Correlation of Reduction in MRP-1/CD9 and KAI1/CD82 Expression with Recurrences in Breast Cancer Patients
1998; Elsevier BV; Volume: 153; Issue: 3 Linguagem: Inglês
10.1016/s0002-9440(10)65639-8
ISSN1525-2191
AutoresCheng‐long Huang, Nobuoki Kohno, Eiji Ogawa, Masashi Adachi, Toshihiko Taki, Masayuki Miyake,
Tópico(s)Sarcoma Diagnosis and Treatment
ResumoMRP-1/CD9,KAI1/CD82, andME491/CD63, have been reported to be associated with the metastatic potential of solid tumors. The aim of this study was to determine whether their expression in tumor tissues is a useful indicator for prognosis in breast cancer patients. We studied 109 breast cancer patients who underwent surgery. Quantitative reverse transcription-polymerase chain reaction analysis was performed to evaluate the expression of these genes. The results were confirmed with immunohistochemistry. All of the carcinomas wereME491/CD63 positive. Thirty-six tumors wereMRP-1/CD9 negative. The disease-free survival rate and the 5-year survival rate of patients withMRP-1/CD9-negative tumors were both significantly lower than that in patients with MRP-1/CD9-positive tumors (P = 0.0005 and P = 0.0380, respectively). Sixty-five tumors wereKAI1/CD82 negative. The disease-free survival rate of patients with KAI1/CD82-negative tumors was significantly lower than that of patients withKAI1/CD82-positive tumors (P = 0.0065). Cox regression analysis demonstrated thatMRP-1/CD9 status (P = 0.0016) andKAI1/CD82 status (P = 0.0234) were useful indicators for the disease-free survival of breast cancer patients. The disease-free survival rate and 5-year survival rate of patients with either MRP-1/CD9-negative orKAI1/CD82-negative tumors were both significantly lower than patients who were positive for both genes (P = 0.0003 and P = 0.0292, respectively). The expression of MRP-1/CD9 and KAI1/CD82genes are useful indicators of a poor prognosis in breast cancer patients. MRP-1/CD9,KAI1/CD82, andME491/CD63, have been reported to be associated with the metastatic potential of solid tumors. The aim of this study was to determine whether their expression in tumor tissues is a useful indicator for prognosis in breast cancer patients. We studied 109 breast cancer patients who underwent surgery. Quantitative reverse transcription-polymerase chain reaction analysis was performed to evaluate the expression of these genes. The results were confirmed with immunohistochemistry. All of the carcinomas wereME491/CD63 positive. Thirty-six tumors wereMRP-1/CD9 negative. The disease-free survival rate and the 5-year survival rate of patients withMRP-1/CD9-negative tumors were both significantly lower than that in patients with MRP-1/CD9-positive tumors (P = 0.0005 and P = 0.0380, respectively). Sixty-five tumors wereKAI1/CD82 negative. The disease-free survival rate of patients with KAI1/CD82-negative tumors was significantly lower than that of patients withKAI1/CD82-positive tumors (P = 0.0065). Cox regression analysis demonstrated thatMRP-1/CD9 status (P = 0.0016) andKAI1/CD82 status (P = 0.0234) were useful indicators for the disease-free survival of breast cancer patients. The disease-free survival rate and 5-year survival rate of patients with either MRP-1/CD9-negative orKAI1/CD82-negative tumors were both significantly lower than patients who were positive for both genes (P = 0.0003 and P = 0.0292, respectively). The expression of MRP-1/CD9 and KAI1/CD82genes are useful indicators of a poor prognosis in breast cancer patients. When compared with other types of solid human cancers, breast cancer is interesting because of its high sensitivity to hormonal therapy and chemotherapy.1Early Breast Cancer Trialists' Collaborative GroupSystemic treatment of early breast cancer by hormonal, cytotoxic, or immune therapy: 133 randomized trials involving 31 000 recurrences and 24 000 deaths among 75 000 women.Lancet. 