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Keywords:

  • acute promyelocytic leukaemia;
  • multidrug resistance;
  • P-glycoprotein;
  • PGP;
  • LRP;
  • MRP1

Abstract

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

We analysed the expression of three drug transporter proteins [p-glycoprotein (PGP), lung resistance-related protein (LRP) and multidrug resistance-associated protein (MRP1)] involved in anthracycline resistance that are frequently overexpressed in poor-risk adult acute non-lymphocytic leukaemia (ANLL), in 23 acute promyelocytic leukaemia (APL) patients at onset managed at a single institution. Cellular daunorubicin accumulation was also evaluated. At onset, no case had PGP or MRP1 expression that exceeded that of non-multidrug-resistant (MDR) cell lines. Only one case showed LRP overexpression. No peculiar MDR features distinguished the seven patients who relapsed from those who maintained complete remission. In the onset vs. first relapse, only one patient showed an increased (threefold) PGP expression at relapse. At second relapse, three out of four patients showed a PGP expression two- to threefold higher than baseline values. These results are consistent with the view that low PGP, LRP and MRP1 expression and the absence of defects in intracellular drug accumulation may account for the peculiarly high sensitivity of APLs to anthracycline. It does not support the screening of MDR markers in APL patients at onset as predicting factors of early relapse. The results suggest that no significant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, PGP expression is likely to increase later in the patient history as a result of additional chemotherapy courses.

The molecular basis of multidrug resistance (MDR) has not yet been described. MDR provoked by the overexpression of P-glycoprotein (PGP) is the most well-known. PGP, also named P-170, is the mdr-1 gene product. It belongs to the ATP-binding cassette of the transmembrane transporter superfamily and acts by increasing the efflux of a number of drugs and xenobiotics including anthracyclines ( Tsuruo, 1988 ; Chin et al, 1989 ; Weinstein et al, 1990 ). The MDR-associated protein (MRP1) is structurally similar to PGP and belongs to the same transmembrane transporter superfamily. It has been shown that, similar to PGP, mrp-1 transfection results in MDR (including anthracyclines) in sensitive cell lines ( Kruth et al, 1994 ; Izquierdo et al, 1996 ). In addition to MRP1, at least four other homologous proteins (MRP2 to MRP5) have been described and ongoing studies continue to clarify their role ( Legrand et al, 1999 ). Lung resistance-related protein (LRP) was isolated in drug-selected cell lines lacking PGP overexpression, which showed MDR associated with a defect in cellular drug accumulation ( Kuiper et al, 1990 ; Scheper et al, 1993 ). LRP was shown to be a normal protein with a striking similarity to the rat major vault protein mVp-α. In humans, the lrp gene maps near the mrp1 gene on chromosome 16. PGP and LRP overexpression and a high ability in effluxing dyes or drugs have been identified as common features in acute non-lymphocytic leukaemia (ANLL), especially in cases that developed after chemo-radiotherapy or myelodysplastic syndrome, or in elderly patients ( Zhou et al, 1995 ; Filipitis et al, 1997 ; Michieli et al, 1997 ; Willman, 1997; Damiani et al, 1998 ; Legrand et al, 1999 ; Leith et al, 1999 ). The contribution of PGP, LRP and MRP1 overexpression to treatment failure in leukaemia is still a question of debate. However, in ANLL, PGP, LRP and MRP1 have increasingly been shown to be prognostic factors predicting poor chemotherapy outcome ( Zhou et al, 1995 ; List et al, 1996 ; Filipitis et al, 1997 ; Willman, 1997; Legrand et al, 1999 ; Leith et al, 1999 ; Michieli et al, 1999 ). Acute promyelocytic leukaemia (APL) is a biological and clinically well-defined subtype of ANLL with specific morphological and karyotypic characteristics. The pml/rarα product resulting from the (15–17) translocation constitutes a unique useful molecular marker of APL. From a clinical perspective, if disseminated intravascular coagulation is controlled, using protocols based on anthracyclines and all-trans retinoic acid (ATRA), a high proportion of APL patients achieve and maintain long-term complete remission (CR) ( Mandelli et al, 1997 ). The role played by anthracyclines in these leukaemias was underlined by Avvisati et al (1990) , who showed that APLs maintained a high CR rate even when treated with anthracyclines as a single therapeutic agent, while Head et al (1995) reported that higher daunorubicin (DNR) doses during induction were significantly associated with higher CR rates in APL.

