Hypoxia Drives Dihydropyrimidine Dehydrogenase Expression in Macrophages and Confers Chemoresistance in Colorectal Cancer.

Colorectal adenocarcinoma is a leading cause of death worldwide, and immune infiltration in colorectal tumors has been recognized recently as an important pathophysiologic event. In this context, tumor-associated macrophages (TAM) have been related to chemoresistance to 5-fluorouracil (5-FU), the first-line chemotherapeutic agent used in treating colorectal cancers. Nevertheless, the details of this chemoresistance mechanism are still poorly elucidated. In the current study, we report that macrophages specifically overexpress dihydropyrimidine dehydrogenase (DPD) in hypoxia, leading to macrophage-induced chemoresistance to 5-FU via inactivation of the drug. Hypoxia-induced macrophage DPD expression was controlled by HIF2α. TAMs constituted the main contributors to DPD activity in human colorectal primary or secondary tumors, while cancer cells did not express significant levels of DPD. In addition, contrary to humans, macrophages in mice do not express DPD. Together, these findings shed light on the role of TAMs in promoting chemoresistance in colorectal cancers and identify potential new therapeutic targets. SIGNIFICANCE: Hypoxia induces HIF2α-mediated overexpression of dihydropyrimidine dehydrogenase in TAMs, leading to chemoresistance to 5-FU in colon cancers.

Severe toxicity of capecitabine in a patient with DPD deficiency after a safe FEC-100 experience: why we should test DPD deficiency in all patients before high-dose fluoropyrimidines.

We report the case of a 44-year-old patient who experienced severe toxicity while being treated with capecitabine at standard dose for metastatic breast cancer. As the patient had already received 5-FU within the FEC protocol (5-FU 500 mg/m2, epirubicin 100 mg/m2, and cyclophosphamide 500 mg/m2) 10 years ago without experiencing any severe adverse event, no DPD deficiency testing was performed before capecitabine treatment. Nevertheless, she experienced severe diarrhea and grade 2 hand-foot syndrome from the first cycle, forcing her to stop the treatment. Phenotypic and genotypic investigation of DPD activity revealed that the patient had a partial deficiency and had therefore been exposed to a higher risk of developing severe toxicities on fluoropyrimidines. This case proves that tolerance to low-dose fluoropyrimidines does not preclude DPD deficiency and the occurrence of severe toxicities if higher doses of fluoropyrimidines are used as a second-line treatment. It emphasizes the role of DPD phenotyping testing based on uracilemia in patients scheduled for fluoropyrimidine drugs, even if previous courses with low-dose 5-FU were safely administered.

Body Surface Area Dosing of High-Dose Methotrexate Should Be Reconsidered, Particularly in Overweight, Adult Patients.

BACKGROUND: High-dose methotrexate is used for treating several types of cancer. However, it is associated with a high risk of acute kidney injury (AKI), especially in patients with high MTX concentrations. Although therapeutic drug monitoring is performed to monitor MTX concentrations, it is unclear what concentration should be considered critical, thus requiring rescue protocols to prevent nephrotoxicity. METHODS: Patients treated with high-dose methotrexate for lymphoma or acute lymphoblastic leukemia and those benefited from therapeutic drug monitoring were included. The relationship between MTX concentrations and the presence or absence of AKI was assessed. MTX concentrations were analyzed using a population pharmacokinetic approach. Specific attention was given to morphological covariates because MTX doses are individualized according to body surface area (BSA). RESULTS: In total, 328 patients and 657 cycles of treatment were analyzed. Higher MTX concentrations were observed in the AKI+ group. For cycle 1, all patients showing an MTX concentration >6 µM at 36 hours or >2 µM at 48 hours after infusion developed nephrotoxicity. The final pharmacokinetic model had 2 compartments and included the effect of age on clearance (CL) and of body weight on peripheral distribution volume. None of the morphological covariates tested on CL led to significant improvement in the model. Higher MTX concentrations were observed in patients with extreme BSA values (≥2 m2) or body mass index (≥25 kg/m2). Patients with AKI who received at least 1 cycle had higher BSA and BMI. CONCLUSIONS: The results from this study provide additional information on the relationship between MTX concentration and nephrotoxicity. Patients with a plasma MTX concentration >6 µM at 36 hours were more likely to manifest AKI. In addition, the results suggest that overweight patients have a high AKI risk and that BSA-based adjustment of MTX dose is not appropriate.

