Trial Information

Idarubicin Overcomes Multidrug Resistant-1(MDR1) Induced Chemoresistance With Higher Induction Remission Rate and Remission Quality Than Daunorubicin in de Novo Acute Myeloid Leukemia Patients

The long-term survival rate of acute myeloid leukemia (AML) has been significantly prolonged
due to the improvement of chemotherapeutic protocols and hemopoietic stem cell
transplantation. However, about 20%-40% of the patients with refractory AML fail to achieve
complete remission (CR) and approximately 50% of the CR patients will relapse eventually.
1-3 The refractoried and relapsed AML patients have very poor prognosis, therefore
refractoriness and relapse are the major challenges for the chemotherapy of AML.

Chemoresistance is one of the major obstacle for the success of AML treatment.4 One of the
main mediators for AML chemoresistance is the multi-drug resistance-1(MDR1) gene and MDR1
protein, also known as permeability-glycoprotein or P-gp.5,6,7 Studies on leukemic blast
cells demonstrated the association of MDR1 expression with a lower intracellular retention
of cytostatic drugs leading to cancer cell resistance to several commonly used anti-cancer
drugs in chemotherapy. Several subsequent studies indicated the influence of MDR1 expression
levels in the treatment response and survival of leukemia patients, the frequency of
resistance to the chemotherapy increased with increasing MDR1 expression.8,9,10 Previous
methods for determination of MDR1 expression have included demonstration of P-glycoprotein
by flow cytometry and/or immunohistochemistry, and molecular polymerase chain reaction
(PCR)-based assays for RNA expression. However, these assays have either proven difficult to
standardize or tedious to perform and they are mainly focused on qualitative or
semi-quantitative assays. 11,12 A real-time fluorescence quantitative PCR will increase the
sensitivity and also maintain the high specificity. The false positivity caused by pollution
of amplification production can also be decreased by hermetization operation. RQ- PCR is a
novel methodology which enables sensitive and quantitative measurement of gene expression.
12We have established RQ-PCR method for determination expression of MDR1 gene. We have
performed a pilot study with a small cohort of AML patients to determine the relationship of
MDR1 expression and CR rate of AML patients. The data shows MDR1 expression has been found
in most cases of AML. However, the MDR1 expression levels vary widely from case to case.
This result indicated that RQ-PCR method would provide an accurate quantitative measurement
for detection of MDR1 expression level in different AML cases who were both positive by
qualitative RT-PCR method. 13,14 The pilot results indicated that the complete response rate
to induction therapy was compromised by the expression of high levels of MDR1. 15

Minimal residual disease (MRD) is the major cause of leukemia relapse. It has been reported
that the treatment intensity in human leukemia can be assessed by the expression status of
MRD gene. However, more than 50% of acute leukemia, especially in AMLs, lack known genetic
lesions or clonegenic markers suitable for MRD monitoring. WT1 gene is located on chromosome
11p13 coding for a zinc-finger transcription factor, has been identified in most AML
patients. It has been reported that detection of WT1 levels can effectively monitor the MRD
expression status in AML.16,17 Therefore, WT1 expression can be used to monitor the MRD and
evaluation the remission quality in AML patients .By deteccting WT1 levels we can evaluate
the efficacy of different induction therapies.

By employing multiple fluorescence quantitative PCR, several target genes can be
simultaneously and quantitatively amplified. Therefore multiple fluorescence quantitative
PCR is more suitable for clinical application. This technology will not only provide higher
accuracy, specificity and reproducibility but also simplify procedure and reduce measurement
cost. The recombinant plasmids of MDR1 and WT1 gene have been constructed and the
methodology of multiple fluorescence quantitative PCR has also been developed in our group
.14

High MDR1 gene expression is a common feature of AML resistance to conventional
chemotherapeutic regimen.18,19 One of the strategies that overcome MDR is to increase the
pharmacal liposolubility and elevate the intracelluar drug concentration, so as to
counteract drug efflux pumped by P-glycoprotein. Idarubicin (IDA), a new anthracycline, has
more strengthened liposolubility and can easily permeate the cell membrane in comparison
with traditional marcellomycin. Also, the in vivo metabolite of IDA (4-IDA) has the same
antineoplasmic activity as IDA and a longer half-life in vivo. It can also permeate blood
brain barrier. As a result, IDA has a stronger antileukemia activity than other
marcellomycin and can partially overcome P-glycoprotein-mediated drug resistance.20, 21 In
our recently pilot study, we have detected the expression of MDR1 in pre-treated bone
marrow samples from 160 de novo AML patients and the median value of MDR1/GAPDH ratio in the
de novo AML cases was 0.016（0-81.865）. The median value of MDR1 gene expression was taken as
the cut-off point of high or low MDR1 gene expression levels. For high MDR1 expression
patients, a lower CR rate (61.3%) was observed compared with low-MDR1 expression patients
(80.0%, P =0.009). In this retrospective pilot study, 123 patients were received
daunorubicin (DNR 35~45mg/m2/d,3 days) or idarubicin (IDA 6~8 mg/m2/d, 3 days) in
combination with cytarabine (Ara-c100~200 mg/m2/d, 7 days) for remission induction and
significant difference were also observed after 2 courses of chemotherapy between those who
receive IA regimen (57 cases) and DA regimen (66 cases) regarding to CR rate (80.7% vs
57.6%, P=0.006). Significant difference could also be discovered between two treatment arm
with regard to CR rate (75.9% vs 43.6%, P=0.011) in those who had high MDR1 expression AML
patients. Our data shows that IDA may overcome MDR1 induced chemoresistance with a higher CR
rate than DNR in de novo high MDR1 expresser AML patients.These results provide rationale
for development of randomized, prospective trial to compare the CR rate of IA regimen and DA
regimen in de novo AML patients with high MDR1 expression and to identify whether Idarubicin
can overcomes MDR1 induced chemoresistance with higher induction remission rate than
daunorubicin in de novo acute myeloid leukemia patients. We speculated that the more
favorable outcome in high-MDR1 patient group with IA regimen may be associated with a higher
remission rate.

It had been reported that AML patients achieved lower MRD level had an extremely lower
relapse rate. It had also been reported that patients treated with IA regimen had a
significant higher long-term survival than those with DA regimen .22-24 Therefore, we
speculated that the more favorable outcome in IA group may be associated with a lower MRD
level and higher remission quality. This study is also to conduct a prospective analysis on
the alteration of WT1 and MDR1 gene expression level after induction therapy with IA or DA
separately to identify whether induction therapy with IA regimen has a lower MRD level and a
higher remission quality than that of DA regimen.