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Functional Plasminogen Activator Inhibitor-1 Gene Variants and Breast Cancer Survival

Authors' Affiliations: ¹ Department of Medicine, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee and ² Department of Epidemiology, Shanghai Cancer Institute, Shanghai, China

Requests for reprints: Xiao-Ou Shu, Department of Medicine, Center for Health Services Research, Vanderbilt University, Medical Center East, Suite 6000, 1215 21st Avenue South, Nashville, TN 37232-8300. Phone: 615-936-0713; Fax: 615-936-1269; E-mail: Xiao-Ou.Shu{at}Vanderbilt.edu.

	Abstract

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Purpose: Plasminogen activator inhibitor-1 (PAI-1) plays an important role in cancer invasion and metastasis. A common polymorphism (4G/5G) in the promoter region of the PAI-1 gene has been reported to influence transcription and plasma levels of PAI-1. We evaluated the association between PAI-1 4G/5G polymorphism and breast cancer survival in a population-based cohort of breast cancer patients.

Experimental Design: Included in this analysis were 1,083 Chinese women diagnosed with stage 0 to III primary breast cancer at age 25 to 64 years who were recruited between 1996 and 1998 for the Shanghai Breast Cancer Study and followed for a median of 5.2 years. The Kaplan-Meier method and Cox model were used to evaluate the genotype and survival association.

Results: After adjustment for known prognostic factors for breast cancer, patients homozygous for the 4G allele had significantly poorer disease-free survival [hazard ratio (HR), 1.7; 95% confidence interval (95% CI), 1.1-2.4] and overall survival (HR, 1.5; 95% CI, 1.0-2.3) than those homozygous for the 5G allele. The association was more evident in patients with advanced disease. The HRs (95% CI) were 3.5 (1.4-9.0) for disease-free survival and 3.1 (1.1-8.3) for overall survival in stage III patients.

Conclusions: The PAI-1 4G/5G polymorphism may be a prognostic marker for young and middle-aged Chinese breast cancer patients.

The human PAI-1 gene is located on chromosome 7 (6). A common single-bp insertion/deletion polymorphism (4G/5G) has been reported in the promoter region of the PAI-1 gene, 675 bp upstream of the transcriptional start site (6, 7). This polymorphism affects the binding of transcription factors; a repressor protein binds to the promoter with the 5G allele but not to the promoter with the 4G allele (6–9). Studies in different populations, including the Chinese, have shown consistently that subjects homozygous for the 4G allele have significantly higher plasma PAI-1 levels than those homozygous for the 5G allele (6, 10, 11).

The 4G/5G polymorphism of the PAI-1 gene has been extensively studied for associations with cardiovascular disease (6, 7, 9, 12); little research, however, has been conducted on its association with cancer. We evaluated the association between PAI-1 4G/5G polymorphism and breast cancer survival in 1,083 breast cancer patients who participated in the Shanghai Breast Cancer Study. Furthermore, we investigated potential interactions of this polymorphism with functional polymorphisms in the genes encoding transforming growth factor-ß1 (TGF-ß1) and vascular endothelial growth factor (VEGF), two factors known to induce PAI-1 expression and be involved in cancer progression (13–16).

	Materials and Methods

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The Shanghai Breast Cancer Study is a population-based case-control study that recruited incident breast cancer cases and community controls between 1996 and 1998. Incident cases were identified through a rapid case ascertainment system, supplemented by the population-based Shanghai Cancer Registry (17). Of the 1,602 eligible cases identified, 1,459 (91.1%) cases were recruited and completed in-person interviews. The main reasons for nonparticipation included refusal (109 cases, 6.8%), death before interview (17 cases, 1.1%), or inability to locate (17 cases, 1.1%). Blood samples (10 mL from each woman) were obtained from 1,193 (82%) cases and processed typically within 6 hours and stored at –70°C until relevant bioassays. Medical charts were reviewed to abstract information on cancer diagnosis, tumor-node-metastasis (TNM) stage, estrogen receptor (ER) and progesterone receptor (PR) status, and cancer treatment. Pathologic slides for all cases were reviewed independently by two senior pathologists to confirm the diagnosis.

