Purpose: CD47 plays a variety of roles in intercellular signaling. Herein, we focused on the clinicopathologic significance of CD47 expression in human breast cancer. Our data suggest that the correlation between CD47 and signal regulatory protein α (SIRPA) expression may play a key role in the progression of breast cancer.

Experimental Design: Quantitative real-time PCR was used to evaluate CD47 mRNA and SIRPA mRNA expression in bone marrow and in peripheral blood from 738 cases of breast cancer.

Results: In patients with high levels of CD47 expression in the bone marrow, survival was significantly poorer compared with patients with low levels of CD47 expression [disease-free survival (DFS), P = 0.0035; overall survival (OS), P = 0.015]. Furthermore, high CD47 expression group in a multivariate analysis showed significance as an independent variable for poorer prognosis in DFS (P = 0.024). In the peripheral blood, however, high CD47 expression in patients was not an independent and significant prognostic factor for DFS and OS in a multivariate analysis. CD47 expression was strongly correlated with SIRPA expression in both the bone marrow (P < 0.0001) and peripheral blood (P < 0.0001) of breast cancer patients.

Conclusions: This is one of the first studies to show that a host factor in bone marrow confers prognostic importance. CD47 is an important biomarker in breast cancer, and functions as a prognostic factor for DFS. Moreover, we suggest that the poor prognosis of breast cancer patients with high expression of CD47 is due to an active CD47/SIRPA signaling pathway in circulating cells. Clin Cancer Res; 16(18); 4625–35. ©2010 AACR.

Translational Relevance

The association between cancer cells and normal host side factors is thought to be important in promoting cancer progression and metastasis. Herein, we focused on the clinicopathologic significance of CD47 gene expression in bone marrow and peripheral blood of breast cancer. Our data strongly suggest that CD47 is a significant prognostic indicator for disease-free survival (DFS). Moreover, CD47 expression is strongly correlated with SIRPA expression in both the bone marrow and peripheral blood of breast cancer, and it indicates that the poor prognosis of breast cancer with high expression of CD47 is due to an active CD47/SIRPA signaling pathway in circulating cells. Regarding patient care after surgery, many cases require postoperative adjuvant chemotherapy. Due to the associated adverse effects of such treatment, reliable prognostic markers for recurrence and metastasis would greatly improve patient management. We suggest that this biomarker may fill that need for enhanced patient care.

The numerous efforts in breast cancer research and care have improved early detection and treatment. However, breast cancer prevalence and mortality remain at a high level every year. The prevention and therapy of breast cancer among Japanese women is a crucial public health concern. The most recent statistics for Japan document over 55,000 cases per year (1), with a mortality surpassing 12,000 per year (2). Even after apparently successful localized treatments, there are long-term risks of recurrence and metastasis. To evaluate the postsurgical risk of recurrence of breast cancer, mammography, echogram, computer tomography, and magnetic resonance imaging are utilized for diagnostic imaging, and carcinoembryonic antigen (CEA), CA15-3, and NCC-ST439 are evaluated in peripheral blood as tumor markers. However, the long-term risk of relapse is largely due to clinically occult microrecurrences and micrometastases that are currently beyond detection by current conventional screening strategies. Therefore, it is important to exploit novel tumor markers that could predict recurrence and metastasis with greater reliability.

Kaplan et al. have shown that bone marrow–derived hematopoietic progenitor cells play an important role in the accumulation of premetastatic niches and the promotion of carcinogenesis and metastasis (3). We confirmed their findings by using clinical samples in which hematogenous metastasis occurred, and within these metastases, hematopoietic progenitor cells and isolated tumor cells (ITC) coexisted. This study showed the necessity of identifying metastasizing cancer cells as well as normal host side factors, such as bone marrow–derived cells and endothelial cells (4).

Recent studies showed that CD47 specifically inhibits phagocytosis and that there was a significant correlation between gene expression of CD47 and leukemia, hematopoietic stem cells, and tumor-initiating cells of bladder cancer (57). It can be inferred from the association between CD47 and cancer stem cells that ITC would elude the immune system by taking advantage of activation and initiation of the signal transduction cascade of CD47, resulting in inhibition of phagocytosis.

CD47 was originally identified in association with the integrin αvβ3, hence its alternative name of integrin-associated protein. It is also a member of the Ig superfamily, possessing a V-type Ig-like extracellular domain, five putative membrane-spanning segments, and a short cytoplasmic tail (8). CD47 seems to carry out several functions. For instance, CD47 functions as a marker of “self” on murine RBC. Erythrocytes lacking CD47 expression are rapidly removed from the bloodstream by splenic red pulp macrophages (9, 10). Signal regulatory protein α (SIRPA), a transmembrane glycoprotein, is a novel intracellular signal transducer when it is engaged by its ligand, CD47. CD47 on normal peripheral blood red cells circumvent elimination by binding to SIRPA (10). The interaction of CD47 with SIRPA occurs between host-derived cells, and is mostly related to cell signaling in the immune and nervous systems (11).

In the present study, we confirmed that high expression of CK19, a marker for ITC, had correlation with high CD47 expression. Therefore, we focused on the clinicopathologic significance of CD47 gene expression in the bone marrow and peripheral blood of breast cancer and its potential utility as a novel and specific biological marker for recurrence and/or overall survival (OS) in breast cancer patients. Investigating the characteristics of CD47 mRNA expression in bone marrow and peripheral blood in this study, we evaluated the correlation between CD47 and clinicopathologic factors in 738 breast cancer cases, and showed that the magnitude of CD47 expression could be used as a new prognostic marker for recurrence and metastasis. Moreover, we found that expression of CD47 and SIRPA were correlated in bone marrow and peripheral blood. This association has potentially important implications for clinicopathologic outcome. We suggest that the correlated expression of CD47 and SIRPA represents a dynamic process involved in the progression of breast cancer cells. This report is one of the first to show that a host factor in bone marrow confers prognostic importance.

