Methods to detect P-glycoprotein-associated multidrug resistance in patients' tumors: consensus recommendations.

Multidrug resistance (MDR), especially that associated with overexpression of MDR1 and its product, P-glycoprotein (Pgp), is thought to play a role in the outcome of therapy for some human tumors; however, a consensus conclusion has been difficult to reach, owing to the variable results published by different laboratories. Many factors appear to influence the detection of Pgp in clinical specimens, including its low and heterogeneous expression; conflicting definitions of detection end points; differences in methods of sample preparation, fixation, and analysis; use of immunological reagents with variable Pgp specificity and avidity and with different recognition epitopes; use of secondary reagents and chromogens; and differences in clinical end points. Also, mechanisms other than Pgp overexpression may contribute to clinical MDR. The combined effect of these factors is clearly important, especially among tumors with low expression of Pgp. Thus, a workshop was organized in Memphis, Tennessee, to promote the standardization of approaches to MDR1 and Pgp detection in clinical specimens. The 15 North American and European institutions that agreed to participate conducted three preworkshop trials with well-characterized MDR myeloma and carcinoma cell lines that expressed increasing amounts of Pgp. The intent was to establish standard materials and methods for a fourth trial, assays of Pgp and MDR1 in clinical specimens. The general conclusions emerging from these efforts led to a number of recommendations for future studies: (a) although detection of Pgp and MDR1 is at present likely to be more reliable in leukemias and lymphomas than in solid tumors, accurate measurement of low levels of Pgp expression under most conditions remains an elusive goal; (b) tissue-specific controls, antibody controls, and standardized MDR cell lines are essential for calibrating any detection method and for subsequent analyses of clinical samples; (c) use of two or more vendor-standardized anti-Pgp antibody reagents that recognize different epitopes improves the reliability of immunological detection of Pgp; (d) sample fixation and antigen preservation must be carefully controlled; (e) multiparameter analysis is useful in clinical assays of MDR1/Pgp expression; (f) immunostaining data are best reported as staining intensity and the percentage of positive cells; and (g) arbitrary minimal cutoff points for analysis compromise the reliability of conclusions. The recommendations made by workshop participants should enhance the quality of research on the role of Pgp in clinical MDR development and provide a paradigm for investigations of other drug resistance-associated proteins.

Phase I trial of doxorubicin with cyclosporine as a modulator of multidrug resistance.

PURPOSE: To study the effects of cyclosporine (CsA), a modulator of multidrug resistance (MDR), on the pharmacokinetics and toxicities of doxorubicin. PATIENTS AND METHODS: Nineteen patients with incurable malignancies entered this phase I trial. Initially patients received doxorubicin alone (60 or 75 mg/m2) as a 48-hour continuous intravenous (i.v.) infusion. Patients whose tumors did not respond received CsA as a 2-hour loading dose of 6 mg/kg and a 48-hour continuous infusion of 18 mg/kg/d with doxorubicin. Target CsA levels were 3,000 to 4,800 ng/mL (2.5 to 4.0 mumol/L). Doxorubicin doses were reduced to 40% of the prior dose without CsA, and then escalated until myelosuppression equivalent to that resulting from doxorubicin alone was observed. Doxorubicin pharmacokinetics were analyzed with and without CsA. RESULTS: Thirteen patients received both doxorubicin alone and the combination of doxorubicin and CsA. Mean CsA levels were more than 2,000 ng/mL for all cycles and more than 3,000 ng/mL for 68% of cycles. Dose escalation of doxorubicin with CsA was stopped at 60% of the doxorubicin alone dose, as four of five patients at this dose level had WBC nadirs equivalent to those seen with doxorubicin alone. Nonhematologic toxicities were mild. Reversible hyperbilirubinemia occurred in 68% of doxorubicin/CsA courses. The addition of CsA to doxorubicin increased grade 1 and 2 nausea (87% v 47%) and vomiting (50% v 10%) compared with doxorubicin alone. There was no significant nephrotoxicity. Paired pharmacokinetics were studied in 12 patients. The addition of CsA increased the dose-adjusted area under the curve (AUC) of doxorubicin by 55%, and of its metabolite doxorubicinol by 350%. CONCLUSION: CsA inhibits the clearance of both doxorubicin and doxorubicinol. Equivalent myelosuppression was observed when the dose of doxorubicin with CsA was 60% of the dose of doxorubicin without CsA. Understanding these pharmacokinetic interactions is essential for the design and interpretation of clinical trials of MDR modulation, and should be studied with more potent MDR modulators.

