Systemic short chain fatty acids limit antitumor effect of CTLA-4 blockade in hosts with cancer.

Gut microbiota composition influences the clinical benefit of immune checkpoints in patients with advanced cancer but mechanisms underlying this relationship remain unclear. Molecular mechanism whereby gut microbiota influences immune responses is mainly assigned to gut microbial metabolites. Short-chain fatty acids (SCFA) are produced in large amounts in the colon through bacterial fermentation of dietary fiber. We evaluate in mice and in patients treated with anti-CTLA-4 blocking mAbs whether SCFA levels is related to clinical outcome. High blood butyrate and propionate levels are associated with resistance to CTLA-4 blockade and higher proportion of Treg cells. In mice, butyrate restrains anti-CTLA-4-induced up-regulation of CD80/CD86 on dendritic cells and ICOS on T cells, accumulation of tumor-specific T cells and memory T cells. In patients, high blood butyrate levels moderate ipilimumab-induced accumulation of memory and ICOS + CD4 + T cells and IL-2 impregnation. Altogether, these results suggest that SCFA limits anti-CTLA-4 activity.

Potentiation of mitotane action by rosuvastatin: New insights for adrenocortical carcinoma management.

Mitotane (also termed o,p'­DDD) is the most effective therapy for advanced adrenocortical carcinoma (ACC). Mitotane­induced dyslipidemia is treated with statins. Mitotane and statins are known to exert anti­proliferative effects in vitro; however, the effects of statins have never been directly evaluated in patients with ACC and ACC cells, at least to the best of our knowledge. Thus, in this study, we aimed to examine the effects of the rosuvastatin on ACC cells. It has been shown that the combined use of mitotane and statins significantly increases the tumor control rate in patients with ACC; however, it would be of interest to elucidate the molecular mechanisms involved in this potentiation. In this study, we examined the effects of mitotane, rosuvastatin and their combination in NCI­H295R human ACC cells using proliferation assays, gene expression analyses and free intracellular cholesterol measurements. The results revealed that mitotane dose­dependently reduced cell viability, induced apoptosis and increased intracellular free cholesterol levels, considered as one of the key features of mitotane action, while rosuvastatin alone reduced cell viability and increased apoptosis at high concentrations. We also demonstrated that rosuvastatin potentiated the effects of mitotane by reducing cell viability, inducing apoptosis, increasing intracellular free cholesterol levels, and by decreasing the expression of 3­hydroxy­3­methylglutaryl­CoA reductase (HMGCR) and ATP binding cassette subfamily a member 1 (ABCA1), genes involved in cholesterol metabolism, and inhibiting steroidogenesis. Collectively, potentiating the effects of mitotane with the use of rosuvastatin may provide novel therapeutic strategies for ACC, given that the combination of these drugs, pending clinical validation, may lead to the better management of ACC.

A Novel Spectroscopically Determined Pharmacodynamic Biomarker for Skin Toxicity in Cancer Patients Treated with Targeted Agents.

Raman spectroscopy is a noninvasive and label-free optical technique that provides detailed information about the molecular composition of a sample. In this study, we evaluated the potential of Raman spectroscopy to predict skin toxicity due to tyrosine kinase inhibitors treatment. We acquired Raman spectra of skin of patients undergoing treatment with MEK, EGFR, or BRAF inhibitors, which are known to induce severe skin toxicity; for this pilot study, three patients were included for each inhibitor. Our algorithm, based on partial least squares-discriminant analysis (PLS-DA) and cross-validation by bootstrapping, discriminated to variable degrees spectra from patient suffering and not suffering cutaneous adverse events. For MEK and EGFR inhibitors, discriminative power was more than 90% in the viable epidermis skin layer; whereas for BRAF inhibitors, discriminative power was 71%. There was a 81.5% correlation between blood drug concentration and Raman signature of skin in the case of EGFR inhibitors and viable epidermis skin layer. Our results demonstrate the power of Raman spectroscopy to detect apparition of skin toxicity in patients treated with tyrosine kinase inhibitors at levels not detectable via dermatological inspection and histological evaluation. Cancer Res; 77(2); 557-65. ©2016 AACR.

Dyslipidemia causes overestimation of plasma mitotane measurements.

UNLABELLED: Mitotane (o,p'-DDD) is the standard treatment for advanced adrenocortical carcinoma (ACC). Monitoring of plasma mitotane levels is recommended to look for a therapeutic window between 14 and 20mg/L, but its positive predictive value requires optimization. We report the case of an ACC patient with a history of dyslipidemia treated with mitotane in whom several plasma mitotane levels >30mg/L were found together with an excellent neurological tolerance. This observation led us to compare theoretical or measured o,p'-DDD and o,p'-DDE levels in a series of normolipidemic and dyslipidemic plasma samples to explore potential analytical issues responsible for an overestimation of plasma mitotane levels. We demonstrate an overestimation of mitotane measurements in dyslipidemic patients. Mitotane and o,p'-DDE measurements showed a mean 20% overestimation in hypercholesterolemic and hypertriglyceridemic plasma, compared with normolipidemic plasma. The internal standard p,p'-DDE measurements showed a parallel decrease in hypercholesterolemic and hypertriglyceridemic plasma, suggesting a matrix effect. Finally, diluting plasma samples and/or using phospholipid removal cartridges allowed correcting such interference. LEARNING POINTS: Hypercholesterolemia (HCH) and hypertriglyceridemia (HTG) induce an overestimation of plasma mitotane measurements.We propose a routine monitoring of lipidemic status.We propose optimized methodology of measurement before interpreting high plasma mitotane levels.