Patients

A total of 738 breast cancer patients were identified based on their pathologic diagnosis before surgery at Kyushu Cancer Center from July 2000 to August 2005. Written informed consent was obtained from all patients according to the guidelines approved by the Institutional Research Board. Patients ranged in age from 24 to 89 years, with a mean age of 55.1 years. No patients received antihormonal treatment, chemotherapy, or radiotherapy before surgery. All patients were closely followed after surgery at regular 3- to 6-month intervals, and the follow-up periods ranged from 2 months to 6 years, with a median of 3.0 years. After surgery, all patients were clearly classified into the category of breast cancer based on the clinicopathologic criteria described by the Japanese Society for Breast Cancer. All data, including age, menopause, tumor stage, lymphatic invasion, lymph node metastasis, vascular invasion, distant metastasis, clinical stage, estrogen receptor (ER), progesterone receptor (PgR), human epidermal growth factor receptor 2 (Her2) score, and recurrence were obtained from the clinical and pathologic records. ER, PgR, and Her2 scores were obtained from immunohistochemistry staining conducted by two well-trained pathologists. Her2 status was scored using the Her2 expression criteria (Supplementary Table S1). These criteria changed in May 2009, but we applied the previous criteria to evaluate Her2 status. The primary tumors with Her2 score (2+) had their immunohistochemistry results additionally validated by fluorescence in situ hybridization. After surgical therapy, all patients were individually treated by antihormonal treatment, chemotherapy, and/or radiotherapy according to breast cancer treatment guidelines in Japan, which were based on American Society of Clinical Oncology and National Comprehensive Cancer Network recommendations. Regarding noncancer patients, we considered that inflammatory and neoplastic diseases, including benign tumors, might affect the result of our experiments, and we therefore excluded those patients from this group. We selected 19 patients who underwent surgery for elective cholecystolithiasis at the Medical Institute of Bioregulation Hospital, Kyushu University, between 1999 and 2003; all patients had a blood test before surgery, and they were confirmed to be without inflammatory symptoms. Written informed consent was obtained from all patients. All control patients had a whole body computer tomography examination to determine whether they had cancer, and their status was confirmed by assessing tumor markers in the peripheral blood. After surgery, they were followed at regular 6-month intervals, and the absence of cancer was confirmed over 3 years following surgery.

Sample collection

Bone marrow and peripheral blood samples were obtained from patients under anesthesia before surgery. Peripheral blood samples were taken from superficial veins on the opposite side of the breast cancer, and bone marrow samples were taken from the sternum with a 15G needle. As there was a potential for contamination by skin, bone marrow specimens were collected with another syringe after the first 2 to 3 mL were aspirated. ISOGEN-LS (Nippon Gene Co., Ltd., Japan) was added and mixed, stored for 5 minutes at room temperature, and was immediately frozen in liquid nitrogen and stored at −80°C until RNA extraction. Samples from noncancer patients were obtained with the same procedure.

Cell lines

The breast cancer cell lines CRL1500, MCF7, MRK-nu1, YMB1, YMB1E, SKBR3, and MDA-MB-231 were obtained from the Cell Resource Center for Biomedical Research Institute of Development, Aging and Cancer (Tohoku University, Japan).

RNA preparation and reverse transcription

Total RNA from bone marrow and peripheral blood was extracted from control and breast cancer patients using the ISOGEN-LS method followed by Isogen-chloroform extraction and isopropanol precipitation (12). As described previously, cDNA was synthesized from 8.0 μg of total RNA (13).

Evaluation of CD47 and SIRPA expression in clinical samples

The sequences of CD47 primers were as follows: sense primer, 5′-GGCAATGACGAAGGAGGTTA-3′; antisense primer, 5′-ATCCGGTGGTATGGATGAGA-3′. The sequences of SIRPA primers were as follows: sense primer, 5′- GTTTAAGTCTGGAGCAGGCACT-3′; antisense primer, 5′-GCAGATGACTTGAGAGTGAACG-3′. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as an internal control and the sequences of GAPDH primers were as follows: sense primer, 5′-TTGGTATCGTGGAAGGACTCTA-3′; antisense primer, 5′-TGTCATATTTGGCAGGTT-3′. cDNA was synthesized from 8.0 μg of total RNA. Real-time monitoring of PCR reactions was done using the LightCycler system (Roche Applied Science) and SYBR-Green I dye (Roche Applied Science). Monitoring was done according to the manufacturer's instructions. Quantitative reverse transcriptase-PCR (RT-PCR) was done with the following cycling conditions: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 10 seconds, annealing at 60°C for 10 seconds, and extension at 72°C for 10 seconds. After amplification, products were subjected to a temperature gradient from 68°C to 95°C at 0.2°C/second, under continuous fluorescence monitoring, to produce a melting curve of the products. All concentrations were calculated relative to the concentration of cDNA from Human Universal Reference total RNA (Takara Bio Inc., Japan). The concentrations of CD47 and SIRPA mRNAs were then divided by the concentration of the endogenous reference (GAPDH) to obtain normalized expression values (1416). Each assay was done twice to verify the results, and the mean normalized value of mRNA expression was used for subsequent analyses.

Statistics

For continuous variables, data were expressed as means ± SD. The relationship between CD47, SIRPA mRNA expression and clinicopathologic factors was analyzed using the χ2 test and Student's t-test. In addition, the data were also analyzed using the nonparametric Wilcoxon rank-sum test. Survival curves were plotted according to the Kaplan-Meier method, and the generalized log-rank test was applied to compare the survival curves. Variables with a value of P < 0.05 in univariate analysis were used in a subsequent multivariate analysis using the Cox regression. All tests were analyzed using JMP 7 software (SAS version 7.0.1, SAS Institute, Inc.), and the findings were considered significant when P < 0.05.