Pharmacokinetic and Pharmacogenetic Study of Etoposide in High-Dose Protocol (TI-CE) for Advanced Germ Cell Tumors.

BACKGROUND: Etoposide dosing is based on body surface area. We evaluated if further dose individualization would be required for high dose (HD) etoposide within the TI-CE (taxol, ifosfamide, carboplatin, and etoposide) protocol. METHODS: Eighty-eight patients received 400 mg/m2/day of etoposide as a 1-hour IV infusion on 3 consecutive days over 3 cycles as part of a phase II trial evaluating efficacy of therapeutic drug monitoring (TDM) of carboplatin in the TI-CE HD protocol. Pharmacokinetic (PK) data were analyzed using population PK model on NONMEM to quantify inter- and intra-individual variabilities. Relationship between etoposide exposure and pharmacodynamic (PD) endpoints, and between selected genetic polymorphisms and tumor response or toxicity were evaluated. RESULTS: The inter-patient, inter- and intra-cycle variabilities of clearance were 16%, 9% and 0.1%, respectively. The PK-PD relationship was not significant despite a trend toward higher etoposide exposure in patients responding to treatment. A significant correlation was found between exposure and extended neutropenia at cycle 3. A significant association between UGT1A1*28 polymorphism and late neutropenia was observed but needs further evaluation. CONCLUSIONS: The present study suggests that neither a priori dose individualization nor dose adaptation using TDM is required validating body surface area dosing of etoposide in the TI-CE protocol.

Therapeutic Drug Monitoring of Carboplatin in High-Dose Protocol (TI-CE) for Advanced Germ Cell Tumors: Pharmacokinetic Results of a Phase II Multicenter Study.

Purpose: We aimed to evaluate the performance of therapeutic drug monitoring (TDM) approach in controlling interpatient variability of carboplatin exposure (AUC) in patients treated with TI-CE high-dose chemotherapy for advanced germ cell tumors and to assess the possibility of using a formula-based dosing method as a possible alternative.Experimental Design: Eighty-nine patients receiving carboplatin for 3 consecutive days during 3 cycles were evaluable for pharmacokinetic study. Blood samples were taken on day 1 to determine the carboplatin clearance using a Bayesian approach (NONMEM 7.2) and to adjust the dose on day 3 to reach the target AUC of 24 mg.min/mL over 3 days. On days 2 and 3, samples were taken for retrospective assessment of the actual AUC. A population pharmacokinetic analysis was also performed on 58 patients using NONMEM to develop a covariate equation for carboplatin clearance prediction adapted for future TI-CE patients, and its performance was prospectively evaluated on the other 29 patients along with different methods of carboplatin clearance prediction.Results: The mean actual AUC was 24.4 mg.min/mL per cycle (22.4 and 26.8 for 10th and 90th percentiles, respectively). The new covariate equation [CL (mL/min) = 130.7 × (Scr/83)-0.826 × (BW/76)+0.907 × (Age/36)-0.223 with Scr in µmol/L, BW in kilograms, age in years] allows unbiased and more accurate prediction of carboplatin clearance compared with other equations.Conclusions: TDM allows controlling and reaching the target AUC. Alternatively, the new equation of carboplatin clearance prediction, better adapted to these young male patients, could be used if TDM cannot be implemented. Clin Cancer Res; 23(23); 7171-9. ©2017 AACR.

Population pharmacokinetics of peritoneal, plasma ultrafiltrated and protein-bound oxaliplatin concentrations in patients with disseminated peritoneal cancer after intraperitoneal hyperthermic chemoperfusion of oxaliplatin following cytoreductive surgery: correlation between oxaliplatin exposure and thrombocytopenia.

