: pLGG with MYB/MYBL1 alterations have good outcomes. Our findings suggest that surgical resectability is a crucial determinant of EFS. Further characterization is required to identify optimal treatment strategies for progressive tumors.

Functional characterization of a novel C-terminal ATP1A2 mutation causing hemiplegic migraine and epilepsy.

BACKGROUND: We describe a four-generation Italian family with familial hemiplegic migraine (FHM) and epilepsy due to a novel ATP1A2 missense mutation (R1007W). CASE RESULTS: Mutational analysis revealed a heterozygous nucleotide substitution c.3019C>T resulting in the missense substitution p.Arg1007Trp (p.R1007W) in seven subjects: Three individuals had hemiplegic migraine, two exhibited a clinical overlap between migraine and epilepsy, one had migraine and one was unaffected. The identified ATP1A2 mutation was not found in an ethnically matched control population of 190 individuals and was not reported in a polymorphisms database. In two-electrode voltage-clamp experiments on XENOPUS oocytes, the ATP1A2 R1007W mutant showed (i) reduced ion pumping activity due to a more profound voltage dependence and (ii) decreased apparent affinity for extracellular K⁺ at voltages around the cellular resting potential. This distinct type of loss of function has not been reported for other FHM2 mutations and can lead to impaired K⁺ clearance and elevated K⁺ levels in the CNS. CONCLUSIONS: The functional data and clinical evidence suggest that in FHM2 migraine and epilepsy may originate from the same pathogenic mechanisms associated with genetically determined alterations of ion channels and pumps. Our data also support the hypothesis that the new mutation R1007W in our family may be a susceptibility factor for epilepsy.

Treatment of children with glioblastoma with conformal radiation, temozolomide, and bevacizumab as adjuncts to surgical resection.

Prognosis for children with glioblastoma is unacceptably poor. Modest improvements in progression-free survival were seen in adults with glioblastoma by combining temozolomide and bevacizumab with conformal radiation. We retrospectively reviewed 3 cases of glioblastoma in children treated using upfront bevacizumab and temozolomide during radiation, followed by 12 cycles of maintenance therapy. All patients completed therapy with minimal toxicity and no delays in treatment. Two patients remain disease free at 38 and 49 months from diagnosis. One patient recurred 14 months off therapy and currently receives salvage therapy 48 months from diagnosis. These results support further investigation of this regimen.

Inhibition of nuclear factor kappa-B signaling reduces growth in medulloblastoma in vivo.

BACKGROUND: Medulloblastoma is a highly malignant pediatric brain tumor that requires surgery, whole brain and spine irradiation, and intense chemotherapy for treatment. A more sophisticated understanding of the pathophysiology of medulloblastoma is needed to successfully reduce the intensity of treatment and improve outcomes. Nuclear factor kappa-B (NFκB) is a signaling pathway that controls transcriptional activation of genes important for tight regulation of many cellular processes and is aberrantly expressed in many types of cancer. METHODS: To test the importance of NFκB to medulloblastoma cell growth, the effects of multiple drugs that inhibit NFκB, pyrrolidine dithiocarbamate, diethyldithiocarbamate, sulfasalazine, curcumin and bortezomib, were studied in medulloblastoma cell lines compared to a malignant glioma cell line and normal neurons. Expression of endogenous NFκB was investigated in cultured cells, xenograft flank tumors, and primary human tumor samples. A dominant negative construct for the endogenous inhibitor of NFκB, IκB, was prepared from medulloblastoma cell lines and flank tumors were established to allow specific pathway inhibition. RESULTS: We report high constitutive activity of the canonical NFκB pathway, as seen by Western analysis of the NFκB subunit p65, in medulloblastoma tumors compared to normal brain. The p65 subunit of NFκB is extremely highly expressed in xenograft tumors from human medulloblastoma cell lines; though, conversely, the same cells in culture have minimal expression without specific stimulation. We demonstrate that pharmacological inhibition of NFκB in cell lines halts proliferation and leads to apoptosis. We show by immunohistochemical stain that phosphorylated p65 is found in the majority of primary tumor cells examined. Finally, expression of a dominant negative form of the endogenous inhibitor of NFκB, dnIκB, resulted in poor xenograft tumor growth, with average tumor volumes 40% smaller than controls. CONCLUSIONS: These data collectively demonstrate that NFκB signaling is important for medulloblastoma tumor growth, and that inhibition can reduce tumor size and viability in vivo. We discuss the implications of NFκB signaling on the approach to managing patients with medulloblastoma in order to improve clinical outcomes.

