Overexpression of Hrad17 Mrna in Human Breast Cancer: Correlation with Lymph Node Metastasis

Vol. 7, 2815–2820, September 2001 Clinical Cancer Research 2815

Overexpression of HRad17 mRNA in Human Breast Cancer: Correlation with Lymph Node Metastasis

Akemi Kataoka, Noriaki Sadanaga, tasis in human breast cancers. Although its function still Koshi Mimori, Hiroaki Ueo, Graham F. Barnard, remains unclear, the expression of HRad17 mRNA could Keizo Sugimachi, Daniel Auclair, Lan Bo Chen,1 open up a new window for the diagnostic staging and treatment of human breast cancers. and Masaki Mori1 Department of Surgery, Medical Institute of Bioregulation, Kyushu INTRODUCTION University, Beppu 874-0838, Japan [A. K., N. S., M. M.]; Kimmel Cancer Institute, Jefferson Medical College, Philadelphia, In Schizosaccharomyces pombe, the products of six genes, Pennsylvania 19107 [K. M.]; Department of Surgery, Oita Prefectural Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1, have been iden- Hospital, Oita 870-8511, Japan [H. U.]; Division of Digestive Disease tified as essential components of checkpoint pathways. Several and Nutrition, University of Massachusetts Medical Center, of these genes have structural homologues in the budding yeasts, Worcester, Massachusetts 01655 [G. F. B.]; Dana-Farber Cancer Institute, Harvard Medical School, Boston, Massachusetts 02115 and additional conservation across eukaryotes has been demon- [D. A., L. B. C.]; and Department of Surgery II, Faculty of Medicine, strated, e.g., human homologues of S. pombe Rad3, ataxia Kyushu University, Fukuoka 812-8582, Japan [K. S.] telangiectasia mutated (1), and ataxia telangiectasia- and Rad3- related; 2, 3); a human homologue of S. pombe Rad9, HRad9 (4); and a human homologue of S. pombe Rad1, HRad1 (5). ABSTRACT Recently, a human homologue of the S. pombe Rad17 check- Purpose: A novel human gene, designated HRad17, was point gene named HRad17 was identified (6–8). S. pombe identified as the human homologue of the Rad17 of Schizos- Rad17 is known to play critical roles in maintaining genomic accharomyces pombe and Rad24 of Saccharomyces cerevi- stability and integrity, as a G2-M checkpoint protein in the cell siae. In yeast, these genes play a critical role in maintaining cycle, and to prevent the development of cancer and hereditary genomic stability. The aim of this study was to evaluate the diseases; however, the function of HRad17 is still unclear. expression of HRad17 in human breast cancer. It was reported independently that HRad17 could be local- Experimental Design: We investigated HRad17 mRNA ized on human chromosome 4q or 5q by fluorescence in situ expression in 64 cases of human breast cancer by means of hybridization analysis (6, 7, 9). These regions are implicated in reverse-transcription-PCR, in situ hybridization, and immu- the etiology of a variety of human cancers, including breast nohistochemistry. cancer, hepatocellular carcinoma, small cell lung cancer, non- Results: The HRad17 mRNA was overexpressed in 35 small cell lung cancer, duodenal adenocarcinoma, and head and cases (54.7%). Twenty-four (68.6%) of 35 cases with neck squamous cell carcinoma (1, 10–13). IHC2 and RT-PCR HRad17 overexpression in cancer tissues were node-posi- analysis indicates elevated levels of expression of HRad17 in tive, whereas only 8 (27.6%) of 29 cases without HRad17 human testis and colon cancer (6). But there are no previous overexpressions were node-positive. The expression of reports studying the clinical significance of HRad17 expression HRad17 mRNA correlated with both lymph node metastasis in human cancer. Thus, we investigated its expression in human ؍ ؍ (P 0.001) and high Ki67 labeling index (P 0.006). breast cancer cell lines and in clinical breast cancers by means Although not significantly different, expression of HRad17 of RT-PCR and IHC using a monoclonal antibody to evaluate its ؍ mRNA tended to correlate with tumor size (P 0.06) and clinical significance. We also determined whether expression of ؍ expression of mutant p53 protein (P 0.10). Furthermore, HRad17 mRNA correlates with p53 mutation, overexpression of expression of HRad17 mRNA was an independent predictor oncoprotein c-erbB-2 (also known as HER2/neu), or Ki67 la- of axillary lymph node metastasis as well as of lymphatic beling index as a marker of proliferation. permeation by multivariate analysis (P < 0.0001). Conclusions: Our study demonstrates that HRad17 MATERIALS AND METHODS might be related to the development of lymph node metas- Tissue Specimens and Cell Lines. Sixty-four matched normal breast and breast cancer tissue samples were obtained from radical mastectomy performed at the Oita Prefectural Hos- pital (Beppu, Japan), the National Beppu Hospital (Beppu, Ja- Received 7/5/00; revised 5/22/01; accepted 5/31/01. pan), the Matsuyama Red Cross Hospital (Beppu, Japan), and The costs of publication of this article were defrayed in part by the the Medical Institute of Bioregulation, Kyushu University payment of page charges. This article must therefore be hereby marked (Beppu, Japan), between June 1998 and May 1999. Patients advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 To whom requests for reprints should be addressed, at Dr. Lan Bo Chen, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115; Phone: (617) 632-3385; Fax: (617) 632-4470; or at Dr. Masaki 2 The abbreviations used are: IHC, immunohistochemistry; RT-PCR, Mori, Department of Surgery, Medical Institute of Bioregulation, reverse-transcription-PCR; GAPDH, glyceraldehyde-3-phosphate dehy- Kyushu University, 4546 Tsurumihara, Beppu 874-0838, Japan. drogenase.

