Marc Maliepaard

Frequent expression of the multi-drug resistance-associated protein BCRP/MXR/ABCP/ABCG2 in human tumours detected by the BXP-21 monoclonal antibody in paraffin-embedded material.

Breast cancer resistance protein (BCRP/MXR/ABCP/ABCG2; hereafter ABCG2) is a member of the ATP‐binding‐cassette family of transporters that causes multi‐drug resistance to various anticancer drugs. The expression of ABCG2 in human tumours and its potential involvement in clinical drug resistance are unknown. Recently, two monoclonal antibodies against human ABCG2 were produced, BXP‐34 and BXP‐21. This study describes an immunohistochemical method using BXP‐21 to study ABCG2 expression in formalin‐fixed, paraffin‐embedded tissues. No staining was seen using BXP‐34 with the same protocols. The expression of ABCG2 was then investigated in a panel of 150 untreated human solid tumours comprising 21 tumour types. Overall, ABCG2 expression was frequent. Specificity of immunohistochemistry was confirmed by the detection of a 72 kD band in western blotting. ABCG2 expression was seen in all tumour types, but it seemed more frequent in adenocarcinomas of the digestive tract, endometrium, and lung, and melanoma. Positive tumours showed membranous and cytoplasmic staining. In certain adenocarcinomas, prominent membranous staining was seen. Endothelial cells frequently displayed moderate to strong staining. ABCG2 is widely present in untreated human solid tumours and may represent a clinically relevant mechanism of drug resistance. Future studies in specific tumour types are needed to ascertain its clinical relevance. BXP‐21 and the immunohistochemical protocol described here will be of value in these investigations. Copyright

Transport of Topoisomerase I Inhibitors by the Breast Cancer Resistance Protein: Potential Clinical Implications

Abstract: The multidrug resistance protein BCRP (breast cancer resistance protein) is a member of the ATP‐binding cassette family of drug transporters. Overexpression of BCRP caused by exposure of cells to mitoxantrone (MX) or doxorubicin/verapamil resulted in a resistance pattern that is different from what is generally seen in the case of P‐glycoprotein and MRP1 overexpression. Recently, the BCRP gene has been described in ovarian, breast, colon,and gastric cancer and fibrosarcoma cell lines. Our human tumor cells T8 and MX3, derived from the ovarian cancer cell line IGROV1 by stepwise increased exposure to topotecan and MX, are resistant to topotecan, CPT11, SN38, and 9‐aminocamptothecin as well as MX. Increased energy‐dependent efflux of affected drugs was noted. BCRP is a very efficient transporter of topotecan. Our recent studies, using the monoclonal antibody (mAb) BXP34, revealed that BCRP is located in the plasma membrane of the T8 and MX3 cell lines. Preliminary results of staining of human tumor cells showed low or absent levels of BCRP in a panel of solid tumors and acute myeloid leukemia cells.

Breast cancer resistance protein expression and resistance to daunorubicin in blast cells from patients with acute myeloid leukaemia

Breast cancer resistance protein (BCRP) is a recently described member of the ATP binding cassette transporter superfamily. It has been shown to confer resistance to mitoxantrone, topotecan, doxorubicin and daunorubicin in human tumour cell lines. We describe a study of BCRP expression in blast cells derived from 20 patients with acute myeloid leukaemia (AML). Twelve samples were from patients who had received previous cytotoxic therapy. BCRP expression was measured by immunocytochemistry using the BXP‐34 monoclonal antibody. In vitro drug sensitivity was assessed using the methyl thiazol tetrazoliumbromide assay. BCRP expression varied between patients, and six out of 22 (27%) samples had > 10% cells staining positively (median 37%, range 13–95%). BCRP positivity was seen in both de novo samples and those from previously treated patients. There was a marked variation in the effect of all drugs tested between patients. Although there was no correlation between BCRP positivity and the effect of mitoxantrone, topotecan or doxorubicin, the median daunorubicin LC50 value of BCRP+ cells was fourfold higher than that of BCRP− cells (0·89 µmol/l compared with 0·21 µmol/l, P < 0·05). These results suggest that BCRP may be involved in resistance to the agents commonly used in AML and may explain some of the anomalous results found when studying other membrane transporters, such as P‐gp or MRP.

Synergistic cytotoxicity of cisplatin and topotecan or SN-38 in a panel of eight solid-tumor cell lines in vitro.

