Mireille Mousseau

Effects of permeability transition inhibition and decrease in cytochrome c content on doxorubicin toxicity in K562 cells

As mitochondria play a key role in the commitment to cell death, we have investigated the mitochondrial consequences of resistance to doxorubicin (DOX) in K562 cells. We found that the permeability transition pore (PTP) inhibitor cyclosporine A (CsA) failed to inhibit PTP opening in the resistant clone. Moreover, the Ca2+ loading capacity in the resistant clone was identical to that observed in the parent cells in the presence of CsA, suggesting that the PTP was already inhibited in a CsA-like manner in the resistant cells. In agreement with this proposal, the mitochondrial target of CsA cyclophilin D (CyD) decreased by half in the resistant cells. The levels of adenine nucleotide translocator, voltage anion-dependent channel, Bax, Bcl-2, Bcl-xL, AIF and Smac/Diablo, were similar in both cell lines, whereas cytochrome c content was divided by three in the resistant cells. Since P-glycoprotein inhibition did not restore DOX toxicity in the resistant cells, while DOX-induced cell death in the parent cells was prevented by either PTP inhibition or siRNA-induced decrease in cytochrome c content, we conclude that the inhibition of PTP opening and the decrease in cytochrome c content participate in the mechanism that makes K562 cells resistant to DOX.

HER2 Overexpression/Amplification and Trastuzumab Treatment in Advanced Ovarian Cancer: A GINECO Phase II Study

Background Variable rates of HER2 protein overexpression and gene amplification have been reported in advanced ovarian cancers (AOC). Trastuzumab, tested only as a single agent, has been shown to achieve 7% response in heavily pretreated patients with AOC with 3+ and 2+ HER2 immunostaining by immunohistochemistry (IHC). The GINECO trial explored the combination of trastuzumab with paclitaxel and carboplatin in patients with resistant AOC (< 6 months) and HER2 gene amplification.

Monitoring Monoclonal Antibody Delivery in Oncology: The Example of Bevacizumab

Developing therapeutic monoclonal antibodies paves the way for new strategies in oncology using targeted therapy which should improve specificity. However, due to a lack of biomarkers, a personalized therapy scheme cannot always be applied with monoclonal antibodies. As a consequence, the efficacy or side effects associated with this type of treatment often appear to be sporadic. Bevacizumab is a therapeutic monoclonal antibody targeting Vascular Endothelial Growth Factor (VEGF). It is used to limit tumor vascularization. No prognosis or response biomarker is associated with this antibody, we therefore assessed whether the administration protocol could be a possible cause of heterogeneous responses (or variable efficacy). To do this, we developed a bevacizumab assay with a broad sensitivity range to measure blood bevacizumab concentrations. We then analyzed bevacizumab concentrations in 17 patients throughout the first quarter of treatment. In line with previously published data, average blood concentrations were 88+/−27 mg/L following the first dose administered, and 213+/−105 mg/L after the last (6th) dose administered. However, the individual values were scattered, with a mean 4-fold difference between the lowest and the highest concentration for each dose administered. We demonstrated that the bevacizumab administration schedule results in a high inter-individual variability in terms of blood concentrations. Comparison of assay data with clinical data indicates that blood concentrations above the median are associated with side effects, whereas values below the median favor inefficacy. In conclusion, bevacizumab-based therapy could benefit from a personalized administration schedule including follow-up and adjustment of circulating bevacizumab concentrations.

New advances in DPYD genotype and risk of severe toxicity under capecitabine

Background Deficiency in dihydropyrimidine dehydrogenase (DPD) enzyme is the main cause of severe and lethal fluoropyrimidine-related toxicity. Various approaches have been developed for DPD-deficiency screening, including DPYD genotyping and phenotyping. The goal of this prospective observational study was to perform exhaustive exome DPYD sequencing and to examine relationships between DPYD variants and toxicity in advanced breast cancer patients receiving capecitabine. Methods Two-hundred forty-three patients were analysed (88.5% capecitabine monotherapy). Grade 3 and grade 4 capecitabine-related digestive and/or neurologic and/or hemato-toxicities were observed in 10.3% and 2.1% of patients, respectively. DPYD exome, along with flanking intronic regions 3’UTR and 5’UTR, were sequenced on MiSeq Illumina. DPD phenotype was assessed by pre-treatment plasma uracil (U) and dihydrouracil (UH2) measurement. Results Among the 48 SNPs identified, 19 were located in coding regions, including 3 novel variations, each observed in a single patient (among which, F100L and A26T, both pathogenic in silico). Combined analysis of deleterious variants *2A, I560S (*13) and D949V showed significant association with grade 3–4 toxicity (sensitivity 16.7%, positive predictive value (PPV) 71.4%, relative risk (RR) 6.7, p<0.001) but not with grade 4 toxicity. Considering additional deleterious coding variants D342G, S492L, R592W and F100L increased the sensitivity to 26.7% for grade 3–4 toxicity (PPV 72.7%, RR 7.6, p<0.001), and was significantly associated with grade 4 toxicity (sensitivity 60%, PPV 27.3%, RR 31.4, p = 0.001), suggesting the clinical relevance of extended targeted DPYD genotyping. As compared to extended genotype, combining genotyping (7 variants) and phenotyping (U>16 ng/ml) did not substantially increase the sensitivity, while impairing PPV and RR. Conclusions Exploring an extended set of deleterious DPYD variants improves the performance of DPYD genotyping for predicting both grade 3–4 and grade 4 toxicities (digestive and/or neurologic and/or hematotoxicities) related to capecitabine, as compared to conventional genotyping restricted to consensual variants *2A, *13 and D949V.

