Characterization and comparison of human glioblastoma models.

Glioblastoma (GBM) is one of the deadliest cancers. Treatment options are limited, and median patient survival is only several months. Translation of new therapies is hindered by a lack of GBM models that fully recapitulate disease heterogeneity. Here, we characterize two human GBM models (U87-luc2, U251-RedFLuc). In vitro, both cell lines express similar levels of luciferase and show comparable sensitivity to temozolomide and lapatinib exposure. In vivo, however, the two GBM models recapitulate different aspects of the disease. U87-luc2 cells quickly grow into large, well-demarcated tumors; U251-RedFLuc cells form small, highly invasive tumors. Using a new method to assess GBM invasiveness based on detecting tumor-specific anti-luciferase staining in brain slices, we found that U251-RedFLuc cells are more invasive than U87-luc2 cells. Lastly, we determined expression levels of ABC transporters in both models. Our findings indicate that U87-luc2 and U251-RedFLuc GBM models recapitulate different aspects of GBM heterogeneity that need to be considered in preclinical research.

A phase I trial of lenalidomide and radiotherapy in children with diffuse intrinsic pontine gliomas or high-grade gliomas.

PURPOSE: This study was performed to determine the maximum tolerated dose (MTD) or recommended phase 2 dose (RP2D) of the immunomodulatory agent, lenalidomide, when administered daily during 6 weeks of radiation therapy to children with newly diagnosed diffuse intrinsic pontine glioma (DIPG) or high-grade glioma (HGG) PATIENTS & METHODS: Children and young adults < 22 years of age with newly diagnosed disease and no prior chemotherapy or radiation therapy were eligible. Children with HGG were required to have an inoperable or incompletely resected tumor. Eligible patients received standard radiation therapy to a prescription dose of 54-59.4 Gy, with concurrent administration of lenalidomide daily during radiation therapy in a standard 3 + 3 Phase I dose escalation design. Following completion of radiation therapy, patients had a 2-week break followed by maintenance lenalidomide at 116 mg/m2/day × 21 days of a 28-day cycle. RESULTS: Twenty-nine patients (age range 4-19 years) were enrolled; 24 were evaluable for dose finding (DIPG, n = 13; HGG, n = 11). The MTD was not reached at doses of lenalidomide up to 116 mg/m2/day. Exceptional responses were noted in DIPG and malignant glioma (gliomatosis cerebri) notably at higher dose levels and at higher steady state plasma concentrations. The primary toxicity was myelosuppression. CONCLUSION: The RP2D of lenalidomide administered daily during radiation therapy is 116 mg/m2/day. Children with malignant gliomas tolerate much higher doses of lenalidomide during radiation therapy compared to adults. This finding is critical as activity was observed primarily at higher dose levels suggesting a dose response.

Autologous Fat Grafting Does Not Increase Risk of Oncologic Recurrence in the Reconstructed Breast.

INTRODUCTION: Autologous fat grafting (AFG) is a popular and effective method of breast reconstruction after mastectomy; however, the oncological safety of AFG remains in question. The aim of this study was to determine whether AFG increases the risk of cancer recurrence in the reconstructed breast. METHODS: A matched, case-control study was conducted from 2000 to 2017 at the senior author's institution. Inclusion was limited to female patients who underwent mastectomy and breast reconstruction with or without AFG. Data were further subdivided at the breast level. χ analyses were used to test the association between AFG status and oncologic recurrence. A Cox proportional-hazards model was constructed to assess for possible differences in time to oncologic recurrence. The probability of recurrence was determined by Kaplan-Meier analyses and confirmed with log-rank testing. RESULTS: Overall, 428 breasts met study criteria. Of those, 116 breasts (27.1%) received AFG, whereas 312 (72.9%) did not. No differences in the rates of oncologic recurrence were found between the groups (8.2% vs 9.0%, P < 1.000). Unadjusted (hazard ratio = 1.03, confidence interval = 0.41-2.60, P < 0.957) and adjusted hazard models showed no statistically significant increase in time to oncologic recurrence when comparing AFG to non-AFG. In addition, no statistical differences in disease-free survival were found (P = 0.96 by log rank test). CONCLUSION: Autologous fat grafting for breast reconstruction is oncologically safe and does not increase the likelihood of oncologic recurrence. Larger studies (eg, meta analyses) with longer follow-up are needed to further elucidate the long-term safety of AFG as a reconstructive adjunct.

