Endocrine-Related Cancer (2003) 10 43–73 REVIEW ATP binding cassette transporters and drug resistance in breast cancer

F Leonessa and R Clarke1

Laboratory of Clinical Investigation,National Institute on Aging,National Institutes of Health,5600 Nathan Shock Drive,Baltimore,Maryland 21224,USA 1Departments of Oncology,Physiology and Biophysics,and Lombardi Cancer Center,Georgetown University School of Medicine,3970 Reservoir Road NW,Washington DC 20007,USA (Requests for offprints should be addressed to R Clarke, Department of Oncology, room W405A, Research Building, 3970 Reservoir Road NW, Washington DC 20007, USA; Email: [email protected])

Abstract Resistance to chemotherapy is a critical issue in the management of breast cancer patients. The nature of clinical drug resistance is likely to be multifactorial. However,in the last decade considerable attention has been dedicated to the role played by membrane transporter proteins belonging to the ATP binding cassette protein superfamily,and in particular by the MDR1 product P-glycoprotein (Pgp) and the multidrug resistance protein (MRP1). Heterogeneity of results is a common feature of studies evaluating the expression and prognostic role of these proteins,due to both methodological and biological factors. Nonetheless,Pgp and MRP1 are detected in a significant proportion of untreated breast cancers (on average 40 and 50% respectively,by immunohistochemistry),without a clear and consistent association with cancer stage. Exposure to chemotherapy increases the expression of both proteins. In vitro studies on primary cultures of breast cancer cells obtained at surgery consistently show an association between Pgp (protein) or MDR1 (mRNA) expression and resistance to chemotherapy. However,the correlation with clinical drug resistance is not as well defined. A stronger association of Pgp/MDR1 with response rates has been observed when expression or an increase in expression are detected immediately following chemotherapy. Correlations with prognosis appear more evident in studies using immunohistochemistry,in adjuvant and neoadjuvant settings. Evidence of clinical reversal of drug resistance by verapamil suggests a functional role of Pgp in drug resistance,although the significance of the evidence is generally weakened by poor trial designs. Future studies should take into account the multifactorial nature of drug resistance in breast cancer and use standardized approaches with adequate controls. Expression studies should be complemented by well-designed trials of drug-resistance reversal using target-specific chemosensitizing agents,and relating the results to the levels of expression of the target proteins. Endocrine-Related Cancer (2003) 10 43–73

shows response rates to first-line chemotherapy of 25–55% Introduction (Winer et al. 2001). Chemotherapy plays an important role in the management Taxanes and anthracyclines represent the most active of breast cancer. Adjuvant chemotherapy, in the early breast agents in metastatic breast cancer, with response rates to cancer setting, decreases the annual odds of recurrence by single-agent treatment of 35–50% (Ellis et al. 2000) and 31% 24% and death by 15% (Early Breast Cancer Trialists’ Col- (Bishop et al. 1997, 1999) respectively, in previously laborative Group 1998). In locally advanced breast cancer untreated patients. The antimetabolites 5-fluorouracil and (Bonadonna et al. 1991, Sapunar & Smith 2000), neoadju- methotrexate and the alkylating agent cyclophosphamide vant chemotherapy administered before surgery can reduce have been classically used for breast cancer treatment. Active breast tumor size in 80–90% of breast cancer patients (Kling agents also include vinca alkaloids, anthraquinones et al. 1997), with pathological complete response (CR) rates (mitoxantrone) and epipodophyllotoxins (etoposide) (Ellis et that approach 20% (Moll & Chumas 1997, Sapunar & Smith al. 2000). Taxane/anthracycline combinations, among other 2000). This reduction can render operable a significant pro- chemotherapeutic regimens, can induce response rates in portion of initially inoperable cancers, and allow for a more excess of 50%, with complete remissions in about 15% of conservative surgical approach. Metastatic breast cancer also cases (Miller & Sledge 1999). In a small percentage of

Endocrine-Related Cancer (2003) 10 43–73 Online version via http://www.endocrinology.org 1351-0088/03/010–043  2003 Society for Endocrinology Printed in Great Britain Downloaded from Bioscientifica.com at 10/01/2021 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer patients (2–3%) long-term remissions are observed (iii) Is there a correlation between ABC protein expression (Greenberg et al. 1996). Even after failure of first-line treat- in breast cancer and prognosis? (iv) Does the expression of ments, a significant response can be achieved with second- these proteins affect breast cancer response to chemotherapy? line anticancer regimens (Sedlacek 1990). Thus, even in the (v) What factors contribute to the heterogeneity of results metastatic setting, many cancer cells are still sensitive to the between different studies? (vi) What suggestions can be cytotoxic effect of chemotherapy. However, beyond response given about the design of future studies? In this review, we rates, systemic treatments offer only a modest prolongation have included clinical trials of MDR1 reversal in breast of survival for some metastatic patients and only palliation cancer. The focus is not on the results of these studies in for most (Miller & Sledge 1999). Breast cancer remains, terms of drug efficacy, but in terms of ‘proof of principle’ for especially in the advanced setting, largely an incurable dis- the functional role of Pgp in causing clinical drug resistance. ease. This review does not provide a formal meta-analysis of The high percentage of non-responders and of failures the literature, as we have done previously for Pgp (Trock et following initial responses highlight the critical role played al. 1997). However, we have attempted to follow some of by drug resistance mechanisms in breast cancer management. the same criteria that were applied to that analysis. In the Conversely, the significant number of initial responses and section dedicated to the expression and role of ABC proteins the impact on survival, especially in early disease, suggests in breast cancer, we provide a summary of the sources and that, at least initially, a limited number of mechanisms of methods of citation searches, the criteria we have used for resistance are involved. Thus, among solid cancers, breast exclusion and inclusion, and for data stratification, to address cancer represents perhaps one of the best fields for the study possible causes of heterogeneity. The same data, updated, of clinical drug resistance and of its reversal. will be part of a formal meta-analysis at a later date. A large number of mechanisms, outlined later in this review, can cause a drug-resistant phenotype, at least in vitro. Mechanisms of drug resistance: the ABC One type of mechanism is based on the function of a group transporters (Pgp, MRP1) of transporter proteins, able to prevent the intracellular accu- mulation of anticancer drugs by an efflux mechanisms. Sev- Many factors may affect the ability of anticancer drugs to eral of these proteins belong to the ATP binding cassette kill cancer cells (Lehne et al. 1997, Gottesman 2002). Some (ABC) protein superfamily. Among these proteins, P-glyco- of the mechanisms involved are summarized in Table 1. protein (Pgp) represents the one most thoroughly studied for Resistance may be due to pharmacological, cancer multi- its ability to confer a multidrug-resistant phenotype in vitro cellular and cancer unicellular mechanisms. Pharmacological and in vivo. In the early 1990s, Pgp generated a lot of mechanisms include mechanisms that affect the ability of the enthusiasm about its possible role in conferring drug resist- drug to reach the target cell (e.g. cell metabolism and ance to cancer in the clinical setting and the possibility of pharmacokinetics). Multicellular mechanisms are related to increasing the efficacy of chemotherapy through its inhi- the three-dimensional structure of a tumor and include bition. Multidrug-resistance protein MRP1 was the second changes in local permeability to drug and the extracellular ABC protein to be identified as a cause of multidrug resist- microenvironment (e.g. changes in pH, hypoxia) (Alabaster ance. More recently, the breast cancer-resistance protein et al. 1989, Teicher 1994), and cellular mechanisms that are (BCRP, MXR, ABCP, ABCG2), was identified. Other ABC regulated by intercellular communication and contact proteins have been initially identified for their physiological (Desoize & Jardillier 2000). Cellular mechanisms include function, but subsequently have been shown to include mechanisms that affect the ability of anticancer drugs to anticancer drugs among their substrates. All of these proteins reach the intracellular target (regulating drug influx, efflux, could potentially play a role in clinical drug resistance. How- intracellular redistribution), mechanisms that regulate drug ever, the great majority of studies on the clinical role of ABC chemical activation/inactivation, qualitative and/or quantita- transporters have been limited to only two of these proteins: tive modifications to drug targets, and alterations in the Pgp and, to a more limited extent, MRP1. molecular pathways regulating the cellular response to drug- The aim of this review is to explore present knowledge induced damage (e.g. apoptosis). about the role of ABC membrane transporters in human Among the cellular mechanisms that regulate the ability breast cancer drug resistance. This paper is organized into of an anticancer drug to reach its intracellular target, the two parts. The first is aimed at providing some background efflux mechanism based on the function of Pgp is perhaps on drug resistance and ABC proteins. In the second part, the most thoroughly studied. Pgp was first studied in the we provide an overview of the published studies that have 1970s, when different groups described a multidrug-resistant addressed the expression and role of Pgp and MRP1 in breast phenotype characterized by resistance to the cytotoxic effect cancer. We have focused our attention on specific questions: of multiple unrelated drugs, such as vinca alkaloids, epipodo- (i) Are ABC transporter proteins expressed in breast tumors? phyllotoxins, anthracyclines and actinomycin D, following (ii) Does treatment affect ABC protein expression and how? cell selection with just one of these drugs (Biedler & Riehm

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Table 1 Cellular mechanisms of drug resistance Table 1 Continued I. SYSTEMIC PHARMACOLOGY I. Other Drug pharmacokinetics STAT Drug metabolism Heat shock proteins Metallothioneins II. CANCER SUPERCELLULAR MECHANISMS GST,glutathione; ATM,ataxia-teleangectasia; DNA-PK, Tumor environment (pH,pO 2,pressure) Multicellular drug resistance DNA-dependent protein kinase.

III. CANCER CELL MECHANISMS 1970, Juliano & Ling 1976). This phenomenon was A. Cell pharmacology Regulation of drug influx accompanied by decreased intracellular concentration of the Reduced folate carrier expression target drugs due to their active efflux from cells (Riehm & Nucleoside transport proteins Biedler 1971, Dano 1972, 1973, Ling & Thompson 1974). Regulation of drug efflux Later, it was defined that the same mechanism also confers Active efflux resistance to taxanes and can also cause intracellular drug ABC transporters redistribution (Bellamy 1996). The protein responsible for Entrapment this phenotype was named Pgp and it was subsequently Regulation of folate polyglutamation Poly(ADP-ribose) polymerase defined to be a 170 kDa plasma membrane glycoprotein Formation of pH gradients (Juliano & Ling 1976). The gene coding for Pgp (in humans Drug sequestration MDR1) was isolated and cloned in the 1980s (Roninson et ABC transporters al. 1984, 1986, Gros et al. 1986). B. Drug activation In the early 1990s another membrane transporter, MRP1, Cytochrome P450 enzymes was identified, characterized, and shown to confer a multi- drug-resistant phenotype similar to but distinct from the one C. Drug inactivation/detoxification conferred by Pgp (Cole et al. 1992, 1994). The MRP1 GST GST enzymes mechanism of action is broadly similar to that of Pgp (active γ-glutamyl cysteine synthetase efflux of substrates from cells and intracellular redistribution) glutathione-S-transferase (Coley et al. 1993, Cole et al. 1994, Slapak et al. 1994, D. Drug target alterations (qualitative,quantitative) Zaman et al. 1994, Binaschi et al. 1995, Breuninger et al. Tubulin 1995, van Luyn et al. 1998). MRP1 confers resistance to and/ Topoisomerase I and II or alters intracellular accumulation of anthracyclines, epipo- Dihydrofolate reductase dophyllotoxins, vinca alkaloids (Cole et al. 1994, Zaman et E. Damage repair mechanisms al. 1994, Breuninger et al. 1995, Lorico et al. 1996, Ruetz Mismatch repair proteins et al. 1996), high concentrations of methotrexate (Hooijberg Direct damage repair et al. 1999), and actinomycin D (Lorico et al. 1996). Unlike alkyltransferases (O6-methyl-guanineethyltransferase) Pgp, MRP1 does not affect resistance to and/or transport of Peroxide dismutases paclitaxel (Cole et al. 1994, Zaman et al. 1994, Lorico et al. Manganese superoxide dismutase 1996), mitoxantrone (Cole et al. 1994), arabinofuranosyl- Photolyase cytosine (Lorico et al. 1996) or m-AMSA (amsacrine) Nucleotide excision repair (Zaman et al. 1994). Evidence of the transport of colchicine, Base excision repair Recombination repair a classic substrate for Pgp, by MRP1 has been inconsistent (Cole et al. 1994, Zaman et al. 1994, Lorico et al. 1996). F. Regulation of apoptotic pathways Pgp and MRP1 have a wide range of substrates (Table 2). Damage recognition Many MRP1 substrates are transported in the form of gluco- High mobility group protein Mismatch repair ronate, sulfate or glutathione conjugates (Leier et al. 1994, ATM Muller et al. 1994, Jedlitschky et al. 1996, Loe et al. 1996a,b, DNA-PK Evers et al. 1997, Heijn et al. 1997, Jedlitschky et al. 1997). Pgp and MRP1 have physiological functions that can be G. Apoptotic pathways p53 inferred by their expression in normal tissues (Table 2), the mdm2 nature and range of their substrates (Table 3), and the results E2F of ‘knock-out’ and pharmacological reversal studies (Lorico BCL2/BAX et al. 1997, Schinkel et al. 1997, Wijnholds et al. 1997, 1998, Death receptors (Fas/CD95,TNFR1,DR3,DR4 and 2000). In particular, these studies suggest that both Pgp and DR5) MRP1 play a role in protecting the organism from excessive H. Apoptotic effectors concentrations of external as well as internal (e.g. steroids) Caspases toxins, through a regulation of their absorption, distribution www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202145 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