Comparison between CK19 and CD47 expression

In our study, we assessed CK19 expression in the bone marrow and peripheral blood of 738 breast cancer patients with quantitative real-time RT-PCR analysis. We detected 57 CK19-positive cases in bone marrow and 57 cases in peripheral blood. We divided patients into two groups: the CK19-negative group and the CK19-positive group. We then evaluated CD47 expression in the bone marrow and peripheral blood of 738 breast cancer patients with quantitative real-time RT-PCR and compared CD47 expression with CK19 status. CD47 expression in the CK19-positive group was higher than that in CK19-negative group (bone marrow, P = 0.04; peripheral blood, P = 0.02; Supplementary Fig. S1). CD47 expression (mean ± SD) in bone marrow was 1.91 ± 2.06 [confidence interval (CI), 1.54-2.28] in the CK19-positive group and 1.51 ± 1.34 (CI, 1.39-1.63) in the CK19-negative group. CD47 expression in peripheral blood was 2.65 ± 3.03 (CI, 2.12-3.18) in the CK19-positive group and 1.97 ± 1.74 (CI, 1.74-2.19) in the CK19-negative group.

High expression levels of CD47 in bone marrow and peripheral blood of breast cancer patients

In this study, we selected 19 patients with cholecystolithiasis as control cases. Quantitative real-time RT-PCR analysis showed higher expression of CD47 mRNA in breast cancer cases than in control cases (Fig. 1A-1). In bone marrow, the mean expression ratio (mean ± SD) of CD47/GAPDH mRNAs in breast cancer, 1.52 ± 1.43 (CI, 1.42-1.63), was significantly higher than that in noncancer cases, 0.80 ± 0.65 (CI, 0.49-1.11; Wilcoxon Rank-Sum test, P = 0.033). In peripheral blood, the mean expression ratio (mean ± SD) of CD47/GAPDH mRNAs in breast cancer, 1.83 ± 1.69 (CI, 1.67-1.99), was significantly higher than that in noncancer cases, 0.48 ± 0.49 (CI, 0.24-0.73; P = 0.0015). In Fig. 1A-2, the histogram shows the number of cases within each range of expression ratios of CD47/GAPDH. We also evaluated CD47 mRNA in 32 noncancer patients; the results of CD47 expression were similar to that from 19 patients as normal controls (Supplementary Fig. S2-a).

Fig. 1.

A-1, comparison of CD47 mRNA expression in breast cancer, breast cancer cell lines, and control samples. The distribution chart shows each expression ratio of CD47/GAPDH mRNA derived from cancer cases in bone marrow (BM; BM cancer), control patients in BM (BM normal), breast cancer cell lines, cancer cases in peripheral blood (PB; PB cancer) and normal patients in PB (PB normal). A-2, number of cases within each range of expression ratios of CD47/GAPDH mRNA in bone marrow derived from breast cancer patients (white bar), breast cancer cell lines (black bar), and normal cases (grey bar). B, comparison of CD47 expression among histologic types of breast cancer. The distribution chart shows each expression ratio of CD47/GAPDH mRNA derived from ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), and other types of breast cancer (other). C and D, the 5-year disease-free survival (DFS) and overall survival (OS) rates in patients with high CD47 mRNA (dash line) and patients with low CD47 mRNA (solid line) in BM and in PB. In DFS, excluding 9 metastatic breast cancer patients, the numbers of patients with high CD47 and low CD47 are 221 and 222, respectively in BM and PB. In OS, 226 patients with high CD47 expression and 226 patients with low CD47 expression are displayed with dash and solid lines, respectively.

Fig. 1.

A-1, comparison of CD47 mRNA expression in breast cancer, breast cancer cell lines, and control samples. The distribution chart shows each expression ratio of CD47/GAPDH mRNA derived from cancer cases in bone marrow (BM; BM cancer), control patients in BM (BM normal), breast cancer cell lines, cancer cases in peripheral blood (PB; PB cancer) and normal patients in PB (PB normal). A-2, number of cases within each range of expression ratios of CD47/GAPDH mRNA in bone marrow derived from breast cancer patients (white bar), breast cancer cell lines (black bar), and normal cases (grey bar). B, comparison of CD47 expression among histologic types of breast cancer. The distribution chart shows each expression ratio of CD47/GAPDH mRNA derived from ductal carcinoma in situ (DCIS), invasive ductal carcinoma (IDC), and other types of breast cancer (other). C and D, the 5-year disease-free survival (DFS) and overall survival (OS) rates in patients with high CD47 mRNA (dash line) and patients with low CD47 mRNA (solid line) in BM and in PB. In DFS, excluding 9 metastatic breast cancer patients, the numbers of patients with high CD47 and low CD47 are 221 and 222, respectively in BM and PB. In OS, 226 patients with high CD47 expression and 226 patients with low CD47 expression are displayed with dash and solid lines, respectively.

Close modal

We assessed CD47 expression in breast cancer cell lines to determine whether the expression ratio of CD47 is affected by the number of ITC in bone marrow or peripheral blood. The human breast cancer cell lines CRL1500, MCF7, MRK-nu1, YMB1, YMB1E, SKBR3, and MDA-MB-231 were assessed. The mean expression ratio of CD47/GAPDH mRNA was 0.24 ± 0.18 (CI, 0.07-0.40) in breast cancer cell lines, and was significantly lower than those found in the bone marrow and peripheral blood samples of breast cancer cases. Next, we compared the CD47/GAPDH ratio of each histologic type of breast cancer in bone marrow (Fig. 1B). We divided patients into three types: ductal carcinoma in situ (DCIS; n = 53), invasive ductal carcinoma (IDC; n = 647), and other types of breast cancer (other; n = 38). The mean expression ratios of CD47/GAPDH mRNAs were as follows: DCIS, 0.68 ± 0.71 (CI, 0.47-0.88); IDC, 1.66 ± 1.47 (CI, 1.54-1.77); others, 1.09 ± 1.31 (CI, 0.69-1.49). The CD47/GAPDH expression ratio in IDC was significantly higher than in DCIS (P < 0.0001) and others (P = 0.03). Such variations in the CD47/GAPDH expression ratios were likely to be reflected in the bone marrow of IDC, so we focused on the IDC subtype for subsequent analyses.