Mdr1 gene expression in childhood acute lymphoblastic leukemias and lymphomas: a critical evaluation by four techniques.

Expression of the multidrug resistance gene mdr1 is reported to be an important determinant of responsiveness to therapy and survival in some cancers. Many different methods have been used to evaluate mdr1 expression in these studies. This paper compares four methods for determination of mdr1 expression. We studied the mdr1 gene expression in 36 freshly established cell lines from 28 children with acute lymphoblastic leukemia (16 T-ALL, six BCP-ALL, two B-ALL (L3), two biphenotypic leukemias, two Burkitt's lymphomas). Leukemic specimens were obtained at the time of diagnosis in 16 cases, and after chemotherapy in 20 cases. In all the samples, mdr1 mRNA was measured by slot blotting and reverse transcriptase polymerase chain reaction (rt-PCR), and the presence of the mdr1 product, P-glycoprotein, was detected by immunohistochemistry with the MRK-16 monoclonal antibody. In situ mdr1 RNA hybridization was performed in 30 cases. Complete agreement was noted between all the techniques in 14 cases (39%). Results differed on a single test result in another 39% of the cases. These 78% of cases were considered assessable, and the consensus result was presumed to be correct. By this consensus criterion, immunohistochemistry yields both false negative (11%), and false positive (11%) results. RNA slot blotting has a high (21%) false positive rate. In situ mRNA hybridization and rt-PCR have the highest concordance, 80%. The 28 patients from whom these cell lines were derived appear to represent a very poor prognosis group, since there are only two patients (with Burkitt's lymphoma) who are long-term survivors. Nonetheless, a complete clinical response to therapy was correlated with absence of mdr1 expression in assessable cases (p = 0.04). These four methods of determining mdr1 expression often yield discordant results. Therefore, the use of at least two methods for evaluating mdr1 expression is advisable. Rt-PCR is recommended because of its relative simplicity and specificity. This should be supplemented by a technique (immunohistochemistry or flow cytometry) able to detect heterogeneity of P-glycoprotein expression among cells.

Clinical trials of modulation of multidrug resistance. Pharmacokinetic and pharmacodynamic considerations.

A growing body of evidence indicates that expression of the mdr1 gene, which encodes the multidrug transporter, P-glycoprotein, contributes to chemotherapeutic resistance of human cancers. Expression of this protein in normal tissues such as the biliary tract, intestines, and renal tubules suggests a role in the excretion of toxins. Modulation of P-glycoprotein function in normal tissues may lead to decreased excretion of drugs and enhanced toxicities. A clinical trial of etoposide with escalating doses of cyclosporine (CsA) as a modulator of multidrug resistance was performed. CsA was delivered as a 2-hour loading dose followed by a 60-hour intravenous infusion, together with etoposide administered as a short infusion daily for 3 days. Patients received one or more courses of etoposide alone before the combined therapy to establish their clinical resistance to etoposide and to study etoposide pharmacokinetics without and then with CsA. Plasma and urinary etoposide was measured by high-performance liquid chromatography and plasma CsA by a nonspecific immunoassay. Conclusions from the initial phase I trial with the use of CsA as a modulator of etoposide are: (1) Serum CsA steady-state levels of up to 4800 ng/ml (4 microM) could be achieved with acceptable toxicity. (2) Toxicities caused by the combined treatment included increased nausea and vomiting, increased myelosuppression, and hyperbilirubinemia, consistent with modulation of P-glycoprotein function in the blood-brain barrier, hematopoietic stem cell, and biliary tract. Renal toxicity was uncommon, but severe in two patients with steady-state plasma CsA levels above 6000 ng/ml. (3) CsA administration had a marked effect on the pharmacokinetics of etoposide, with a doubling of the area under the concentration-time curve as a result of both decreased renal and nonrenal clearance, necessitating a 50% dose reduction in patients with normal renal function and hepatic function. (4) The recommended dose of CsA is a 6-7 mg/kg loading dose administered as a 2-hour intravenous infusion followed by a continuous infusion of 18-21 mg/kg/day for 60 hours with adjustments in the infusion rate to maintain steady-state serum levels of 3000-4800 ng/ml (2.5-4.0 M). We are performing additional phase I trials combining CsA with single-agent doxorubicin and taxol, and the CsA analog PSC-833 with various multidrug-resistant-related cytotoxins.