Table 2 Some normal tissues expressing Pgp www.med.rug.nl/mdl/humanabc.htm). The ABC superfamily Tissue References has been described in previous reviews (Klein et al. 1999, Dean et al. 2001). Based on sequence homology, ABC pro- Intestinal mucosa (brush Thiebaut et al. (1987) border of intestinal teins can be grouped into seven different subfamilies (A–G). epithelial cells) All ABC proteins share an ATP-binding domain of 200–250 Liver (hepatocytes,Fojo et al. (1987),Thiebaut amino acids (nucleotide-binding domain, NBD). The NBD epithelial cells lining small et al. (1987) unit includes two short, conserved peptide motifs (Walker A biliary ductules) and Walker B), involved in ATP binding, and a third conser- Pancreas (small pancreatic Fojo et al. (1987),Thiebaut ved sequence, located between the other two, that has been ductules) et al. (1987) called the ‘ABC signature’, and is unique to the NBD unit. Kidney (renal proximal tubule Thiebaut et al. (1987) cells) Many of the ABC proteins are transporters and are char- Adrenal cortex Fojo et al. (1987),Thiebaut acterized by the basic structural module ‘TMD-NBD’ (or et al. (1987),Croop et al. NBD-TMD) consisting of one ABC unit and a hydrophobic (1989) transmembrane domain (TMD). The TMD is usually com- Placenta Sugawara (1990) posed of six transmembrane segments (helices). To be func- Central nervous system Cordon-Cardo et al. (1989), tional, ABC transporters require a minimum of two modules, (endothelial cells of Thiebaut et al. (1989) capillaries) i.e. two TMDs and two NBD units. ABC proteins can be Testes (endothelial cells of Cordon-Cardo et al. (1990) classified according to the number and arrangement of the capillaries) basic structural modules (Fig. 1). In ‘full transporters’ the Blood (including pluripotent Neyfakh et al. (1989), two TMD-NBD units are part of the same polypeptide chain: stem cells and Holmes et al. (1990), (TMD-NBD)2. Some full transporters, e.g. several members lymphocytes) Chaudhary & Roninson of the ABC-C subfamily, including MRP1 (ABCC1), have a (1991),Chaudhary et al. TMD L (TMD-NBD) format. This format includes a third (1992),Klimecki et al. 0 0 2 (1994) membrane domain (TMD0), with transmembrane helices con- nected through a linker region (L0) to the (TMD-NBD)2 double module (Klein et al. 1999, Borst et al. 2000). The Table 3 Some Pgp substrates structure of ‘half transporters’ comprises only one TMD- Anthracyclines (doxorubicin,daunomycin,epirubicin) NBD or NBD-TMD module; their activity requires aggre- Acridines (m-AMSA) gation in multiprotein complexes. Full transporters are usu- Azatoxins (azatoxin) ally located in the plasma membrane and half transporters in Benxzoheptalene compounds (colchicine) intracellular membranes, the half-transporter BCRP being Benzothiazepines (diltiazem) one exception (Rocchi et al. 2000). Dihydropyridines (azidopine,nicardipine) Epipodophyllotoxins (etoposide) ABC transporters exhibit ATPase activity and translocate Isoquinolines (cepharantine) different substrates across the cell membrane and/or between Macrolides (FK506) various cell compartments. Transport of substrates is ensured Organometallic cations (99mTc-sestamibi) by a tight molecular coupling of the TMDs to the NBD units, Peptides (actinomycin D,bleomycin,cyclosporin A, and a positive cooperation of the two NBD units. Besides valinomycin) Pgp and MRP1 (renamed according to the new ABC protein Phenothiazines (chlorpromazine) nomenclature, respectively ABCB1 and ABCC1), at least Phenylalkylamines (verapamil,tiapamil) Pyrrolo-indoles (mitomycin C) another ABC protein, the half transporter BCRP (also known Quinolines (chloroquine,quinidine) as MXR, ABCP and ABCG2) has been initially characterized Rhodamines (rhodamine 123) for its ability to confer a multidrug-resistant phenotype Steroids (aldosterone,corticosterone,cortisol, (Allikmets et al. 1998, Doyle et al. 1998, Miyake et al. dexamethasone,testosterone) 1999). Other ABC proteins have been primarily identified for Taxanes (paclitaxel,docetaxel) other physiological functions but are able to transport Vinca alkaloids (vincristine,vinblastine) Other (digoxin,Hoechst 33342,etc.) anticancer substrates, and consequently represent potential drug-resistance proteins. These include the T-antigen presenting proteins TAP1/TAP2 (better known for their and excretion. Among other things, Pgp appears to be an immunological function) (Izquierdo et al. 1996, Lankat- important component of the blood–brain barrier (De Boer et Buttgereit & Tampe 1999), the bile salt export protein (also al. 2003). known as a sister of Pgp) (Childs et al. 1998, Lecureur et al. Pgp and MRP1 are part of a larger family of proteins 2000), the canalicular multispecific organic anion transporter called the ABC protein superfamily, which in humans is (cMOAT, also known as MRP2), MRP3, MRP4 and MRP5 known to include 48 ABC proteins (see web site: (Borst et al. 1999, 2000).

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Figure 1 Structure of ABC proteins. The white vertical bars represent transmembrane helices. Multiple transmembrane helices (generally six),form TMDs. Based on Klein et al. (1999).

Pgp appears approximately cylindrical in electron cry- cell membrane and its cytoplasmic end corresponds to the omicroscopy studies, with a diameter of about 10 nm and a apex of the cone and is virtually closed (Loo & Clarke depth (perpendicular to the plasma membrane) of about 8 nm 2001a). The Pgp substrate binding area is located around the and with two lobes of about 3 nm each projecting from the mid-level of the pore (Loo & Clarke 1997, 1999a, 2000). cytoplasmic end (Rosenberg et al. 1997). Pgp has a central The results of cysteine scanning mutagenesis and cross- pore that, in the resting phase, is conical in shape. The base linking studies are consistent with a ‘cyclone model’ of Pgp, of the cone, which is open to the extracellular space, is 5 whereas the two homologous halves of Pgp are arranged in nm wide (Rosenberg et al. 1997, Loo & Clarke 2001a). The the cell membrane in an antiparallel fashion, with transmem- diameter of the pore is 0.9–2.5 nm at mid-level through the brane segments 4–6 and 10–12 more directly contributing to www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202147 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer delimit Pgp’s central pore (Loo & Clarke 1999b). Pgp sub- corresponding to the minimum definition of positivity pro- strates most likely diffuse from the inner leaflet of the cell vided by the authors. For immunohistochemical (IHC) evalu- membrane (hydrophobic vacuum cleaner model) (Raviv ations, when more than one antibody was used we considered 1990, Higgins 1992), and between Pgp’s transmembrane a case as positive when immunostained by any of the anti- α-helices to the Pgp’s substrate binding area located inside bodies. In studies where more than one antibody was used, the pore, where they interact mostly with non-polar amino we have considered the results of the antibodies that gave the acids (Loo & Clarke 1997, 1999a, 2000). Interaction with the highest sensitivity. Where possible, we have also calculated binding site in the presence of ATP induces ATP hydrolysis, the average detection rate across studies that included 20 followed by a rearrangement of the protein conformation: patients or more per target subgroup. In the previous meta- movement of Pgp’s N-terminal and C-terminal halves in analysis (Trock et al. 1997) we observed that detection opposite directions closes the pore (Loo & Clarke 2002), methods and previous chemotherapy are significant factors while rotation of the transmembrane α-helices (Loo & Clarke in the heterogeneity of Pgp expression among human breast 2001b) may contribute to decrease the affinity of the site for cancers. The highly sensitive RT-PCR approach has been the substrates. Ultimately, the substrate is pushed either to increasingly used to evaluate Pgp (MDR1) mRNA levels. the extracellular phase (‘hydrophobic vacuum cleaner’ Thus, some studies now report MDR1 detection rates of model) or to the membrane’s outer leaflet (‘flippase model’). 100% in breast tumors with this method. RT-PCR and IHC Two-dimensional crystal data from purified MRP1 have data are considered separately in this review. been obtained and evaluated by electron microscopy, MDR1/Pgp and MRP expression studies were identified revealing an overall dimension of 8–10 nm and the associa- through literature searches in databases including PubMed tion of MRP1 in dimers (Rosenberg et al. 2001). Infrared Medline (National Library of Medicine, National Institutes spectroscopy has revealed a secondary structure made of of Health, Bethesda, MD, USA), CANCERLIT (National 46% α-helix, 26% β-sheet, 12% β-turns and 17% random Library of Medicine), Current Contents (Institute for Scien- coil. The secondary structure of MRP1 is not changed by tific Information, Philadelphia, PA, USA), Current Advances binding of ATP, its hydrolysis, or GSH-dependent transport in Cancer Research (Current Awareness in Biological of substrates. Binding of ATP (but not its hydrolysis), Sciences, Elsevier Science Inc., New York, NY, USA), changes the protein’s tertiary structure. The tertiary structure Knowledge Finder (Aries Systems Corp., North Andover, is then reversed to its original conformation when the MA, USA), Science Citation Index (Institute for Scientific hydrolyzed inorganic phosphate is released. Binding of sub- Information), and through cross-references in published strates modifies the tertiary structure of MRP1’s TMDs, but papers. modification of the tertiary structure of the cytoplasmic The studies considered in this review represent the domains with an associated increase in ATP binding and results of original research involving measurement of Pgp hydrolysis requires the presence of glutathione (Manciu et al. or MRP1 mRNA and/or protein expression in human breast 2000, 2002). Transmembrane segments 10–11, in membrane- cancers. Gene amplification studies were not included. Only spanning domain 1 (MSD1), and transmembrane segments peer-reviewed publications were considered (with the excep-

16–17, in MSD2, appear to be particularly critical for sub- tion of a study whose results were presented as a letter to strate interaction, as demonstrated by both mutation and Lancet (Nooter et al. 1997a)). Reviews were excluded as a photo-affinity labeling studies (Daoud et al. 2001, Ito et al. source of data but were used as a source of references, as 2001, Mao et al. 2002). were data publications in a language other than English. Where the same study was reported in different papers, only Expression and role of Pgp and MRP1 in the most recent publication was considered. We cannot breast cancer exclude the possibility that some cases were included in dif- ferent papers from the same research group. Previously, we completed a formal meta-analysis of pub- lished data on the expression and functional significance of MDR1/Pgp in breast cancer (Trock et al. 1997). Here, we Pgp and breast cancer provide an updated review of both Pgp/MDR1 and MRP1 and their clinical role in breast cancer. We have not carried Pgp/MDR1 expression in untreated breast cancer out a formal meta-analysis of published data. To obtain a and normal tissues more synthetic and encompassing view of results, whenever In almost all studies on expression in non-neoplastic breast feasible, we have pooled the data from comparable studies. tissue, Pgp/MDR1 could be detected in normal breast tissues Papers often report different levels of positivity for protein at both the protein and mRNA level. Pgp was readily expression. In some studies, specimens have been defined as detected by IHC in four studies (Cordon-Cardo et al. 1990, positive even if Pgp was detected only in the cytoplasm or Ro et al. 1990, van der Valk et al. 1990, Scala et al. 1995). in a small number of cells. For each study, we used the data Van der Valk et al. (1990) found Pgp expression to be weak