1992; 339 (71–85): 1-15Abstract PubMed Google Scholar However, the prognosis varies according to the extent of the disease and its biological behavior. Although the histopathological presence of axillary lymph node metastases is considered to be the most informative parameter for predicting the occurrence of relapses and the prognosis in breast cancer patients, there is a possibility of recurrence after resection even in patients with an early stage of node-negative breast cancer.2Allred DC Clark GM Elledge R Fuqua SA Brown RW Chamness GC Osborne CK McGuire WL Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.J Natl Cancer Inst. 1993; 85: 200-206Crossref PubMed Scopus (741) Google Scholar Therefore, it is important to understand the biological behavior of each individual tumor and to determine which types of tumor will have a more malignant course and thus need more intensive adjuvant therapy. It is widely accepted that several kinds of cancers are caused by the accumulation of genetic alterations.3Hartmann LC Ingle JN Wold LE Farr GH Grill JP Su JQ Maihle NJ Krook JE Witzig TE Roche PC Prognostic value of c-erbB2 overexpression in axillary lymph node positive breast cancer.Cancer. 1994; 74: 2956-2963Crossref PubMed Scopus (104) Google Scholar, 4Giai M Roagna R Ponzone R Bortoli MD Dati C Sismondi P Prognostic and predictive relevance of c-erbB-2 and ras expression in node positive and negative breast cancer.Anticancer Res. 1994; 14: 1441-1450PubMed Google Scholar, 5Berns EMJJ Klijn JGM Smid M Staveren IL Look MP Putten WLJ Foekens JA TP53, and MYC gene alterations independently predict poor prognosis in breast cancer patients.Genes Chromosomes Cancer. 1996; 16: 170-179Crossref PubMed Scopus (64) Google Scholar, 6Bland KI Konstadoulakis MM Vezeridis MP Wanebo HJ Oncogene protein co-expression: value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma.Ann Surg. 1995; 221: 706-720Crossref PubMed Scopus (109) Google Scholar Recent investigations have revealed that some of these genetic changes can be used as prognostic factors for predicting a poor prognosis. For example, the amplification of some oncogenes such as c-erbB-2,3Hartmann LC Ingle JN Wold LE Farr GH Grill JP Su JQ Maihle NJ Krook JE Witzig TE Roche PC Prognostic value of c-erbB2 overexpression in axillary lymph node positive breast cancer.Cancer. 1994; 74: 2956-2963Crossref PubMed Scopus (104) Google Scholar, 4Giai M Roagna R Ponzone R Bortoli MD Dati C Sismondi P Prognostic and predictive relevance of c-erbB-2 and ras expression in node positive and negative breast cancer.Anticancer Res. 1994; 14: 1441-1450PubMed Google Scholarmyc,5Berns EMJJ Klijn JGM Smid M Staveren IL Look MP Putten WLJ Foekens JA TP53, and MYC gene alterations independently predict poor prognosis in breast cancer patients.Genes Chromosomes Cancer. 1996; 16: 170-179Crossref PubMed Scopus (64) Google Scholar ras,6Bland KI Konstadoulakis MM Vezeridis MP Wanebo HJ Oncogene protein co-expression: value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma.Ann Surg. 1995; 221: 706-720Crossref PubMed Scopus (109) Google Scholar and c-fos6Bland KI Konstadoulakis MM Vezeridis MP Wanebo HJ Oncogene protein co-expression: value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma.Ann Surg. 1995; 221: 706-720Crossref PubMed Scopus (109) Google Scholar have been found to be associated with a poor prognosis in patients with breast cancers. Mutations of p53, a well known tumor suppressor gene, also may be important for the prognosis of breast cancer patients.5Berns EMJJ Klijn JGM Smid M Staveren IL Look MP Putten WLJ Foekens JA TP53, and MYC gene alterations independently predict poor prognosis in breast cancer patients.Genes Chromosomes Cancer. 1996; 16: 170-179Crossref PubMed Scopus (64) Google Scholar, 7Kovach JS Hartmann A Blaszyk H Cunningham J Schaid D Sommer SS Mutation detection by highly sensitive methods indicates that p53 gene mutations in breast cancer can have important prognostic value.Proc Natl Acad Sci USA. 1996; 93: 1093-1096Crossref PubMed Scopus (132) Google Scholar In addition, assessing a combination of these genetic alterations may enable the more precise prediction of the outcomes of patients with breast cancers.6Bland KI Konstadoulakis MM Vezeridis MP Wanebo HJ Oncogene protein co-expression: value of Ha-ras, c-myc, c-fos, and p53 as prognostic discriminants for breast carcinoma.Ann Surg. 1995; 221: 706-720Crossref PubMed Scopus (109) Google Scholar Recently, three members of the transmembrane 4 superfamily MRP-1/CD9,8Ikeyama S Koyama M Yamaoko M Sasada R Miyake M Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.J Exp Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar, 9Miyake M Nakano K Ieki Y Adachi M Huang C Itoi S Koh T Taki T Motility related protein 1 (MRP-1/CD9) expression: inverse correlation with metastases in breast cancer.Cancer Res. 1995; 55: 4127-4131PubMed Google ScholarKAI1/CD82,10Dong JT Lamb PW Rinker-Schaeffer CW Vukanovic J Ichikawa T Isaacs JT Barrett JC KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.Science. 