ANTICANCER RESEARCH 26: 1809-1814 (2006) Clinical Role of ABCF2 Expression in Breast Cancer YOSHINARI OGAWA1,2, HIROSHI TSUDA2,3, EISHU HAI4, NOZOMI TSUJI2, SHIGETO YAMAGATA1, SHINYA TOKUNAGA5, KAZUNORI NAKAZAWA1, YUTAKA TAMAMORI1, MASAO OGAWA1, SADATOSHI SHIMIZU1, TAKESHI INOUE4 and YUKIO NISHIGUCHI1 Departments of 1Surgery, 3Gynecology, 4Pathology and 5Clinical Oncology and 2Institute of Education and Clinical Research, Osaka City General Hospital, Osaka, 534-0021 Japan Abstract. Background: The ATP binding cassette (ABC) drug accumulation are important mechanisms (3, 4). The family acts as efflux pumps and some members are related to ATP binding cassette (ABC) transporter family acts as an chemoresistance in breast cancer. The clinical role of ABCF2 efflux pump and its members are related to chemoresistance expression, a member of the ABC family, was analyzed. (5). ABCF2 is a member, which has a chromosome gain at Materials and Methods: One hundred and ninety-one patients 7q 34-36 (6). Overexpression of ABCF2 was found in a with breast cancer were enrolled. The median follow-up term chemoresistance clear cell ovarian carcinoma (7) and it was was 76 months. ABCF2 expression was examined by also suggested that ABCF2 may contribute to the immunohistochemistry. Results: Ninety percent of the breast chemoresistant phenotype. However, the ABCF2 protein cancer cases displayed immunoreactivity for ABCF2. The has a structure different from other members of the ABC positive rate of ABCF2 expression was 63%. ABCF2 had a transporter family. While the ABCF2 protein contains a negative relationship to distant metastasis. ABCF2-positive pair of nucleotide binding domains (NBD), it does not tumors had longer disease-free survival (DFS) than -negative contain trans-membrane domains (8, 9). The actual function tumors in patients with lymph node metastasis (p=0.001). In of ABCF2 in cancer cells is still unclear. patients treated with endocrine therapy, ABCF2-positive With the exception of investigations into ovarian cancer, tumors had a longer DFS when the tumors were estrogen few studies have addressed ABCF2 expression in clinical receptor-negative or progesterone receptor-negative (p=0.0019 samples. In this pilot study, ABCF2 protein expression was and 0.005, respectively). Conclusion: ABCF2 was initially retrospectively analyzed in breast cancer to evaluate the thought to be related to drug resistance. ABCF2 may play a value of ABCF2 in breast cancer treatment. role in tumor suppression at metastatic sites and in the endocrine pathway for breast cancer. Materials and Methods Compared to other types of cancer, breast cancer is often Patients. The patient group comprised 191 females with primary breast cancer, who had been surgically treated at Osaka City responsive to anticancer agents. Adjuvant chemo- and General Hospital, Japan, from 1997 to 1999. These patients were endocrine-therapy following surgery have improved the selected consecutively from those who had had no other survival of patients with breast cancer, resulting in 20% to malignancy at the time of breast cancer diagnosis and who had not 30% proportional reductions in mortality (1, 2). However, received any pre-operative chemotherapy. One hundred and tumor cells exposed to cytotoxic drugs can acquire twenty-two patients underwent radical mastectomy and 69 patients resistance to these drugs, and this resistance is an obstacle received breast-conserving surgery followed by radiation. After in the treatment of breast cancer. surgery, 180 out of the 191 patients received systemic therapy in a routine clinical setting as follows: 118 patients received endocrine Several explanations for drug resistance have been therapy, 58 patients received chemo-endocrine therapy and four reported. Enhanced drug efflux and decreased intracellular patients received only chemotherapy. Tamoxifen 20 mg per day was administered as endocrine therapy for 2 or 3 years. Twenty-one patients received goserelin 3.6 mg every 4 weeks for 2 years. Fifty patients received 5FU or its derivatives orally for 2 years and Correspondence to: Yoshinari Ogawa, Department of Surgery, twelve received combination chemotherapy, such as a classical type Osaka City General Hospital, 2-13-22 Miyakojima-hondori, of CAF or CMF. Miyakojima-ku, Osaka, 534-0021 Japan. Tel: +81-6-6929-1221, The patient characteristics are summarized in Table I. The Fax: +81-6-6929-2041 clinicopathological factors were determined from the initial surgical pathology reports. The mean age of the patients was 54.8 Key Words: Breast cancer, ABCF2, ABC family, endocrine therapy, years old (range, 29 to 87). One hundred and seventy-six out of the chemoresistance. 