Implementation fidelity of village health and nutrition days in Hardoi District, Uttar Pradesh, India: a cross-sectional survey.

BACKGROUND: Village Health and Nutrition Days (VHNDs) are a cornerstone of the Government of India's strategy to provide first-contact primary health care to rural areas. Recent government programmes such as the Janani Suraksha Yojana (JSY) and Mission Indradhanush (MI) have catalysed important changes impacting VHNDs. To learn how VHNDs are currently being delivered, we assessed the fidelity of services provided as compared to government norms in a priority district of Uttar Pradesh. METHODS: We fielded a cross-sectional study of VHNDs to provide a snapshot of health services functioning. Process evaluation data were collected via administrative sources, non-participant observation using a standardised form, and structured questionnaires. Questionnaires were designed using a framework to assess implementation fidelity. Key respondents were VHND participants, front-line workers involved in VHND delivery, and VHND non-participants (pregnant women due for antenatal care or children due for vaccination as per administrative records). Results were summarised as counts, frequencies, and proportions. RESULTS: In the 30 villages randomly selected for inclusion, 36 VHNDs were scheduled but four (11.1%) were cancelled and one VHND was not surveyed. Vaccination and antenatal care were offered at 96.8% (30/31) and child weighing at 83.9% (26/31) of VHNDs. Other normed services were infrequently provided or completely absent. Health education and promotion were particularly weak; institutional delivery was the only topic discussed in a majority of VHNDs. The true proportion of any serious problem impeding vaccine delivery was 47.2% (17/36), comprising 4 VHND cancellations and 13 VHNDs experiencing vaccine shortages. Of the 13 incidents of vaccine shortage, 11 related to an unexpected global shortage of injectable polio vaccine (IPV). Over the 31 VHNDs, 37.8% (171 of the 452 scheduled beneficiaries) did not participate. Analysis of missed opportunities for vaccination highlighted inaccuracies in beneficiary identification and tracking and demand side-factors. CONCLUSIONS: The transformative potential of VHNDs to improve population health is only partially being met. A core subset of high-priority services for antenatal care, institutional delivery, and vaccination associated with high-priority government programmes (JSY, MI) is now being provided quite successfully. Other basic health promotion and prevention services are largely not provided, constituting a critical missed opportunity.

Plasma and cerebrospinal fluid pharmacokinetics of select chemotherapeutic agents following intranasal delivery in a non-human primate model.

The blood-brain barrier (BBB) limits entry of most chemotherapeutic agents into the CNS, resulting in inadequate exposure within CNS tumor tissue. Intranasal administration is a proposed means of delivery that can bypass the BBB, potentially resulting in more effective chemotherapeutic exposure at the tumor site. The objective of this study was to evaluate the feasibility and pharmacokinetics (plasma and CSF) of intranasal delivery using select chemotherapeutic agents in a non-human primate (NHP) model. Three chemotherapeutic agents with known differences in CNS penetration were selected for intranasal administration in a NHP model to determine proof of principle of CNS delivery, assess tolerability and feasibility, and to evaluate whether certain drug characteristics were associated with increased CNS exposure. Intravenous (IV) temozolomide (TMZ), oral (PO) valproic acid, and PO perifosine were administered to adult male rhesus macaques. The animals received a single dose of each agent systemically and intranasally in separate experiments, with each animal acting as his own control. The dose of the agents administered systemically was the human equivalent of a clinically appropriate dose, while the intranasal dose was the maximum achievable dose based on the volume limitation of 1 mL. Multiple serial paired plasma and CSF samples were collected and quantified using a validated uHPLC/tandem mass spectrometry assay after each drug administration. Pharmacokinetic parameters were estimated using non-compartmental analysis. CSF penetration was calculated from the ratio of areas under the concentration-time curves for CSF and plasma (AUCCSF:plasma). Intranasal administration was feasible and tolerable for all agents with no significant toxicities observed. For TMZ, the degrees of CSF drug penetration after intranasal and IV administration were 36 (32-57) and 22 (20-41)%, respectively. Although maximum TMZ drug concentration in the CSF (Cmax) was lower after intranasal delivery compared to IV administration due to the lower dose administered, clinically significant exposure was achieved in the CSF after intranasal administration with the lower doses. This was associated with lower systemic exposure, suggesting increased efficiency and potentially lower toxicities of TMZ after intranasal delivery. For valproic acid and perifosine, CSF penetration after intranasal delivery was similar to systemic administration. Although this study demonstrates feasibility and safety of intranasal drug administration, further agent-specific studies are necessary to optimize agent selection and dosing to achieve clinically-relevant CSF exposures.