1995; 268: 884-886Crossref PubMed Scopus (762) Google Scholar andME491/CD6311Radford KJ Mallesch J Hersey P Suppression of human melanoma cell growth and metastasis by the melanoma-associated antigen CD63 (ME491).Int J Cancer. 1995; 62: 631-635Crossref PubMed Scopus (91) Google Scholar have been reported to be associated with the biological behavior of solid tumors, especially with their metastatic potential. Initially, we found that MRP-1/CD9 was recognized by the murine MAb M31–15, which inhibited cell motility.12Miyake M Koyama M Seno M Ikeyama S Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31–15, which inhibits cell motility.J Exp Med. 1991; 174: 1347-1354Crossref PubMed Scopus (190) Google Scholar After the transfection of MRP-1/CD9 into human lung cancer cell lines, we demonstrated that cell motility was suppressed in theMRP-1/CD9-expressing cells.8Ikeyama S Koyama M Yamaoko M Sasada R Miyake M Suppression of cell motility and metastasis by transfection with human motility-related protein (MRP-1/CD9) DNA.J Exp Med. 1993; 177: 1231-1237Crossref PubMed Scopus (274) Google Scholar In addition, we showed that reduced MRP-1/CD9 protein expression was associated with metastasis and a poor prognosis in breast cancer patients13Miyake M Nakano K Itoi S Koh T Taki T Motility-related protein-1 (MRP-1/CD9) reduction as a factor of poor prognosis in breast cancer.Cancer Res. 1996; 56: 1244-1249PubMed Google Scholar and that reduced MRP-1/CD9 gene expression was also correlated with a poor prognosis in non-small cell lung cancer patients.14Higashiyama M Taki T Ieki Y Adachi M Huang C Koh T Kodama K Doi O Miyake M Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer.Cancer Res. 1995; 55: 6040-6044PubMed Google Scholar KAI1/CD82 gene is located on human chromosome 11p11.2–13, and encodes a protein of 267 amino acids.10Dong JT Lamb PW Rinker-Schaeffer CW Vukanovic J Ichikawa T Isaacs JT Barrett JC KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.Science. 1995; 268: 884-886Crossref PubMed Scopus (762) Google Scholar Initially, it was identified by cDNA cloning as the R2 antigen, which was strongly up-regulated in mitogen-activated human T cells.15Gaugitsch HW Hofer E Huber NE Schnabl E Baumruker T A new superfamily of lymphoid and melanoma cell proteins with extensive homology to Schistosoma mansoni antigen Sm23.Eur J Immunol. 1991; 21: 377-383Crossref PubMed Scopus (86) Google Scholar KAI1/CD82 gene was also found to suppress tumor metastasis in a prostate cancer cell line10Dong JT Lamb PW Rinker-Schaeffer CW Vukanovic J Ichikawa T Isaacs JT Barrett JC KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.Science. 1995; 268: 884-886Crossref PubMed Scopus (762) Google Scholar and a breast cancer cell line,16Yang X Welch DR Phillips KK Weissman BE Wei LL KAI1, a putative marker for metastatic potential in human breast cancer.Cancer Lett. 1997; 119: 149-155Abstract Full Text PDF PubMed Scopus (91) Google Scholar, 17Phillips KK White AE Hicks DJ Welch DR Barrett JC Wei LL Weissman BE Correlation between reduction of metastasis in the MDA-MB-435 model system and increased expression of the Kai-1 protein.Mol Carcinog. 1998; 21: 111-120Crossref PubMed Scopus (44) Google Scholar and thus may function as a metastatic suppressor gene.10Dong JT Lamb PW Rinker-Schaeffer CW Vukanovic J Ichikawa T Isaacs JT Barrett JC KAI1, a metastasis suppressor gene for prostate cancer on human chromosome 11p11.2.Science. 