We hypothesized that the high sensitivity of APL to anthracyclines may account for low expression of drug transporter proteins and for a lack of drug or dye accumulation in blasts. Therefore, the expression of PGP, LRP and MRP1 and the ability of blast cells to accumulate DNR were evaluated in 23 newly diagnosed APL patients. Protein expression was studied using MRK-16 (anti-PGP), LRP 56 (anti-LRP) and MRPm6 (anti-MRP1) monoclonal antibodies, while blast cell ability to accumulate drugs was evaluated by measuring the intracellular anthracycline content after a 2-h incubation of samples in 1000 ng/ml DNR. During the study period, seven patients relapsed. The onset vs. relapse expression of PGP, LRP and MRP1 and the relationship with the chemotherapy administered to the patients have also been described.

PATIENTS and METHODS

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Patients We studied 23 patients with newly diagnosed APL who were managed at a single institution, the Department of Haematology at the Udine University Hospital, between April 1989 and May 1999. Eight patients were males and 15 females. Their median age was 32 years (range 16–72 years). The APL diagnosis was based on French–American–British guidelines criteria ( Bennett et al, 1985 ) and confirmed with karyotypic and/or molecular evidence of the t(15–17) in leukaemic blast cells. The median white blood cell (WBC) count at diagnosis was 3·0 × 109/l with a range of 0·8–134 × 109/l. Only three patients had a peripheral WBC count ≥ 20 × 109/l. Induction and consolidation treatment was not homogeneous and varied according to the institution's ongoing first-line therapy guidelines. Therefore, for the first patient (case 1) enrolled in 1989, induction consisted of one course of arabinosyl cytosine (AC) 200 mg/m2/day (days 1–7) given as a continuous intravenous (i.v.) infusion and daunorubicin (60 mg/m2, i.v., days 1–3), and a second course of high-dose AC (2000 mg/m2, i.v. twice daily, days 1–3) and daunorubicin (60 mg/m2, i.v., days 4–6) (HD AC/DNR). For this patient, consolidation consisted of three courses of HD AC/DNR. Eleven patients were treated according to the GIMEMA LAP 0389 protocol ( Rotoli, 1992) and 11 according to the GIMEMA AIDA protocol ( Mandelli et al, 1997 ). All patients except two, who died during the induction at days +1 and +10, achieved CR after the first (19 cases) or the second (two cases) course of therapy. As an additional consolidation therapy, five out of 11 patients treated according to the GIMEMA LAP 0389 protocol underwent autologous bone marrow transplantation (ABMT) after 8–12 months of continuous CR. Among the six patients treated with the same protocol, one relapsed before stem cell harvest, two refused the procedure and three failed the bone marrow harvest. No case treated with the GIMEMA AIDA protocol underwent ABMT at first CR. Seven patients relapsed during the study period. One patient underwent ABMT in second CR (case 2), and one patient (case 5) underwent an allogeneic bone marrow transplant (BMT) in second CR. Median follow-up was 30 months (range 1–111 months).