Comparison with tumor markers

Table 1 shows the results of tumor markers that were analyzed in peripheral blood samples taken before surgery. The mean values (mean ± SD) of tumor markers in breast cancer were as follows: CEA, 2.01 ± 3.37; CA15-3, 12.19 ± 11.19; and NCC-ST439, 7.45 ± 31.50. NCC-ST439 was only slightly higher than normal levels, whereas the values of CEA and CA15-3 were within normal levels. As a result, these tumor markers may not be useful as biomarkers in breast cancer cases before surgery.

Table 1.

Clinical magnitude of CD47/GAPDH in breast cancer cases prior to surgery, and comparison with known serum tumor markers

Normal valueAverage (SD)CI
CEA (serum) <5.0 ng/mL 2.01 (3.37) 1.69-2.33 
CA15-3 (serum) <25.0 U/mL 12.19 (11.19) 11.13-13.26 
NCC-ST439 (serum) <7.0 U/mL 7.45 (31.50) 1.06-13.83 
CD47/GAPDH (BM) (0.80, average of BM in noncancer cases) 1.52 (1.43) 1.42-1.63 
    High-expression group  2.95 (1.11) 2.80-3.09 
    Low-expression group  0.62 (0.42) 0.56-0.67 
CD47/GAPDH (PB) (0.48, average of PB in noncancer cases) 1.83 (1.69) 1.67-1.99 
    High-expression group  3.86 (1.86) 3.60-4.12 
    Low-expression group  0.58 (0.63) 0.50-0.66 
Normal valueAverage (SD)CI
CEA (serum) <5.0 ng/mL 2.01 (3.37) 1.69-2.33 
CA15-3 (serum) <25.0 U/mL 12.19 (11.19) 11.13-13.26 
NCC-ST439 (serum) <7.0 U/mL 7.45 (31.50) 1.06-13.83 
CD47/GAPDH (BM) (0.80, average of BM in noncancer cases) 1.52 (1.43) 1.42-1.63 
    High-expression group  2.95 (1.11) 2.80-3.09 
    Low-expression group  0.62 (0.42) 0.56-0.67 
CD47/GAPDH (PB) (0.48, average of PB in noncancer cases) 1.83 (1.69) 1.67-1.99 
    High-expression group  3.86 (1.86) 3.60-4.12 
    Low-expression group  0.58 (0.63) 0.50-0.66 

Abbreviations: BM, bone marrow; PB, peripheral blood.

Next, we divided breast cancer patients into two groups according to their CD47/GAPDH ratios. Thus, bone marrow and peripheral blood values were divided into those greater or less than the median CD47/GAPDH ratio: the high-expression group (n = 226) and the low-expression group (n = 226). In both bone marrow and peripheral blood, the mean expression ratios of CD47/GAPDH mRNAs in cancer cases were significantly higher than in control patients. Furthermore, the mean ratio of CD47/GAPDH mRNAs in the high-expression group of cancer cases was three to five times higher than in noncancer cases.

Clinicopathologic factors, disease-free survival, and overall survival of breast cancer patients

In this study, all the data were obtained from clinical and pathologic records. However, due to lack of data from breast cancer patients, particularly Her2 scores, we limited the analysis to the 452 clinical cases in which data were complete. Clinicopathologic significance of the CD47/GAPDH mRNA expression ratio in bone marrow is shown in Table 2. The incidence of triple negatives for ER, PgR, and Her2 status was significantly higher (P = 0.0097) in the high-expression group than in the low-expression group. The incidence of premenopausal patients was significantly higher (P = 0.01) in the high-expression group than in the low-expression group. The incidence of Her2 score was significantly higher (P = 0.03) in the high-expression group than in the low-expression group. The incidence of recurrence was significantly lower (P = 0.04) in the high-expression group than in the low-expression group. Conversely, no significant differences were observed regarding age, tumor stage, lymph node metastasis, lymphatic invasion, venous invasion, distant metastasis, clinical stage, estrogen receptor, and progesterone receptor.

Table 2.

Clinicopathologic significance of the CD47/GAPDH mRNA expression ratio in bone marrow and in peripheral blood