Abstract The cytotoxicity of cisplatin alone and in combination with topotecan (TPT) or SN-38, two novel topoisomerase I (topo I) inhibitors, was determined in a panel of eight well-characterized human solid-tumor cell lines. Interactions between cisplatin and these topo I inhibitors were investigated using three different administration schedules: (1) simultaneous incubation (C + T and C + S), (2) cisplatin followed by TPT or SN-38 (C → T and C → S), and (3) TPT or SN-38 followed by cisplatin (T → C and S → C). Median-effect analysis revealed synergistic cytotoxicity in seven of the eight cell lines used. In addition, a significant schedule-dependent synergistic cytotoxicity was found in three of the cell lines used, with C → T (or C → S) being the most active schedule. The formation and repair of total cisplatin-DNA adducts in the IGROV-1 ovarian cancer cell line and its cisplatin-resistant subline IGROVCDDP was not significantly affected by TPT on simultaneous incubation. In contrast, the number of cisplatin-DNA interstrand cross-links detected in the IGROV-1 and IGROVCDDP lines at certain time points was significantly lower after coincubation of the cells with TPT. Assessment of the cell-cycle distribution revealed an accumulation of cells in the G2/M phase after exposure to cisplatin. After exposure to TPT a different pattern was observed that was cell-type-specific and dependent upon the TPT concentration. Although up to 4-fold differences in topo I activity were observed in this panel of cell lines, these differences did not appear to be related to the synergy observed between cisplatin and TPT or SN-38. The observed synergy may at least partly be explained by the increased retention of cisplatin-DNA interstrand cross-links in the presence of topo I inhibitors.

Abrogated energy-dependent uptake of cisplatin in a cisplatin-resistant subline of the human ovarian cancer cell line IGROV-1

Abstract The parental IGROV-1 human ovarian adenocarcinoma cell line was intermittently exposed to increasing concentrations of cisplatin to obtain resistant sublines. A stable resistant subline with a resistance factor of 8.4 had been developed after 9 months and 28 passages, which was denoted IGROVCDDP. A high correlation coefficient of 0.97 was found between the log cell survival and the DNA-adduct peak level during the process of resistance development. IGROVCDDP was strongly cross-resistant to carboplatin and doxorubicin and moderately cross-resistant to etoposide, docetaxel, and topotecan. Only minor resistance against 5-fluorouracil was observed, whereas IGROVCDDP was not cross-resistant to methotrexate. Intracellular accumulation of cisplatin was 65% lower in IGROVCDDP as compared with parental IGROV-1 at 37  °C under normal conditions. Coincubation of cisplatin with the Na+/K+-ATPase inhibitor ouabain resulted in a more pronounced decrease in platinum accumulation in IGROV-1 (44% decrease) than in IGROVCDDP (26% decrease). Under energy-depleting conditions the accumulation of cisplatin in the parental cell line was approximately 60% lower than that observed under normal (energy [i.e., ATP] rich) culture conditions. In contrast, the accumulation in IGROVCDDP was not affected by ATP-depletion. There appeared to be no significant difference between the intracellular accumulation of platinum in the resistant and sensitive cells under conditions of energy deprivation or when the uptake was studied at 0  °C. In conclusion, abrogation of energy-dependent accumulation in IGROVCDDP seems to be a major mechanism of resistance to cisplatin in this cell line.

Adaptive intrapatient dose escalation of cisplatin in combination with low-dose vp16 in patients with nonsmall cell lung cancer

The objective of this phase II and pharmacologic study was to explore the feasibility, toxicity and activity of adaptive intrapatient dose escalation of cisplatin in a dose-intensive weekly schedule using predefined levels of exposure, with the ultimate aim to improve the antitumour activity of the therapy in patients with nonsmall cell lung cancer (NSCLC). Platinum DNA-adduct levels in peripheral white blood cells during treatment were used as the primary parameter for adaptive dosing. If DNA-adduct levels were not available, the area under the concentration–time curve (AUC) of unbound platinum in plasma was used for dose adaptation. Target levels for DNA-adducts and AUC have been defined in a previously performed pharmacologic study. The feasibility of adaptive dosing was tested in 76 patients with stage IIIB and IV NSCLC, who were planned to receive 6 weekly courses of cisplatin at a starting dose of 70 mg m−2, together with daily low oral dose of 50 mg VP16. In total, 37 patients (49%) who were given more than one course received a dose increase varying from 10 to 55%. The majority of patients reached the defined target levels by a dose increase during course two. Relevant grade 2 neurotoxicity was observed in eight (10%) patients and reversible ototoxicity grade 2 in 14 (18%) patients. The strategy of adaptive intrapatient dose adjustment of cisplatin is practically feasible in a research setting even when results for dose adaptation have to be reported within a short time-period of 1 week. The toxicity appeared to be manageable in this cohort of patients. In some patients, exposure after the standard dose was substantially lower than the defined target level and significant dose escalations of more than 50% had to be applied. The response rate (RR) was relatively high: overall 40% (29 out of 72 patients) partial remission (PR), in patients with stage IIIB the RR was 60% (15 out of 25 patients) and with stage IV 30% (14 out of 47 patients). Randomised studies are needed to determine whether the adaptive dosing strategy results in better efficacy than standard dosing.