1995; 268: 884-886Crossref PubMed Scopus (762) Google Scholar A clinical analysis of patients with non-small cell lung cancers also revealed that reduced KAI1/CD82 gene expression was associated with the metastasis of these tumors.18Adachi M Taki T Ieki Y Huang C Higashiyama M Miyake M Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer.Cancer Res. 1996; 56: 1751-1755PubMed Google Scholar In addition, it has been reported that ME491/CD63 is strongly expressed on the cell surface during the early stage of malignant melanoma but becomes weaker in the advanced stages.19Hotta H Ross AH Huebner K Isobe M Wendeborn S Chao MV Ricciardi RP Tsujimoto Y Croce CM Koprowski H Molecular cloning and characterization of an antigen associated with early stages of melanoma tumor progression.Cancer Res. 1988; 48: 2955-2962PubMed Google Scholar These findings are similar to those observed for MRP-1/CD9 and KAI1/CD82. Thus, of the many genetic markers available for evaluating the prognosis in breast cancers, MRP-1/CD9, KAI1/CD82, andME491/CD63 gene expression may have significant value as prognostic indicators in breast cancer patients. In the present study, we investigated whether the levels of MRP-1/CD9,KAI1/CD82, and ME491/CD63 gene expression in tumor tissues are of value as prognostic factors in predicting the clinical behavior of breast cancer. Therefore, we performed a reverse transcription-polymerase chain reaction (RT-PCR) analysis to quantify the expression of these genes in tumor tissues from 109 patients with breast cancer. Immunohistochemical assays were also performed to confirm the results of the RT-PCR. From February 1987 to December 1995, 109 patients who underwent surgery at the Department of Thoracic Surgery of Kitano Hospital, Medical Research Institute of Osaka in Japan, were studied. The complete clinical records of all patients were available, and their histopathological diagnoses were fully documented. The postsurgical stage of each tumor was classified according to the Union International Contre Cancer TNM system.20Hermanek P Sobin LH TNM Classification of Malignant Tumors.in: Hermanek P Sobin LH ed 4. Springer-Verlag, Berlin1987: 103-109Google Scholar In total, 109 patients with breast cancer up to stage IIIB were investigated. Ninety-five patients had undergone a mastectomy, and 14 patients had undergone a quadrantectomy followed by immediate radiotherapy. Adjuvant systemic chemotherapy was given according to the patients' estrogen receptor (ER) status. Patients who were node positive or premenopausal (n = 73) underwent chemotherapy with oral 5-fluorouracil (200 mg/day) for 2 years, and eight patients with N2 disease were also treated with six cycles of cyclophosphamide/Adriamycin. Fifty-two ER-positive patients were treated with tamoxifen (20 mg/day) for 2 years or before recurrence. Sixteen postmenopausal patients of node-negative and receptor-negative status did not have any further adjuvant treatment. Thirty-three patients had recurrences during the observation period. After the recurrence, the locoregional tumor or lymph nodes were principally resected, followed by radiotherapy. Patients with distant metastases were treated with more effective adjuvant chemotherapies, including cisplatin and pirarubicin. This report includes follow-up data as of May 1, 1997. The median follow-up period was 48.5 months. To ascertain the presence of cancer cells, one-half of each fresh tumor tissue specimen was immediately embedded in optimum cutting temperature compound (Miles, Kankakee, IL), and frozen sections were then cut on the cryostat to a thickness of 6 μm and immediately stained with hematoxylin and eosin. After the connective tissues were trimmed off, the other half of the tumor specimen containing greater than 80% cancer cells of all tissue cells was selected for the RT-PCR analysis. Total cellular RNA was extracted from the frozen tumor tissues by the acid guanidinium thiocyanate procedure.21Chomczynski P Sacchi N Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.Anal Biochem. 