191 cases presented with invasive ductal carcinoma, while the 0250-7005/2006 $2.00+.40 1809 ANTICANCER RESEARCH 26: 1809-1814 (2006) Table I. Patient characteristics and ABCF2 expression. micrometer sections were cut from formalin-fixed, paraffin- embedded primary tumors for staining. After deparaffinization and ABCF2 expression hydration, the sections were placed in 3% hydrogen peroxide for 10 min to quench endogeneous peroxidase activity. The sections No. of cases Positive Negative p-value# were reacted with the ABCF2 antibody (1:4000 dilution in 10 mM TBS) at 4ÆC overnight. The sections were processed according to Total 191 121 70 the manufacturer’s instructions for the Envision kit. Diaminobenzidine was used as a chromogen, followed by Menopausal status hematoxylin counter staining. TBS 10 mM was substituted for the Pre- 76 48 28 0.96 primary antibody as a negative control. Post- 115 73 42 The slides were evaluated under microscopy without knowledge Tumor size of the patient characteristics. ABCF2 protein expression in tumor ~20 mm 93 63 30 0.20 50 mm 91 56 35 cells was seen mostly in the cytoplasm and occasionally in the ~ nucleus. Normal epithelial cells were not stained, or were stained 51 mm ~ 624 No. of lymph node focally or weakly. The tumors were scored by the proportion of metastases cytoplasmic stained cancer cells as follows: 0, none; 1, <1/10; 2, 0 104 62 42 0.11 1/10 to 1/2; 3, 1/2 to 9/10; 4, >9/10 (Figure 1). 1~3483612 4~ 33 18 15 Statistical analysis. The Chi-square test was used to evaluate the Distant metastasis correlation between ABCF2 expression and clinicopathological – 182 118 64 0.056 factors. The relationship between the ABCF2 expression and +936survival was examined by constructing Kaplan-Meier survival Mitotic count curves and analyzing differences by the log-rank test. For 1 111 76 35 0.44 multivariate analysis, Cox’s hazard regression model was used. 2352015P<0.05 was considered statistically significant. 3291811 Nuclear grade 1 103 69 34 0.82 Results 2301911 3422616The frequency of staining proportion scores were as follows: Estrogen receptor 19 cases scored 0, 51 cases scored 1, 68 cases scored 2, 42 + 111 73 38 0.18 cases scored 3 and 11 cases scored 4. ABCF2 –583226 Progesterone receptor immunoreactivity was detected in 90.1% of the breast + 95 60 35 0.76 cancer specimens (172/191). As a precedent for analysis, –744529every staining score was evaluated as a cut-off point. The greatest prognostic difference between the two groups when # p-values were examined by the Chi-square test. score 2 was used as a cut-off point. Therefore, tumors with over 10% cytoplasmically-stained cells were classified as ABCF2-positive. Using this cut-off point, the positive rate remainder were invasive lobular carcinoma, medullary carcinoma, for ABCF2 expression was 63.3% (121/191). mucinous carcinoma or non-invasive ductal carcinoma. The TNM The correlations between ABCF2 expression and analysis was performed according to the UICC criteria and the clinicopathological factors are illustrated in Table I. The nuclear grade was scored with nuclear atypia and mitotic counts positive rate of ABCF2 was relatively low in tumors with (10). The tumor estrogen receptor (ER) and progesterone receptor distant metastasis (p=0.056). There was no correlation (PR) status were analyzed by enzyme-immunoassay methods. The between ABCF2 expression and menopausal status, tumor cut-off value for receptor status positivity was determined as size, lymph node metastasis, mitotic count, nuclear grade or 13 fmol/mg protein for ER and 10 fmol/mg protein for PR. Prognostic analysis was performed in 181 curatively-operated ER and PR status. patients. The survival period was defined as the interval between Disease-free survival (DFS) was analyzed in 181 the date of surgery and the date of first relapse. The median follow- curatively-operated patients. The ABCF2-positive group up term was 76 months (range, 9 to 107 months). Over this term, had a significantly longer DFS than the ABCF2-negative 41 out of 181 patients relapsed after surgery: 20 at distant sites, 11 group (p=0.033). Stratification by nodal status revealed that at locoregional sites and 10 at both distant and locoregional sites. ABCF2 expression was related to longer DFS in lymph node-positive patients (p=0.0010) (Figure 2a), while the Immunohistochemistry. The immunohistochemical staining for ABCF2 protein was performed using a polyclonal anti-ABCF2 difference in DFS between the ABCF2-positive and antibody and the Envision kit (Dako, CA, USA). The anti-ABCF2 -negative groups was lost in lymph node-negative patients antibody was generated by injecting the purified full-length ABCF2 (p=0.79) (Figure 2b). Multivariate analysis revealed that fusion protein into rabbits, as previously described (7). Four- ABCF2 was an independent prognostic factor (Table II). 1810 Ogawa et al: ABCF2 Expression in Breast Cancer Figure 1. Expression of ABCF2 is found in the cytoplasm of cancer cells. a) ABCF2-positive tumor (Score 3). Most cancer cells in this specimen are clearly stained. b) ABCF2-negative tumor (Score 1). Weakly-stained cancer cells are sporadically seen in this specimen. Bar, 50 Ìm. As for adjuvant therapy, 167 out of 181 patients were and PR status. The ABCF2-positive group had a longer DFS treated with endocrine or chemo-endocrine therapy.