Background

Primary chemotherapy for patients with AML leads to high complete response rates in younger patients with significantly lower rates in older patients as well as those with leukemia secondary to antecedent myelodysplastic syndromes (MDS) or previous chemoradiotherapy. For patients who achieve complete remission (CR), response durability is also significantly lower in older patients or those with a clinically apparent secondary onset (1-7). AML features in older patients and those with previous MDS include an increased incidence of cytogenetic abnormalities associated with a poor prognosis, surface phenotypes suggestive of a more primitive etiology, and increased incidence of inherent biologic resistance. Of the cellular mechanisms of drug resistance described for patients with acute leukemia, the best characterized resistance profile is the phe-notype of multidrug resistance (MDR) mediated by Pgp. Pgp expression consistently emerges as a clinically significant marker of resistance in patients with AML (8-15), although biologic resistance can be attributed to several different mechanisms. Although the role of Pgp as a marker for resistance in adult ALL has not been as rigorously evaluated, there is increasing evidence that Pgp resistance plays an important role in clinical outcome (16-18).

Pgp confers cross-resistance to a variety of mechanistically and structurally unrelated cytotoxic drugs, such as anthracyclines, taxanes, Vinca alkaloids, and epipodophyllotoxins (8,9,19). All anthracyclines are subject to Pgp-mediated resistance, despite evidence that idarubicin has greater cellular retention and is less susceptible to Pgp-mediated efflux (20,21). Pgp is a member of the ABC gene superfamily, which is conserved throughout species evolution. Common to ABC transporters is a heterodimeric transmembrane glycoprotein complex, each with six transmembrane domains and an ATP binding site with conserved sequences known as the Walker A and Walker B domains (19,22). ABC transporters are involved in a range of transport functions from the epithelial transporter mutated in cystic fibrosis (CTFR), the canalicular multispecific organic anion (cMOAT) and canalicular bile acid (cBAT) transporters of liver, mono-

cyte secretion of IL-ip (ABC1), antigen processing in T-cells (TAP), cholesterol and phospholipid efflux (ABCA1), and non-Pgp-mediated anthracycline transport (BCRP), also known as the mitoxantrone resistance gene (MXR), ABCP, and ABCG2. Current ABC nomenclature has been reviewed (19,22,23) and is available on the Web (24). The phylogenetic conservation of ABC transporters suggests a broad role in protection from naturally occurring toxic compounds (xenobiotics). In mammalian species, the systemic elimination of xenobiotics by the liver and kidney, the integrity of the blood-brain barrier, the isolation of germ cells from the systemic circulation, fetal isolation from maternal circulation, and protection of the stem cell compartment are all mediated in part by ABC transporters.

To standardize assays, consensus recommendations have outlined criteria for determining Pgp in clinical samples (25), encouraging both immunophenotypic assessment as well as functional assays. Anthracyclines and, alternatively, fluorescent dyes such as rhodamine 123 (Rh123), Di(OC)2, or calcein are used to determine functional efflux (11,12,26), calculated as the decrease in cellular fluorescence compared with baseline assessment. Efflux measurements and pheno-typic determinations on different cell populations can be compared using the Kolmogorov-Smirnov (KS) statistic. Expression profiling for resistance genes is an intriguing research tool (27) that may provide future clinical use.

Multidrug-resistance associated protein (MRP) is also a member of the ABC superfamily and is capable of efflux and intracellular sequestration in conjunction with glutathione conjugation or cotransport (26,28). MRP describes a group of transporters, of which MRP1 is the only member implicated in drug transport. The substrate specificity of MRP1 is similar but more limited than Pgp, and its normal physiologic role may be detoxification of intracellular oxi-dants. It has been suggested that the location of MRP1 genes on chromosome 16 may contribute to the favorable prognosis found in patients with AML with inv(16) abnormalities. MRP1 is typically assessed using flow cytometry. Functional assays using fluorescent dyes in the presence or absence of reversers, such as cyclosporine and probenecid (26), or after glutathione depletion (11), can specifically assess MRP-mediated efflux.

Lung-resistance protein was initially identified in a lung cancer cell line during in vitro selection for drug resistance (29-32). It has significant homology with rodent vault proteins, which are subcellular organelles likely involved in nuclear-cytoplasmic transport. Enforced expression of LRP in transfection experiments is not sufficient to confer resistance, suggesting that other cofac-tors or posttranslational assembly is necessary for biologic function (29). Of interest, LRP expression was increased in patients with relapsed AML after response to induction therapy that included cyclosporine to overcome Pgp resistance (32), suggesting that modulating Pgp-mediated resistance may result in selection or upregulation of LRP as a secondary resistance mechanism after

Pgp reversal. LRP evaluation can be assessed with flow cytometry, immunocy-tochemistry, and reverse transcriptase-polymerase chain reaction (RT-PCR), although given the subcellular cytoplasmic localization, permeabilizing agents must be used with conventional flow cytometric techniques. Furthermore, post-translational regulation can make immunologic detection inconsistent. These techniques have been compared on clinical samples and cell lines, and the relative advantages and highlights of each have been described (33).

The BCRP/MXR/AB CP/ABCG2 transporter was initially described in breast cancer cell lines and represents a "half-transporter," consisting of six transmembrane domains and an ATP binding site. It is believed that the functional complex involves formation of a homodimer or heterodimeric partnering with another subunit (19,34,35). BCRP is characterized by high affinity for mitoxantrone and topoisomerase I inhibitors, as well as a resistance spectrum that includes other anthracyclines but unlike Pgp does not include Vinca alkaloids or taxanes (19,36). BCRP has been assessed using RT-PCR, monoclonal antibodies, and selective efflux inhibitors, although optimal evaluation of clinical samples has not been determined.

With the availability of many small molecules capable of reversing Pgp, several clinical trials have been designed and executed to overcome this specific resistance. Despite initial promise from encouraging preclinical data, many of these trials have been unsuccessful, attributable to toxicities arising from the pharmacokinetic effects of Pgp modulation on the cytotoxic anticancer drugs.

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