CD47/GAPDH clinicopathologic factorsBMPB
Low ratio (%)High ratio (%)PLow ratio (%)High ratio (%)P
Age, y (mean ± SD) 55.7 ± 11.2 55.1 ± 11.5 n.s. 55.8 ± 10.9 54.9 ± 11.8 n.s. 
Menopause status   0.01*   n.s. 
    Pre 71 (31.4) 96 (42.5)  76 (33.6) 91 (40.3)  
    Post 155 (68.6) 130 (57.5)  150 (66.4) 135 (59.7)  
Tumor stage   n.s.   0.0011* 
    T1 103 (45.6) 123 (54.4)  96 (42.8) 130 (57.2)  
    T2-4 123 (54.4) 103 (45.6)  130 (57.2) 96 (42.8)  
Lymph node metastasis   n.s.   n.s. 
    Absent 138 (61.1) 137 (60.6)  135 (59.7) 140 (61.9)  
    Present 88 (38.9) 89 (39.4)  91 (40.3) 86 (38.1)  
Lymphatic invasion   n.s.   n.s. 
    Absent 127 (56.2) 146 (64.6)  130 (58.0) 143 (62.3)  
    Present 99 (43.8) 80 (35.4)  96 (42.0) 83 (36.7)  
Venous invasion   n.s.   n.s. 
    Absent 215 (95.1) 208 (92.0)  215 (95.1) 208 (92.0)  
    Present 11 (4.9) 18 (8.0)  11 (4.9) 18 (8.0)  
Distant metastasis   n.s.   n.s. 
    Absent 221 (97.8) 222 (98.2)  221 (98.2) 222 (97.8)  
    Present 5 (2.2) 4 (1.8)  5 (1.8) 4 (2.2)  
Stage   n.s.   n.s. 
    Stage I 78 (34.5) 86 (38.1)  73 (32.3) 91 (40.7)  
    Stage II-IV 148 (65.5) 140 (62)  153 (67.7) 135 (59.3)  
ER   n.s.   n.s. 
    Absent 56 (24.8) 74 (32.7)  68 (30.1) 62 (27.0)  
    Present 170 (75.2) 152 (67.3)  158 (69.9) 164 (73.0)  
PgR   n.s.   n.s. 
    Absent 102 (45.1) 101 (44.7)  95 (42.0) 108 (47.8)  
    Present 124 (54.9) 125 (55.3)  131 (58.0) 118 (52.2)  
Her2 score   0.03*   n.s. 
    0-1 157 (69.5) 135 (59.7)  152 (67.3) 140 (61.9)  
    2-3 69 (30.5) 91 (40.3)  74 (32.7) 86 (38.1)  
ER, PgR, Her2 status   0.0097*   n.s. 
    Triple negative 26 (11.5) 46 (20.4)  26 (11.5) 46 (20.4)  
    Either one positive 200 (88.5) 180 (79.7)  200 (88.5) 180 (79.7)  
Recurrence   0.04*   <0.0001* 
    Absent 189 (83.6) 204 (90.3)  181 (80.1) 212 (93.8)  
    Present 37 (16.4) 22 (9.7)  45 (19.9) 14 (6.2)  
CD47/GAPDH clinicopathologic factorsBMPB
Low ratio (%)High ratio (%)PLow ratio (%)High ratio (%)P
Age, y (mean ± SD) 55.7 ± 11.2 55.1 ± 11.5 n.s. 55.8 ± 10.9 54.9 ± 11.8 n.s. 
Menopause status   0.01*   n.s. 
    Pre 71 (31.4) 96 (42.5)  76 (33.6) 91 (40.3)  
    Post 155 (68.6) 130 (57.5)  150 (66.4) 135 (59.7)  
Tumor stage   n.s.   0.0011* 
    T1 103 (45.6) 123 (54.4)  96 (42.8) 130 (57.2)  
    T2-4 123 (54.4) 103 (45.6)  130 (57.2) 96 (42.8)  
Lymph node metastasis   n.s.   n.s. 
    Absent 138 (61.1) 137 (60.6)  135 (59.7) 140 (61.9)  
    Present 88 (38.9) 89 (39.4)  91 (40.3) 86 (38.1)  
Lymphatic invasion   n.s.   n.s. 
    Absent 127 (56.2) 146 (64.6)  130 (58.0) 143 (62.3)  
    Present 99 (43.8) 80 (35.4)  96 (42.0) 83 (36.7)  
Venous invasion   n.s.   n.s. 
    Absent 215 (95.1) 208 (92.0)  215 (95.1) 208 (92.0)  
    Present 11 (4.9) 18 (8.0)  11 (4.9) 18 (8.0)  
Distant metastasis   n.s.   n.s. 
    Absent 221 (97.8) 222 (98.2)  221 (98.2) 222 (97.8)  
    Present 5 (2.2) 4 (1.8)  5 (1.8) 4 (2.2)  
Stage   n.s.   n.s. 
    Stage I 78 (34.5) 86 (38.1)  73 (32.3) 91 (40.7)  
    Stage II-IV 148 (65.5) 140 (62)  153 (67.7) 135 (59.3)  
ER   n.s.   n.s. 
    Absent 56 (24.8) 74 (32.7)  68 (30.1) 62 (27.0)  
    Present 170 (75.2) 152 (67.3)  158 (69.9) 164 (73.0)  
PgR   n.s.   n.s. 
    Absent 102 (45.1) 101 (44.7)  95 (42.0) 108 (47.8)  
    Present 124 (54.9) 125 (55.3)  131 (58.0) 118 (52.2)  
Her2 score   0.03*   n.s. 
    0-1 157 (69.5) 135 (59.7)  152 (67.3) 140 (61.9)  
    2-3 69 (30.5) 91 (40.3)  74 (32.7) 86 (38.1)  
ER, PgR, Her2 status   0.0097*   n.s. 
    Triple negative 26 (11.5) 46 (20.4)  26 (11.5) 46 (20.4)  
    Either one positive 200 (88.5) 180 (79.7)  200 (88.5) 180 (79.7)  
Recurrence   0.04*   <0.0001* 
    Absent 189 (83.6) 204 (90.3)  181 (80.1) 212 (93.8)  
    Present 37 (16.4) 22 (9.7)  45 (19.9) 14 (6.2)  

Abbreviation: n.s., not significant.

*P < 0.05, statistical significance.

The 5-year disease-free survival (DFS) and OS rates in patients with high CD47/GAPDH mRNA in bone marrow and patients with low CD47/GAPDH mRNA in bone marrow are shown in Fig. 1C. The survival difference between these two groups was statistically significant (DFS, P = 0.0035, log-rank test; OS, P = 0.015, log-rank test). The patients received at least one postoperative therapy (antihormonal treatment, chemotherapy, or radiotherapy). Univariate and multivariate analyses of clinicopathologic factors affecting DFS rate in bone marrow are shown in Table 3. Univariate analysis revealed a significant relationship between DFS and the following factors: lymphatic invasion, lymph node metastasis, venous invasion, estrogen receptor, progesterone receptor, Her2 score, and CD47 expression. Multivariate analysis indicated that the high expression ratio of CD47 was found to be an independent and significant prognostic factor for survival (P = 0.024). Univariate and multivariate analyses of clinicopathologic factors affecting OS rate in bone marrow are shown in Table 3. Univariate analysis revealed a significant relationship between OS and the following factors: menopause, lymph node metastasis, estrogen receptor, progesterone receptor, recurrence, and CD47 expression. Multivariate analysis indicated that the high expression ratio of CD47 was not an independent and significant prognostic factor for survival (P = 0.41).

Table 3.