were excluded if they had received preoperative neoadjuvant Table 1 Clinico-pathological data of 64 patients chemotherapy or radiotherapy. Specimens were frozen in liquid HRad17 mRNA nitrogen immediately after surgical resection and kept at Ϫ90°C until the extraction of RNA. Human breast cancer cell lines Negative Positive Factors (%) (%) P MCF-7, MRK-nu-1, YMB-1-E, and YMB-1 were used. RNA Extraction and RT-PCR. Total RNA was ex- Mean age (yr) 29 (45.3%) 35 (54.7%) Menopause 54.6 Ϯ 12.6 56.1 Ϯ 12.0 NSa tracted by the acid guanidium thiocyanate-phenol-chloroform Premenopausal 11 (34.5%) 11 (32.8%) NS extraction procedure. The cDNA was synthesized from 2.5 ␮g Postmenopausal 19 (65.5%) 24 (67.2%) of total RNA as described previously (14). HRad17-specific Tumor size (cm) PCR was performed with primers 5Ј-TCCTTAGAACAGATT- Յ2.0 14 (48.3%) 9 (25.7%) 0.06 Ј Ј Ն2.1 15 (51.7%) 26 (74.3%) TATGGTTTA-3 and 5 -ATACTTTACATGAAGTTCTA- Ϯ Ϯ Ј Mean 2.4 1.4 2.8 1.6 AGGA-3 to amplify a 523-bp fragment. To prevent amplifica- Histological subtype tion from eventual contamination genomic DNA, these primers NIDC 1 (3.5%) 0 (0%) NS are located in different exons. PCR was performed for 26 cycles IDC 27 (93.0%) 30 (88.6%) (45sat94°C; 45 s at 60°C; and 45 s at 72°C). Five-␮l aliquots Other 1 (3.5%) 4 (11.4%) Lymph node metastasis of the PCR products were size-fractionated on a 1.5% agarose Negative 21 (72.4%) 11 (31.4%) 0.001 gel and visualized after ethidium bromide staining. The PCR Positive 8 (27.6%) 24 (68.6%) products were subcloned into the pCR II vector (Invitrogen) and Lymphatic permeation verified by sequencing (ABI100 version 3.3; ABI PRISM). Negative 17 (58.6%) 16 (45.7%) NS RNA quality was verified by running RT-PCR reactions for Positive 12 (41.4%) 19 (54.3%) Vascular invasion GAPDH on each RNA sample (14). Primer sequences for Negative 25 (86.2%) 29 (82.9%) NS GAPDH were 5GTCAACGGATTTGGTCTGTATT-3Ј and 5Ј- Positive 4 (13.8%) 6 (17.1%) AGTCTTCTGGGTGGCAGTGAT-3Ј(product size, 560 bp), ER status and PCR was performed for 22 cycles (60 s at 94°C; 60 s at Negative 8 (27.6%) 15 (42.9%) NS Positive 20 (69.0%) 19 (54.3%) 56°C; and 60 s at 72°C). Each series of RT-PCR reactions had Unknown 1 (3.5%) 1 (2.9%) a sample without RNA as a negative control, and all specimens PR status were analyzed at least twice. Negative 10 (34.5%) 17 (48.6%) NS In Situ RNA Hybridization. In situ RNA hybridization Positive 18 (62.1%) 17 (48.6%) was performed using a nonradioactive RNA color kit Unknown 1 (3.5%) 1 (2.9%) p53 proteinb (RPN3300; Amersham Pharmacia). A 523-bp PCR product was Negative 20 (69.0%) 17 (48.6%) 0.10 subcloned into TA vectors (pCR II; Invitrogen). The plasmid Positive 9 (31.0%) 18 (51.4%) DNA was isolated and purified using the RPM kit (BIO 101; Overexpression of c-erbB-2c Biotechnologies, Inc.), and fragments corresponding to the Negative 26 (89.7%) 27 (77.1%) NS Ј Positive 3 (10.3%) 8 (22.9%) COOH terminus and 3 region of Hrad17 were excised using Ki67 labeling index (%)d XhoI (Takara Co., Kyoto, Japan). Riboprobes were generated Ͻ8.3 20 (69.0%) 12 (34.2%) 0.006 with T7 and SP6 RNA polymerase. Ն8.3 9 (31.0%) 23 (65.8%) IHC. We used mouse monoclonal antibodies 31E9 for a NS, not significant; NIDC, non-invasive ductal carcinoma; IDC, HRad17 protein (6, 15), DO-7 (Dako Co.) for p53, and NCL- invasive ductal carcinoma; ER, estrogen receptor; PR, progesterone Ki67-MM1 (NovoCastra Laboratories, Ltd.) for Ki67. Rabbit receptor. b Ͼ polyclonal antibody (Nichirei Co., Tokyo, Japan) was used for p53 protein was judged as positive when 10% of cancer cells ␮ were stained with DO-7 by IHC. the detection of overexpression of c-erbB-2. Five- m-thick c Overexpression of c-erbB-2, judged as positive when Ͼ10% of sections of formalin-fixed and paraffin-embedded tissue sam- cancer cells were stained membrane with polyclonal antibody. ples were deparaffinized by incubation in xylene and rinsed in d Ki67 labeling index ranged from 3.3–66.5% (median value, graded ethanol-water solutions. Antigen retrieval for p53 and 8.3%; mean value, 12.5%). Ki67 (16) staining was performed by heating the samples in a microwave oven for 20 min at 98°C, in citric buffer (pH 6.0). Samples were blocked with normal horse serum, and endoge-