PURPOSE: First, to evaluate the peritoneal (IP), plasma ultrafiltrated (UF) and protein-bound (B) pharmacokinetics (PK) of oxaliplatin after intraperitoneal hyperthermic chemoperfusion (HIPEC) following cytoreductive surgery. Second, to evaluate the relationship between oxaliplatin exposure and observed toxicity. METHODS: IP, UF, and B concentrations from 75 patients treated by 30-min oxaliplatin-based HIPEC procedures were analysed according to a pharmacokinetic modelling approach using NONMEM. Oxaliplatin was administered in a 5 % dextrose solution (2 L/m(2)) at 360 (n = 58) or 460 mg/m(2) (n = 17). The most frequently observed toxicities were related to the peritoneal, systemic exposures and to the parameters corresponding to the oxaliplatin absorption from peritoneal cavity into plasma. RESULTS: IP (n = 536), UF (n = 669) and B (n = 661) concentrations were simultaneously described according to a five-compartment PK model with irreversible nonlinear binding from UF to B according to a Michaelis-Menten equation. The mean (±SD) maximum fraction of dose absorbed and elimination half-life from the peritoneum was 53.7 % (±8.5) and 0.49 h (±0.1), respectively. The mean (±SD) ratio AUC(IP)/AUC(UF) was 5.3 (±2) confirming the pharmacokinetic advantage of the procedure. Haemoperitoneum (22.7 %), neuropathy (18.7 %), grade 3/4 thrombocytopenia (13.3 %) were the most frequently reported toxicities. AUC(UF) accounts for approximately 12 % of the variation in the maximum percentage of platelet decrease (r = 0.35, p = 0.002). Thrombocytopenia was correlated with higher AUCUF, partly dependent on the extent and rate of oxaliplatin absorption. CONCLUSIONS: Despite a common dose administered, variability in peritoneal and systemic oxaliplatin exposures are observed, leading to differences in haematological toxicity between patients.

Vitamin B6 metabolism influences the intracellular accumulation of cisplatin.

Vitamin B6 metabolism influences the adaptive response of non-small lung carcinoma (NSCLC) cells to distinct, potentially lethal perturbations in homeostasis, encompassing nutrient deprivation, hyperthermia, hypoxia, irradiation as well as the exposure to cytotoxic chemicals, including the DNA-damaging agent cisplatin (CDDP). Thus, the siRNA-mediated downregulation of pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6, protects NSCLC cells (as well as a large collection of human and murine malignant cells of distinct histological derivation) from the cytotoxic effects of CDDP. Accordingly, the administration of pyridoxine, one of the inactive precursors of vitamin B6, exacerbates cisplatin-induced cell death, in vitro and in vivo, but only when PDXK is expressed. Conversely, antioxidants such as non-oxidized glutathione (GSH) are known to protect cancer cells from CDDP toxicity. Pyridoxine increases the amount of CDDP-DNA adducts formed upon the exposure of NSCLC cells to CDDP and aggravates the consequent DNA damage response. On the contrary, in the presence of GSH, NSCLC cells exhibit near-to-undetectable levels of CDDP-DNA adducts and a small fraction of the cell population activates the DNA damage response. We therefore wondered whether vitamin B6 metabolism and GSH might interact with CDDP in a pharmacokinetic fashion. In this short communication, we demonstrate that GSH inhibits the intracellular accumulation of CDDP, while pyridoxine potentiates it in a PDXK-dependent fashion. Importantly, such pharmacokinetic effects do not involve plasma membrane transporters that mediate a prominent fraction of CDDP influx, i.e., solute carrier family 31, member 1 (SLC31A1, best known as copper transporter 1, CTR1) and efflux, i.e., ATPase, Cu ( 2+) transporting, ß polypeptide (ATP7B).

Technical note: Hapten synthesis, antibody production and development of an enzyme-linked immunosorbent assay for detection of the natural steroidal alkaloid Dendrogenin A.