1987; 162: 156-159Crossref PubMed Scopus (62920) Google Scholar First-strand cDNA synthesis was performed with 5 μg of total RNA using a cDNA synthesis kit (Pharmacia, Piscataway, NJ) according to the manufacturer's protocol. All of the subsequent assays were then carried out using the same procedures as described previously.14Higashiyama M Taki T Ieki Y Adachi M Huang C Koh T Kodama K Doi O Miyake M Reduced motility related protein-1 (MRP-1/CD9) gene expression as a factor of poor prognosis in non-small cell lung cancer.Cancer Res. 1995; 55: 6040-6044PubMed Google Scholar, 18Adachi M Taki T Ieki Y Huang C Higashiyama M Miyake M Correlation of KAI1/CD82 gene expression with good prognosis in patients with non-small cell lung cancer.Cancer Res. 1996; 56: 1751-1755PubMed Google Scholar The generated cDNAs were amplified using primers for MRP-1/CD9 (5′-TGCATCTGTATCCAGCGCCA-3′ and 5′-CTCAGGGATGTAAGCTGACT-3′), KAI1/CD82(5′-AGTCCTCCCTGCTGCTGTGTG-3′ and 5′-TCAGTCAGGGTGGGCAAGAGG-3′) andME491/CD63 (5′-CCCGAAAAACAACCACACTGC-3′ and 5′-GATGAGGAGGCTGAGGAGACC-3′). The internal control was β-actin (5′-GAGAAGATGACCCAGATCATGT-3′ and 5′-ACTCCATGCCCAGGAAGGAAGG-3′).22Nakajima-Iijima S Hamada H Reddy P Kakunaga T Molecular structure of the human cytoplasmic β-actin gene: interspecies homology of sequences in the introns.Proc Natl Acad Sci USA. 1985; 82: 6133-6137Crossref PubMed Scopus (692) Google Scholar All of the subsequent assays were then carried out under conditions that yielded amplifications of MRP-1/CD9, KAI1/CD82,ME491/CD63, and β-actin within a linear range. Twenty-six cycles of PCR amplification were performed as follows: denaturation at 94°C for 40 seconds, annealing at 60°C for 40 seconds, and extension at 72°C for 90 seconds, followed by the final extension at 72°C for 7 minutes. The same PCR conditions were used to amplify the β-actin DNA. Tubes containing all of the ingredients except templates were included in all runs and served as negative controls. The human endothelial cell line ECV304 was used as a positive control, which has positive expression ofMRP-1/CD9, KAI1/CD82, andME491/CD63.23Takahasi K Sawasaki Y Rare spontaneously transformed human endothelial cell line provides useful research tool.In Vitro Cell Dev Biol. 1992; 28A: 380-382Crossref PubMed Scopus (60) Google Scholar The amplified PCR products were electrophoresed on a 1% agarose gel containing ethidium bromide, and the bands were visualized under ultraviolet light followed by densitometric analysis (Figure 1). Because it has been difficult to quantitate the absolute amount of specific mRNA without an internal standard of known concentration, the adjustment with a housekeeping gene has been used for the precise quantitation of mRNA of a specific gene in Northern blotting. Recently, quantitative RT-PCR has been developed using this method.24Coen DM Quantitation of rare DNAs by PCR. Current Protocols in Molecular Biology.in: Ausubel FM Brent R Kingston RE Moore DD Seidman JG Smith JA Struhl K John Wiley & Sons, New York1995: 15.3.1-15.3.8Google Scholar The densitometric values obtained for MRP-1/CD9,KAI1/CD82, and ME491/CD63 bands in a given tumor tissue sample were divided by the corresponding value of β-actin for normalization, and the ratio was referred to as the gene expression ratio for each gene. The expression ratio of the tumor was divided by the expression ratio of the human endothelial cell line ECV304 to obtain the gene conservation rates. When the conservation rate of a given specimen was ≥1.0, it was considered to indicate conserved (positive) gene expression. If the value was <1.0, this denoted nonconserved (reduced) gene expression. To confirm the results of MRP-1/CD9 andKAI1/CD82 gene expression on RT-PCR, immunohistochemical studies were performed as described previously.25Huang C Taki T Adachi M Yagita M Sawada S Takabayashi A Inufusa H Yoshie O Miyake M MRP-1/CD9 and KAI1/CD82 expression in normal and various cancer tissues.Int J Oncol. 1997; 11: 1045-1051PubMed Google Scholar Because MRP-1/CD9 and KAI1/CD82 are not well preserved in formalin-fixed, paraffin-embedded tissues, frozen sections were used instead. After quenching the endogenous peroxidase activity with 0.3% H2O2 (in absolute methanol) for 30 minutes, the sections were blocked for 2 hours at room temperature with 5% bovine serum albumin. Subsequently, duplicate sections were incubated for 2 hours with the anti-MRP-1/CD9 monoclonal antibody M31-1512Miyake M Koyama M Seno M Ikeyama S Identification of the motility-related protein (MRP-1), recognized by monoclonal antibody M31–15, which inhibits cell motility.J Exp Med. 1991; 174: 1347-1354Crossref PubMed Scopus (190) Google Scholar and the anti-KAI1/CD82 monoclonal antibody C33,26Imai T Fukudome K Takagi S Nagira M Furuse M Fukuhara N Nishimura M Hinuma Y Yoshie O C33 antigen recognized by monoclonal antibodies inhibitory to human T cell leukemia virus type 1-induced syncytium formation is a member of a new family of transmembrane proteins including CD9, CD37, CD53, and CD63.J Immunol. 