Leukaemic blast cells Peripheral blood (three cases at onset and five at relapse) and marrow samples (20 at onset and nine at relapse) anticoagulated with heparin were collected during diagnostic procedures after receiving the patients' informed consent. Mononuclear cells were separated by sedimentation on Ficoll. Dual-colour immunfluorescence analysis of the surface antigens was performed to evaluate sample purity using a panel of monoclonal antibodies (mAbs) including anti-CD13PE, anti-CD33PE, anti-HLA-DRFITC, anti-CD34, anti-CD2 FITC and anti-CD3FITC (all from Becton Dickinson, Mountain View, CA, USA). Samples with more than 88% blast cells (range 88–96%) were cryopreserved at 5 × 106 cells/ml in RPMI 1640 (Biochrom KG, Belgium)/DMSO 10% (Carlo Erba, Milan, Italy) solution. Before the analysis, the samples were thawed and the cells suspended in RPMI 1640/FCS 20% (Biochrom KG) at 37°C in 5% CO2 for 12 h. Then, cells were checked for viability using the trypan blue exclusion test. Dead cells were removed by sedimentation on Ficoll. Before functional and immunophenotyping studies, blast cells washed in PBS were suspended in medium or in PBS at the required concentration. Blast cells were gathered using scatter parameter, and only viable blasts (propidium iodide-negative) were used for the studies. No samples had more than 5% CD3-positive cells on the blasts' gathered region. According to these criteria, seven samples (four bone marrow and one peripheral blood of patients at onset and two samples at relapse) were rejected. Thus, seven cases (five at onset and two at relapse) managed at our institute during the study period were not included in this work.

PGP, LRP and MRP1 expression PGP, LRP and MRP1 expression was evaluated by flow cytometry using the anti-PGP MRK-16, the anti-LRP LRP-56 and the anti-MRP1 MRPm6 mAbs (Kamiya) as described previously ( Michieli et al, 1997 ). To study PGP, 1 × 106 blast cells were fixed in periodate lysine paraformaldehyde (PLP), washed twice and incubated for 30 min in 50 μl of a PBS–saponine solution (0·02%) containing 2 μg/ml MRK-16. After two washes in PBS, cells were incubated with 2·5 μl of fluorescein isothiocyanate (FITC) rabbit anti-mouse antibody (Dako, Denmark). To study MRP1 and LRP, 1 × 106 blast cells were fixed and permeabilized in the Becton Dickinson lysing solution and, after two washes, incubated with 2·0 μl of MRPm6 or 10 μl of LRP-56 for 1 h at 4°C in a PBS–saponine solution, as recommended by the manufacturer. Staining was revealed using 2·5 μl of a FITC goat anti-mouse antibody (Dako). Control samples were carried out simultaneously by replacing the primary mAb with an IgG2a or IgG2b, as appropriate. Samples of cell lines that overexpress PGP (CEMVLB300 and HL60 DNR), LRP (SW1573/2R120) and MRP1 (GLC4ADR 150) and their negative parental counterparts (CCRF CEM, HL60, GLC4 and SW1573) were investigated simultaneously. Data acquisition and analysis were made using a FACScan and the Lysis II software program (Becton Dickinson). Results were expressed by the mean fluorescence index (MFI = the ratio between the mean fluorescence intensity of the cells incubated with MRK-16, LRP-56 and MRPm6 and the mean fluorescence intensity of the respective controls) rather than by the percentage of stained cells. As reported previously, leukaemic cells were defined as positive (= overexpressing) when the MFI exceeded the highest MFI of non-MDR cell lines (CCRF CEM, GLC4 and SW1573 respectively) and normal peripheral or marrow mononuclear cells. Therefore, APLs were classified as PGP, LRP or MRP1 overexpressing when the MFI was higher than 6 for MRK-16, 5 for LRP-56 and 3 for MRPm6 ( Michieli et al, 1997 ).