Biophysical properties of human medulloblastoma cells.

Medulloblastoma is a pediatric high-grade cerebellar malignancy derived from neuronal precursors. Although electrophysiologic characteristics of cerebellar granule neurons at all stages of cell development have been well described, such characterization has not been reported for medulloblastoma. In this study we attempt to characterize important electrophysiologic features of medulloblastoma that may distinguish it from the surrounding cerebellum. Using patient-derived cell lines and tumor tissues, we show that medulloblastoma cells have no inward Na+ current or transient K+ current involved in action potential generation and propagation, typically seen in granule neurons. Expression and function of calcium-activated, large-conductance K+ channels are diminished in medulloblastoma, judged by electrophysiology and Western analysis. The resting membrane potential of medulloblastoma cells in culture is quite depolarized compared to granule neurons. Interestingly, medulloblastoma cells express small, fast-inactivating calcium currents consistent with T-type calcium channels, but these channels are activated only from hyperpolarized potentials, which are unlikely to occur. Additionally, a background acid-sensitive K+ current is present with features characteristic of TASK1 or TASK3 channels, such as inhibition by ruthenium red. Western analysis confirms expression of TASK1 and TASK3. In describing the electrophysiologic characteristics of medulloblastoma, one can see features that resemble other high-grade malignancies as opposed to normal cerebellar granule neurons. This supports the notion that the malignant phenotype of medulloblastoma is characterized by unique changes in ion channel expression.

A second conserved GAF domain cysteine is required for the blue/green photoreversibility of cyanobacteriochrome Tlr0924 from Thermosynechococcus elongatus.

Phytochromes are widely occurring red/far-red photoreceptors that utilize a linear tetrapyrrole (bilin) chromophore covalently bound within a knotted PAS-GAF domain pair. Cyanobacteria also contain more distant relatives of phytochromes that lack this knot, such as the phytochrome-related cyanobacteriochromes implicated to function as blue/green switchable photoreceptors. In this study, we characterize the cyanobacteriochrome Tlr0924 from the thermophilic cyanobacterium Thermosynechococcus elongatus. Full-length Tlr0924 exhibits blue/green photoconversion across a broad range of temperatures, including physiologically relevant temperatures for this organism. Spectroscopic characterization of Tlr0924 demonstrates that its green-absorbing state is in equilibrium with a labile, spectrally distinct blue-absorbing species. The photochemically generated blue-absorbing state is in equilibrium with another species absorbing at longer wavelengths, giving a total of 4 states. Cys499 is essential for this behavior, because mutagenesis of this residue results in red-absorbing mutant biliproteins. Characterization of the C 499D mutant protein by absorbance and CD spectroscopy supports the conclusion that its bilin chromophore adopts a similar conformation to the red-light-absorbing P r form of phytochrome. We propose a model photocycle in which Z/ E photoisomerization of the 15/16 bond modulates formation of a reversible thioether linkage between Cys499 and C10 of the chromophore, providing the basis for the blue/green switching of cyanobacteriochromes.

Functional analysis of human Na(+)/K(+)-ATPase familial or sporadic hemiplegic migraine mutations expressed in Xenopus oocytes.

