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Address reprint requests to Dr. Masayuki Miyake, Department of Thoracic Surgery and Department V of Oncology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, 13-3, Kamiyama-cho, Kita-ku, Osaka 530-0026, Japan
Department of Thoracic Surgery and Department V of Oncology, Kitano Hospital, Tazuke Kofukai Medical Research Institute, Osaka
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.
Early Breast Cancer Trialists' Collaborative Group Systemic 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.
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.
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.
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,
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.
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.
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.
Materials and Methods
Clinical Characteristics of the Patients
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.
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.
Quantitative RT-PCR Analysis
Total cellular RNA was extracted from the frozen tumor tissues by the acid guanidinium thiocyanate procedure.
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.
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′).
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.
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.
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.
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 M31-15
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.
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,
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 χ
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.
MRP-1/CD9, KAI1/CD82 and ME491/CD63 Gene Expression in Breast Cancer Tissues Analyzed by RT-PCR
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.
MRP-1/CD9 and KAI1/CD82 Protein Expression Analyzed by Immunohistochemistry
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.
Association of Tumor MRP-1/CD9 Status with Disease-Free and 5-Year Survival of Breast Cancer Patients
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 Status
Association of Tumor KAI1/CD82 Status with Disease-Free and 5-Year Survival of Breast Cancer Patients
The disease-free survival rate of patients withKAI1/CD82-negative tumors was significantly lower than that of patients with KAI1/CD82-positive tumors (38.2%versus 80.2%, P = 0.0065) (Table 2 and Figure 4C). In particular, the disease-free survival rate of patients withKAI1/CD82-negative, early-stage tumors (ie, T1, T2, N0, stage I, and stage II) was significantly lower than that of patients with KAI1/CD82-positive, early-stage tumors (P = 0.0226, P = 0.0105,P = 0.0243, P = 0.0429, andP = 0.0108, respectively). However, there was no significant difference between the 5-year survival rates of patients with KAI1/CD82-negative tumors and patients withKAI1/CD82-positive tumors (Table 2 and Figure 4D).
Table 2Disease-Free Survival Rate and 5-Year Survival Rate of 109 Patients with Breast Cancer According to Their Clinicopathological Characteristics and KAI1/CD82 Gene Status
Prognostic Value of MRP-1/CD9 and KAI1/CD82 Status
The Cox regression model was used to evaluate the disease-free survival and overall survival as shown in Table 3. The MRP-1/CD9 status (hazard ratio, 3.332; P = 0.0016), KAI1/CD82status (hazard ratio, 2.778; P = 0.0234), ER status (hazard ratio, 2.242; P = 0.0336), and nodal status (hazard ratio, 3.089; P = 0.0003) were found to be useful indicators for the disease-free survival of breast cancer patients. On the other hand, only two variables, ER status (hazard ratio, 6.358; P = 0.0230) and nodal status (hazard ratio, 3.376; P = 0.0221), were significant factors for predicting the overall survival of breast cancer patients.
Table 3Multivariate Regression Analysis in Predicting the Disease-Free Survival and Overall Survival of 109 Patients with Breast Cancer
Classification of Breast Cancer According to MRP-1/CD9 and KAI1/CD82 Gene Expression
Initially, the 109 breast cancer patients were divided into four groups according to their MRP-1/CD9 and KAI1/CD82gene status; 34 patients had both MRP-1/CD9- andKAI1/CD82-positive tumors, 10 patients hadMRP-1/CD9-negative but KAI1/CD82-positive tumors, 39 patients had MRP-1/CD9-positive butKAI1/CD82-negative tumors, and 26 patients had bothMRP-1/CD9- and KAI1/CD82-negative tumors. The disease-free survival rates of these patients were 94.1%, 40.0%, 26.9%, and 37.6%, respectively. The disease-free survival rate of patients with tumors positive for both genes was significantly higher than that of patients with the other three types of tumors. On the other hand, there were no significant differences among the latter three groups. Therefore, the 109 breast cancer patients were reclassified into two subgroups: one subgroup with bothMRP-1/CD9- and KAI1/CD82-positive tumors and the other subgroup with either or both negative tumors. The disease-free survival rate of the former double-positive subgroup was significantly higher than the latter subgroup (94.1% versus 38.4%,P = 0.0003) (Table 4 and Figure 4E). In addition, the former subgroup also had a higher 5-year survival rate than the latter subgroup (100.0% versus84.4%, P = 0.0292) (Table 4 and Figure 4F). The Cox multivariate regression analysis of disease-free survival is shown in Table 5. This simultaneous evaluation for both MRP-1/CD9 and KAI1/CD82 expression was found to be a significant indicator of a poor prognosis (P = 0.0006), and nodal status is also a significant indicator of a poor prognosis. The other variables (age at surgery, ER status, and T status) did not correlate with theMRP-1/CD9 and KAI1/CD82 gene status or its prognostic value. However, because no patients in the double-positive subgroup have died, it is impossible to perform a Cox multivariate regression analysis for their 5-year survival.