Optimization, Characterization, and Comparison of Two Luciferase-Expressing Mouse Glioblastoma Models.

Glioblastoma (GBM) is the most aggressive brain cancer. To model GBM in research, orthotopic brain tumor models, including syngeneic models like GL261 and genetically engineered mouse models like TRP, are used. In longitudinal studies, tumor growth and the treatment response are typically tracked with in vivo imaging, including bioluminescence imaging (BLI), which is quick, cost-effective, and easily quantifiable. However, BLI requires luciferase-tagged cells, and recent studies indicate that the luciferase gene can elicit an immune response, leading to tumor rejection and experimental variation. We sought to optimize the engraftment of two luciferase-expressing GBM models, GL261 Red-FLuc and TRP-mCherry-FLuc, showing differences in tumor take, with GL261 Red-FLuc cells requiring immunocompromised mice for 100% engraftment. Immunohistochemistry and MRI revealed distinct tumor characteristics: GL261 Red-FLuc tumors were well-demarcated with densely packed cells, high mitotic activity, and vascularization. In contrast, TRP-mCherry-FLuc tumors were large, invasive, and necrotic, with perivascular invasion. Quantifying the tumor volume using the HALO® AI analysis platform yielded results comparable to manual measurements, providing a standardized and efficient approach for the reliable, high-throughput analysis of luciferase-expressing tumors. Our study highlights the importance of considering tumor engraftment when using luciferase-expressing GBM models, providing insights for preclinical research design.

Plasma and cerebrospinal fluid pharmacokinetics of the DNA methyltransferase inhibitor, 5-azacytidine, alone and with inulin, in nonhuman primate models.

BACKGROUND: Epigenetic modifiers are being investigated for a number of CNS malignancies as tumor-associated mutations such as isocitrate dehydrogenase mutations (IDH1/IDH2) and H3K27M mutations, which result in aberrant signaling, are identified. We evaluated the CNS exposure of the DNA methyltransferase inhibitor, 5-azacytidine (5-AZA), in preclinical nonhuman primate (NHP) models to inform its clinical development for CNS tumors. METHODS: 5-AZA and 5-AZA+Inulin pharmacokinetics (PK) were evaluated in NHPs (n = 10) following systemic (intravenous [IV]) and intrathecal (intraventricular [IT-V], intralumbar [IT-L], and cisternal [IT-C]) administration. Plasma, cerebrospinal fluid (CSF), cortical extracellular fluid (ECF), and tissues were collected. 5-AZA levels were quantified via ultra-high-performance liquid chromatography with tandem mass spectrometric detection assay and inulin via ELISA. PK parameters were calculated using noncompartmental methods. RESULTS: After IV administration, minimal plasma exposure (area under the curve [AUC] range: 2.4-3.2 h*µM) and negligible CSF exposure were noted. CSF exposure was notably higher after IT-V administration (AUCINF 1234.6-5368.4 h*µM) compared to IT-L administration (AUCINF 7.5-19.3 h*µM). CSF clearance after IT administration exceeded the mean inulin CSF flow rate of 0.018 ± 0.003 ml/min as determined by inulin IT-V administration. 5-AZA IT-V administration with inulin increased the 5-AZA CSF duration of exposure by 2.2-fold. IT-C administration yielded no quantifiable 5-AZA ECF concentrations but resulted in quantifiable tissue levels. CONCLUSIONS: IT administration of 5-AZA is necessary to achieve adequate CNS exposure. IT administration results in pronounced and prolonged 5-AZA CSF exposure above the reported IC50 range for IDH-mutated glioma cell lines. Inulin administered with 5-AZA increased the duration of exposure for 5-AZA.