Patients were followed up by home visits, telephone interviews, and record linkage to the death certificates kept by the Shanghai Center for Disease Control and Prevention. Of the 1,459 enrolled patients, 1,290 (88.4%) were successfully followed for their disease and survival status by either home visits (n = 1, 241, 85%) or telephone interviews (n = 49, 3.4%) between March 2000 and December 2002. For the remaining 169 patients who could not be contacted in person or by phone, survival status was determined in June 2003 through linkage to the death registry. Only four patients were lost to follow-up because of insufficient information for the record linkage. The study was approved by the institutional review boards of all participating institutes. Informed consent was obtained from each participant.

DNA extraction and genotyping. Genomic DNA was extracted from buffy coat fractions using a Puregene DNA Purification kit (Gentra Systems, Minneapolis, MN) following the manufacture's protocol. DNA concentration was measured using a PicoGreen dsDNA Quantitation kit (Molecular Probes, Eugene, OR). The allelic discrimination of the PAI-1 4G/5G polymorphism (rs1799889) was assessed with the ABI Prism 7900 Sequence Detection Systems (Applied Biosystems, Foster City, CA) using the fluorogenic 5'nuclease assay with primers and probes obtained from ABI Assay-by-Design services. The primers were 5'-AGCCAGACAAGGTTGTTGACA-3' and 5'-GCCGCCTCCGATGATACAC-3'. The probes were VIC-CTGACTCCCCCACGTGT for 5G allele and FAM-CTGACTCCCCACGTGT for 4G allele. PCR was done in a total volume of 5 µL containing 5 ng DNA, 1x Taqman Universal PCR Master Mix, 900 nmol/L of each primer, and 200 nmol/L of each probe. The thermal cycling conditions were as follows: 95°C for 10 minutes to activate the AmpliTaq Gold enzyme followed by 40 cycles of 92°C for 15 seconds and 60°C for 1 minute. The fluorescence level was measured with an ABI Prism 7900HT Sequence Detector (Applied Biosystems). Genotypes were determined by ABI SDS software. Genotyping data were obtained from 1,095 (91.8%) of the cases who provided a blood sample. The major reasons for incomplete genotyping were insufficient DNA used in the assay and unsuccessful PCR amplification.

The laboratory staff was blind to the identity of the subjects. Quality control samples were included in the genotyping assays. Each 384-well plate contained four water, eight CEPH 1347-02 DNA, and eight blinded and eight unblinded quality control samples. The PAI-1 genotypes determined for the quality control samples were in complete agreement with the genotypes determined for the study samples.

Outcomes and statistical analysis. The primary outcomes for this analysis were disease-free survival (DFS) and overall survival (OS). The end points included cancer recurrence/metastasis or death due to breast cancer for the analysis of DFS and death from any cause for the analysis of OS. We excluded from the analysis patients who were lost to follow-up (n = 4) or had missing genotyping data (n = 360). We further excluded patients with distant metastatic (TNM stage IV) disease (n = 12) because of the difficulty in assessing DFS among these patients and because genetic polymorphisms may have a minimal effect on their survival. After these exclusions, 1,083 stage 0 to III patients with complete information for both survival and genotype remained for the analysis. Survival time was calculated as the time from cancer diagnosis until the occurrence of the study end points, censoring at the date of last contact or noncancer death (for DFS only). The Kaplan-Meier method was used to estimate the survival function, and the log-rank test was used to examine the difference in survival. The Cox regression model was used to compute hazard ratios (HR) and 95% confidence intervals (95% CI), adjusting for known prognostic factors for breast cancer, including age and TNM stage at diagnosis, ER and PR status, cancer treatment, and body mass index (weight in kilograms divided by the square of height in meters). Further analyses stratified by major prognostic factors were conducted to examine potential effect modification. Graphic evaluation of Schoenfeld's residual plot suggested no evidence of violation of the proportional hazards assumption for the use of the Cox model. Statistical analyses were done using SAS version 9.1 (SAS Institute, Cary, NC). All tests were based on two-sided probability.