Phase I trial of etoposide with cyclosporine as a modulator of multidrug resistance.

PURPOSE: To determine the maximum-tolerated dose (MTD) of cyclosporine (CsA) infusion administered with etoposide for 3 days in patients with cancer. PATIENTS AND METHODS: Of the 72 registered patients, 26 were treated initially with CsA and etoposide. Forty-six received etoposide alone until disease progression, and 31 of these proceeded to CsA and etoposide. CsA was administered as a 2-hour loading dose (LD) and as a 3-day continuous infusion (CI); doses were escalated from 2 to 8 mg/kg LD and 5 to 24 mg/kg/d CI. RESULTS: Fifty-seven patients were treated with 113 cycles of CsA with etoposide. Steady-state serum CsA levels (nonspecific immunoassay) more than 2,000 ng/mL were achieved in 91% of the cycles at CsA doses > or = 5 mg/kg LD and > or = 15 mg/kg/d CI. The major dose-related toxicity of CsA was reversible hyperbilirubinemia, which occurred in 78% of the courses with CsA levels > 2,000 ng/mL. Myelosuppression and nausea were more severe with CsA and etoposide. Other CsA toxicities included hypomagnesemia, 60%; hypertension, 29%; and headache, 21%. Nephrotoxicity was mild in 12% and severe in 2% of the cycles. Tumor regressions occurred in four patients after the addition of CsA (one non-Hodgkin's lymphoma, one Hodgkin's disease, and two ovarian carcinomas). Biopsy procedures for tumors from three of the four patients who responded were performed, and the results were positive for mdr1 expression. CONCLUSIONS: Serum CsA levels of up to 4 mumol/L (4,800 ng/mL) are achievable during a short-term administration with acceptable toxicities when administered in combination with etoposide. The CsA dose that is recommended in adults is a LD of 5 to 6 mg/kg, followed by a CI of 15 to 18 mg/kg/d for 60 hours. CsA blood levels should be monitored and the doses should be adjusted to achieve CsA levels of 2.5 to 4 mumol/L (3,000 to 4,800 ng/mL). Reversible hyperbilirubinemia may be a useful marker of inhibition by CsA of P-glycoprotein function. When used with high-dose CsA, etoposide doses should be reduced by approximately 50% to compensate for the pharmacokinetic effects of CsA on etoposide (Lum et al, J Clin Oncol, 10:1635-1642, 1992).

Alteration of etoposide pharmacokinetics and pharmacodynamics by cyclosporine in a phase I trial to modulate multidrug resistance.