Characterizing the pharmacokinetics of panobinostat in a non-human primate model for the treatment of diffuse intrinsic pontine glioma.

PURPOSE: Diffuse intrinsic pontine glioma (DIPG) is one of the deadliest forms of childhood cancers. To date, no effective treatment options have been developed. Recent drug screening studies identified the HDAC inhibitor panobinostat as an active agent against DIPG cells lines and animal models. To guide in the clinical development of panobinostat, we evaluated the CNS pharmacokinetics of panobinostat using CSF as a surrogate to CNS tissue penetration in a pre-clinical nonhuman primate (NHP) model after oral administration. METHODS: Panobinostat was administered orally to NHP (n = 3) at doses 1.0, 1.8, 2.4, and 3.0 mg/kg (human equivalent dose: 20, 36, 48, 60 mg/m2, respectively). The subjects served as their own controls where possible. Serial, paired CSF and plasma samples were collected for 0-48 h. Panobinostat was quantified via a validated uHPLC-MS/MS method. Pharmacokinetic (PK) parameters were calculated using non-compartmental methods. RESULTS: CSF penetration of panobinostat after systemic delivery was low, with levels detectable in only two subjects. CONCLUSION: The CSF penetration of panobinostat was low following oral administration in this pre-clinical NHP model predictive of human PK.

Flow Rate and Apparent Volume of Cerebrospinal Fluid in Rhesus Macaques (Macaca mulatta) Based on the Pharmacokinetics of Intrathecally Administered Inulin.

Cerebrospinal fluid (CSF) flow rate and volume are fundamental to the design and interpretation of preclinical pharmacokinetics and pharmacodynamics studies in NHP. To determine the values of CSF flow rate and volume, we evaluated the plasma and CSF pharmacokinetics of inulin, an inert polysaccharide tracer, in 5 rhesus macaques with CSF ventricular res- ervoirs and lumbar ports; these reservoirs and ports facilitate humane intrathecal administration and serial CSF sampling in unanesthetized macaques. Inulin was administered intrathecally via the CSF ventricular reservoir (n = 3), followed by the collection of lumbar CSF via the lumbar port and plasma. The contribution of dietary inulin was evaluated by using pre- and postprandial inulin plasma concentrations (n = 2) and a feed analysis of the NHP diet. Inulin concentrations were quantified using ELISA. Pharmacokinetic parameters were calculated by using noncompartmental methods. Daily diet was analyzed for inulin by using Official Method no. 997.08 of AOAC International. In male rhesus macaques, the mean CSF flow rate, established via inulin clearance after IT administration, was 0.018 ± 0.003 mL/min; mean CSF volume, established based on apparent volume of distribution, was 10.17 ± 0.63 mL. In plasma, inulin was quantifiable in all pre-administration samples and increased over the sampling period, precluding interpretation of plasma pharmacokinetics. Evaluation of the effect of diet on plasma concentrations established quantifiable inulin levels that showed minimal variation relative to the prandial state. Analysis of the feed detected 5 inulin types ranging from 1100 to 1440 mg per100 g. The diet was the source of detectable pre-administration inulin plasma concentrations, whereas inulin was not detected in CSF before inulin administration.

Diagnosis, staging, and risk stratification in prostate cancer: Utilizing diagnostic tools to avoid unnecessary therapies and side effects.