1992; 149: 2879-2886PubMed Google Scholar respectively, and were then incubated for 1 hour with biotinylated horse anti-mouse immunoglobulin G (Vector Laboratories Inc., Burlingame, CA). The sections were incubated with the avidin-biotin-peroxidase complex (Vector) for 1 hour, and the antibody binding was visualized with 3,3′-diaminobenzidine tetrahydrochloride. Finally, the sections were lightly counterstained with Mayer's hematoxylin (Figure 2). Specimens of fibroadenoma of the breast were used as positive controls. All of the immunostained sections were reviewed by two pathologists who had no knowledge of the patients' clinical status. Slides were examined under low power (×4 objective) to identify regions containing low-staining invasive tumors cells. In cases of multiple areas of low intensity, five areas selected at random were scored, and in sections where all of the staining appeared intense, one random field was selected. The proportion of high- and low-staining tumor cells in each selected field was determined by counting individual tumor cells at high magnification. At least 200 tumor cells were scored per ×40 field. Positive tumor cells were stained equivalent to normal breast glands and benign fibroadenoma tumor cells. All sections were scored in a semiquantitative fashion according to the method described previously,27McCarty Jr, KS Szabo E Flowers JL Cox EB Leight GS Miller L Konrath J Soper JT Budwit DA Creasman WT Seigler HF McCarty Sr, KS Use of a monoclonal anti-estrogen receptor antibody in the immunohistochemical evaluation of human tumors.Cancer Res. 1986; 46: 4244s-4248sPubMed Google Scholar which considers both the intensity and percentage of cells staining at each intensity. Intensities were classified as 0 (no staining), +1 (weak staining), +2 (distinct staining), and +3 (very strong staining), whereas 10% groupings were used for the percentage of cells that stained positive. For each slide, a value designated HSCORE was obtained by application of the following algorithm: HSCORE = Σ(I ×PC), where I and PC represent intensity and percentage cells that stain at each intensity, respectively, and corresponding HSCOREs were calculated separately. Specimens with an HSCORE of ≥50 were classified as MRP-1/CD9 or KAI1/CD82-positive (+), and when HSCORE was <50, specimens were classified as reduced (−). The statistical significance of differences betweenMRP-1/CD9 or KAI1/CD82 gene expression and several other clinical pathological parameters was assessed by the χ2Allred DC Clark GM Elledge R Fuqua SA Brown RW Chamness GC Osborne CK McGuire WL Association of p53 protein expression with tumor cell proliferation rate and clinical outcome in node-negative breast cancer.J Natl Cancer Inst. 1993; 85: 200-206Crossref PubMed Scopus (741) Google Scholar test. The disease-free survival and the overall survival curves were constructed according to the Kaplan-Meier method,28Kaplan EL Meier P Nonparametric estimation from incomplete observations.J Am Stat Assoc. 1958; 53: 457-481Crossref Scopus (47504) Google Scholar and differences in the survival of subgroups of patients were compared using Mantel's log-rank test.29Mantel N Evaluation of survival data and two new rank order statistics arising in its consideration.Cancer Chemother Rep. 1966; 50: 163-170PubMed Google Scholar Multivariate analyses were performed using the Cox regression model to study the effects of different variables on survival,30Cox DR Regression models and life-tables.J R Stat Soc B. 1972; 34: 187-220Google Scholar and six factors (MRP-1/CD9 status, KAI1/CD82status, ER status, age at surgery, T status, and N status) were studied. Scores were assigned to each variable for the regression analysis. All P values were based on two-tailed statistical analyses, and a P value < 0.05 was considered to indicate statistical significance. Of all 109 breast cancers studied, ME491/CD63 gene expression was preserved and