nous peroxidases were quenched with 0.3% H2O2 in methanol. tively stained cells in 1000 cancer cells. For p53, tumors were Sections were incubated with anti-HRad17 (1:200), anti-p53 considered positive if Ͼ10% of all cancer cells were stained. For (1:100), anti-Ki67 (1:200) or anti-c-erbB-2 (1:100) antibodies Ki67, tumors were classified as high or low labeling index by for 30 min at room temperature, rinsed, and then incubated comparing their value with the median of all samples. Overex- further with the peroxidase-conjugated secondary antibody for pression of c-erbB-2 was judged as positive when Ͼ10% of 30 min at room temperature. Detection substrate was 3,3-dia- cancer cells were stained with the antibody. Two independent minobenzidine, and all sections were counterstained with pathologists observed all specimens. Meier’s Hematoxylin before mounting. Negative controls were Clinicopathological Data and Statistical Methods. The run simultaneously with preimmune immunoglobulin. The per- clinical variables considered in this study, including the stage of centage of breast cancer cells showing a positive immunohisto- disease and various pathological factors, are shown in Table 1. chemical reaction in a representative section of each tumor was The relationship between HRad17 mRNA expression and the determined for p53 and Ki67 by counting the number of posi- clinicopathological factors were analyzed using the ␹2 test and