48 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:06:29PM via free access Endocrine-Related Cancer (2003) 10 43–73 and confined to breast epithelial cells, while absent from the (Wishart 1990 et al., De la Torre et al. 1994, Decker et al. stroma. Scala et al. (1995) observed Pgp expression in 21/24 1995, Linn et al. 1995, 1997, Wang et al. 1997, Gago et (88%) normal or benign breast tissues. Pgp expression was al. 1998, Yang et al. 1999). Of these, six studies used two confined to the luminal surface of ductal epithelium, where antibodies, and two (Linn et al. 1995, Wang et al. 1997) used it was located in cell membranes. Ro et al. (1990) found Pgp three antibodies. Different antibodies were used on separate expressed in the adjacent normal or hyperplastic tissue of samples, with one exception where two antibodies were used 67% of 40 locally advanced breast cancers. These authors as a ‘cocktail’ (Gago et al. 1998). Only four of the above also reported Pgp expression in 69% of the corresponding papers report the results in a way that allowed us to calculate cancer tissues. In 35% of cases expression in cancerous and the detection rate when positivity is defined as staining with normal tissues was discrepant. Only one study used an both of two antibodies: the pooled rate is 24% (or 30/124) immunoblot approach: no Pgp could be detected in five heal- (Wishart et al. 1990, De la Torre et al. 1994, Decker et al. thy breast tissue samples, as compared with a detection rate 1995, Wang et al. 1997). Unfortunately, there are not enough of 10/34 in untreated breast cancers (Sanfilippo et al. 1991). data available to allow an evaluation of whether this more At the mRNA level, an RT-PCR study showed higher aver- stringent definition of positivity improves the correlation of age levels in the neoplastic as compared with the associated Pgp expression with other clinical and pathological param- normal tissue component of 40 locally advanced breast can- eters, including treatment outcome. cers before treatment with neoadjuvant chemotherapy. Fol- A study by Del Vecchio et al. (1997) represents a single lowing treatment, the significance of this difference was lost, instance in which in vitro quantitative autoradiography, with largely due to a higher level of induction of MDR1 in benign 125I-MRK16 antibody, was used for the in situ detection of tissues (Arnal et al. 2000). Pgp expression. In this study, Pgp was detected in all 27 Expression of Pgp/MDR1 protein in untreated breast cases studied (100%). cancer has been most often evaluated by IHC. We have Detection of Pgp-positive (Pgp + ) cells by fluor- found 29 papers reporting the results of IHC evaluations of escence-activated cell sorting has been used in two separate Pgp expression in previously untreated patients. The data studies (Brotherick et al. 1996, Gregorcyk et al. 1996). Only from these papers are summarized in Tables 4–6. When all the paper by Gregorcyk et al. provides an actual Pgp detec- data are pooled, the detection rate of Pgp by IHC is 40% tion rate: 43.6% (24/55) of untreated breast cancers. (744/1840) in untreated patients. Overall, detection in these Protein expression has also been evaluated by a ‘bulk’ studies ranged from 0 (Yang et al. 1999) to 100% (Del Vec- method, such as Western blot, with a pooled rate of detection chio et al. 1997). When only studies with more than 20 of 32.6% (80/245) across four studies. This pooled number patients are considered (28 studies), the average rate of detec- does not reflect the difference in detection reported in an tion is 45.9% (95% confidence interval: 35.6–56.2). early study by Merkel et al. (1989), using the C219 antibody Various different antibodies have been used for Pgp (where Pgp was detected in 0/125 cases), and a later study detection in breast cancer. Six different monoclonal anti- by Buser et al. (1997), who detected Pgp in 70/84 cases using bodies were used on breast cancer tissues: JSB-1, C219, a polyclonal antibody. MRK16, C494, 4E3 and UIC2. The characteristics of these Detection of Pgp/MDR1 mRNA by RT-PCR studies antibodies have been previously reviewed (van der Heyden shows a higher level of sensitivity than IHC. Across 12 et al. 1995, Beck et al. 1996). JSB-1 recognizes a cytoplas- mic epitope of Pgp and does not cross-react with MDR3 or studies, MDR1 expression was detected in 334/531 cases with the heavy chain of muscle myosin, as does the C219 (63%) (Table 5). Detection rates range from 0 to 100%. Ear- antibody. The 16 studies using JSB-1 as the primary antibody lier evaluations of MDR1 mRNA levels had been based on show a pooled detection rate of 34% (361/1075). C219 also the use Northern blotting techniques, yielding a pooled detec- recognizes a cytoplasmic epitope and it has been the second tion rate of 28% (52/185) across four studies (Table 5). most frequently used antibody for the detection of Pgp in Four studies have considered the agreement between breast cancer (eight studies). In studies where only C219 was IHC- and RT-PCR-based evaluations of MDR1/Pgp expres- used, it detected Pgp in 111/321 cases (35%). MRK16 (seven sion in untreated breast cancer. Two studies showed a con- studies), which recognizes an external epitope and does not cordance rate of 73% (Chevillard et al. 1996, Filipits et al. cross-react with MDR3, gave a pooled detection rate of 36% 1996). However, Hegewisch-Becker et al. (1998) found a (110/307). Polyclonal antibodies had a higher detection rate wider disagreement between the two techniques, with a con- (59/94, or 63%). cordance rate of only 34%. Discrepancy was interpreted as at One of the recommendations from the St Jude MDR least partly due to lymphocyte contamination in the RT-PCR Workshop on Methods to Detect Pgp-associated Multidrug samples. A concordance rate as low as 31% was also Resistance (Beck et al. 1996) was that more than one anti- reported by Wang et al. (1997), although this might simply body be used for Pgp detection. Eight studies on Pgp expres- reflect the difference in sensitivity between the two tech- sion in untreated breast cancer used more than one antibody niques. www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202149 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

Table 4 Pgp expression in untreated breast cancer. The table includes data only from those studies providing the ratio of positive/total cases. Studies where previous treatment status was uncertain were excluded. For IHC-based studies,when more than one antibody was used,any positivity (with either) would be considered as a sign of overall positivity. Where different levels of positivity were provided,we used the data for the lowest provided cut-off of positivity Reference Setting Antibodies Pgp + /total IHC studies Salmon et al. (1989) Metastatic JSB-1 1/5 Sugawara et al. (1990) Not described MRK16 2/20 Wishart et al. (1990) Operable* C219,MRK16 23/29 Ro et al. (1990) Locally advanced C219 6/9 Verrelle et al. (1991) Locally advanced,metastatic C494 14/17,3/3 Koh et al. (1992) Locally advanced JSB-1 1/11 Charpin et al. (1994) Not described JSB-1 113/213 De la Torre et al. (1994) Operable C219,MRK16 6/41 Keen et al. (1994) Operable Polyclonal 14/43 Veneroni et al. (1994) Locally advanced C219 18/39 Decker et al. (1995) Locally advanced (w/o mts***) C219,C494,polyclonal 8/9 Locally advanced (with mts) 1/2 Metastatic (from mts) 8/9 He et al. (1995) Operable C219 23/50 Schneider & Romero (1995) Mixed Polyclonal 28/31 Seymour et al. (1995) Locally advanced C219 18/40 Chevillard et al. (1996) Locally advanced C219,JSB1,C494 9/54 Filipits et al. (1996) Operable C219 36/63 Bargou et al. (1997) Operable JSB-1 9/27 Chung et al. (1997) Locally advanced JSB-1 6/23 Kostakoglu et al. (1997) Not described JSB-1 15/19 Linn et al. (1997) Operable JSB-1 (C219) 2/20 Locally advanced 16/25 Wang et al. (1997) Locally advanced 4ES,JSB1,C494 12/48 (with and w/o mts) Gago et al. (1998) Operable C494,JSB-1 70/177 Hegewisch-Becker et al. (1998) Operable UIC-2 44/61 (MRK16) (42/57)** Honkoop et al. (1998) Locally advanced JSB-1 27/42 Mechetner et al. (1998) Not described JSB-1 28/244 Yang et al. (1999) Primary JSB-1,MRK16 0/39 Bottini et al. (2000) Mixed JSB-1 42/141 Sun et al. (2000) Mixed JSB-1 8/24 Faneyte et al. (2001) Mixed JSB-1 44/62 Liu et al. (2002) Mixed JSB-1 12/30 Del Vecchio et al. (1997) Mixed MRK16 27/27 Schneider et al. (2001) Locally advanced C494 9/23

Immunoblot studies Merkel et al. (1989) Operable C219 0/125 Sanfilippo et al. (1991) Operable C219 10/34 Buser et al. (1997) Operable (early) Polyclonal 44/53 Locally advanced 15/20 Metastatic 11/11 Yang et al. (1999) Operable C219 0/2

Immuno-flowcytometric studies Gregorcyk et al. (1996) Mixed C219 24/55

In vitro quantitative autoradiography Del Vecchio et al. (1997) Mixed MRK16 27/27 *It generally refers to patients undergoing breast surgery for early breast cancer. **Information provided does not allow calculation of how many cases are positive with either antibody. The detection ratio for the less sensitive antibody is provided in parentheses. ***mts = metastases.

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Table 5 MDR1 mRNA expression in untreated breast cancer. Table 7 Pgp expression in early operable vs locally The table includes data only from those studies providing the advanced untreated breast cancer ratio of positive/total cases. Studies where previous treatment IHC + IHC RT-PCR status was uncertain were excluded. Where different levels of RT-PCR positivity were provided,we used the data for the lowest provided cut-off of positivity Operable early breast cancer Number of studies 13 9 4 Reference State MDR1 + / Pgp(MDR1) + /total (%) 453/843 227/511 226/332 total (53.7%) (44.4%) (68.1%) RT-PCR Locally advanced breast cancer Chevillard et al. (1996) Locally advanced 20/73 Number of studies 13* 11 3 Filipits et al. (1996) Operable 80/134 Pgp(MDR1) + /total (%) 234/459 145/342 101/165 O’Driscoll et al. (1996) Not described 4/6 (51.0%) (42.4%) (61.2%) Wang et al. (1997) Locally advanced 44/52 Dexter et al. (1998) Mixed 72/72 *Wang et al. (1997) used both RT-PCR and IHC to analyze Ito et al. (1998) Operable 25/27 Pgp/MDR1 expression: in ‘IHC + RT-PCR’ only the RT-PCR Hegewisch-Becker et al. Mixed 38/61 data are considered. (1998) Lacave et al. (1998) Operable 61/74 Yang et al. (1999) Operable 0/39 When the results of these studies are pooled, Pgp appears to Arnal et al. (2000) Locally advanced 37/40 be expressed in 23% (5/22) of T1 (tumors 2 cm or less), 47% Ferrero et al. (2000) Operable 85/85 (30/64) of T2 (between 2 and 5 cm), 50% (10/20) of T3 Faneyte et al. (2001) Mixed 25/25 (more than 5 cm in size), and 42% (5/12) of T4 cases (tumors Northern blot with a direct extension to chest wall or skin). These data Goldstein et al. (1989) Not described 9/57 appear to suggest that untreated tumors larger than 2 cm are Kacinski et al. (1989) Mixed 11/16 more likely to express Pgp than smaller tumors. Merkel et al. (1989) Operable 0/53 Keith et al. (1990) Not described 25/49 Overall the results from six IHC, one flow cytometric, Wallner et al. (1991) Operable 27/59 and one Western blot study are not supportive of a correla- Hennequin et al. (1993) Operable 0/24 tion between Pgp expression and nodal status. This is partly due to the high heterogeneity of results and the low number of cases in certain subgroups. Two of the IHC studies (Ro et Pgp/MDR1 and breast cancer stage al. 1990, Charpin et al. 1994), and the isolated flow cytomet- Pooling the results of IHC studies of Pgp protein expression ric study (Brotherick et al. 1996) found no correlation and breast cancer stage gives a Pgp detection rate of about between nodal status and Pgp expression. Buser et al. (1997) 42% (145/342, from 11 studies) in locally advanced as com- detected Pgp by Western blot in 20/26 (78%) node-negative pared with 44% (227/511, from nine studies) in primary and in 26/30 (87%) node-positive early breast cancer cases. operable breast cancers (Tables 4, 5 and 7). The average In the locally advanced setting, Pgp was expressed respect- detection rates in studies that included 20 cases or more were ively in two of four (50%) node-negative and in 26/30 node- respectively 40.8% (95% confidence interval: 26.0–55.6; positive cases. eight studies) and 42.7% (95% confidence interval: 24.5– The small number of node-negative cases in the locally 60.9; nine studies). Only one study directly compared Pgp advanced setting does not allow any conclusion as to the rate expression at the early and at the advanced stage: using IHC, of Pgp expression. A similar problem affects the evaluation Linn et al. (1997) detected a statistically significant differ- of the study by De La Torre et al. (1994), who reported a ence in Pgp expression between early operable (2/20, 10%) higher incidence of nodal involvement in Pgp-negative and locally advanced breast cancers (18/30, 60%). (Pgp−, 23/35 cases, or 65%) than in Pgp+ cases (one of six, Four studies considered the correlation between Pgp or 17%). Two studies (Schneider & Romero 1995, Sun et al. expression and T staging (De La Torre et al. 1994, Schneider 2000) considered Pgp expression’s correlation with N stag- & Romero 1995, Sun et al. 2000, Schneider et al. 2001). ing. Pooling the data from the two studies provides what

