INVESTIGATION OF MRP-1 PROTEIN AND MDR-1 P-GLYCOPROTEIN EXPRESSION IN INVASIVE BREAST CANCER: A PROGNOSTIC STUDY Annemarie LARKIN 1 * , Lorraine O’DRISCOLL 1 , Susan KENNEDY 2,3 , Rachel PURCELL 2,3 , Elizabeth MORAN 1 , John CROWN 4 , Michael PARKINSON 5 and Martin CLYNES 1 1 National Institute for Cellular Biotechnology, Dublin City University, Dublin, Ireland 2 Department of Pathology, Royal Victoria Eye and Ear Hospital, Dublin, Ireland 3 Department of Pathology, St. Vincent’s University Hospital, Dublin, Ireland 4 Department of Medical Oncology, St Vincent’s, University Hospital, Dublin, Ireland 5 School of Biotechnology, Dublin City University, Dublin, Ireland The efficacy of breast cancer treatment is limited by the development of resistance to various chemotherapeutic agents. We conducted a retrospective study of the expres- sion of 2 drug resistance efflux pumps, MRP-1 and MDR-1 Pgp, in 177 invasive breast carcinomas. Immunohistochemi- cal expression of these proteins was correlated with clinico- pathologic characteristics as well as relapse-free survival (RFS) and overall survival (OS) times. MDR-1 Pgp was asso- ciated strongly with higher histologic grade (grade III). A highly significant association was shown between MDR-1 Pgp and MRP-1 expression (p < 0.01), 47.4% of patients express- ing both proteins; MRP-1 was expressed in approximately 61% of patients and MDR-1, in approximately 66% of patients. No association was shown in the overall group between ei- ther MDR-1 Pgp or MRP-1 and any of the other clinicopath- ologic features. Kaplan-Meier analysis revealed that in a sub- set of patients with either high-grade (grade III) stage 1 (node-negative) or stage 2 (node-positive) tumours who were treated with surgery followed by adjuvant chemother- apy, MRP-1 expression in <25% of tumour cells at diagnosis was significantly associated with improved RFS (p < 0.02) and OS (p < 0.02). Using multivariate analysis, MRP-1 ex- pression in <25% of tumour cells at diagnosis was identified as an independent, significant prognostic factor for RFS (p < 0.01) and OS (p < 0.01) in this patient group but not in other groups. In this subgroup, no significant correlation was ob- served between expression of MDR-1 Pgp and MRP-1. While the number of patients with high-grade tumours treated with adjuvant chemotherapy was small and further confirmatory research is warranted, it appears that assessment of MRP-1 expression at diagnosis may offer useful prognostic informa- tion in subgroups of patients with stage 1 or stage 2 high- grade tumours who receive CMF-based adjuvant chemother- apy. Given the known substrate specificities of MRP-1, any mechanistic relationship between MRP-1 expression and CMF resistance remains unclear. No association was shown between MDR-1 Pgp expression and either RFS or OS time in any subgroup of patients. © 2004 Wiley-Liss, Inc. Key words: breast cancer; MRP-1; MDR-1; Pgp; immunohistochem- istry; prognosis; relapse-free survival; overall survival Breast cancer is the most common cause of cancer death in the female population in the Western world. 1 Management of breast carcinoma has changed considerably over the past 20 years. The number of available therapeutic options has widened considerably, and there is an increasing range of hormonal, cytotoxic and more recently MAb-targeted drug regimes available for both the adju- vant and neoadjuvant settings. 2 However, despite the advances in both detection and treatment, 40 –50% of patients diagnosed will eventually die of the disease. 3 It is thus vital to determine the optimal treatment modality for each individual patient and to identify subgroups of patients who might benefit from individual- ised treatment strategies. Identification of biologic markers which might predict clinical outcome (prognostic markers) and the like- lihood of a response to a particular type of adjuvant therapy (predictive markers) will facilitate this. Treatment of breast cancer patients with operable disease is stage-dependent. Stage 2 patients (those with involvement of ax- illary nodes) have a 10-year average survival of 50% and will receive adjuvant chemotherapy following surgery. 4 Stage 1 pa- tients (node-negative) are cured by surgery in 70% of cases. Because of the 30% risk of relapse, many of these patients will also be treated with chemotherapy or hormonal therapy. The emergence of drug resistance is one of the main obstacles to successful chemotherapy in this disease. MDR is a phenomenon whereby tumour cells acquire resistance to a broad range of structurally and functionally diverse chemotherapeutic drugs, in- cluding anthracyclines, vinca alkaloids, epipophyllotoxins and paclitaxel (Taxol), following exposure to a single agent. 5 Most attention has been directed to the role played by membrane transporter proteins belonging to the ATP binding cassette super- family in MDR observed in breast cancer, particularly by the MDR-1 Pgp drug transporter 6 and another efflux pump, MRP-1, which encodes a 190 kDa membrane-bound glycoprotein. 7 Since the initial discovery of MRP-1, additional members of this family have been described (MRPs 2– 8). 8 –11 The specific role of these proteins in clinical drug resistance has not been fully elucidated. Studies have reported conflicting results with regard to the prognostic and/or predictive role of MDR-1 Pgp in breast cancer. MDR-1 Pgp is expressed in approximately 41% of untreated breast cancers, and prior exposure to chemotherapy increases this expres- sion. 12 There is some evidence supporting a correlation between MDR-1 Pgp expression at diagnosis and long-term outcome in both the adjuvant and neoadjuvant settings. 13 Some studies have suggested that evaluation of MDR-1 Pgp expression following Abbreviations: ABC, avidin– biotin complex; BCRP, breast cancer re- sistance protein; CMF, cyclophosphamide, methotrexate and 5-fluoroura- cil; DAB, 3,3'-diaminobenzidine; DCIS, ductal carcinoma in situ; DPX, dextropropoxyphene; ER, oestrogen receptor; HRP, horseradish peroxi- dase; LN, lymph node; MAb, monoclonal antibody; MDR-1, multiple drug resistance protein-1; MRP-1, multiple drug resistance–associated pro- tein-1; OS, overall survival; Pgp, P-glycoprotein; PR, progesterone recep- tor; RFS, relapse-free survival; TBS, TRIS-buffered saline. Grant sponsor: Atlantic Philanthropies; Grant sponsor: Higher Education Authority (PRTLI Cycle 3 Programme); Grant sponsor: Bioresearch Ire- land; Grant sponsor: Cancer Research Ireland; Grant sponsor: Health Research Board of Ireland. *Correspondence to: National Institute for Cellular Biotechnology, Dub- lin City University, Glasnevin, Dublin 9, Ireland. Fax: +353-1-7005484. E-mail: annemarie.larkin@dcu.ie Received 20 January 2004; Accepted after revision 31 March 2004 DOI 10.1002/ijc.20369 Published online 10 June 2004 in Wiley InterScience (www.interscience. wiley.com). Int. J. Cancer: 112, 286 –294 (2004) © 2004 Wiley-Liss, Inc. Publication of the International Union Against Cancer