PURPOSE: To determine the effects of high-dose cyclosporine (CsA) infusion on the pharmacokinetics of etoposide in patients with cancer. PATIENTS AND METHODS: Sixteen patients were administered 20 paired courses of etoposide and CsA/etoposide. Etoposide was administered daily for three days, alone or with CsA, which was delivered by a loading dose and 3-day infusion. Etoposide was measured by high-performance liquid chromatography (HPLC) and serum CsA by nonspecific immunoassay. Etoposide pharmacokinetics included area under the concentration-time curve (AUC), total and renal clearance (CL), half-life (T1/2), and volume of distribution at steady state (Vss). RESULTS: CsA concentrations more than 2,000 ng/mL produced an increase in etoposide AUC of 80% (P less than .001), a 38% decrease in total CL (P < .01), a > twofold increase in T1/2 (P < .01), and a 46% larger Vss (P = .01) compared with etoposide alone. CsA levels ranged from 297 to 5,073 ng/mL. Higher CsA levels (< 2,000 ng/mL v > 2,000 ng/mL) resulted in greater changes in etoposide kinetics: Vss (1.4% v 46%) and T1/2 (40% v 108%). CsA produced a 38% decrease in renal and a 52% decrease in nonrenal CL of etoposide. Etoposide with CsA levels > 2,000 ng/mL produced a lower WBC count nadir (900/mm3 v 1,600/mm3) compared with baseline etoposide cycles. CONCLUSIONS: High-dose CsA produces significant increases in etoposide systemic exposure and leukopenia. These pharmacokinetic changes are consistent with inhibition by CsA of the multidrug transporter P-glycoprotein in normal tissues. Etoposide doses should be reduced by 50% when used with high-dose CsA in patients with normal renal and liver function. Alterations in the disposition of other multidrug resistance (MDR)-related drugs should be expected to occur with modulation of P-glycoprotein function in clinical trials.

Expression of multidrug resistance gene mdr1 mRNA in a subset of normal bone marrow cells.

The multidrug resistance gene mdr1, encoding P-glycoprotein (P-gp), can be expressed at high levels in tumour cells derived from normal tissues with constitutive high expression of this gene. In myelogenous leukaemia, the incidence of increased expression of mdr1 gene contrasts with the low expression of this gene in normal bone marrow (b.m.). To detect cells expressing mdr1 gene in normal and post-chemotherapy b.m., we used in situ RNA hybridization and RNA phenotyping by the polymerase chain reaction for mdr1 mRNA detection. The presence of P-gp was evaluated by immunocytochemistry with MRK16. Fifteen b.m. (eight normal and seven post chemotherapy) were tested by in situ hybridization and either PCR (three b.m.) or immunocytochemistry (11 b.m.) or both (one b.m.). With in situ mRNA hybridization, a subset (7.7% +/- 3.1%) of b.m. cells expressed mdr1 mRNA in all cases tested, with no significant differences between normal b.m. and post chemotherapy b.m. 18% of myeloid recognizable cells and 7% of the cells with lymphoid morphology expressed mdr1 mRNA. By RNA phenotyping, the four samples tested for in situ hybridization and two additional post chemotherapy b.m. expressed mdr1. MRK16 was unable to detect a significant number of cells expressing P-gp either by immunocytochemistry in the 12 b.m. tested for in situ hybridization (0% in nine cases; 0.4%, 1% and 3% of positive cells in three cases), or by flow cytometry in six additional normal b.m. (0-1.4% positive cells).

Therapy of patients with malignant melanoma using a monoclonal antimelanoma antibody-ricin A chain immunotoxin.

We conducted a trial of a murine monoclonal antimelanoma antibody-ricin A chain immunotoxin (XOMAZYME-MEL) in 22 patients with metastatic malignant melanoma. The dose of immunotoxin administered ranged from 0.01 mg/kg daily for 5 days to 1 mg/kg daily for 4 days (total dose: 3.2 to 300 mg). Side effects observed in most patients were a transient fall in serum albumin with an associated fall in serum protein, weight gain, and fluid shifts resulting in edema. In addition, patients experienced mild to moderate malaise, fatigue, myalgia, decrease in appetite, and fevers. There was a transient decrease in voltage on electrocardiograms without clinical symptoms, change in serial echocardiograms or elevation of creatine phosphokinase MB isozyme levels. Symptoms consistent with mild allergic reactions were observed in three patients. The side effects were related to the dose of immunotoxin administered and were generally transient and reversible. Encouraging clinical results were observed, even after a single course of a low dose of immunotoxin. In addition, localization of antibody and A chain to sites of metastatic disease was demonstrated by immunoperoxidase staining of biopsy specimens. Additional studies are being conducted to continue the evaluation of safety and efficacy of immunotoxin therapy for malignancy.