Table 6 Pgp expression in untreated breast cancer — summary. Some studies used more than one approach to Pgp detection (Chevillard 1996,Fillipits et al. 1996,Hegewisch-Becker et al. 1998,Merkel et al. 1989,Wang et al. 1997,Yang et al. 1999). Only the approach that was used on most cases was considered when calculating the expression of Pgp across all assays All assays IHC RT-PCR Northern blot Western blot Other Number of studies 49 32 12 6 4 2 Pgp + /total 1136/2584 744/1840 334/531 72/258 80/245 52/82 (%) (44.0%) (40.4%) (62.9%) (27.9%) (32.7%) (63.4%)

www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202151 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer appears to be a certain inverse correlation between Pgp results of this study show that MDR1 mRNA levels expression and N stage: Pgp expression is observed in measured by RT-PCR at the primary site, are lower in meta- 17/21 (81%) N0 cases, 13/19 (68.4%) N1 cases, and 6/15 static disease than in non-metastatic disease. However, the (40%) N2 cases. Another study, however, showed that 14/23 evaluation included only four metastatic cases and the differ- (60.9%) Pgp+ and 14/27 (40.7%) Pgp− breast cancers have ences were not significant (Dexter et al. 1998). more than four axillary lymph nodes involved (He et al. 1995). Effect of chemotherapy on Pgp expression Data on Pgp expression in untreated metastatic breast Both the processes of induction and of selection could be cancer are infrequent. All studies are based on the use of involved in an effect of chemotherapy on Pgp expression. In IHC. Four of these studies compare the expression of Pgp in vitro studies have shown that treatment of neoplastic cells the primary lesion of metastatic and non-metastatic patients. with chemotherapeutic agents can induce the expression of When the results of these four studies are pooled, no differ- Pgp (Chaudhary & Roninson 1993). This induction appears ence in Pgp expression is observed between metastatic and to be independent of whether the anticancer drugs are sub- non-metastatic cases, Pgp being detected in 55/74 (74.3%) strates for Pgp, and may be part of a response to different non-metastatic vs 16/22 (72.7%) metastatic cases (Verrelle stress stimuli, including irradiation, genotoxic stress and et al. 1991, Decker et al. 1995, Schneider & Romero 1995, inflammation (Sukhai & Piquette-Miller 2000). Conversely, Sun et al. 2000). Most of the M0 cases were represented by treatment with anticancer drugs to which Pgp confers resist- cancers with primaries larger than 2 cm, if not locally ance would be expected to enrich cancer in Pgp+ cells by a advanced. One study compared the expression of Pgp in the process of selection and should be specific for drugs that are primary lesions of non-metastatic cancers and in the metast- MDR1 substrates. ases of metastatic cancers (apparently, all untreated): Pgp Twenty-six studies have evaluated the expression of Pgp was detected in 7/12 metastatic samples (58%) and in only following either recent or remote anticancer treatment 27/92 (29%) primary cancers (P = 0.055) (Linn et al. 1995). (Tables 8a–d), mostly including MDR1 substrates. IHC The results of this study suggest an increased expression of evaluations detected Pgp in 46% of previously treated cases Pgp in metastases as compared with the primary sites. How- (256/556; 19 studies), as compared with the 41% detection ever, to the best of our knowledge, expression of Pgp has rate reported for untreated cases (see above). When only never been compared in the primary and metastatic sites of those cases are included whose treatment certainly included the same patients. MDR1 substrates (i.e. also excluding studies where treatment In considering MDR1 mRNA expression and breast was not defined), Pgp appears to be detected by IHC in 172/ cancer stage, there is no difference in pooled MDR1 mRNA 329 cases (52%, across 16 studies). Pgp is detected in only detection rates between primary operable (183/314 cases, or 37% (84/227, four studies) of those cases whose treatment 58%, in five studies) and locally advanced cases (101/165 was either not described or did not include MDR1 substrates. cases, or 61%, in three studies) from studies where RT-PCR The rate of IHC detection appears be lower in remotely (42/ was used (Chevillard et al. 1996, Filipits et al. 1996, O’Dris- 117, or 36%, across six studies) than in recently treated coll et al. 1996, Wang et al. 1997, Hegewisch-Becker et al. cancer (170/306, or 56%, across 11 studies), suggesting that 1998, Lacave et al. 1998, Yang et al. 1999, Arnal et al. 2000) the effect of chemotherapy is transient. RT-PCR detected (Table 7). MDR1 in 71% (135/190 cases, in five studies) of treated vs As observed in IHC studies, data on the correlation about 61% of untreated cases. Western blotting detected Pgp between MDR1 mRNA and nodal status appear highly in 9/16 (56%) cases in two small studies (Merkel et al. 1989, heterogeneous and inconclusive. No difference in MDR1 Sanfilippo et al. 1991), while two small Northern blotting mRNA levels or in the percentage of MDR1+ and MDR1− studies did not detect MDR1 mRNA in any of 16 patients cases (as evaluated by slot blot analysis) was observed in (Merkel et al. 1989, Hennequin et al. 1993). cases with different nodal stages (N0, N1 and N2) (Wallner A more adequate evaluation of the effect of chemo- et al. 1991). In an RT-PCR study, MDR1 mRNA levels were therapy may be obtained in studies where the same cancers not different in node-positive and node-negative cases are evaluated for Pgp expression before and after chemo- (Lacave et al. 1998). In another RT-PCR study, levels of therapy. These studies represent only a fraction of those MDR1 mRNA seem to decrease from N0 to N2 cases, but considered above, and they have been carried out almost the differences are not statistically significant (Dexter et al. exclusively in patients being treated with neoadjuvant 1998). The only statistically significant difference was chemotherapy for locally advanced breast cancer. The results reported by Schneider et al. (2001) who observed a higher of these studies are summarized in Table 9. In seven IHC rate of nodal involvement in MDR1+ (18/22; 81.8%) than in studies, involving 176 patients, Pgp was detected in 71 cases MDR1− cases (13/24; 54.2%). (40%) before and in 102 cases (58%) after systemic treat- Only one study compared MDR1 mRNA levels in ment (Ro et al. 1990, Koh et al. 1992, Keen et al. 1994, untreated metastatic and non-metastatic breast cancer. The Decker et al. 1995, Chevillard et al. 1996, Chung et al. 1997,

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Table 8a Pgp/MDR1 expression in treated breast cancer: IHC studies Reference Treatment Treatment Pgp (positive/total) Remoteness setting of treatment* Pre- Post- treatment treatment Salmon et al. Adjuvant Undefined cycles of treatment combinations 1/5 – Remote (1989) including vinca alkaloids and doxorubicin – 4/8 Ro et al. (1990) Neoadjuvant Undefined no. cycles of Dox,Ctx,Vcr,Pdn 6/9 4/9 Recent – 2/2 van Kalken (1991) Advanced Doxorubicin- or epirubicin-containing – 0/7 Undefined† regimens Dixon et al. (1992) Neoadjuvant Mitox,MitC,Mtx × 4 – 0/24# Recent Koh et al. (1992) Neoadjuvant FAC × 3 1/11 7/11 Recent – 7/15 Botti et al. (1993) Neoadjuvant CEF – 19/25 Recent Keen et al. (1994) Neoadjuvant Tam 14/43 17/43 Recent Bates et al. (1995) Metastatic Undefined chemotherapy: with and w/o – 9/21 Remote disease antracyclines Decker et al. (1995) Mixed FAC × 2–6 5/5 5/5 Recent 17/20 – Seymour et al. Metastatic Anthracycline-containing treatment – 13/17 Remote (1995) disease Chevillard et al. Neoadjuvant FAC × 4orCFT× 4 5/48 23/48 Recent** (1996) Mruren et al. (1996) Advanced Anthracycline- or anthracene-refractory – 14/16 Remote disease patients Tolcher et al. Metastatic Paclitaxel 3/16*** 13/17 Recent*** (1996) disease Chung et al. (1997) Neoadjuvant Undefined cycles of Ctx/Dox/FU ± Tam 6/23 13/23 Recent – 10/17 Relapse CAF ± Tam – 2/6 Remote Kostakoglu et al. Advanced Varied treatment (with Mtx,FU,Dox,Tax, 15/19 – Not described (1997) VP16),always containing an MDR1 – 9/11 substrate Honkoop et al. Neoadjuvant Dox,Ctx,GM-CSF × 6 18/27 17/30 Recent (1998) Mechetner et al. Not described Not described 28/244 – Not described (1998) – 35/115 Vargas-Roig et al. Neoadjuvant FAC × 3–4 34/37 33/37 Recent (1999) Yang et al. (1999) Advanced Dox-containing treatment – 0/49* Remote disease CAF,cyclophosphamide,adriamycin,5-fluorouracil; ctx,cyclophosphamide. Table 8b Pgp/MDR1 expression in treated breast cancer: immunoblot studies Reference Treatment Treatment Pgp (positive/total) Remoteness setting of treatment* Pre- Post- treatment treatment Merkel et al. (1989) Metastatic Doxorubicin – 0/2 Recent disease Sanfilippo et al. Not described CMF × 3orFAC× 3–4 or 6 cycles of 10/34 9/14 Not described (1991) iododeoxyrubicin or a Dox-containing regimen

Vargas-Roig et al. 1999). When the study by Keen et al. the rates of detection of MDR1 mRNA were 50% (57/113) (1994) (treatment with tamoxifen only) is excluded, detection before and 73% (82/113) after treatment with MDR1 sub- rates are respectively 43% (57/133) and 64% (85/133). This strate-containing regimens, with an induction rate (change means that 37% of previously Pgp− cases were rendered from negative to positive) of 45% (Chevillard et al. 1996, positive following chemotherapy. In two RT-PCR studies, Arnal et al. 2000). One study of particular interest is that by www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202153 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

Table 8c Pgp/MDR1 expression in treated breast cancer: RT-PCR studies Reference Treatment Treatment Pgp (positive/total) Remoteness setting of treatment* Pre- Post- treatment treatment Chevillard et al. Neoadjuvant FAC × 4,or CFT × 4 20/73 42/73 Recent** (1996) O’Driscoll et al. Not described CMF and/or Tam 4/6 4/4 Not described (1996) Arnal et al. (2000) Neoadjuvant FAC × 6 37/40 40/40 Recent Faneyte et al. Mixed Anthracycline-containing treatment 25/25 27/27 Mixed (2001) Schneider et al. Neoadjuvant FAC or docetaxel,Dox or CMF – 21/44 Recent (2001) – 1/1 – 0/1

Table 8d Pgp/MDR1 expression in treated breast cancer: Northern blot studies Reference Treatment Treatment Pgp (positive/total) Remoteness setting of treatment* Pre- Post- treatment treatment Merkel et al. (1989) Metastatic disease Dox-containing treatment – 0/11 Recent Hennequin et al. (1993) Neoadjuvant CEF × 6 – 0/5 Recent Table 8a–d. CEF,cyclophosphamide,epirubicin,fluorouracil; FAC,fluorouracil,doxorubicin,cyclophosphamide; CFT, cyclophosphamide,fluorouracil,thiotepa; Dox,doxorubicin; MitC,mitomycin C; Mitox,mitoxantrone; Mtx,methotrexate; Pdn, prednisone; Tam,tamoxifen; TAx,Taxol (paclitaxel),Vcr,vincristine; VP16,etoposide,GM-CSF,granulocyte-macrophate colony-stimulating factor; CMF,cyclophosphamide,methotrexate and fluorouracil; FU,fluouroracil. *Whether recent or remote with respect to post-treatment evaluations. Remote treatment usually means that post-treatment Pgp evaluation was carried out at release. **Evaluated 28 days after systemic treatment. ***All patients had been remotely treated with ‘conventional’ chemotherapy. †Advanced patients progressing on anthracycline-containing regimens (time of treatment with respect to Pgp evaluation is presumably recent,but undefined). #Staining observed in 4/26 cases was stromal. §Staining was observed in interstitial mononuclear cells.

Table 9 Effect of neoadjuvant treatment on Pgp/MDR1 expression* Method Pgp + Pgp + Induction** n References (pre-treatment) (post-treatment) All treatments IHC 71/176 (40.3%) 102/176 (58.0%) 31/105 (29.5%) 7 Ro et al. (1990),Koh et al. (1992), Keen et al. (1994),Decker et al. (1995),Chevillard et al. (1996), Chung et al. (1997),Vargas-Roig et al. (1999) RT-PCR 57/113 (50.4%) 82/113 (72.6%) 25/56 (44.6) 2 Chevillard et al. (1996),Arnal et al. (2000) Total 128/289 (44.3%) 184/289 (63.7%) 56/161 (34.8%) 9 – Only treatments including MDR1 substrates IHC 57/133 (42.9%) 85/133 (63.9%) 28/76 (36.8%) 6 Ro et al. (1990),Koh et al. (1992), Decker et al. (1995),Chevillard et al. (1996),Chung et al. (1997), Vargas-Roig et al. (1999) RT-PCR 57/113 (50.4%) 82/113 (72.6%) 25/56 (44.6%) 2 Chevillard et al. (1996),Arnal et al. (2000) Total 114/246 (46.3%) 167/246 (67.9%) 53/132 (40.1%) 8 – *Includes only those cases where Pgp/MDR1 expression was evaluated both before and after treatment. **Number of initially negative cases that turned positive following adjuvant treatment.