Table 4Disease-Free Survival Rate and 5-Year Survival Rate of 109 Patients with Breast Cancer according to Their MRP-1/CD9 andKAI-1/CD82 Gene Status
As part of our evaluation of the members of the transmembrane 4 superfamily as possible prognostic predictors for breast cancer, we have extended our study to other genes, such asME491/CD63 and KAI1/CD82. It has been reported that ME491/CD63 is strongly expressed on the cell surface during the early stage of malignant melanoma, but is weaker or absent in the more malignant stages and in the normal melanocyte.
However, the ME491/CD63 mRNA levels in almost all of the breast cancers were well preserved, and no cases with a reduction inME491/CD63 mRNA levels were detected. This suggests thatME491/CD63 may limit the progression only in malignant melanomas and that its expression might have no effect on the characteristics of breast cancers. On the other hand, our present study demonstrated that a reduction in not only MRP-1/CD9 gene expression but also KAI1/CD82 gene expression in breast tumors correlated with a poor prognosis. In addition, a simultaneous classification according to both MRP-1/CD9 andKAI1/CD82 gene expression was a very useful indicator for predicting the recurrence of breast cancer. Of patients with N0 status or stage I diseases, those with both MRP-1/CD9- andKAI1/CD82-positive tumors had no recurrences. However, among patients with either MRP-1/CD9- orKAI1/CD82-negative tumors, the disease-free survival rates were only 55.1% for patients with N0 status and 72.9% for patients with stage I. Therefore, when breast cancer patients have eitherMRP-1/CD9- or KAI1/CD82-negative tumors, they should be carefully observed after surgery even in early pathological stages. Furthermore, these patients might need additional adjuvant chemotherapy and/or hormonal therapy. In contrast, of the 34 patients with both MRP-1/CD9- and KAI1/CD82-positive tumors, only 2 patients (T2N2 and T3N1) had recurrences, and all of the patients were alive for 5 years after their surgery. These results suggested that the tumors positive for both MRP-1/CD9 andKAI1/CD82 may have a lower-grade malignancy and that adjuvant chemotherapy might not be necessary before recurrence.
Both MRP-1/CD9 and KAI1/CD82 are cell surface membrane glycoproteins that are widely expressed in various normal epithelia.
Their extracellular loops usually have some N-glycosylation sites. This type of structure suggests that these cell surface glycoproteins might play an important role in signal transduction and may regulate cell growth, cell differentiation, cell adhesion, and cell motility.
this family may organize the positioning of cell surface proteins and thus play a role in signal transduction. In addition, MRP-1/CD9, KAI1/CD82, CD63, and CD81 are considered to form complexes with each other.
These results suggest that MRP-1/CD9 or KAI1/CD82 gene expression might be declining as the malignant tumors advance and could disrupt the tetraspan network and enable the malignant cells to acquire their metastatic potential. In fact, MRP-1/CD9 gene has been reported to be more highly expressed in a primary colon cancer as compared with its corresponding metastatic tumor using differential display cloning.
We also have found no mutations of MRP-1/CD9 gene in 143 resected lung tumor specimens (data not shown). To date, we believe that the mechanism underlying the reduction in the expression of these genes in tumor tissues might be an abnormal gene promoter or an abnormal down-regulation somewhere upstream in their signal pathway. Various oncogenes and oncosuppressor genes have their actions relatively upstream of the signal pathway. During the progression of these tumors, the accumulations of abnormalities might act as triggers inducing the tumors to become more aggressive.
Another interesting aspect of the current research is that our study showed that the results of MRP-1/CD9 andKAI1/CD82 gene expression as evaluated by RT-PCR agreed well quantitatively with their protein expression as evaluated by immunohistochemistry. In addition, because the results in larger-scale studies might also depend on the quality of the mRNA, it may be relatively easy to quantitate MRP-1/CD9 and KAI1/CD82 protein expression in an actual clinical setting. Our present study demonstrated that MRP-1/CD9 and KAI1/CD82 gene expression in breast cancers are important factors for predicting recurrence. The classification of breast cancers according to bothMRP-1/CD9 and KAI-1/CD82 gene expression will prove to be useful for the clinical treatment of breast cancers patients.
We thank Tatsuya Hirai for his technical assistance and Drs. Tadashi Obayashi and Sadayuki Matsumoto for the pathological examination.
Early Breast Cancer Trialists' Collaborative Group
Systemic 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.
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.