	Results

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Patients included in the analyses did not differ from those who were excluded due to missing genotype data with respect to major clinical prognostic factors, such as TNM stage and ER status. Among the 1,083 patients, 320 (29.6%) had the 4G/4G genotype, 550 (50.8%) had the 4G/5G genotype, and 213 (19.7%) had the 5G/5G genotype. The allele frequencies for 4G and 5G were 54.9% and 45.1%, respectively. These data are consistent with the distribution predicted by the Hardy-Weinberg equilibrium (P = 0.40).

After a median follow-up of 5.2 years (ranging from a minimum of 0.3 to a maximum of 6.3 years), 174 deaths from any cause and 224 relapses, metastases, or deaths due to breast cancer were documented. Table 1 shows the 5-year DFS and OS rates according to known prognostic factors for breast cancer. TNM stage and body mass index were significant predictors of both DFS and OS. Age at diagnosis and menopause (defined as natural or surgically induced cessation of menstrual periods for at least 12 consecutive months before cancer diagnosis) also seemed to be related to survival. No difference in survival by ER or PR status was found.

View larger version (13K): [in this window] [in a new window]   Fig. 1. DFS as a function of PAI-1 genotype and disease stage at diagnosis.

View larger version (12K): [in this window] [in a new window]   Fig. 2. OS as a function of PAI-1 genotype and disease stage at diagnosis.

	Discussion

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High tissue levels of PAI-1 have been found repeatedly to be associated with unfavorable breast cancer prognosis (1, 4, 5). This observation apparently contradicts the theory that increased proteolytic activity by urokinase-type plasminogen activator promotes tumor invasion and metastasis (1, 4, 5). It is now known that PAI-1 is synthesized by multiple types of cells and has multiple molecular interactions, and its overall effect goes beyond that of a simple protease inhibitor (1, 2, 18). By interacting with urokinase-type plasminogen activator receptor, integrin, and vitronectin, PAI-1 may play a crucial role in governing tumor cell adhesion and cell migration (1, 2, 18). PAI-1 is also believed to be essential for sustained angiogenesis (19, 20). By protecting the extracellular matrix against excessive degradation, PAI-1 may stabilize the matrix, which acts as a scaffold required for endothelial cell migration and tube formation (20).

Little is known about the prognostic effect of functional polymorphisms in the PAI-1 gene. A recent study reported that the PAI-1 4G allele was associated with high tissue levels of PAI-1 and less favorable characteristics of breast cancer (such as an advanced histologic grade), a finding that is in line with our results (21). However, in an earlier study of breast cancer patients, no association was found between PAI-1 4G/5G genotype and lymph node status (22). One study of colorectal cancer found a link between PAI-1 4G/4G genotype and more advanced tumors (Dukes' C and D), suggesting that the PAI-1 genotype may also be a prognostic factor for colorectal cancer (23). None of these studies evaluated survival as an outcome.

To our knowledge, our study is the first to investigate the association of PAI-1 4G/5G polymorphism and breast cancer survival. The overall effect of the genotype on survival outcomes was only moderate. However, a strong association was observed in stage III patients. Because patients with locally advanced disease have elevated PAI-I levels (5), it can be speculated that the presence of both 4G/4G genotype and an advanced stage may lead to excessive increases in PAI-1 levels than any one of these factors present alone. Similarly, the association between the 4G/4G genotype and coronary heart disease was found to be strengthened with the additional presence of cardiovascular risk factors that have been associated with increased PAI-1 levels (12).