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Chevillard et al. (1996). In this investigation, both Pgp (by no association with tumor grade was observed in four studies, IHC) and MDR1 mRNA (by RT-PCR) were evaluated two of which used IHC, one flow cytometry and one RT- before, and 8 and 28 days after the first cycle of neoadjuvant PCR, to detect Pgp/MDR1expression (Charpin et al. 1994, treatment with either a doxorubicin-containing regimen Brotherick et al. 1996, Chevillard et al. 1997, Dexter et al. (FAC – fluorouracil, doxorubicin, and cyclophosphamide), 1998). or a regimen where doxorubicin was substituted with the For ploidy, a correlation between Pgp/MDR1− status non-MDR1 substrate thiotepa. The authors evaluated not and aneuploidy is supported by two out of three studies. only the number of cases changing from MDR1− to Chevillard et al. (1997) observed a significant correlation MDR1+, but also whether there was an increase in MDR1 between MDR1 negativity and aneuploidy in a series of expression in tumors already MDR1+ prior to treatment. locally advanced breast cancers, evaluated before neoadju- Independently of the treatment applied, MDR1 mRNA vant chemotherapy. A similar trend can be observed in the expression was increased in 23/73 (32%) of the tumors on data reported by Lacave et al. (1998). However, in the latter day 8, and in 35/73 (48%) on day 28, of the first treatment study the correlation did not reach statistical significance. In cycle. The increase on day 8 included both the acquisition of a third study, De la Torre et al. (1994) found no difference in an MDR1+ phenotype in 18/35 (51%) initially ploidy between Pgp+ and Pgp− cases. In their study, diploid MDR1−cases, and an increased expression in 5/20 (25%) cancers represented 31% of 35 Pgp− cases, and 33% of six already MDR1+ cancers. The increase by day 28 included Pgp+ cases. an acquired MDR1+ phenotype in 22/35 (63%) of initially In comparison with histology, two RT-PCR-based MDR1− cases, and an increased expression in 13/20 (65%) studies report a higher incidence of MDR1 expression in already MDR1+ cancers. lobular than in ductal breast carcinomas. In the first, MDR1 The two chemotherapy regimens used in this study had mRNA was detected in three of four cases of lobular carci- somewhat different effects on the rapidity of induction of noma and in 15/60 ductal carcinomas. Notwithstanding the MDR1 expression. Induction by the doxorubicin-containing small numbers, this difference was statistically significant regimen was evident in 18/50 cases (36%) on day 8 and 24/ (Chevillard et al. 1997). Similarly, the second study reported 50 cases (48%) on day 28. The thiotepa-containing regimen significantly higher levels of MDR1 mRNA in lobular than induced MDR1 in ten (43%) and in 11 (48%) out of 23 cases in ductal carcinomas (Dexter et al. 1998). In contrast, Lacave on day 8 and 28 respectively. Changes in the levels of protein et al. (1998) found no difference in MDR1 expression expression were similar to those observed for MDR1 mRNA between 63 cases of ductal carcinomas and ten cases of lobu- expression. Hence, the level of Pgp induction is comparable lar carcinomas. for both regimens, independent of the presence of MDR1 In receptor studies, overall, there is no clear indication substrates. However, as the results of this study show, induc- of a correlation between Pgp and ER, and between Pgp and tion following treatment with doxorubicin may occur more PgR expression. A direct correlation between ER and Pgp slowly than after treatment with thiotepa. The apparent inde- expression was observed in two studies. An IHC study of 101 pendence of the observed effect from the presence of MDR1 biopsies from accessible recurrent or metastatic sites found a anticancer drugs would appear to support a non-specific Pgp/ significant association between ER and Pgp positivity. ER+ MDR1 induction, rather than a selection effect. tumors were more often Pgp+ (69%) than ER− tumors (46%) (Seymour et al. 1995). In the second study, evaluation Association of Pgp expression with other clinical, by RT-PCR reported MDR1 to be expressed at higher levels pathological and molecular markers in ER+ (9.22 molecules/µg × 106) than in ER− cases (4.00 The expression of MDR1/Pgp may correlate with that of molecules/µg × 106) but the difference was not statistically other known clinical, pathological, and molecular markers. significant (Dexter et al. 1998). By contrast, an inverse Parameters evaluated for their association with Pgp expres- correlation is suggested by the results of two other IHC- sion include, among others, tumor grade, ploidy, histology, based studies. In the first of these studies it was observed estrogen receptor (ER) and progesterone receptor (PgR), and that ER− tumors were more often Pgp+ (30%) than Pgp− p53. (15%), the difference being statistically significant (Charpin With tumor grade, overall, there is no consistent or con- et al. 1994). A similar trend was reported by Wang et al. vincing evidence for a significant correlation between MDR1 (1997), who reported Pgp positivity in 15% (4/26) of ER+ expression and grade. Ferrero et al. (2000), measuring and 39% (7/18) of ER− cases with borderline statistical sig- MDR1 expression by RT-PCR, reported a modest association nificance (P = 0.078). of MDR1 expression with grade 1 cancers. A similar trend For PgR, a trend to a direct correlation with Pgp is sug- is evident from the RT-PCR data reported by Beck et al. gested by the RT-PCR-based study by Dexter et al. (1998) (1998). In contrast, the opposite trend was observed in two and the IHC study by Seymour et al. (1995). However, a additional studies, one using RT-PCR (Lacave et al. 1998), statistically significant inverse correlation between PgR and and the other using IHC (De la Torre et al. 1994). Finally, Pgp proteins was reported by Wang et al. (1997). www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202155 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

We found seven additional studies reporting no correla- as a cause of drug resistance, correlative clinical studies tion between Pgp expression and expression of ER and/or should demonstrate a significant relationship between the PgR (Ro et al. 1990, Wallner et al. 1991, De la Torre et al. protein (or its mRNA) expression and resistance to systemic 1994, He et al. 1995, Brotherick et al. 1996, Lacave et al. treatments that include drugs that are substrates for the drug- 1998, Ferrero et al. 2000). resistance proteins. Evaluations in the neoadjuvant setting Turning to mutant p53 proteins, these are often more can consider expression before, during and after treatment, stable and are readily detected by IHC methods (Elledge & and by comparison, the amount of induction. Expression in Allred 1998). Most studies show a direct correlation between this setting and at these different times in relation to treat- Pgp and p53. In a group of 15 archival breast cancer speci- ment, can be compared with response rates (clinical, mens, overexpression of Pgp was most often accompanied pathological) as well as survival parameters. Studies in the by high levels of p53 (Bodey et al. 1997). In another IHC adjuvant setting allow evaluation only before treatment and study, p53 was positive in 88/113 (78%) of Pgp+ cases, with its correlation with longer-term outcomes (survival and dis- cells staining with both Pgp and p53 antibodies mostly ease-free survival (DFS)), but have the potential advantage coinciding (Charpin et al. 1994). Linn et al. (1996) reported of a lower number of coexpressed and possibly redundant a statistically significant correlation between IHC detected mechanisms of resistance. Conversely, in the advanced set- Pgp and nuclear accumulation of p53, with Pgp staining in ting, the role of a specific protein is more likely to be masked 5/26 p53− tumors (19%) and in 15/24 p53+ cases (62%). by the multifactoriality of drug resistance. Similarly, Schneider & Romero (1995) observed cell mem- Clinical trials of drug resistance reversal represent the brane staining for Pgp in 8/22 p53− (36%) and in six out of ultimate test of a protein’s functional role in clinical drug nine p53+ breast cancers (67%) resistance. Such a role may be established by randomized or In contrast to IHC, which is expected to detect mutant cross-over trials that can demonstrate statistically significant forms of p53, RT-PCR has been used to quantitate the cancer sensitization. To be meaningful, this sensitization expression of normal p53. Ferrero et al. (2000) observed a should be specific to drugs which are the resistance protein’s slight but significant inverse correlation between cytosolic substrates, it should be obtained with chemosensitizing p53 levels and MDR1 expression as detected by RT-PCR in agents that specifically target the protein, and specifically in 85 node-positive patients undergoing surgery and adjuvant cancers where it is expressed. treatment. Another study failed to detect any association between p53 mutations and de novo expression of MDR1 in Pgp/MDR1 expression and response to treatment 64 patients undergoing neoadjuvant chemotherapy Most published studies relating MDR1/Pgp expression to (Chevillard et al. 1997). prognosis in the adjuvant setting have used IHC to detect Overall, with the only exception of the study by Chevil- Pgp. The results of most evaluations support a correlation lard et al. (1997), these studies are consistent with basic between Pgp expression and prognosis. research studies indicating the ability of normal p53 to Among 205 patients undergoing a variety of treatments, repress the transcription of MDR1 and conversely an increase Gago et al. (1998) observed a higher incidence of subsequent in the expression of MDR1 in the presence of p53 mutants metastatic recurrences in the group of patients whose primary (Goldsmith et al. 1995, Strauss & Haas 1995, Strauss et al. tumor had been Pgp+ than in those with Pgp− tumors (27 vs 1995, Chevillard et al. 1997, Thottassery et al. 1997). How- 16% respectively). The prognostic association was statisti- ever, these basic research data have sometimes been contra- cally significant by univariate but not by multivariate ana- dicted by the results of other studies (Li et al. 1997, Zhou & lysis. The correlation of Pgp expression with recurrence rates Kuo 1998). Both at the in vitro and at the clinical level, the did not consider the kind of treatment, whether or not MDR1 relationship between p53 and MDR1/Pgp is likely to be com- substrates were used. plex and affected by both the kind of p53 mutations and the In another study, evaluating 50 patients treated with particular cell signaling environment. MDR1 substrate-containing regimens (doxorubicin or vincristine), overall survival (OS) was significantly better for Clinical role of Pgp 27 Pgp− than for 23 Pgp+ patients, survival rates at 60 Ultimately, the purpose of measuring Pgp expression is in its months being 70 and 26% respectively. Statistical signifi- potential clinical role, either as a marker of prognosis and/or cance of the difference was confirmed by multivariate ana- as a predictor of response to treatment. If Pgp plays such a lysis (He et al. 1995). role, knowledge of its expression may be used for the selec- Linn et al. (1995) compared Pgp− breast cancers (73 tion of more effective treatments, for example including cases) with cancers where both the cancer cell and the inter- anticancer drugs that are not substrates for Pgp, or inhibitors mixed desmoplastic stroma components stained positive for of Pgp function. Pgp (four cases). This study demonstrated differences at 3 The clinical role of a drug-resistance protein can be eval- years in both DFS (respectively 80 and 25%) and OS rates uated in different ways. To support the specific protein’s role (respectively 92 and 25%). These differences are significant