Lipoprotein-Free Mitotane Exerts High Cytotoxic Activity in Adrenocortical Carcinoma.

CONTEXT: Mitotane (o,p'-DDD), the only approved drug for advanced adrenocortical carcinoma (ACC), is a lipophilic agent that accumulates into circulating lipoprotein fractions and high-lipid-containing tissues. OBJECTIVE: The aim of our study was to evaluate the in vivo and in vitro biological implication of serum lipoproteins on pharmacological action of mitotane. Distribution and concentration of mitotane were studied in plasma and adrenal tissue samples from mitotane-treated patients. The effect of lipoprotein-bound or lipoprotein-free (LP-F) mitotane was analyzed on proliferation and apoptosis of human adrenocortical H295R cells. A retrospective study of patients with ACC treated or not with statins was also performed. RESULTS: o,p'-DDD distribution among very low-density lipoprotein, low-density lipoprotein (LDL), high-density lipoprotein (HDL), and LP-F fractions obtained after plasma ultracentrifugation of 23 of mitotane-treated patients was widely distributed in each subfraction. A positive correlation was observed between mitotane levels in plasma and in LDL, HDL, but also LP-F compartment. Intratumor o,p'-DDD concentrations in five ACC samples of mitotane-treated patients were found to be independent of cholesterol transporter expression, scavenger receptors, and LDL receptors. In vitro studies showed significant higher antiproliferative and proapoptotic effects and higher cell and mitochondrial uptake of mitotane when H295R cells were grown in LP-F medium. Finally, retrospective study of an ACC cohort of 26 mitotane-treated patients revealed that statin therapy was significantly associated with a higher rate of tumor control. CONCLUSIONS: Altogether, our in vitro and in vivo studies provided compelling evidence for a greater efficacy of LP-F mitotane. Patients with ACC may thus benefit from therapeutic strategies that aim to increase LP-F mitotane fraction.

Safety and efficacy compared between irinotecan-loaded microspheres HepaSphere and DC bead in a model of VX2 liver metastases in the rabbit.

PURPOSE: To compare irinotecan-eluting HepaSphere (BioSphere Medical, Roissy-en-France, France) and DC Bead (Biocompatibles UK Ltd, London, United Kingdom) embolization microspheres for distribution in tumors, release properties, tolerance, and antitumor effects in a model of liver metastases in the rabbit. MATERIALS AND METHODS: Multiple liver tumors were created by injection of a VX2 cell suspension in the portal vein of rabbits. After 2 weeks, embolization of the proper hepatic artery was performed with a fixed volume of bland HepaSphere (n = 5), irinotecan-loaded HepaSphere (n = 6), or irinotecan-loaded DC Bead (n = 5) microspheres. Untreated animals injected with VX2 cells served as control animals (n = 5). Plasma pharmacokinetics of irinotecan and its metabolite SN38 were assessed. Histopathology and gene expression analysis were performed 3 days after treatment. RESULTS: Among all treated groups, there was no significant difference in liver enzymes or liver damage on histology. Irinotecan-loaded HepaSphere microspheres showed a faster release of drug than DC Bead microspheres leading to a twofold higher concentration of drug in plasma for HepaSphere microspheres. HepaSphere microspheres were less frequently found inside tumor nodules on histology than DC Bead microspheres (11% vs 48%, P < .001) because of their larger size. Tumor necrosis was significantly greater for rabbits given irinotecan-loaded HepaSphere microspheres (69% of total tumor surface) and rabbits given DC Bead microspheres (50% of total tumor surface) compared with control animals (24% of total tumor surface, P = .006 and P = .047). CONCLUSIONS: HepaSphere and DC Bead microspheres loaded with irinotecan caused significant necrosis of tumor nodules in a model of VX2 liver metastases. This outcome was mostly due to irinotecan delivery rather than vascular occlusion by the microspheres and was greater for HepaSphere microspheres compared with DC Bead microspheres.

The lack of antitumor effects of o,p'DDA excludes its role as an active metabolite of mitotane for adrenocortical carcinoma treatment.