Table 2 Statistical associations between lymph node metastasis and examined variables (n ϭ 64) Lymph node metastasis Negative Positive Univariate Multivariate Factors n ϭ 32 n ϭ 32 P P Tumor size (cm) Յ2.0a 14 9 NSb Ն2.1 18 23 Յ3.0 28 20 0.02 NS Ն3.1 4 12 Fig. 1 The results of HRad17-specific RT-PCR in human breast cancer Stage cell lines. Expression of HRad17 mRNA was observed in all four human I/II 31 24 0.01 NS breast cancer cell lines, I–IV (I, MCF-7; II, MRK-nu-1; III, YMB-1; IV, III/IV 1 8 YMB-1-E); M, size marker; C, negative control. Expression of GAPDH Lymph node palpability served as internal control. Nonpalpable 30 18 0.0005 NS Palpable 2 14 Lymphatic permeation Negative 25 8 Ͻ0.0001 Ͻ0.0001 Positive 7 24 Vascular invasion Negative 31 23 0.006 NS Positive 1 9 Overexpression of c-erbB-2 Negative 27 26 NS Positive 5 6 Ki67 labeling index (%) Ͻ8.3a 18 14 NS Ͼ8.3 14 18 HRad17 mRNA Negative 21 8 0.001 Ͻ0.0001 Positive 11 24 a Tumor size (Յ2.0 cm versus Ն2.1 cm), overexpression of c- erbB-2 and Ki67 labeling index were excluded by multivariate analysis because there were no significant differences with respect to lymph node metastasis by univariate analysis. b NS, not significant. Fig. 2 Representative results of six node-negative and seven node- positive breast cancer patients. Of the upper six node-negative cases, cases 1, 2, and 6 overexpressed HRad17 mRNA. Of the lower seven node-positive cases, cases 1–6 overexpressed Hrad17 mRNA, and case normal tissues (Fig. 2). As shown in Table 1, there were no 7 did not express it. M, size marker; T, tumor; N, matched normal breast significant differences between the HRad17 mRNA expression tissue control; B, breast cancer cell line MCF-7; C, negative control. status and each clinical factor of age, menopausal state, histo- Expression of GAPDH served as internal control. logical subtype, lymphatic permeation, vascular invasion, or overexpression of c-erbB-2 and estrogen receptor or progesterone receptor status. However, the expression of HRad17 mRNA showed a tendency to correlate with tumor size and expression Student’s t test. A stepwise logistic regression model was used of p53 protein, although it did not reach a statistically significant for the multivariate analysis of independent predictors of axil- difference (P ϭ 0.06 and P ϭ 0.10, respectively). Interestingly, lary lymph nodes metastasis using the StatView software pack- the expression of HRad17 mRNA was significantly associated age (Abacus Concepts, Inc.). All tests were considered signifi- with lymph node metastasis and Ki67 index (P ϭ 0.001 and P ϭ Ͻ cant when the P was 0.05. 0.006, respectively). Twenty-four (68.6%) of 35 cases with HRad17 mRNA expression showed lymph node metastasis, RESULTS whereas only 8 (27.6%) of 29 cases without HRad17 mRNA HRad17 mRNA Expression in Cell Line. HRad17 expression showed lymph node metastasis. Twenty-three mRNA was expressed in all of four human breast cancer cell (65.8%) of 35 cases with HRad17 mRNA expression showed a lines, MCF-7, MRK-nu-1, YMB-1-E, and YMB-1, by RT-PCR high Ki67 labeling index, whereas only 9 (31.1%) of 29 cases (Fig. 1). without HRad17 mRNA expression showed a high Ki67 label- Expression of HRad17 mRNA and Clinicopathological ing index. The Ki67 labeling index ranged from 0.3% to 66.5% Data. A total of 64 matched normal and primary breast tumors (median, 8.4%; mean, 12.5%). The average Ki67 labeling index were examined for expression of HRad17 mRNA. HRad17 was 15.4 Ϯ 14.1% with HRad17 mRNA expression group, mRNA was expressed in 35 of 64 tumor specimens (54.7%), whereas it was higher than that of 9.0 Ϯ 13.9% without HRad17 whereas no amplified product was recognized in the matched mRNA expression group (P ϭ 0.07).