Univariate and multivariate analyses of clinicopathologic factors affecting disease-free survival and overall survival rate in bone marrow

Clinicopathologic factorsDisease-free survivalOverall survival
No. of patientsUnivariate analysis HR (CI)Multivariate analysisNo. of patientsUnivariate analysis HR (CI)Multivariate analysis
Relative risk (CI)PRelative risk (CI)P
Age (years) 
    55 233 0.98 (0.58-1.67) — — 237 0.55 (0.22-1.32) — — 
    >55 210 215 
Menopause 
    Pre 165 0.86 (0.47-1.49) — — 167 0.21 (0.03-0.72) 0.25 (0.04-0.92) 0.035* 
    Post 278 285  
Tumor stage 
    T1 226 0.60 (0.33-1.04) — — 226 0.61 (0.22-1.52) — — 
    T2-4 217 226 
Lymphatic invasion 
    Present 172 2.08 (1.22-3.70) 1.22 (0.66-2.32) 0.54 179 1.85 (0.78-4.55) — — 
    Absent 271  273 
Lymph node metastasis 
    Present 170 3.60 (2.04-6.67) 2.63 (1.31-5.56) 0.0057* 177 3.97 (1.61-11.17) 1.72 (0.07-5.00) 0.27 
    Absent 273  275  
Venous invasion 
    Present 29 3.64 (1.23-8.33) 2.50 (0.84-6.25) 0.1 29 4.00 (0.62-15.02) — — 
    Absent 414  423 
Distant metastasis 
    Present — — — 5.32 (0.83-19.23) — — 
    Absent 443 443 
ER 
    Present 318 0.49 (0.29-0.84) 0.80 (0.38-1.69) 0.56 322 0.27 (0.11-0.64) 1.67 (0.48-5.39) 0.40 
    Absent 125  130  
PgR 
    Present 245 0.55 (0.32-0.93) 0.71 (0.35-1.49) 0.36 249 0.21 (0.07-0.54) 0.19 (0.04-0.79) 0.022* 
    Absent 198  203  
Her2 score 
    0-1 285 0.42 (0.25-0.72) 0.69 (0.38-1.26) 0.22 292 0.47 (0.19-1.14) — — 
    2-3 158  160 
Recurrence 
    Present 56 — — — 59 19.08 (5.26-33.33) 1955 <0.0001* 
    Absent 387 393   
CD47 expression 
    High 221 2.32 (1.28-4.17) 2.00 (1.10-3.61) 0.024* 226 2.99 (1.18-7.61) 1.54 (0.55-4.30) 0.41 
    Low 222  226  
Clinicopathologic factorsDisease-free survivalOverall survival
No. of patientsUnivariate analysis HR (CI)Multivariate analysisNo. of patientsUnivariate analysis HR (CI)Multivariate analysis
Relative risk (CI)PRelative risk (CI)P
Age (years) 
    55 233 0.98 (0.58-1.67) — — 237 0.55 (0.22-1.32) — — 
    >55 210 215 
Menopause 
    Pre 165 0.86 (0.47-1.49) — — 167 0.21 (0.03-0.72) 0.25 (0.04-0.92) 0.035* 
    Post 278 285  
Tumor stage 
    T1 226 0.60 (0.33-1.04) — — 226 0.61 (0.22-1.52) — — 
    T2-4 217 226 
Lymphatic invasion 
    Present 172 2.08 (1.22-3.70) 1.22 (0.66-2.32) 0.54 179 1.85 (0.78-4.55) — — 
    Absent 271  273 
Lymph node metastasis 
    Present 170 3.60 (2.04-6.67) 2.63 (1.31-5.56) 0.0057* 177 3.97 (1.61-11.17) 1.72 (0.07-5.00) 0.27 
    Absent 273  275  
Venous invasion 
    Present 29 3.64 (1.23-8.33) 2.50 (0.84-6.25) 0.1 29 4.00 (0.62-15.02) — — 
    Absent 414  423 
Distant metastasis 
    Present — — — 5.32 (0.83-19.23) — — 
    Absent 443 443 
ER 
    Present 318 0.49 (0.29-0.84) 0.80 (0.38-1.69) 0.56 322 0.27 (0.11-0.64) 1.67 (0.48-5.39) 0.40 
    Absent 125  130  
PgR 
    Present 245 0.55 (0.32-0.93) 0.71 (0.35-1.49) 0.36 249 0.21 (0.07-0.54) 0.19 (0.04-0.79) 0.022* 
    Absent 198  203  
Her2 score 
    0-1 285 0.42 (0.25-0.72) 0.69 (0.38-1.26) 0.22 292 0.47 (0.19-1.14) — — 
    2-3 158  160 
Recurrence 
    Present 56 — — — 59 19.08 (5.26-33.33) 1955 <0.0001* 
    Absent 387 393   
CD47 expression 
    High 221 2.32 (1.28-4.17) 2.00 (1.10-3.61) 0.024* 226 2.99 (1.18-7.61) 1.54 (0.55-4.30) 0.41 
    Low 222  226  

Abbreviation: HR, hazard ratio.

*P < 0.05.

The clinicopathologic factors analyzed in relation to CD47 mRNA expression in peripheral blood are shown in Table 2. The incidence of recurrence was significantly lower (P < 0.0001) in the high-expression group than in the low-expression group. The incidence of tumor stage was significantly lower (P = 0.0011) in the high-expression group than in the low-expression group. Conversely, no significant differences were observed regarding age, menopause, lymph node metastasis, lymphatic invasion, venous invasion, distant metastasis, clinical stage, estrogen receptor, progesterone receptor, Her2 score, and ER, PgR, Her2 status. The 5-year DFS and OS rates in patients with high CD47 mRNA and patients with low CD47 mRNA are shown in Fig. 1D. The survival difference between these two groups was not statistically significant for DFS (P = 0.18, log-rank test) and OS (P = 0.87, log-rank test). Univariate and multivariate analyses of clinicopathologic factors affecting DFS rate in peripheral blood are shown in Table 4. Univariate analysis revealed a significant relationship between OS and the following factors: tumor stage, lymphatic invasion, lymph node metastasis, venous invasion, estrogen receptor, progesterone receptor, and Her2 score, but CD47 expression was not included. Multivariate analysis indicated that the presence of lymph node metastasis was found to be an independent and significant prognostic factor for survival (P = 0.0055). Univariate and multivariate analyses of clinicopathologic factors affecting OS rate in peripheral blood are shown in Table 4. Univariate analysis revealed a significant relationship between OS and the following factors: menopause, lymph node metastasis, estrogen receptor, progesterone receptor, and recurrence. Univariate analysis indicated that the high expression ratio of CD47 was not an independent and significant prognostic factor for survival.