We have recently discovered the existence of 5α-Hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-ol, called Dendrogenin A (DDA), as the first endogenous steroidal alkaloid ever described in mammals. We found that the DDA content of tumors and cancer cell lines was low or absent compared with normal cells showing that a deregulation in DDA biosynthesis was associated with cancer and therefore suggesting that DDA could represent a metabolomic cancer biomarker. This prompted us to produce antibodies that selectively recognize DDA. For this purpose, the hapten 5α-hydroxy-6ß-[2-(1H-imidazol-4-yl)ethylamino]cholestan-3ß-o-hemisuccinate with a carboxylic spacer arm attached to the 3ß-hydroxyl group of DDA was synthesized. The hapten was coupled to bovine serum albumin and keyhole limpet hemocyanin for antibody production to develop an enzyme-linked immunosorbent assay (ELISA). The protein conjugates were injected into BALB/c mice to raise antibodies. The monoclonal antibodies that were secreted from the hybridoma cell lines established were assessed with indirect ELISA by competitive assays using dilutions of a DDA standard. The antibodies from the selected hybridomas had an IC(50) value ranging from 0.8 to 425 ng/ml. Three antibodies showed no cross-reactivity with structurally related compounds including histamine, cholesterol, ring B oxysterols and a regio-isomer of DDA. In this study, high-affinity and selective antibodies against DDA were produced for the first time, and a competitive indirect ELISA was developed.

Prognostic impact of vitamin B6 metabolism in lung cancer.

Patients with non-small cell lung cancer (NSCLC) are routinely treated with cytotoxic agents such as cisplatin. Through a genome-wide siRNA-based screen, we identified vitamin B6 metabolism as a central regulator of cisplatin responses in vitro and in vivo. By aggravating a bioenergetic catastrophe that involves the depletion of intracellular glutathione, vitamin B6 exacerbates cisplatin-mediated DNA damage, thus sensitizing a large panel of cancer cell lines to apoptosis. Moreover, vitamin B6 sensitizes cancer cells to apoptosis induction by distinct types of physical and chemical stress, including multiple chemotherapeutics. This effect requires pyridoxal kinase (PDXK), the enzyme that generates the bioactive form of vitamin B6. In line with a general role of vitamin B6 in stress responses, low PDXK expression levels were found to be associated with poor disease outcome in two independent cohorts of patients with NSCLC. These results indicate that PDXK expression levels constitute a biomarker for risk stratification among patients with NSCLC.

Tipifarnib plus tamoxifen in tamoxifen-resistant metastatic breast cancer: a negative phase II and screening of potential therapeutic markers by proteomic analysis.

PURPOSE: Tipifarnib, a farnesyltransferase inhibitor, has antitumor activity in heavily pretreated metastatic breast cancer patients. Preclinical data suggest that FTIs could restore tamoxifen responsiveness in tamoxifen-resistant disease. Thus, combining FTIs and tamoxifen may be a promising clinical approach after relapse or progression on tamoxifen. EXPERIMENTAL DESIGN: Postmenopausal patients with measurable estrogen receptor- and/or progesterone receptor-expressing metastatic breast cancers were enrolled. Only patients with disease progression on tamoxifen were eligible, but there was no limitation regarding prior chemotherapy or hormone therapy regimens. Patients were immediately treated with 300 mg (n = 12) or 200 mg (n = 10) tipifarnib twice daily for 21 of 28-day cycles plus tamoxifen once daily. Serum was collected at baseline and after 8 weeks of treatment to enable proteomic comparison and identify possible predictive response markers. RESULTS: Twenty patients were enrolled and evaluated for efficacy: one patient had an objective response (liver metastasis) and nine had stable disease after 6 months for a clinical benefit rate of 50%; median duration of benefit was 10.3 (range, 7.4-20.2) months. The proteomic analysis by SELDI-TOF and LTQ-FT-Orbitrap identified a known peptide of fibrinogen alpha, the intensity of which was significantly increased in patients with progression compared with patients who benefited from the combined treatment after 8 weeks. CONCLUSIONS: Because the primary end point of efficacy (three objective responses) was not achieved, the study is negative. Nevertheless, the identified peptide could be of interest in discriminating, at 8 weeks of treatment, responders from nonresponders.