Fig. 3 Expression of HRad17 mRNA in breast cancer and in normal tissues. In situ RNA hybridization was performed on paraffin-embedded sections from breast carcinoma tissues (a) and normal tissue (c) using the HRad17 PCR fragment as a probe. Corresponding H&E stainings are shown in (b) and (d).

The independent predictors of lymph node metastasis were nucleus of the cancer cells, but faint staining was seen in the determined by stepwise logistic regression analysis. The follow- cytoplasm of the surrounding normal breast epithelial cells (Fig. ing variables were subjected to the multivariate analyses: (a) 4). There was a difference in staining intensity and localization tumor size (Յ3.0 cm versus Ն3.1 cm); (b) nodal palpability in of HRad17 protein between cancer cells and the surrounding the axilla (nonpalpable versus palpable); (c) clinical stage (stage normal epithelial cells. In most cases, strong staining of the I versus stages II–IV); (d) lymphatic permeation (negative ver- nucleus was observed frequently in an advancing margin rather sus positive); (e) vascular invasion (negative versus positive); than in the central area of the tumors. A representative case of and (f) expression of HRad17 mRNA (negative versus positive). immunohistochemical staining for HRad17, p53, and Ki67 is As shown in Table 2, all of these parameters correlate with shown in Fig. 4. lymph node metastasis by univariate analysis. Tumor size (Յ2.0 cm versus Ն2.1 cm), overexpression of c-erbB-2, and Ki67 labeling index were excluded because there were no significant DISCUSSION differences with respect to lymph node metastasis. Of these In human malignancy, identifying new genes that are as- variables, the lymphatic permeation and the expression of sociated with tumor growth, metastasis, and prognosis is very HRad17 mRNA were the only ones proven to be the independ- important in advancing the understanding of cancer biology. ent predictors of the lymph node metastasis (Ps Ͻ 0.0001). The size of the tumor and the number of metastatic lymph nodes In situ hybridization was also performed to confirm that the are the most powerful prognostic markers for breast cancer expression of the gene represented by the PCR fragment is much patients (17). Consequently, most breast cancer patients are higher in breast carcinoma tissues than in normal tissues. High offered postoperative chemoendocrine therapy according to expression of the gene represented by the PCR fragment was their metastatic nodal status, tumor size, and hormone receptor observed in breast carcinoma tissues, whereas very little expres- status (17). Recently, several molecular markers, such as Ki67 sion was found in normal breast (Fig. 3). These results con- (18, 19), p53 (20), c-erbB-2 (21), vascular endothelial growth firmed that the PCR fragment-containing gene is indeed over- factor (22), and several others, were reported as predictors of expressed by breast carcinoma tissues. lymph nodes metastasis or prognosis for breast cancer patients. Expression of HRad17 Protein. HRad17 protein was Most of these markers were investigated by IHC or enzyme detected by IHC. Strong staining was frequently seen in the immunoassay using resected specimens postoperatively. With a

Fig. 4 Immunohistochemical expression of HRad17 protein (a), Ki67 (c), and p53 (d) in breast cancer, invasive ductal carcinoma. Breast cancer cells show brown staining mainly in the nucleus. b, H&E staining. This representative case was considered to be p53-positive with a high Ki67 index.