Table 4.

Univariate and multivariate analyses of clinicopathologic factors affecting disease-free survival and overall survival rate in peripheral blood

Clinicopathologic variableDisease-free survivalOverall survival
No. of patientsUnivariate analysis HR (CI)Multivariate analysisNo. of patientsUnivariate analysis HR (CI)Multivariate analysis
Relative risk (CI)PRelative risk (CI)P
Age (years) 
    55 233 0.98 (0.58-1.67) — — 237 0.55 (0.22-1.32) — — 
    >55 210 215 
Menopause 
    Pre 165 0.86 (0.47-1.49) — — 167 0.21 (0.03-0.72) 0.26 (0.04-0.94) 0.039* 
    Post 278 285  
Tumor stage 
    Tis-1 226 0.60 (0.33-1.04) — — 226 0.61 (0.22-1.52) — — 
    T2-4 217 226 
Lymphatic invasion 
    Present 172 2.08 (1.22-3.70) 1.20 (0.65-2.27) 0.56 179 1.85 (0.78-4.55) — — 
    Absent 271  273 
Lymph node metastasis 
    Present 170 3.60 (2.04-6.67) 2.63 (1.32-5.43) 0.0055* 177 3.97 (1.61-11.17) 1.58 (0.63-4.51) 0.34 
    Absent 273  275  
Venous invasion 
    Present 29 3.64 (1.23-8.33) 2.76 (0.92-6.67) 0.07 29 4.00 (0.62-15.02) — — 
    Absent 414  423 
Distant metastasis 
    Present — — — 5.32 (0.83-19.23) — — 
    Absent 443 443 
ER 
    Present 318 0.49 (0.29-0.84) 0.69 (0.34-1.41) 0.3 322 0.27 (0.11-0.64) 1.50 (0.45-4.47) 0.49 
    Absent 125  130  
PgR 
    Present 245 0.55 (0.32-0.93) 0.71 (0.36-1.43) 0.35 249 0.21 (0.07-0.54) 0.18 (0.04-0.68) 0.011* 
    Absent 198  203  
Her2 score 
    0-1 285 0.42 (0.25-0.72) 0.69 (0.38-1.24) 0.21 292 0.47 (0.19-1.14) — — 
    2-3 158  160 
Recurrence 
    Present 56 — — — 59 19.08 (5.26-33.33) 2075 <0.0001* 
    Absent 387 393   
CD47 expression 
    High 221 1.57 (0.79-3.03) — — 226 1.10 (0.30-3.22) — — 
    Low 222 226 
Clinicopathologic variableDisease-free survivalOverall survival
No. of patientsUnivariate analysis HR (CI)Multivariate analysisNo. of patientsUnivariate analysis HR (CI)Multivariate analysis
Relative risk (CI)PRelative risk (CI)P
Age (years) 
    55 233 0.98 (0.58-1.67) — — 237 0.55 (0.22-1.32) — — 
    >55 210 215 
Menopause 
    Pre 165 0.86 (0.47-1.49) — — 167 0.21 (0.03-0.72) 0.26 (0.04-0.94) 0.039* 
    Post 278 285  
Tumor stage 
    Tis-1 226 0.60 (0.33-1.04) — — 226 0.61 (0.22-1.52) — — 
    T2-4 217 226 
Lymphatic invasion 
    Present 172 2.08 (1.22-3.70) 1.20 (0.65-2.27) 0.56 179 1.85 (0.78-4.55) — — 
    Absent 271  273 
Lymph node metastasis 
    Present 170 3.60 (2.04-6.67) 2.63 (1.32-5.43) 0.0055* 177 3.97 (1.61-11.17) 1.58 (0.63-4.51) 0.34 
    Absent 273  275  
Venous invasion 
    Present 29 3.64 (1.23-8.33) 2.76 (0.92-6.67) 0.07 29 4.00 (0.62-15.02) — — 
    Absent 414  423 
Distant metastasis 
    Present — — — 5.32 (0.83-19.23) — — 
    Absent 443 443 
ER 
    Present 318 0.49 (0.29-0.84) 0.69 (0.34-1.41) 0.3 322 0.27 (0.11-0.64) 1.50 (0.45-4.47) 0.49 
    Absent 125  130  
PgR 
    Present 245 0.55 (0.32-0.93) 0.71 (0.36-1.43) 0.35 249 0.21 (0.07-0.54) 0.18 (0.04-0.68) 0.011* 
    Absent 198  203  
Her2 score 
    0-1 285 0.42 (0.25-0.72) 0.69 (0.38-1.24) 0.21 292 0.47 (0.19-1.14) — — 
    2-3 158  160 
Recurrence 
    Present 56 — — — 59 19.08 (5.26-33.33) 2075 <0.0001* 
    Absent 387 393   
CD47 expression 
    High 221 1.57 (0.79-3.03) — — 226 1.10 (0.30-3.22) — — 
    Low 222 226 

*P < 0.05.

Correlation with SIRPA

We investigated SIRPA expression in the same breast cancer patients and controls. Figure 2 shows the correlation between CD47 expression and SIRPA expression. In cancer cell lines, CD47 expression did not correlate with SIRPA expression (Spearman correlation = 0.0319; P = 0.95; data not shown). In control patients, CD47 expression was correlated with SIRPA in both bone marrow (P < 0.0001) and peripheral blood (P = 0.0044; Fig. 2A and B). In 32 noncancer cases, CD47 expression was correlated with SIRPA in both bone marrow (P = 0.004) and peripheral blood (P < 0.0001; Supplementary Fig. S2-b).