56 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:06:29PM via free access Endocrine-Related Cancer (2003) 10 43–73 by univariate analysis. Interestingly, when patients were In terms of correlation with survival parameters, the pub- compared on the basis of Pgp expression only in cancer cells, lished data show results that range from none to a significant there was no difference in DFS, and a much smaller differ- survival advantage for Pgp− cases. Verrelle et al. (1991) ence in OS. It is difficult to relate this prognostic impact to observed that progression-free survival was significantly an anticancer drug treatment, as this was limited to a fraction worse in the seven patients (out of a total of 17) expressing of the patients and it was quite heterogeneous: of 27 patients the highest levels of Pgp (more than 75% cells staining treated in the adjuvant setting, 11 were treated with a CMF strongly for Pgp), following neoadjuvant treatment with a (cyclophosphamide, methotrexate and fluorouracil) regimen, chemotherapy regimen containing MDR1 drugs (doxorubicin only one with a regimen containing an MDR1 substrate drug, and vincristine). The study by Vargas-Roig et al. (1999), who and 15 were treated with tamoxifen or a chemo-hormonal used a quite stringent criterion for Pgp positivity (more than combination. Interestingly, the clearest negative prognostic 66% of cancer cells staining positive), reports 22 Pgp+ cases impact of Pgp was on the group of tamoxifen-treated out of a total of 37. Following neoadjuvant treatment with patients. doxorubicin- or epirubicin-containing chemotherapy, there In the only study where MDR1 mRNA expression was was no significant difference in OS and DFS based on Pgp evaluated by RT-PCR, no significant correlation was found status, although, in both cases a trend for a worse survival between MDR1 mRNA levels and progression-free survival can be noticed for Pgp+ cases. Honkoop et al. (1998) or OS of 85 node-positive patients treated with anthracyc- observed no significant difference in DFS and OS in patients line-containing adjuvant regimens (Ferrero et al. 2000). treated with doxorubicin and cyclophosphamide, based on Effect of pre- and post-treatment Pgp expression on the pre-treatment Pgp status only (Pgp+ status being defined as response of locally advanced breast cancer to neoadjuvant the presence of < 20% positively stained cells; 9/27 cases chemotherapy is summarized in Table 10, which shows the were considered positive). In the same study, nuclear accu- results of IHC studies evaluating the correlation of pre- and mulation of p53 did not appear to have an impact on prog- post-treatment Pgp expression with the rates of response to nosis. However, when cancers were positive for both Pgp neoadjuvant chemotherapy in locally advanced breast and p53, both DFS and OS were significantly worse. cancers. Pgp expression after treatment with anthracycline- Comparing pooled data, the presence of detectable containing chemotherapy appears to be more predictive of amounts of Pgp before treatment appears to correlate with response than its expression before chemotherapy (Ro et al. the CR rate (14%, or 10/72 in Pgp+, vs 36%, 40/112 in 1990, Koh et al. 1992, Botti et al. 1993, Decker et al. 1995, Pgp− cases), but not the OR rate (about 63% in both Chevillard et al. 1996, Chung et al. 1997) (Table 10). The cases, or 34/54 in Pgp+ vs 60/95 in Pgp−, across seven pooled objective response rates are 93% (56/60 patients) studies) following treatments containing anticancer drugs among Pgp− cases vs 52% (45/82) among Pgp+ cases. that are MDR1 substrates (Verrelle et al. 1991, Decker et Pooled CR rates are respectively 17 and 7.5%. In terms of al. 1995, Chevillard et al. 1996, Chung et al. 1997, Wang impact on survival parameters, a statistically significant asso- et al. 1997, Honkoop et al. 1998, Bottini et al. 2000, ciation between post-chemotherapy Pgp expression and DFS Faneyte et al. 2001, Schneider et al. 2001). In the only is suggested by one IHC study (Botti et al. 1993). As in the study considering the response to non-MDR1 chemotherapy case of pre-treatment expression, in the study by Honkoop et (cyclophosphamide, methotrexate and 5-fluorouracil, plus al. (1998) an association of post-treatment Pgp expression tamoxifen), nearly identical percentages of complete with OS was significant only when combined with positivity responders were observed (respectively 29 and 30%) in for p53. Pgp+ and Pgp− cancers (Bottini et al. 2000). The apparently higher predictive value of Pgp expres- sion, when it is detected after chemotherapy, suggests the possibility that inducibility of Pgp expression by treatment Table 10 Effect of cancer Pgp status on the response to may be a more important predictor of response to chemo- neoadjuvant chemotherapy including MDR1 substrates therapy than measuring only Pgp expression, whether before or after chemotherapy. Results of the study by Chevillard Response rate No. of et al. (1996) indicate a significant correlation between rapid Pgp + Pgp − studies induction of Pgp (rapid Pgp increase by 8 days after start of Pgp evaluated before treatment treatment) and both lower DFS and OS. Interestingly, this OR 34/54 (63.0%) 60/95 (63.2%) 7 correlation was not statistically significant when a slower CR 10/72 (13.9%) 40/112 (35.7%) 7 induction (by day 28) was considered. The neoadjuvant treat- ment in this study included an MDR1 substrate Pgp evaluated after treatment (anthracycline) in most cases (42/53). OR 43/82 (52.4%) 56/60 (93.3%) 6 The results of RT-PCR studies of MDR1 mRNA expres- CR 5/67 (7.5%) 7/42 (16.7%) 5 sion in the neoadjuvant setting are inconsistent and overall www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202157 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer fail to support a correlation between higher pre- and post- was no apparent difference in response rates and survival in treatment MDR1 mRNA levels and clinical resistance to Pgp+ and Pgp− patients. Of the other, smaller studies con- chemotherapy. sidering metastatic patients, three (Verrelle et al. 1991, In one study considering the relationship between pre- Schneider & Romero 1995, Buser et al. 1997) detected Pgp treatment MDR1 mRNA and clinical response, a signifi- in all cases, and two (Chevillard et al. 1997, Linn et al. 1997) cantly higher response rate was observed in MDR1− (22/35) in none. Among the remaining, Cox proportional hazard than in MDR1+ (4/15) cases, following treatment with four regression analysis of the data from Gregorcyk et al. (1996) cycles of an anthracycline-containing regimen suggests a 5-year recurrence-free survival of 57% in stage (cyclophosphamide, 5-fluorouracil, doxorubicin) (Chevillard IV Pgp− patients vs 2% in stage IV Pgp+ patients. It has to et al. 1996). In contrast, MDR1 expression was not predictive be noted, however, that these conclusions are based on four of the response to treatment with a combination of cyclopho- metastatic patients, two of whom are Pgp+ and two Pgp−. sphamide, 5-fluorouracil and thiotepa (none of which is an Overall, there are no grounds to support a significant impact MDR1 substrate). These data suggest a direct role for Pgp in of Pgp, by itself, on the prognosis and response to chemo- affecting response to chemotherapy (Chevillard et al. 1996). therapy of metastatic patients. Wang et al. (1997) observed that MDR1 detection by RT-PCR predicted the response to tamoxifen but not to chemotherapy with doxorubicin. Arnal et al. (2000) did not Pgp expression and response to endocrine observe any significant correlation between RT-PCR therapy detected pre-treatment MDR1 mRNA levels and clinical Linn et al. (1995), measured Pgp expression before treatment response rate in 40 patients. and found that early breast cancer patients treated with As for the MDR1 mRNA expression following treat- tamoxifen had a 3-year survival of 27% in five Pgp+ cases, ment, in the study by Schneider et al. (2001), clinical and 85% in 15 Pgp−. Another study (Keen et al. 1994) eval- response rates following treatment with anthracycline- uated the correlation between Pgp immunostaining, before containing regimens were paradoxically somewhat higher for and after a 3-month treatment with tamoxifen (prior to loco- MDR1+ (18 responses out of 22, including two CRs) than regional treatment), and response rates in 43 patients, aged for MDR1− cases (15 responses out of 23, with no CRs). over 70. Patients whose tumors did not express Pgp after The study by Arnal et al. (2000) shows no association treatment were significantly more likely to respond to tamox- between post-treatment MDR1 mRNA expression and clini- ifen, response rates being 85% (22/26) in Pgp− vs 35% (6/ cal response, nor between MDR1 mRNA expression and 17) in Pgp+ cases. Differences in response rate based on Pgp DFS and OS, following neoadjuvant treatment with expression before tamoxifen treatment were less striking and 5-fluorouracil, doxorubicin and cyclophosphamide. These statistically not significant. Pre-treatment expression of results are essentially confirmed by the outcome of the study MDR1 mRNA, as detected by RT-PCR, but not IHC detec- of Faneyte et al. (2001) who observed no significant differ- tion of Pgp, appeared to correlate significantly with response ence in MDR1 mRNA levels between 12 responders and 11 to tamoxifen treatment in 15 locally advanced breast cancers non-responders. (Wang et al. 1997). Since Pgp does not confer resistance to tamoxifen (Clarke et al. 1992), and tamoxifen, although an Chemotherapy in metastatic breast cancer inhibitor of the MDR1 phenotype (Leonessa et al. 1994), is Ten studies evaluated the prognostic impact of different fac- not effluxed by Pgp (Callaghan & Higgins 1995) in in vitro tors in breast cancer, included the evaluation of Pgp in meta- models, it is unclear whether and how Pgp may have a causal static/recurrent cancers (Verrelle et al. 1991, Hennequin et role in resistance to tamoxifen. al. 1993, Decker et al. 1995, Linn et al. 1995, Schneider & Binding and transport by Pgp has been demonstrated in Romero 1995, Seymour et al. 1995, Gregorcyk et al. 1996, vitro for several steroids (Qian & Beck 1990, Wolf & Buser et al. 1997, Chevillard et al. 1997, Linn et al. 1997). Horwitz 1992, van Kalken et al. 1993, Gruol & Bourgeois Taken together, these studies include 162 metastatic and/or 1994, Barnes et al. 1996). Moreover, several steroids and recurrent (mostly metastatic) cases, some of whom had pre- steroid derivatives appear to reverse the multidrug resistance viously been treated with chemotherapy. Ninety-three (57%) conferred by Pgp, possibly by a competitive mechanism of these cases resulted positive for Pgp/MDR1, 91/156 at the (Naito et al. 1989, Yang et al. 1989, Hu et al. 1991, Abraham protein and two of six at the mRNA level. Only one study et al. 1993, Zalcberg et al. 1993, Gruol & Bourgeois 1994, (Seymour et al. 1995), included more than 12 metastatic Gruol et al. 1994, Yang et al. 1994, Barnes et al. 1996, patients. In this study, Pgp expression was detected in 56/ Gruol & Bourgeois 1997, Tansan et al. 1997, Leonessa et 101 (55%) of metastatic or recurrent patients, a minority of al. 2002). Among steroids (and derivatives), glucocorticoids whom had previously been treated with tamoxifen with or generally represent good substrates, while progesterone, without adjuvant chemotherapy. Following treatment with medroxyprogesterone acetate, testosterone and androstene- chemotherapy (mostly containing MDR1 substrates), there dione are poor substrates (Barnes et al. 1996). Pgp’s role in

58 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:06:29PM via free access Endocrine-Related Cancer (2003) 10 43–73 the clinical resistance to steroidal agents has been explored levels of Pgp expression, as defined by IHC, and the intensity in one study: the results show no correlation between Pgp of inhibition of thymidine incorporation, was observed for expression before treatment and response rate and survival the MDR1 substrates doxorubicin (171 cases) and paclitaxel of patients with metastatic disease treated with different (131 cases), but not for 5-fluorouracil (164 cases), which is endocrine therapies, including gestagens and testosterone not an MDR1 substrate. (Seymour et al. 1995). As in most other instances, the study Finally, using an anchorage-dependent clonogenic assay was not specifically aimed at investigating the role of Pgp in for the evaluation of resistance to doxorubicin, Keith et al. resistance to hormonal treatments, and included only 20 (1990) observed a non-significant correlation between patients treated with gestagens and six with testosterone. doxorubicin IC50 and the level of MDR1 mRNA expression in cell cultures from 14 breast cancer biopsies.

Correlation of clinical Pgp expression with in vitro chemosensitivity Clinical reversal of the MDR1 phenotype Studies relating in vitro drug resistance of primary breast The results of clinical trials of MDR1-reversing agents in cancer cell cultures to MDR1/Pgp expression are few and drug-resistant breast cancer are summarized in Table 11 heterogeneous for both models and methodological approach. and are reviewed here with a focus not on the efficacy of Nonetheless, the results obtained in these studies consistently the test agents, but as a possible proof of principle for show a correlation between Pgp expression and in vitro Pgp’s causative role in breast cancer’s clinical drug resist- resistance to the MDR1 anticancer substrate doxorubicin, and ance. Most of these studies are represented by ‘cross-over’ are supportive of a functional role of Pgp in clinical drug trials, whereas patients refractory to a specific treatment resistance. are then treated again with the same treatment, but with the Salmon et al. (1989) used IHC to evaluate the levels of addition of an MDR1-reversing agent. Two are randomized Pgp expression in 35 breast cancer specimens. Chemosensi- studies. tivity was evaluated by an anchorage-independent clonogenic Verapamil has been the drug most widely investigated assay on primary cultures from 13 cancers, and defined as a as an MDR1− reversing agent in breast cancer. Results of reduction of the clonogenic efficiency of untreated controls a single randomized study (48 patients) showed no differ- to less than 50% following treatment with 0.1 µg/ml doxoru- ence in response rate following treatment with epirubicin bicin. Resistance to doxorubicin was observed in two out of with and without verapamil (Mross et al. 1993). However, eight (20%) Pgp− and five out of five (100%) Pgp+ cases. a small, but positive number of objective responses were This study also included 13 cases of lymphoma and myel- consistently observed in four different cross-over trials, oma. When all the cases were considered, the correlation where verapamil (mostly R-verapamil) was added to between Pgp staining and in vitro resistance to doxorubicin anthracycline-containing regimens. Response rates of 17% was statistically significant. (4/23) and 14% (2/14) were respectively reported in the IHC was used also by Veneroni et al. (1994). A signifi- cross-over arms by Lehnert et al. (1998) and by Thurlim- cant association was observed between Pgp expression of 39 ann et al. (1995) following treatment with a combination breast cancers and their in vitro drug resistance, as measured of R-verapamil and epirubicin. A similar result, with a by [3H]uridine incorporation. response rate of 10% (2/20) was observed by Warner et Sanfilippo et al. (1991) analyzed Pgp expression in 35 al. (1998), who used a regimen that included R-verapamil breast cancers using Western blotting. Chemosensitivity was and a variety of anthracyclines. Taylor et al. (1997) treated defined in terms of inhibition of [3H]thymidine incorporation 27 advanced breast cancer patients, refractory to a regimen into tissue fragments from different areas of the tumors. of cyclophosphamide, vincristine, doxorubicin and dexame- Single drug concentrations were used, and 20% inhibition of thasone, with the same regimen plus verapamil and quinine, incorporation was used as the cut-off to define sensitivity obtaining a 19% (5/27) objective response rate. The results of or resistance. In this study, a comparatively high fraction of a fifth verapamil trial are consistent with the others (3/16 samples was classified as resistant, perhaps because the cyto- responses), but their interpretation is uncertain because of a toxic drugs were tested at relative concentrations. Resistance flawed cross-over design: in this trial, anthracycline-resistant to doxorubicin was observed in ten out of ten Pgp+ and in patients where treated with verapamil and doxorubicin, but 17/25 Pgp− cases. Ten out of ten Pgp+ and 19/25 Pgp− cases also with vincristine (Ries & Dicato 1991). Overall, when the were classified as resistant to vincristine. Simultaneous data from the first four studies are pooled, verapamil appears resistance to both drugs was observed in all ten Pgp+ cases to re-sensitize 15% (13/84) of advanced breast cancer and in 14/25 Pgp− cases. patients refractory to anthracycline-containing regimens. By A thymidine incorporation endpoint was also used by comparison, the response rate to an alternative second-line Mechetner et al. (1998) in the largest study of the kind pub- chemotherapy in patients already exposed to doxorubicin is lished to date. A significant inverse correlation between about 21% (Henderson 1987). www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202159 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