Mitotane (o,p'DDD) is the most effective treatment of advanced adrenocortical carcinoma (ACC) but its mechanism of action remains unknown. Previous studies suggested that o,p'DDA may represent the active metabolite of mitotane. We aimed at reevaluating the potential role and pharmacological effects of o,p'DDA. Functional consequences of o,p'DDA exposure were studied on proliferation, steroidogenesis, and mitochondrial respiratory chain in human H295R and SW13 adrenocortical cells. Mitotane and its metabolites were quantified using high-performance liquid chromatography combined to an ultraviolet detection in these cells treated with o,p'DDD or o,p'DDA and in human adrenal tissues. Dose-response curves up to 300 µM showed that, as opposed to o,p'DDD, o,p'DDA did not inhibit cell proliferation nor alter respiratory chain complex IV activity, gene expression nor induce mitochondrial biogenesis, oxidative stress, or apoptosis. However, whereas mitotane drastically decreased expression of genes involved in steroidogenesis, o,p'DDA slightly reduced expression of some steroidogenic enzymes and exerts weak anti-secretory effects only at high doses. While o,p'DDD concentration was significantly reduced by 40 % in H295R cell supernatants after 48 h incubation, o,p'DDA levels remained unchanged suggesting that o,p'DDA was not efficiently transported into the cells. o,p'DDA was not detected in cell homogenates or supernatants after 48 h exposure to o,p'DDD, consistent with the absence of o,p'DDA production in these models. Finally, unlike o'p'DDD, we found that o,p'DDA content was undetectable in two ACC and one normal adrenal gland of mitotane-treated patients, suggesting a lack of cellular uptake and in situ production. Our results demonstrate that o,p'DDD, but not o,p'DDA, induces functional alterations in adrenal cells.

Comparative transplacental transfer of taxanes using the human perfused cotyledon placental model.

OBJECTIVES: The use of taxanes (paclitaxel and docetaxel) in pregnant cancer patients is increasing. We aimed to compare their transplacental transfer using the gold standard human placental perfusion model, to guide drug selection. STUDY DESIGN: Term placentas were perfused with paclitaxel or docetaxel and 2 different albumin concentrations. Main transfer parameters such as fetal transfer rate (FTR), clearance index, and placental uptake of taxanes were assessed. RESULTS: Twelve placentas were perfused, 6 with paclitaxel and 6 with docetaxel. Mean FTR of paclitaxel decreased significantly from 5.67 ± 0.02% in low albumin conditions to 1.72 ± 0.09% in physiological albumin conditions. Similarly, mean clearance index decreased significantly from 0.22 ± 0.02 to 0.09 ± 0.01. Regarding docetaxel, mean FTR were similar in low albumin and physiological conditions (5.03 ± 0.60% and 4.04 ± 0.22%, respectively) while mean clearance index decreased significantly from 0.18 ± 0.02 to 0.13 ± 0.01. Taxanes accumulation in cotyledon was similar for docetaxel and paclitaxel: 4.54 ± 1.84% vs 3.31 ± 1.88%, respectively. CONCLUSION: Transplacental transfer and placental accumulation of paclitaxel and docetaxel were low and similar, especially in physiological conditions of albumin. Further studies are warranted to optimize the selection of a taxane in pregnant cancer patients.

Quantification of dimethyl-ifosfamide and its N-deschloropropylated metabolites in mouse plasma by liquid chromatography-tandem mass spectrometry.

Among antitumor oxazaphosphorine drugs, the prodrug ifosfamide (IFO) and its analogs require metabolic activation by specific liver cytochrome P450 (CYP) enzymes to become therapeutically active. New 7,9-dimethyl-ifosfamide analogs have shown greater cytotoxic activity than IFO, whereas side-chain oxidation still occurred leading to monochloroacetone after N-dechloropropylation. A sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay was developed and validated for the simultaneous quantitation of the prodrug 7S,9S-dimethyl-ifosfamide (diMeIFO) and its two inactive metabolites, N(2)- and N(3)-deschloropropyl-dimethylifosfamide (N(2)-DCP-diMeIFO and N(3)-DCP-diMeIFO) in mouse plasma. After protein precipitation with methanol, the analytes were separated by isocratic reversed-phase chromatography with (methanol/ammonium formate pH 5.5, 60:40, v/v) and detected by tandem mass spectrometry using multiple reaction monitoring of transitions ions m/z 289→168 for diMeIFO, m/z 213→168 for N(2)-DCP-diMeIFO, m/z 213→92 for N(3)-DCP-diMeIFO and m/z 261→154 for IFO (internal standard). The calibration curves were linear over the concentration range of 20-10,000ng/mL for the three analytes. Mean extraction recoveries from mouse plasma were 99, 96, 99 and 100% for diMeIFO, N(2)-DCP-diMeIFO, N(3)-DCP-diMeIFO and IFO, respectively. The lower limit of quantitation for diMeIFO and its metabolites was 20 ng/mL in 50 µL plasma. The method was accurate with calculated bias from -5.8 to 4.0% for diMeIFO, from -1.1 to 10.6% for N(2)-DCP-diMeIFO and from -6.9 to 9.8% for N(3)-DCP-diMeIFO, and precise with coefficients of variation lower than 6.8%, 7.8% and 14.3%, respectively. The assay was successfully applied to a preliminary pharmacokinetic study of diMeIFO and of its metabolites in mice.