view to clinical usefulness, early detection techniques that tool. We are now starting a clinical investigation to assess the clearly predict lymph node metastasis before an operation may concordance rate of RT-PCR expression of HRad17 mRNA improve the planning of disease treatment. However, as men- between preoperative needle biopsy samples and subsequent tioned above, information on important predictive factors for resected specimens. First, this is needed to evaluate the expres- lymph node metastasis are obtained by studying resected spec- sion status of HRad17 in various specimens from breast cancer imens after operation. We thus focused on RT-PCR expression tissues, because the display of breast cancer is often complex, of HRad17 mRNA in the resected specimens as a possible involving intraductal, microinvasive, multifocal neoplastic and useful molecular predictor of lymph node metastasis. In this preneoplastic lesions in many samples. Then, the use of a study, only resected samples were available. If it proves to be a marker on a small biopsy sample would seem to be more useful marker in the resected samples, the methodology can be informative than that in a complete surgical resection specimen. applied to preoperative biopsy samples. We would then be able Furthermore, if adequate information about axillary nodal status to get relevant prognostic information before surgery. was obtained before surgery, we could avoid axillary node We demonstrated that the expression of HRad17 mRNA dissection and its uncomfortable complications. A small breast was correlated significantly with both histological lymph node tumor without overexpression of HRad17 could be treated by metastases and high Ki67 index; moreover, it tended to correlate local resection of the primary site only and without axillary node with tumor size and p53 status. This suggested that the tumors dissection or, at most, a sentinel node biopsy only, in place of a with overexpression of HRad17 mRNA might have more po- complete axillary dissection (24, 25). According to our findings, tential to recur after operation. The multivariate analysis dem- many early-staged small breast cancer patients may be curable onstrated that HRad17 expression status and lymphatic vessel with much less invasive surgery. permeation were independent factors for lymph node metastasis. Although little was known about the function of HRad17, The status of the lymphatic vessel permeation is diagnosed by there are some experimental reports suggesting that HRad17 and pathological examination of the resected primary tumors. Thus, p53 may have a role in G2-M checkpoint control of the cell it is impossible to obtain the information before operation. On cycle in human cancer (7, 26). Interestingly, our results showed the other hand, the expression status of HRad17 mRNA can be a tendency for a correlation between the expression of p53 performed by RT-PCR using preoperative needle biopsy sam- mutant protein and HRad17 mRNA (P ϭ 0.100) and a signifi- ples (23). Thus, for preoperative evaluation of lymph node cant correlation between the Ki67 labeling index and HRad17 metastasis, the study of Hrad17 mRNA could become a useful mRNA (P Ͻ 0.01). Ki67 is a proliferation protein expressed in

late G1,S,G2, and M phases of the cell cycle; and Ki67 has 11. Petersen, I., Langreck, H., Wolf, G., Schwendel, A., Psille, R., shown prognostic utility in a number of studies (18, 19). To Vogt, P., Reichel, M. B., Ried, T., and Dietel, M. Small-cell lung cancer clarify the molecular interaction between lymph node metastasis is characterized by a high incidence of deletions on chromosomes 3p, 4q, 5q, 10q, 13q and 17p. Br. J. Cancer, 75: 79–86, 1997. and cell cycle checkpoint proteins, additional studies are 12. Tirkkonen, M., Johannsson, O., Agnarsson, B. A., Olsson, H., needed. Biomolecular analysis of the properties of interactions Ingvarsson, S., Karhu, R., Tanner, M., Isola, J., Barkardottir, R. B., between HRad17 and other G2-M checkpoint proteins or DNA Borg, A., and Kallioniemi, O. P. Distinct somatic genetic changes replication proteins are now required to fully establish the role associated with tumor progression in carriers of BRCA1 and BRCA2 that this novel gene plays role in cell cycle checkpoint control. germ-line mutations. Cancer Res., 57: 1222–1227, 1997. 13. Verhaegh, G. W., Jongmans, W., Jaspers, N. G., Natarajan, A. T., Oshimura, M., Lohman, P. H., and Zdzienicka, M. Z. A gene that Note Added in Proof regulates DNA replication in response to DNA damage is located on human chromosome 4q. Am. J. Hum. Genet., 57: 1095–1103, 1995. Since the original submission of this paper, overexpression of 14. Mori, M., Mimori, K., Inoue, H., Barnard, G. F., Tsuji, K., Nanbara, HRad17 by RT-PCR and IHC has been shown to be correlated with S., Ueo, H., and Akiyoshi, T. Detection of cancer micrometastases in lymph node metastasis in lung cancer (27). lymph nodes by reverse transcriptase-polymerase chain reaction. Cancer Res., 55: 3417–3420, 1995. ACKNOWLEDGMENTS 15. 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Downloaded from clincancerres.aacrjournals.org on September 29, 2021. © 2001 American Association for Cancer Research. Overexpression of HRad17 mRNA in Human Breast Cancer: Correlation with Lymph Node Metastasis

Akemi Kataoka, Noriaki Sadanaga, Koshi Mimori, et al.

Clin Cancer Res 2001;7:2815-2820.

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