Table 11 Clinical studies of MDR1 reversal Reference Trialdesign No. of Treatment* Results patients

Ries & Dicato (1991) Anthracycline-resistant patients 16 Dox,Vcr + verapamil RR = 3/16 Mross et al. (1993) Randomized 48 Epirub ± verapamil No difference in RR and survival Thurlimann et al. (1995) Cross-over 14 Eprib + R-verapamil RR = 2/14** Taylor et al. (1997) Cross-over 27 CVAD + verapamil and RR = 5/27 quinine Lehnert et al. (1998) Cross-over 23 Epirub + R-verapamil RR = 4/23 Warner et al. (1998) Cross-over 20 Anthracycline + R-verapamil RR = 2/20 Belpomme et al. (2000) Anthracycline-resistant patient 99 Vds/FU RR = 5/47 Vds/FU + verapamil RR = 14/42*** Bates et al. (1995) Patients generically refractory 33 Dox or Vbl + amiodarone RR = 9/33 to chemotherapy van Kalken et al. (1991) Cross-over 5 Epirub or Dox + bepridil RR = 0/5 Wishart et al. (1994) Randomized 213 Epirub and No difference in RR Prdnsl + quinidine and survival Toppmeyer et al. (2002) Cross-over 35 Paclitaxel + biricodar RR = 4/35 Murren et al. (1996) Anthracycline-refractory patients 16 Vbl + trifluoperazine RR = 1/6 RR,response rate. *First-line: anticancer agents; second line: MDR1-reversing agent. CVAD,cyclophosphamide,vincristine,adriamycin and dexamathasone; Dox,doxorubicin; epirub,epirubicin; Vbl,vinblastine; Vcr,vincristine; Prdnsl,prednisolone; FU,fluorouracil. **Data from this study may have been included in the subsequent paper by Lehnert et al. (1998). ***P = 0.04.

In a second randomized study (213 patients), quinidine mRNA levels, detected by RT-PCR, were significantly higher failed to improve the response to a combination of epirubicin in breast cancer than in normal breast tissues. and prednisolone (Wishart et al. 1994). Another MDR1− MRP1 mRNA was detected by RT-PCR in 98% (429) of reversing agent, biricodar, when added to paclitaxel, obtained 437 untreated breast cancers (data pooled from six studies). a partial response in 4/35 (11%) patients with locally advanced In five out of these six studies MRP1 was detected in 100% or metastatic disease refractory to paclitaxel (Toppmeyer et al. of cases (Table 12). When an IHC approach was used, MRP1 2002). A study by Bates et al. (1995) reported a 27% (9/33) was detected, overall, in 49% of cases (213/432, across five response rate to amiodarone (chemosensitizer) in combination studies) (Table 12). In one of these studies (Dexter et al. with doxorubicin or vinblastine. However, patients in this 1998), MRP1 was detected in all 20 cases. study were selected for a generic resistance to chemotherapy, There is no convincing evidence of a correlation between as opposed to a specific resistance to respectively doxorub- MRP1 expression and tumor size, lymph node status and icin and vinblastine. In two more cross-over studies, bepridil metastatic status, histological grade and type, ER and PgR and trifluoperazine were ineffective in sensitizing breast status, age and menopausal status. Three studies used IHC to cancer patients to respectively epirubicin or doxorubicin address the correlation between MRP1 tumor size. No corre- = = (response rate 0/14), and vinblastine (response rate 1/16). lation was found in the study by Nooter et al. (1997a): MRP1 was found to be expressed in 23/76 (30%) T1, 50/148 MRP1 and breast cancer (34%) T2, and in 12/33 (36%) T3 tumors. Filipits et al. (1999) observed no significant difference in T stage distribu- Expression in normal breast and in breast tion between 20 MRP1− and 80 MRP1+ primary breast cancer, effect of treatment and association with cancer patients. However, MRP1 was detected more often in other markers operable primary (16/20) than in locally advanced cancers MRP1 is readily detected by both RT-PCR and IHC in both (two of ten before neoadjuvant treatment) in the study by normal breast and breast cancer (Table 12). In a study by Linn et al. (1997). In an isolated RT-PCR study, there was Dexter et al. (1998), MRP1 mRNA was detected by RT-PCR no correlation between T stage and MRP1 mRNA expression in the normal tissues adjacent to all of 55 breast cancers exam- (Kanzaki et al. 2001). ined. Using IHC, Linn et al. (1997) found MRP1 expression in No correlation was observed between MRP1 protein the epithelial component, but not in the stroma, of three of six (Nooter et al. 1997a, Filipits et al. 1999) or mRNA (Lacave normal breast samples. Ito et al. (1998) reported that MRP1 et al. 1998, Kanzaki et al. 2001) expression in the primary

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Table 12 MRP1 expression in untreated breast cancer protein or mRNA level, and histological grade. However, Reference Setting Antibodies Pgp + / in one study significantly higher levels of MRP1 mRNA total were reported by Beck et al. (1998) in grade 2 than in IHC studies grade 3 cancers. Linn et al. (1997) Operable MRPr1 16/20 Two studies evaluating the correlation between MRP1 Locally 2/10 expression and ER and PgR expression found opposite advanced trends, although correlations were not statistically signifi- Nooter et al. (1997a) Operable MRPr1 87/259 cant in either study (Dexter et al. 1998, Filipits et al. Nooter et al. (1997b) Recurrence MRPr1 8/23 1999). A third study, with 43 patients, by Kanzaki et al. Dexter et al. (1998) Mixed QCRL1 20/20 (MRPm6) (19/19) (2001) found no correlation between MRP1 mRNA levels Filipits et al. (1999) Primary,no QCRL1 80/100 and ER and PgR status. metastases* QCRL3 No correlation was observed between MRP1 expression Total 213/432 and age (Nooter et al. 1997a, Dexter et al. 1998, Lacave (49.3%) et al. 1998, Filipits et al. 1999, Kanzaki et al. 2001), RT-PCR studies menopausal status (Filipits et al. 1996, Nooter et al. Filipits et al. (1996) Primary 134/134 1997a), and histological type (Dexter et al. 1998, Lacave cancer et al. 1998, Filipits et al. 1999). (mostly operable)** A correlation between MRP1 and MDR1 mRNA Dexter et al. (1998) Mixed 74/74 expression has been reported in two studies (Beck et al. Ito et al. (1998) Primary 19/27 1998, Kanzaki et al. 2001), but not in a third (Lacave et operable al. 1998). No correlation was found at the level of protein Lacave et al. (1998) Primary 74/74 expression (Filipits et al. 1996). A direct correlation has operable been observed between MRP1 and the expression of drug- Ferrero et al. (2000) Primary 85/85 operable resistance proteins LRP (Beck et al. 1998) and GSTB Kanzaki et al. (2001) Primary 43/43 (Lacave et al. 1998). Single studies have also reported a operable direct correlation between the expression of MRP1 and Total 429/437 PKCη (Beck et al. 1998), and an inverse correlation with (98.2%) the cancer S-phase fraction (Ferrero et al. 2000). *The number of cases includes three patients who had Only in one study was the effect of chemotherapy on undergone induction chemotherapy. MRP1 expression considered. Using IHC, Linn et al. **Assumed to be untreated (not described in manuscript). (1997) detected MRP1 respectively in two of ten (20%) and in 9/16 (56%) locally advanced breast cancer cases before and following neoadjuvant chemotherapy with an lesion and lymph node status. Zochbauer-Muller et al. (2001) anthracycline-containing regimen. The results, although observed a higher MRP1 protein staining intensity in lymph limited by the low number of cases, suggest an inducing nodes than in their corresponding primary tumors. effect of treatment, similar to that observed for MDR1/Pgp Four studies evaluated MRP1 expression in metastatic and non-metastatic patients, but the number of metastatic expression. patients involved was always small. Filipits et al. (1996), MRP1 expression and prognosis detected strong MRP1 staining in 12/57 non-metastatic and in three out of four metastatic patients. Linn et al. (1997) The effect of MRP1 expression on the short-term response detected MRP1 in one out of five metastatic patients vs to chemotherapy was considered in two small studies. Linn two out of ten in the locally advanced (untreated) and 16/ et al. (1997) observed no correlation between pre-treatment 20 in operable primary setting. In the RT-PCR study by MRP1 protein expression and the pathological response of Dexter et al. (1998), only four patients were metastatic: locally advanced cancers to a neoadjuvant regimen includ- the average level of MRP1 mRNA was apparently lower ing doxorubicin and cyclophosphamide. Only two of the in these patients than in less advanced cases, but the differ- cases were MRP1+. The second study, by Nooter et al. ence was not statistically significant. Using RT-PCR, Kan- (1997b), evaluated the impact of MRP1 expression on the zaki et al. (2001) observed no statistically significant differ- prognosis and response to chemotherapy of recurrent breast ence in metastatic involvement between 21 MRP1+ and cancer. In previously untreated patients, the presence of 22 MRP1− cases. MRP1 protein was associated with a lower response rate In most cases (Filipits et al. 1996, 1999, Dexter et al. (one of eight in MRP+ cases vs 6/15 in MRP− cases). No 1998, Lacave et al. 1998, Ferrero et al. 2000), no correla- association between MRP1 and response rate was ob- tion was observed between MRP1 expression, at either the served in patients who had already been treated with www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202161 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer chemotherapy previously. This paper does not specify the a multidrug-resistant phenotype (Piwnica-Worms et al. 1993, anticancer drugs used in the study. Ballinger et al. 1995, Barbarics et al. 1998, Hendrikse et al. A correlation of MRP1 expression with survival para- 1998, Moretti et al. 1998, Vergote et al. 1998, Chen et al. meters appears to be supported by the results of most, but 2000). not all studies. Relapse-free survival and OS were reported Several clinical studies have confirmed an inverse correla- to inversely correlate with MRP1 protein expression by tion between the tumor/background ratio or fractional reten- both Filipits et al. (1999) and Nooter et al. (1997a). Filipits tion of both 99mTc-sestamibi and 99mTc-tetrofosmin and Pgp et al. observed MRP1 expression in 80% of 100 non- expression in human breast cancer (Del Vecchio et al. 1997, metastatic cases: MRP1 expression correlated with shorter Kostakoglu et al. 1998, Yoon et al. 1999, Sun et al. 2000, OS (statistically significant) and DFS (nearly significant), Cayre et al. 2002, Mubashar et al. 2002). In one study, the but results were not analyzed in relation to treatment. In maximum inverse correlation with 99mTc-sestamibi accumula- the study by Nooter et al., MRP1 expression was associ- tion was observed in cancers that expressed both Pgp and ated with an increased risk for failure in the subgroups of MRP1 (Kao et al. 2001). patients with T1 cancers, with node-negative cancers and If Pgp and MRP1 play a role in breast cancer resistance with node-positive cancers treated with CMF (as opposed to chemotherapy, the accumulation/wash-out of 99mTc- to node-positive patients receiving hormonal or no adjuvant sestamibi and 99mTc-tetrofosmin at the tumor level should be treatment). Similarly, the risk of death correlated with predictive of clinical responsiveness to anticancer drugs that MRP1 expression in T1 cancer, and in N+ cancers treated are Pgp and/or MRP1 substrates. Ciarmiello et al. (1998) with CMF. In another study by Nooter et al. (1997b), observed pathological responses (described as the absence of previously untreated relapsed patients whose cancers were any macroscopic and microscopic evidence of disease) to a MRP+ had a shorter time to disease progression. neoadjuvant treatment with epirubicin in only 2/17 (12%) Two studies out of three reported no correlation breast cancer patients showing a rapid clearance of 99mTc- between MRP1 mRNA expression (evaluated using sestamibi from their tumors (i.e. decreased tumor retention), RT-PCR) and prognosis. Ferrero et al. (2000) observed no as compared with 14/22 (64%) patients whose tumors correlation between MRP1 mRNA levels and progression- exhibited prolonged retention. Similarly, in a study by Cayre free survival and OS of patients treated with anthracycline- et al. (2002), including 45 patients, a negative 99mTc- containing adjuvant regimens. Similarly, Kanzaki et al. sestamibi scintimammography predicted resistance to anthra- (2001) observed no correlation between MRP1 mRNA cycline-containing neoadjuvant chemotherapy, as evaluated expression and relapse after adjuvant treatment with in terms of both clinical and pathological response. Alonso doxorubicin. However, in a study by Ito et al. (1998), et al. (2002) observed that tumor/background tissue uptake including 27 patients, an increased risk of relapse in the 10 ratios of 99mTc-sestamibi were significantly higher in 43 years following adjuvant chemotherapy (mostly including patients who responded than in six patients who did not mitomycin C) was observed in patients whose primary respond to treatment with fluorouracil, doxorubicin and tumors expressed higher levels of MRP1 mRNA. cyclophosphamide. Tumor retention of 99mTc-tetrofosmin Overall the above studies appear to indicate a trend appears to be predictive of breast cancer response to chemo- for a correlation between early expression of MRP1 protein therapy as well — Takeuchi et al. (2002) observed response and a worse prognosis. As in the case of Pgp, and even rates of 77% (10/13) in breast cancer patients with high more so, the question of a direct causal role of MRP1 on retention, but of only 8% (1/12) in patients with low retention drug resistance remains open. of 99mTc-tetrofosmin, following treatment with a polychemo- therapy regimen including an anthracycline. There are two reasons why scintigraphic evaluation of Functionalassessment of Pgp and MRP1 in the tumor retention of technetium-99m substrates of Pgp may breast cancers by technetium-99m be expected to be more predictive of clinical drug resistance radiopharmaceuticals than just the evaluation of Pgp expression. First, retention/ 99mTc-sestamibi and 99mTc-tetrofosmin are two lipophilic wash-out of these substrates provides a measure of function 99mTc-labeled agents originally used for the evaluation of which is not provided by expression studies. Pgp might be myocardial perfusion (Wackers et al. 1989, Maddahi et al. expressed without necessarily being functional, although the 1991). These agents are also used for the scintigraphic studies mentioned show a good correlation between Pgp detection of malignant tissues, including breast and lung expression and technetium-99m complex retention. Sec- cancer and sarcomas (Hassan et al. 1989, Caner et al. ondly, it is evident that both 99mTc-sestamibi and 99mTc- 1991, Kostakoglu et al. 1998, Berghammer et al. 1999). tetrofosmin represent substrates not only for Pgp, but also for Studies carried out on cell lines, in vitro and in vivo, have MRP1. Indeed expression of both proteins appears to shown that both agents are substrates for both Pgp and MRP1, inversely correlate with tumor retention better than expres- but not for BCRP/MXR, another ABC protein able to confer sion of either protein by itself (Kao et al. 2001). That these