Fig. 2.

The correlation between CD47 expression and SIRPA expression. In control patients, CD47 expression was correlated with SIRPA in both bone marrow (A) and peripheral blood (B). In breast cancer cases, CD47 expression was even more strongly correlated with SIRPA in both bone marrow (C) and peripheral blood (D).

Fig. 2.

The correlation between CD47 expression and SIRPA expression. In control patients, CD47 expression was correlated with SIRPA in both bone marrow (A) and peripheral blood (B). In breast cancer cases, CD47 expression was even more strongly correlated with SIRPA in both bone marrow (C) and peripheral blood (D).

Close modal

In breast cancer cases, CD47 expression is more strongly correlated with SIRPA in both bone marrow (P < 0.0001) and peripheral blood (P < 0.0001; Fig. 2C and D).

CD47 is expressed on the surface of a wide variety of cells such as hematopoietic cells, keratinocytes, and cells of the brain (17). CD47 is associated with αvβ3 integrin and is implicated in the modulation of integrin functions, such as cell adhesion, phagocytosis, and cellular migration (1820). It is known that CD47 is a marker of self on RBC. CD47 could work as a marker of self on cancer cells, and breast cancer cells may express high levels of CD47 by themselves. Our results showed that the CD47/GAPDH expression ratio in breast cancer cell lines was significantly lower than those found in the bone marrow and peripheral blood samples of breast cancer cases. This may indicate that CD47 has various functions, and that the level of CD47 expression was affected by the cell environment rather than by the number of cancer cells. Therefore, the high expression of CD47 in the bone marrow and peripheral blood of breast cancer patients may represent the characteristic appearance of breast cancer and some evidence of a cancer-specific mechanism in the bone marrow and peripheral blood of breast cancer.

Recent reports have shown that CD47 plays a role in inhibiting macrophage phagocytosis of cancer stem cells and tumor-initiating cells (57). In the same manner as a cancer stem cell, the cancer cell itself may circumvent immune system surveillance by expressing CD47 as a marker of self, thereby evading natural killer cells (21, 22). In our study, we found that high CD47 expression had a correlation with high CK19 expression in the bone marrow and peripheral blood of breast cancer. This result strongly suggests that ITC of breast cancer patients may utilize the function of CD47 in circulating circumstances such as bone marrow and peripheral blood. Moreover, expression of CD47 in the bone marrow and peripheral blood of breast cancer patients was significantly higher than in control patients. Because expression of CD47 in circulating tumor cells increases exponentially with the progress of the cancer stage, CD47 derived from ITC may be an upregulating factor of breast cancer.

CD47 also promotes apoptosis, and the CD47 ligand thrombospondin (TSP) has been implicated as an antitumor and antimetastatic factor in breast cancer (2333). Both TSP1 and a CD47 agonist peptide (4N1K, derived from TSP1) can induce a novel form of apoptosis in transformed and activated normal T cells (34, 35), chronic lymphocytic leukemia cells (36), erythroleukemia cells, and primary arterial smooth muscle cells (35). We supposed that CD47 may have a role not only as a marker of self but also as an inducer of apoptosis to inhibit phagocytosis.

In the present study, the mean ratio of CD47/GAPDH mRNAs in the high-expression group of cancer cases was three to five times higher than in noncancer cases. We suggest that CD47 may be specifically expressed in the bone marrow and peripheral blood of breast cancer patients and that CD47 expression may represent an important biomarker in breast cancer patients. As a result of the identification of the clinical significance of CD47 expression in bone marrow and peripheral blood, we found that overexpression of CD47 in bone marrow and peripheral blood correlated with the aggressiveness of breast cancer. This result might suggest that the more there are circulating tumor cells expressing increased CD47 in bone marrow and peripheral blood, the more active the primary immune system is in inducing apoptosis in tumor cells in the circulating systems. Therefore, it is important to clarify the level of CD47 expression in bone marrow and peripheral blood to indicate whether micrometastasis exists in the breast cancer cases. Thus, CD47 may be a novel biological marker that predicts the number of highly malignant circulating tumor cells that escape from the immune systems in breast cancer.

To further characterize the function of CD47 in bone marrow and peripheral blood, we examined SIRPA expression in the same breast cancer samples. In doing so, we obtained the novel finding that the expressions of CD47 and SIRPA are markedly associated. The correlation between CD47 and SIRPA was significantly stronger in breast cancer patients than in control cases. In control cases, the CD47-SIRPA signaling system is activated in bone marrow and in peripheral blood, reflecting homeostatic regulation in the hematopoietic system. In the breast cancer cases, carcinogenicity may promote the CD47-SIRPA cell signaling system in bone marrow and in peripheral blood, thereby possibly promoting micrometastases. We suggest that expression of the CD47/SIRPA signal system indicates the presence of cancer-specific microenvironmental areas that support micrometastasis.

In conclusion, our data indicate that CD47 is a significant prognostic indicator for DFS, and our study is one of the first to report a host factor in bone marrow with prognostic significance. With regard to patient care, many cases require postoperative adjuvant chemotherapy. Due to the associated adverse effects of such treatment, reliable prognostic markers for recurrence and metastasis would greatly improve patient management. We suggest that this biomarker may fill that need for enhanced patient care.

No potential conflicts of interest were disclosed.

We thank T. Shimooka, K. Ogata, M. Kasagi, Y. Nakagawa, and T. Kawano for their technical assistance, and Kelly K. Chong for her editorial assistance.

Grant Support: CREST, Japan Science and Technology Agency (JST); Japan Society for the Promotion of Science (JSPS) Grant-in-Aid for Scientific Research, grant numbers 19591509, 19390336, 20390360, 20591547, 20659209, 20790960, 20790961 and 21791295.

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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