Abstract P3-15-04: A French prospective pilot study to identify dihydropyrimidine dehydrogenase (DPD) deficiency in breast cancer patients receiving capecitabine

Background: Health Authorities point out that DPD deficiency confers a significant risk of major toxicity for patients receiving capecitabine. Identification of at-risk patients is thus of major concern. This multicentric prospective study of the French GPCO group (Groupe de Pharmacologie Clinique Oncologique, Unicancer) evaluated the sensitivity, specificity and predictive values of DPD phenotyping and genotyping to predict severe cap-related toxixity in metastatic breast cancer patients. Methods: 303 patients were included between February 2009 and February 2011 (15 institutions). Eighty-eight% received capecitabine as monotherapy, 28% were treated as first line (mean dose at 1 st cycle 1957 mg/m 2 /d). Pre-treatment uracil (U, physiological DPD substrate) plasma concentration was measured in 286 patients (HPLC assay). DPD genotyping (IVS14+1G>A, 2846A>T, 1679T>G, 464T>A) was performed on 281 patients. Severe toxicity (G3-4 CTCAE v3 criteria) was measured over cycles 1-2. Results: Grade 3-4 toxicity (diarrhea, vomiting, hematoxicity, hand-foot syndrome) has been observed in 19.6% of patients (one toxic death). A marked trend for higher U concentrations has been noted in patients developing severe toxicity vs those who didn9t (median 12.7 ng/ml (Q1-Q3 9-17) vs median 10.2 ng/ml (range 8-13), respectively, p = 0.014). However, ROC curve has showed that this difference was too small for use as a reliable toxicity predictor. The patient with toxic death had an elevated U concentration at 17 ng/ml. Among the 7 patients with a DPD mutation (3 pts IVS14+1, 3 pts 2846A>T, one 1679T>G, all heterozygous), 5 developed severe toxicity (including the toxic death, 2846A>T), one did not, and the last one was not documented. Relative risk for developing severe toxicity was 4.60 in mutated patients vs wild-type patients (95%CI 2.95-7.16, p = 0.001); positive and negative predictive values were 83.3% and 81.9%, respectively; specificity was 99.5% and sensitivity was 9.8%. Conclusions : Breast cancer patients harbouring a DPD variant allele are at risk to develop severe, up to lethal, capecitabine-related toxicity. Pre-treatment U measurement remains to be more firmly established as a reliable predictor of capecitabine toxicity. These observations are of major interest for breast cancer patients candidate for capecitabine therapy. Citation Information: Cancer Res 2013;73(24 Suppl): Abstract nr P3-15-04.

Non-toxic and short treatment with gemcitabine inhibits in vitro migration of HT-1080 cells.

Gemcitabine has demonstrated clinical activity in solid tumors. Several in vitro studies were carried out regarding its metabolism, toxicity and cell cycle effects, but none was done on the anti-metastasis potential of the drug. We sought to determine the effect of gemcitabine concentrations on migration velocity of HT-1080 cells at concentrations which do not alter cell cycle progression and proliferation. Cells were treated for 1 or 5 h at IC10–70 of gemcitabine in order to estimate its effects on viability, proliferation and migration capacity using flow cytometry and microscopy imaging, respectively. The gemcitabine treatment for 1 h had no effect on cell proliferation, viability, cycle or migration on HT-1080 cells. Even though the 5 h of exposure at IC10, IC20 and IC50 concentrations did not affect cell viability, proliferation and cell cycle repartition, the mean velocity of HT-1080 dramatically decreased by 50 and 30%, respectively. Gemcitabine at IC70 concentrations for 5 h of exposure first induced a time course inhibition of proliferation, together with a decrease in viability and altered cell morphology, and then inhibited cell migration by 50%. These data suggest the possibility to couple the anti-migratory property of gemcitabine with the known anti-tumoral effect in the treatment of tumors with high metastatic potential.