TGF-ß1 and VEGF are important growth factors involved in cancer progression (15, 16). PAI-1 regulates the bioavailability and activity of these factors (24, 25), which in turn induce PAI-1 expression (13, 14). Apparently, polymorphisms of these genes may have a synergetic effect on breast cancer prognosis. We have reported previously in the same cohort a moderate association of TGF-ß1 (TGF-ß1 T+29C) and VEGF (VEGF C-460T) gene variants with a HR for DSF of 1.4 to 1.5 for TGF-ß1 TC/CC and VEGF CC genotypes (15, 16). When the joint effect of these polymorphisms was evaluated, treating PAI-1 4G/4G, TGF-ß1 TC/CC, and VEGF CC as risk genotypes, we found that the hazards increased substantially as the number of risk genotypes present increased. Carrying any one, two, or three risk genotypes was associated with a HR for DFS of 1.7 (95% CI, 1.0-2.8), 2.4 (95% CI, 1.4-4.0), and 3.3 (95% CI, 1.3-8.4), respectively. Again, a much stronger association was seen in patients with advanced disease (stage II-III) with respective HRs (95% CIs) being 2.1 (1.1-4.1), 3.2 (1.6-6.3), and 6.0 (2.0-18.0) for carriers of one, two, and three risk genotypes. Although they need to be verified in larger studies, these results suggest that the risk genotypes in the PAI-1, TGF-ß1, and VEGF genes may contribute synergistically to breast cancer progression.

This study has several strengths. The study hypothesis has a strong biological basis. The main effect of PAI-1 genotype on survival found in this study followed a dose-response pattern. Sources of potential selection bias were minimized because of the population-based study design and the high participation and follow-up rates. Population stratification is unlikely to be a concern because >98% of the participants belong to a single ethnic group. The genotyping was undertaken with the laboratory staff blinded to the identity of participants and the allele frequencies were consistent with those published in the literature (11).

Limitations of the study also need to be kept in mind when interpreting the results. Tissue levels of PAI-1 have been shown to predict responses to systemic therapy in breast cancer (26–28), suggesting that potential confounding/modifying effects of cancer treatments need to be considered. However, our ability to account for or evaluate these effects was limited due to a lack of specific chemotherapy drug information and missing data on tamoxifen use for a large proportion of the patients. The information on tamoxifen use was collected during the study follow-up period and could not be obtained for many deceased patients, which may, at least in part, explain the observed low survival rates in these patients. We conducted additional analyses restricted to patients who received tamoxifen therapy and found that the association of PAI-1 genotype with survival was strengthened slightly (data not shown). To better understand the effects of genotype on cancer prognosis, it would be desirable to evaluate the association and potential interaction between genotype and tumor characteristics that have important prognostic value, such as histologic grade and lymph node and hormone receptor status. However, our study is not well powered enough to address these issues because of the quickly diminishing sample size within each subcategory of patients and, sometimes, because of a lack of relevant information.

Certain characteristics of the study population also need to be considered when interpreting the study findings. For example, adjuvant chemotherapy is more widely used in Shanghai than in the United States. In the current study, 94% of patients received adjuvant chemotherapy. In another on-going study of 2,245 breast cancer survivors (age at diagnosis between 25-70 years) in Shanghai, the chemotherapy rate is 92%. The 5-year survival rates of our study population (diagnosed between 1996-1998) seemed to be slightly better than those reported in a Taiwanese study that also involved relatively young Chinese breast cancer patients (diagnosed between 1990-1997; ref. 29). The survival rates in our study were comparable with or a little lower than those of U.S. women as reported by the American Cancer Society for breast cancer cases diagnosed between 1992 to 1999, where the 5-year OS rate was 83% to 87% for women under age 55 years. The findings of our study may not be extrapolated directly to other populations.

In summary, this study provides the first evidence that the PAI-1 functional variant, alone or in combination with the TGF-ß1 and VEGF genotypes, may predict outcome for young and middle-aged Chinese breast cancer patients, particularly for those with locally advanced disease.

	Acknowledgments

 
We thank all the participants and research staff of the Shanghai Breast Cancer Study, Regina Courtney and Qing Wang for their laboratory technical assistance, and Bethanie Hull for her assistance in the preparation of this manuscript.

	Footnotes

 
Grant support: R01CA64277 and R01CA90899.

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.

Received 12/30/05; revised 6/20/06; accepted 8/ 4/06.

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