Cellular daunorubicin accumulation The amount of DNR that the blast cells were able to accumulate was evaluated by flow cytometry as described previously ( Michieli et al, 1996 ). Briefly, mononuclear cells separated on Ficoll-Hypaque were suspended at 2·5 × 106 cells/ml in medium (RPMI 1640; Biochem, KG Seromed) and incubated at 37°C with 5% CO2 for 2 h with 1000 ng/ml DNR (Pharmacia, Milan, Italy) in the presence or absence of the MDR reversal agent SDZPSC 833 (PSC,1·6 μm; Sandoz, Basel, Switzerland). After two washes in cold PBS, cells were kept on ice and immediately analysed for their FL2 and FSC, using a FACScan equipped with an argon laser tuned at 488 nm and the Lysis II software (Becton Dickinson). Results were expressed by the normalized mean fluorescence index (NMFI), calculated according to Luk & Tannock (1989). PSC activity in increasing DNR accumulation was expressed by the PSC index calculated by dividing the mean DNR NMFI value of cells incubated in the presence of anthracycline plus PSC for one of the samples treated in the presence of anthracycline but without PSC.

Statistical analysis Statistical analysis was carried out with the winstat software. Comparison between intracellular drug accumulation in the presence or absence of MDR reversal agents was estimated using the Wilcoxon matched pair test.

Results

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

PGP, LRP and MRP1 expression at onset

PGP, LRP and MRP1 expressions were evaluated on the blast cells of 23 de novo APL patients at onset and before any drug administration. MRK-16 (anti-PGP) stained a median of 36% cells (range 15 −77%). LRP- and MRP1-positive cells were detected in 15/23 (65%) and of 8/23 (35%) cases with a median of 29% and 15% positive cells respectively (range for the LRP-56 26–58%; range for MRPm6 9–37%). Figure 1 reports PGP, LRP and MRP1 expression evaluated as the mAbs MFI. Only the mAb MFI of those cases with positive cells is reported. No APL patients had PGP or MRP1 expression that exceeded those of parental non-PGP, -LRP or -MRP overexpressing cell lines used as negative controls, while only one case had LRP levels higher than the negative controls.

image

Figure 1. PGP, LRP and MRP1 expression in 23 APL patients evaluated at onset. All the 23 patients had some MRK-16 (anti-PGP)-positive cells with a median of 36% positive cells. Fifteen out of 23 (65%) and eight out of 23 (35%) cases had LRP- and MRP1-positive cells with a median of 29% and 15%. Data on the mean fluorescence intensity (MFI) of each case with PGP-, LRP- and MRP1-positive cells is shown and compared with MDR and non-MDR cell lines used as negative (parental non-MDR lines) and positive (PGP-, LRP- and MRP1-overexpressing sublines) controls. In leukaemic cells, the median MRK-16 MFI was 3·6, the median LRP-56 MFI was 2·4 and the median MRPm6 MFI was 1·3. No case had a PGP or MRP1 expression that exceeded the controls, while one case had an LRP expression higher than the controls.

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Cellular daunorubicin accumulation at onset

The amount of DNR that blast cells were able to accumulate after a 2-h incubation in 1000 ng/ml anthracycline was evaluated by flow cytometry and reported as the DNR-associated NMFI. The median NMFI was 402 (range 160–548) ( Fig 2). When compared with a previously tested group of 22 leukaemic patients lacking PGP, LRP and MRP1 overexpression ( Michieli et al, 1997 ), only one APL case showed a low cellular DNR accumulation (DNR NMFI = 160). This was the same patient whose LRP expression exceeded the negative controls. The Wilcoxon matched pair test showed no difference between DNR NMFI evaluated in the presence or absence of PSC (P = 0·86). The median PSC index (ratio between the mean DNR NMFI with and the mean DNR NMFI without PSC) was 1·019 (range 0·941–1·141).

image

Figure 2. Cellular daunorubicin (DNR) accumulation expressed as the DNR-associated normalized mean fluorescence index (NMFI) in 23 APL patients at onset. The median cellular DNR accumulation was 402 (range 160–548). Only one case had a markedly low NMFI value. This patient also showed a high LRP expression.