no reduced levels of ME491/CD63DNA were detected (Figure 1). All of the carcinomas were evaluated to be ME491/CD63 positive, and no statistically significant relationships were found between ME491/CD63 gene expression and other known prognostic factors (Figure 1C). Thus,ME491/CD63 might play a different role from the other two transmembrane 4 superfamily members in breast cancer. On the other hand, of the 109 breast cancer patients, 73 tumors (67.0%) were evaluated as MRP-1/CD9 positive, and 36 tumors (33.0%) wereMRP-1/CD9 negative (Figure 1A). Forty-four tumors (40.4%) were evaluated as KAI1/CD82 positive, and 65 tumors (59.6%) were KAI1/CD82 negative (Figure 1B). However, no relationship was found between MRP-1/CD9 expression andKAI1/CD82 expression (r = 0.138,P = 0.3526, data not shown), and the expression of these genes were independent of each other. Of the 109 breast cancers studied using the immunohistochemical method, 72 (66.0%) were classified as MRP-1/CD9 positive. In these cases, the MRP-1/CD9 expression resembled that of benign fibroadenomas, and the immunostaining was intense and uniform on the cell surface membrane (Figure 2A). There were 37 cases (34.0%) with reduced MRP-1/CD9 expression (Figure 2, B and C), and the immunostaining from most of these tumors was heterogeneous. The MRP-1/CD9gene expression was readily evident in those primary tumors that were classified as positive in the immunohistochemical assays. In contrast, the MRP-1/CD9 gene expression was weak or entirely absent in those breast cancers that had reduced immunohistochemically detectable MRP-1/CD9. The MRP-1/CD9 gene expression evaluated by RT-PCR was highly associated with MRP-1/CD9 protein expression, as determined by immunohistochemical staining (r = 0.755,P < 0.0001) (Figure 3A). Overall, the immunohistochemical results agreed well with those from the RT-PCR assays, and 89.9% of the samples coincided exactly. On the other hand, there were 44 cases (40.4%) with positive KAI1/CD82 expression and 65 cases (59.6%) with reduced KAI1/CD82 expression (Figure 2, D to F). The KAI1/CD82 gene expression evaluated by RT-PCR was also associated with KAI1/CD82 protein expression, as determined by immunohistochemical staining (r = 0.803, P < 0.0001) (Figure 3B). These results also agreed well with those from the RT-PCR assays, and 90.8% of the samples coincided exactly. In cases of discrepancy, the results from the RT-PCR analysis were used in the specimen classification. As described in our results from the Western blotting analysis, comparing survival of 109 patients with breast cancer demonstrated that the disease-free survival rate of patients withMRP-1/CD9-negative tumors was significantly lower than that of patients with MRP-1/CD9-positive tumors (37.7%versus 65.7%, P = 0.0005) (Table 1 and Figure 4A). In particular, MRP-1/CD9was an effective indicator of patients with early-stage tumors such as T1, N0, stage I, and stage II (P = 0.0005,P = 0.0007, P = 0.0168, andP = 0.0249, respectively). In addition, the 5-year survival rate of patients with MRP-1/CD9-negative tumors was significantly lower than that of patients withMRP-1/CD9-positive tumors (80.8% versus 94.0%,P = 0.0380) (Table 1 and Figure 4B).Table 1Disease-Free Survival Rate and 5-Year Survival Rate of 109 Patients with Breast Cancer According to Their Clinicopathological Characteristics and MRP-1/CD9 Gene StatusDisease-free survival rate (%)5-year survival rate (%)CharacteristicsMRP-1/CD9 +MRP-1/CD9 −P valueMRP-1/CD9 +MRP-1/CD9 −P valueAge at surgery (years) ≤5068.845.90.040996.283.70.1855 >5063.928.60.001392.876.20.0815ER status +66.245.00.0244100.090.00.4000 −64.434.30.008188.770.30.0587Tumor status T195.244.40.0005100.0100.0>0.9999 T252.052.90.453990.383.70.4179 T380.025.00.0570100.075.0>0.9999 T4100.00.0>0.9999100.033.3>0.9999Nodal status N093.045.00.0007100.085.60.2000 N152.533.90.043890.185.10.6659 N240.00.00.253666.70.00.0445Pathological stage I93.837.50.0168100.0100.0>0.9999 II69.546.30.024993.281.50.1302 III55.614.30.088785.768.60.3759Total number of patients65.737.70.000594.080.80.0380 Open table in a new tab The disease-free survival rate of patients withKAI1/CD82-negative tumors was significantly lower than that o
Referência(s)