AIM: Functional characterization of ATP1A2 mutations that are related to familial or sporadic hemiplegic migraine (FHM2, SHM). METHODS: cRNA of human Na(+)/K(+)-ATPase α2- and ß1-subunits were injected in Xenopus laevis oocytes. FHM2 or SHM mutations of residues located in putative α/ß interaction sites or in the α2-subunit's C-terminal region were investigated. Mutants were analyzed by the two-electrode voltage-clamp (TEVC) technique on Xenopus oocytes. Stationary K(+)-induced Na(+)/K(+) pump currents were measured, and the voltage dependence of apparent K(+) affinity was investigated. Transient currents were recorded as ouabain-sensitive currents in Na(+) buffers to analyze kinetics and voltage-dependent pre-steady state charge translocations. The expression of constructs was verified by preparation of plasma membrane and total membrane fractions of cRNA-injected oocytes. RESULTS: Compared to the wild-type enzyme, the mutants G900R and E902K showed no significant differences in the voltage dependence of K(+)-induced currents, and analysis of the transient currents indicated that the extracellular Na(+) affinity was not affected. Mutant G855R showed no pump activity detectable by TEVC. Also for L994del and Y1009X, pump currents could not be recorded. Analysis of the plasma and total membrane fractions showed that the expressed proteins were not or only minimally targeted to the plasma membrane. Whereas the mutation K1003E had no impact on K(+) interaction, D999H affected the voltage dependence of K(+)-induced currents. Furthermore, kinetics of the transient currents was altered compared to the wild-type enzyme, and the apparent affinity for extracellular Na(+) was reduced. CONCLUSION: The investigated FHM2/SHM mutations influence protein function differently depending on the structural impact of the mutated residue.

Measuring cation transport by Na,K- and H,K-ATPase in Xenopus oocytes by atomic absorption spectrophotometry: an alternative to radioisotope assays.

Whereas cation transport by the electrogenic membrane transporter Na(+),K(+)-ATPase can be measured by electrophysiology, the electroneutrally operating gastric H(+),K(+)-ATPase is more difficult to investigate. Many transport assays utilize radioisotopes to achieve a sufficient signal-to-noise ratio, however, the necessary security measures impose severe restrictions regarding human exposure or assay design. Furthermore, ion transport across cell membranes is critically influenced by the membrane potential, which is not straightforwardly controlled in cell culture or in proteoliposome preparations. Here, we make use of the outstanding sensitivity of atomic absorption spectrophotometry (AAS) towards trace amounts of chemical elements to measure Rb(+) or Li(+) transport by Na(+),K(+)- or gastric H(+),K(+)-ATPase in single cells. Using Xenopus oocytes as expression system, we determine the amount of Rb(+) (Li(+)) transported into the cells by measuring samples of single-oocyte homogenates in an AAS device equipped with a transversely heated graphite atomizer (THGA) furnace, which is loaded from an autosampler. Since the background of unspecific Rb(+) uptake into control oocytes or during application of ATPase-specific inhibitors is very small, it is possible to implement complex kinetic assay schemes involving a large number of experimental conditions simultaneously, or to compare the transport capacity and kinetics of site-specifically mutated transporters with high precision. Furthermore, since cation uptake is determined on single cells, the flux experiments can be carried out in combination with two-electrode voltage-clamping (TEVC) to achieve accurate control of the membrane potential and current. This allowed e.g. to quantitatively determine the 3Na(+)/2K(+) transport stoichiometry of the Na(+),K(+)-ATPase and enabled for the first time to investigate the voltage dependence of cation transport by the electroneutrally operating gastric H(+),K(+)-ATPase. In principle, the assay is not limited to K(+)-transporting membrane proteins, but it may work equally well to address the activity of heavy or transition metal transporters, or uptake of chemical elements by endocytotic processes.

Response of preclinical medulloblastoma models to combination therapy with 13-cis retinoic acid and suberoylanilide hydroxamic acid (SAHA).