A lack of appropriate diagnostic tools for prostate cancer has led to overdiagnosis and over treatment. In a recent publication in the New England Journal of Medicine, Hamdy et al showed no difference in the outcomes of patients that had undergone either radical prostatectomy, radiotherapy, or active monitoring. In an effort to enhance clinical stratification, the development of improved, more accurate diagnostic tools is actively being pursued. Herein, we explore recent advances in prostate cancer screening, including biomarker assays, genetic testing, and specialized fields, such as mathematical oncology. These newly developed, highly sensitive diagnostic assays may potentially aid clinicians in selecting appropriate therapies for patients in the very near future.

Population pharmacokinetic analyses of the effect of carboplatin pretreatment on olaparib in recurrent or refractory women's cancers.

PURPOSE: Combining olaparib with carboplatin was recently shown to be active in both BRCA and non-BRCA mutant cancers in a recent phase I/Ib combination trial. The optimal drug sequence recommended was carboplatin 1-day before olaparib. However, carboplatin pre-treatment induced a ~50% faster olaparib clearance. METHODS: To further explore this drug interaction, a population pharmacokinetic (PK) model was designed that included a lag time parameter, a second absorption compartment from tablet formulation, a single distribution/elimination compartment, and covariance among the clearance and volume parameters. RESULTS: Clearance (6.8 L/h) and volume (33 L) estimates were comparable with literature. The only significant covariate was the presence of carboplatin on olaparib clearance, consistent with published noncompartmental PK and in vitro data. CONCLUSIONS: Simulations predicted lower steady-state peak/trough olaparib exposure through 24-36 h post carboplatin pre-treatment, but this effect was lost by day 2 and thus no dose adjustment is recommended.

The dual mTOR kinase inhibitor TAK228 inhibits tumorigenicity and enhances radiosensitization in diffuse intrinsic pontine glioma.

Diffuse intrinsic pontine glioma (DIPG) is an invasive and treatment-refractory pediatric brain tumor. Primary DIPG tumors harbor a number of mutations including alterations in PTEN, AKT, and PI3K and exhibit activation of mammalian Target of Rapamycin Complex 1 and 2 (mTORC1/2). mTORC1/2 regulate protein translation, cell growth, survival, invasion, and metabolism. Pharmacological inhibition of mTORC1 is minimally effective in DIPG. However, the activity of dual TORC kinase inhibitors has not been examined in this tumor type. Nanomolar levels of the mTORC1/2 inhibitor TAK228 reduced expression of p-AKTS473 and p-S6S240/244 and suppressed the growth of DIPG lines JHH-DIPG1, SF7761, and SU-DIPG-XIII. TAK228 induced apoptosis in DIPG cells and cooperated with radiation to further block proliferation and enhance apoptosis. TAK228 monotherapy inhibited the tumorigenicity of a murine orthotopic model of DIPG, more than doubling median survival (p = 0.0017) versus vehicle. We conclude that dual mTOR inhibition is a promising potential candidate for DIPG treatment.

Quantification of Temozolomide in Nonhuman Primate Fluids by Isocratic Ultra-High Performance Liquid Chromatography-Tandem Mass Spectrometry to Study Brain Tissue Penetration Following Intranasal or Intravenous Delivery.

A sensitive and selective ultra-high performance liquid chromatography-tandem mass spectrometric method was developed for the quantification of temozolomide (TMZ) in nonhuman primate (NHP) plasma, cerebrospinal fluid (CSF), and brain extracellular fluid (ECF) following microdialysis. Ethyl acetate was used to extract the plasma and CSF samples, using theophylline as the internal standard (IS). ECF samples were diluted with acetonitrile prior to analysis. TMZ was separated on a Waters UPLC® BEH C18 column with an isocratic mobile phase of ammonium acetate (10 mM)-0.1% formic acid/acetonitrile (30:70, v/v) in a positive-ion multi pie reaction monitoring mode (m/z 195.5 →137.6 for TMZ; m/z 181.5→124.2 for IS). The retention time of TMZ and theophylline was 0.45 min with a total run time of 2.5 min. The method was validated over the range from 5-2000 ng/mL in NHP plasma, CSF, and ECF with respect to linearity, accuracy, precision, selectivity, and stability. This method was successfully applied toward the measurement of pharmacokinetic samples following various routes of drug administration.