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PGP, LRP and MRP1 expression at relapse

The 23 APL patients were followed up for a median of 30 months (range 1–111 months). Table I summarizes the main features of the seven patients who relapsed during the study period. Table I reports PGP, LRP and MRP1 expression at diagnosis and relapse, the total dose of MDR-related (anthracyclines, mitoxantrone, etoposide) and -unrelated (arabinosyl cytosine, cyclophosphamide, busulphan, 6-thioguanine) drugs delivered as induction and consolidation therapy or administered in between the MDR evaluations, the duration of CR, present disease status and overall survival. Patients 3, 5, 6 and 7 received additional intermittent courses of ATRA throughout the treatment period; patients 4 and 2 received intermittent courses of ATRA only at the times of second and subsequent relapses. Patient 1 never received ATRA. There were no relevant differences in onset vs. first relapse MDR phenotype, except for patient 1 who showed a threefold increase in the baseline (i.e. expression at onset) PGP expression at first relapse. At the time of the second relapse (four cases), patient 1 maintained his MDR profile, patients 2 and 3 increased their PGP expression by a factor of 3·5 and 1·8, respectively, compared with their protein expression at onset. Patient 3 also showed a simultaneous 1·6-fold increase in LRP expression compared with baseline. Patient 4 was the only case who showed an LRP expression that exceeded the negative controls and a low cellular DNR accumulation at onset. This patient's MDR phenotype was stable throughout follow-up.