PURPOSE: Current medulloblastoma therapy, surgery, radiation, and chemotherapy, is unacceptably toxic. However, 13-cis retinoic acid (RA) and SAHA, a histone deacetylase inhibitor, have each been shown to induce apoptosis in medulloblastoma cultures and mouse models. Both drugs cross the blood brain barrier, have been given safely to children, and achieve brain concentrations that are at or near therapeutic levels. Retinoic acid acts by transcriptionally activating bone morphogenetic protein-2 (BMP-2) and SAHA facilitates transcriptional activity through chromatin accessibility. We tested the hypothesis that these drugs additively induce BMP-2 transcription and apoptosis. EXPERIMENTAL DESIGN: RA + SAHA induction of BMP-2 transcription and apoptosis in medulloblastoma cultures was evaluated. Subsequently the response of mouse medulloblastomas to these two agents in the presence and absence of cisplatin was evaluated. RESULTS: BMP-2 transcription multiplied 3-fold with addition of RA to culture, and 7-fold with both agents. The IC50 of SAHA was reduced by 40% when low dose RA was added. Interestingly, a p38 MAP kinase inhibitor that partially blocks RA-induced apoptosis did not inhibit the activity of RA + SAHA. Flank D283 tumors in athymic mice had slower growth in the RA + SAHA arm than single drug or control arms. Intracranial tumors in ND2:SmoA1 mice treated with RA + SAHA + cisplatin showed a 4-fold increase in apoptosis over controls, and a 2-fold increase over animals receiving only SAHA or RA + SAHA. CONCLUSIONS: RA + SAHA additively induce BMP-2 transcription and medulloblastoma apoptosis. The combination may act through a p38 MAPK independent mechanism. Efficacy increased with cisplatin, which has implications for clinical trial design.

Suberoylanilide hydroxamic acid is effective in preclinical studies of medulloblastoma.

PURPOSE: Suberoylanilide hydroxamic acid (SAHA) has been studied in adult solid and hematologic malignancies. However, little information has been reported on the effects of SAHA on central nervous system (CNS) tumors including medulloblastoma, the most common malignant brain tumor in children. We investigated SAHA in preclinical medulloblastoma models to determine its anti-cancer efficacy as well as its ability to affect intracranial lesions when administered systemically. EXPERIMENTAL DESIGN AND RESULTS: Tissue culture studies were performed treating primary human fibroblasts, established medulloblastoma cell lines, and primary human medulloblastoma tumors with SAHA. At 10 microM concentration, SAHA had little effect on normal fibroblasts but caused >90% apoptosis in cultured medulloblastoma cells. Primary medulloblastomas from patients were sensitive to SAHA compared to vehicle alone in ex vivo studies. In athymic mice with medulloblastoma xenograft tumors, oral SAHA resulted in apoptosis of tumor tissue and significantly slowed tumor growth. In the ND2:Smo transgenic mouse medulloblastoma model, SAHA treatment caused significant apoptosis in these cerebellar tumors. CONCLUSIONS: SAHA effectively induces cell death in established medulloblastoma cell lines, human patient primary tumor cultures, medulloblastoma xenografts and intracranial spontaneous medulloblastomas. Fibroblasts in culture and mice treated with SAHA did not reveal prohibitive toxicity profiles. These findings support the advancement of SAHA to pediatric clinical trials.

Metastatic malignant melanoma presenting as pancytopenia in a three-year-old boy.

Malignant melanoma is rare in childhood and has never been reported to cause pancytopenia due to bone marrow metastases in a child. We report a 3-year-old boy with a large congenital melanocytic nevus who presented with bone pain and pancytopenia due to diffuse bone and bone marrow infiltration with metastatic melanoma without an identifiable primary site. Despite treatment with imatinib mesylate there was no response and the patient died with progressive disease. This case illustrates an unusual presentation of bone marrow failure secondary to malignant melanoma in a young child with symptomatic metastatic marrow infiltration, a rarely reported site of melanoma involvement in adults or children.