62 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:06:29PM via free access Endocrine-Related Cancer (2003) 10 43–73 agents may also be substrates for other transporters cannot suggests a similarly inducing effect of chemotherapy on be excluded at present. Retention of technetium-99m tracers MRP1 expression. may actually provide a measure of the cumulative function Is this effect of chemotherapy on Pgp expression the of different transporters involved in cancer drug resistance. result of a true induction or of a selective mechanism? Since Overall, the available evidence on the relationship increased expression is more evident following treatment between retention of 99mTc-sestamibi and of 99mTc- with drugs that are MDR1 substrates, a selective effect is tetrofosmin and response to chemotherapy is consistent with certainly possible. However, the lower expression of Pgp in a role of Pgp and MRP1 as factors in breast clinical drug remotely treated cancers suggests that the increased expres- resistance. A more convincing assessment of the role of each sion that follows treatment is a transient effect. Moreover, transporter will be provided by use of scintigraphic substrates the results of at least one study demonstrate an increase in that are more specific for each transporter. For example, leu- MDR1 mRNA expression following both an MDR1 sub- kotrienes are more specific substrates for MRP proteins and strate-containing and an MDR1 substrate-devoid neoadjuvant could be used, in an ‘inactivated’ form, to more specifically chemotherapeutic regimen (Chevillard et al. 1996). In this image MRP1 and MRP2 function. Other substrates, that are study, the only difference between the two regimens was that more specific for Pgp, may be represented by [11C]colchicine the induction appeared slower in the presence of the MDR1 and [11C]verapamil (Hendrikse et al. 1999). substrate. This non-specific induction may represent a con- firmation at the clinical level of the ability of different kinds of ‘stress’ stimuli to induce MDR1 expression, which has Conclusions already been observed in several in vivo and in vitro models (Chin et al. 1990, Fairchild & Cowan 1991, Miyazaki et al. Since 1997, Pgp and MRP1 have been evaluated in breast 1992, Chaudhary & Roninson 1993, Uchiumi et al. 1993a,b, cancer specimens mostly by IHC and RT-PCR. Moreover, Ohga et al. 1998, Hu et al. 1999, 2000, Stein et al. 1999). some of the more recent studies are based on the evaluation A similar inducing effect of chemotherapy has also been of the proteins’ function. This has been done in terms of in reported for MRP1 (Ishikawa et al. 1996, Linn et al. 1997). vivo evaluation of the tumor accumulation and wash-out of The data reviewed in this paper appear to confirm that, in technetium-99m radiopharmaceuticals. The data pooled from breast cancer, the expression of the mutant form of p53, a the available literature demonstrate that IHC detects Pgp and well-known factor in stress-induced cell response (Soussi MRP1 respectively in 41 and in 49% of untreated breast can- 2000), correlates with Pgp expression. In the study by Chev- cers. The figures for RT-PCR-based detection of MDR1 and illard et al. (1996), the correlation of a more rapid increase MRP1 mRNA are respectively 63 and almost 98%. In these in Pgp/MDR1 levels with clinical drug resistance appears to cancers, Pgp and MRP1 expression inversely correlate with further support an induction effect. Finally, while the overall the tumor/background ratio or fractional retention of 99mTc- available data seem to support an induction phenomenon, it sestamibi and 99mTc-tetrofosmin. Pgp and MRP1 are also is still possible that selection of Pgp+ cells by anticancer detected in normal breast tissue, although usually at lower drugs is also involved. levels than in cancer. Correlations of Pgp expression with other clinical and Pooling the results of older and more recent studies pathological parameters are much less compelling. There is essentially confirms the effect of previous chemotherapy on no difference in the expression of either Pgp protein or Pgp expression, reported in our earlier meta-analysis (Trock mRNA between primary operable and locally advanced can- et al. 1997). Overall, IHC detects Pgp in 52% of cancers cers, although there appears to be at least a trend to lower previously treated with anticancer drugs that are MDR1 sub- rates of Pgp protein expression in primary tumors smaller strates (vs 40% in the untreated cases). This effect is evident than 2 cm (T1). Small trends have also been reported for a in recently treated (Pgp expression rate: 56%), but not in correlation of lower MDR1 mRNA levels with aneuploidy remotely treated patients (expression rate: 36%). There (two out of three studies) and of higher levels with lobular appears to be no overall increase in Pgp expression rate carcinoma (two out of three studies). Overall, data are mixed, (37%) in the pool of cases whose treatment was either unde- negative, or insufficient to support a correlation between Pgp fined or did not include MDR1 substrates. The effect of and tumor grade, ER or PgR expression. Correlative data are, chemotherapy is particularly striking when Pgp expression is if anything, weaker for MRP1. Overall, the data do not sup- evaluated in the same cancers before and after chemotherapy port a correlation of MRP1 with tumor size, lymph node and in a neoadjuvant setting. Pooled data show expression rates metastatic status, histological grade and type, age, meno- of respectively 43 and 64%, with an induction rate pausal status, ER and PgR. (percentage of cases turning from negative to positive) of Several kinds of evidence appear to support a correlation 37%. Similar evaluations by RT-PCR demonstrate an induc- between Pgp (and MRP1) and clinical drug resistance. Cor- tion rate of 45% (from 50% before to 73% after treatment relative studies based on IHC show an impact of Pgp expres- with Pgp substrate-containing regimens). At least one study sion on the long-term outcome of the treatment of both early www.endocrinology.org Downloaded from Bioscientifica.com at 10/01/202163 05:06:29PM via free access Leonessa and Clarke: ABC transporters and breast cancer

(adjuvant setting) and locally advanced (neoadjuvant setting) Pgp expressed in normal tissues, or through cross-reaction breast cancer. In the neoadjuvant setting, the correlation with P450 enzymes involved in anticancer drug metabolism between Pgp expression and short-term (response rates) and (Harris et al. 1994, Relling et al. 1994, Fischer et al. 1998, long-term (DFS and OS) treatment outcome is stronger when Kim et al. 1999, Yao et al. 2000). The cross-reactivity with Pgp is detected after treatment. Data from one study suggest other ABC proteins (such as MRP1 and MRP2/cMOAT) of that rapid Pgp inducibility during treatment may be a more most presently available MDR1− reversing agents important predictor of drug resistance than post-treatment (Breuninger et al. 1995, Davey et al. 1996, Germann et al. expression alone (Chevillard et al. 1996). This kind of corre- 1997) further complicates the interpretation of trials of lation cannot be discerned in the metastatic setting, partly MDR1 reversal. due to the low numbers of cases considered and to data het- More and better-designed studies are needed to define erogeneity. It is also likely that the contribution of specific whether Pgp, MRP1, and possibly other ABC proteins, play factors to drug resistance is more difficult to discriminate in a significant role as causes of drug resistance, and conse- more advanced stages, where drug resistance may be quently represent valid pharmacological targets in breast expected to involve a larger number of redundant mecha- cancer. nisms. Although fewer data are available about MRP1, The data we have reviewed here, as well as theoretical results of correlative studies also appear to support a correla- considerations, indicate that these studies should be prefer- tion between treatment outcome and MRP1 expression. ably based on the use of methods for the in situ detection of A correlation between Pgp and MRP1, and treatment protein expression (such as IHC). The need for standardiz- outcome is further supported by the observed correlation ation of methodology has been well described (Beck et al. between retention of 99mTc-sestamibi (and 99mTc-tetrofosmin), 1996): calibration of the detection method should be an indirect measure of Pgp and MRP1 activity, and clinical achieved through the use of well-characterized ABC protein- response rates. Pgp’s role is supported as well by the results positive cell lines and tissue positive and negative controls, of ex vivo evaluations, which consistently (in five out of five as well as using adequate antibody controls. Detection by studies) show a correlation between Pgp expression and in multiple antibodies should help increase the specificity of vitro resistance of breast cancers explants to the MDR1 sub- results. A major question which remains to be clarified is that strate doxorubicin. of the definition of adequate ‘cut-offs’ for ‘positivity’. The While a correlation between Pgp expression and treat- criteria used so far have been most heterogeneous, preventing ment outcome appears relatively well defined, it is less clear an adequate comparison of the results of different studies. that Pgp plays a significant role in directly causing clinical Adequate statistical criteria should be applied to define the drug resistance. Imperfect trial design largely contributes to size of the study populations, in order to achieve sufficient this lack of clarity. A direct mechanistic role of Pgp in caus- statistical power. Too many of the published studies include ing clinical drug resistance would imply that the observed too small a number of cases. Future correlative studies evalu- correlation with drug resistance is specific for MDR1 sub- ating the role of an ABC transporter protein should, if pos- strates. Results in this sense are insufficient and contradic- sible, compare the outcome of treatment according to the tory. Most of the correlative studies have been carried out absence or presence of the target protein in cancers, and to only with treatments including Pgp substrates, in the absence the use of anticancer drugs that are or are not substrates for of a control group treated without Pgp substrates. Only in the protein. A causal role for the protein under investigation three studies was such a control present. In two of these will be supported by evidence of a decreased efficacy of sub- studies, one in the adjuvant and the other in the neoadjuvant strate drugs specifically in those cancers which express the setting, correlation of Pgp expression with a worse treatment protein. Functional studies, such as those based on the in vivo outcome was observed in the presence of both kinds of treat- use of tracer substrates, should use agents specific for the ment. Only in the third study was correlation with Pgp target proteins, or use parallel evaluations of protein expres- expression before neoadjuvant treatment specific for the sub- sion. group of patients whose regimen included MDR1 substrates. Ex vivo evaluations, based on the use of explants or pri- We also looked at clinical trials of MDR1− reversing mary cultures obtained from surgical specimens, may need agents for a proof of principle of Pgp’s role in causing drug to be further validated. These methods provide the theoretical resistance in breast cancer. The results of four out of four advantage of controlled conditions in which drug toxicity and cross-over studies where verapamil was added to treatment pharmacokinetics, and certain ethical issues, are not factors. consistently show a small, but positive response to anthracyc- While this approach has been used mostly in correlational line-containing regimens in anthracycline-refractory patients studies, these experimental models might help to provide (average response rate = 15%). However, results were not even stronger proof of principle if used as part of ex vivo evaluated in relation to the cancers’ Pgp expression status trials of drug-resistance reversal. Proof of principle would be and the effect reported in these studies may be due to phar- provided by evidence of chemosensitization which is specific macokinetic interactions through drug-induced inhibition of in cultures/explants that express the target protein and are

64 Downloaded from Bioscientifica.comwww.endocrinology.org at 10/01/2021 05:06:29PM via free access Endocrine-Related Cancer (2003) 10 43–73 resistant to the protein substrates, and that is obtained using Arnal M, Franco N, Fargeot P, Riedinger JM, Brunet-Lecomte P & chemosensitizing agents that are specific for the protein. Lizard-Nacol S 2000 Enhancement of MDR1 gene expression in The definitive proof of the relevance of an ABC protein normal tissue adjacent to advanced breast cancer. Breast Cancer Research and Treatment 61 13–20. or of any other mechanism as a cause of drug resistance will Ballinger JR, Hua HA, Berry BW, Firby P & Boxen I 1995 come from the evidence of drug-resistance reversal in clinical 99Tcm-sestamibi as an agent for imaging P-glycoprotein- trials. 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