Discussion

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This study was undertaken to investigate the expression patterns of three proteins (PGP, LRP and MRP1) that confer in vitro multidrug resistance, including anthracyclines, in APL based on reports by Head et al (1995) and Avvisati et al (1990) , who showed the major role of DNR in affecting the outcome of APL. It included 23 adult APL cases at onset who were diagnosed, managed and followed up at a single institution. A second point was to evaluate onset vs. relapse MDR phenotype in paired samples and to compare possible increases in the proteins' expression with the amount of cytotoxic drugs delivered to the patients. Similar to 96 de novo ANLL cases evaluated at onset with FAB cytotypes other than M3 ( Michieli et al, 1999 ), the majority of the APL cases had low amounts of PGP and LRP. However, in APL, the evidence of PGP or MRP-1 expression exceeding the negative controls was a much rarer event. In this series, no APL case at onset had PGP levels higher than the negative controls, whereas LRP was elevated in only one out of 23 cases. Low amounts of MRP1 were found in less than one-third of our APL patients, and no case had an MRP1 expression that exceeded the negative controls. In accordance with these data, a reduced cellular DNR accumulation was only observed in the case showing LRP overexpression. This is a relevant point as it confirmed the immunophenotypic finding but suggests that, besides PGP, LRP and MRP1, other proteins involved in anthracycline transport (such as MRP2, 3, 4 or 5) are unlikely to be overexpressed and active in APL cases at onset. Concerning PGP expression in APL, the data are in line with the work of several authors who used different reagents and techniques for PGP evaluation ( Paietta et al, 1994; Drach et al, 1995 ; Guerci et al, 1995 ; Del Poeta et al, 1997 ). As in our work, Paietta et al (1994) showed a low PGP activity in 11 APL cases. Up to now, only sporadic data have been available on LRP expression in APL. Filipitis et al (1998) reported one out of nine LRP-overexpressing cases, while List et al (1996) did not find any APL-positive cases in four samples analysed. Concerning ANLL with a FAB cytotype other than M3, several authors have reported a higher PGP and LRP overexpression compared with APLs ( Zhou et al, 1995 ; Filipitis et al, 1997 ; Leith et al, 1997 , 1999; Michieli et al, 1997 ; Willman, 1997; Damiani et al, 1998 ; Legrand et al, 1999 ). Few, and sometimes conflicting, data are available on the relationship between PGP, LRP or MRP expression and cytogenetics in ANLL other than M3 ( Del Poeta et al, 1996 , 1997; List et al, 1996 ; Leith et al, 1997, 1999; Michieli et al, 1999 ). A marginal non-significant association between poor-prognosis cytogenetics and PGP overexpression has been reported ( Del Poeta et al, 1996 , 1997; Leith et al, 1997 , 1999), as well as a trend towards a lower frequency of high PGP and LRP expression in patients with a good-prognosis karyotype ( List et al, 1996 ; Michieli et al, 1999 ). At present, no data are available on MDR phenotype in M3 cases at relapse. By comparing the immunophenotypic MDR features of the seven patients who ultimately relapsed vs. those of the patients who achieved and are maintaining CR, we failed to identify any particular functional or immunophenotypical feature useful in distinguishing high- from low-risk relapsing patients. The case that initially showed LRP overexpression associated with low cellular drug accumulation and relapsed at 8 months into CR was only a sporadic finding. Serial analysis undertaken in the seven patients who ultimately relapsed revealed overexpression of PGP in one out of seven at first relapse; however, the acquisition of an MDR phenotype appeared to become increasingly frequent with subsequent relapses (three out of four cases overexpressed PGP at the second relapse). Notably, the only case that substantially increased the baseline PGP expression at first relapse was the only one who received DNR instead of idarubicin or mitoxantrone as first-line therapy. This observation, although occasional and requiring confirmation, suggests that, compared with idarubicin or mitoxantrone, DNR, the toxicity and transport of which has the strongest relationship with PGP action, may also have the highest activity in stimulating PGP expression ( Berman & McBride, 1992 ; Michieli et al, 1993 ). In addition, it must be stressed that the patient who showed an increased PGP expression at first relapse received the highest dose of arabinosyl cytosine (AC), which has been shown to have a high activity in inducing PGP expression ( Grimaz et al, 1998 ). The suggestion that promyelocytic blasts may require multiple courses of drug exposure to become PGP overexpressing was also supported by Su et al (1994) and Chaudhary & Roninson (1993) who carried out experiments on HL60 cell lines. In fact, whereas Su et al (1994) showed that PGP-overexpressing sublines could be obtained after multiple 18-h drug pulsing, Chaudhary & Roninson (1993) failed to obtain PGP-overexpressing HL60 sublines after a single transient high-dose drug exposure. As for PGP, no changes in onset vs. first relapse LRP expression were found. MRP1 expression did not vary significantly during follow-up of the seven relapsing patients. In the literature, the onset vs. relapse data are still limited and sometimes conflicting even in ANLL without a restriction according to the FAB cytotype. Based on non-paired samples, a number of authors have concluded that PGP, LRP and MRP1 expression was lower at onset than in relapsed ANLL patients ( Schneider et al, 1995 ; Hart et al, 1997 ; Nuessler et al, 1997 ; Legrand et al, 1999 ). In paired samples, Hart et al (1997) showed no significant difference in PGP, LRP and MRP1 expression in eight ANLL cases, while Schneider et al (1995) showed an increase in MRP1 expression at relapse in more than 80% of 17 acute leukaemic samples evaluated.

In conclusion, this work is consistent with the view that low PGP, LRP and MRP1 expression and a lack of defects in cell drug accumulation may account for the high sensitivity of APL to anthracyclines. It suggests that the analysis of the MDR phenotype at onset is unlikely to help in screening high- from low-risk APL relapsing patients. Because of the small series, data on PGP, LRP and MRP1 expression in paired onset vs. relapse samples are preliminary and require confirmation. Our data suggest that no relevant changes in PGP, LRP or MRP1 expression are likely to occur at first relapse. In contrast, relevant increases in baseline PGP expression are likely to develop later in the patient's history as a consequence of the delivery of additional chemotherapy courses.

Acknowledgments

  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by AIRC Milano and by Murst 1998 Cofinanziamento Programmi di Ricerca ‘Biological and molecular bases of therapy of ANLL’.

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  1. Top of page
  2. Abstract
  3. PATIENTS and METHODS
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
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