Abstract
Purpose: The prognostic significance of the Wilms’ tumor suppressor gene(WT1) mRNA expression was evaluated in patients with invasive breast cancer.
Experimental Design: WT1 mRNA expression in tumor tissues (n = 99) was examined by a quantitative, real-time PCR assay.
Results: No significant association was observed between WT1 mRNA levels and clinicopathological parameters such as menopausal status, tumor size, lymph node status, histological grade, and estrogen receptor status. Five-year disease-free survival rate of patients with high WT1 mRNA levels (62.6%) was significantly (P < 0.05) poorer than those with low WT1 mRNA levels (77.2%). Lymph node metastasis (P < 0.05), high histological grade (P < 0.01), and estrogen receptor negativity (P < 0.05) were also significantly associated with poor prognosis, respectively. Multivariate analysis revealed that WT1 mRNA levels were a significant prognostic factor, independent of the other conventional prognostic factors.
Conclusions: These results suggest that measurement of WT1 mRNA levels in tumor tissues might be useful as a new prognostic factor in breast cancer patients.
INTRODUCTION
The WT13 gene was originally identified as a tumor suppressor gene responsible for Wilms’ tumor, a kidney neoplasm of childhood (1 , 2) . In addition to germ-line abnormalities, somatic mutations of WT1 as well as loss of heterozygosity at the 11p13 locus harboring WT1 have been reported in sporadic Wilms’ tumors (1, 2, 3, 4, 5) . WT1 encoding a zinc finger polypeptide acts as a transcription factor for many genes regulating renal cell differentiation and growth, and its tumor-suppressive function is supposed to be attributable to transcriptional repression of growth factors including IGF-II, IGF-I receptor, platelet-derived growth factor-A, transforming growth factor-β, c-myc, and bcl-2 (6, 7, 8, 9, 10) .
Recent studies have shown that WT1 plays an important role in the pathogenesis and progression of leukemia (11, 12, 13) . Inoue et al. (11) reported that the WT1 mRNA expression was significantly up-regulated in leukemia as compared with the normal hematopoietic cells, and that leukemia with high WT1 mRNA expression showed a significantly lower CR rate and a significantly worse overall survival than that with a low WT1 expression. These observations, however, seem to be inconsistent with the thesis derived from the studies on Wilms’ tumors that WT1 is a tumor suppressor gene. Rather, these observations seem to suggest that WT1 is an oncogene in leukemia. This speculation is further substantiated by the in vitro experiments, which showed that WT1 mRNA expression was down-regulated during differentiation in leukemia cell lines, and that treatment with antisense oligonucleotides for WT1 mRNA resulted in the growth inhibition in these cell lines (14) . In addition, growth of myeloid progenitor cells is stimulated, and differentiation of these cells is inhibited by inducing WT1 expression in these cells (12) . These results strongly indicate an oncogenic function of WT1 in leukemia.
WT1 expression has rarely been studied in breast cancers. Recently, Loeb et al. (15) have demonstrated that WT1 mRNA and protein are expressed in nearly 90% of breast cancers but not in most normal breast tissues. These results suggest that WT1 plays a certain role in the pathogenesis of breast cancer as an oncogene but not a tumor suppressor gene in analogy to leukemia. Silberstein et al. (16) studied the WT1 expression by immunohistochemistry and demonstrated an association of WT1 protein expression with a biologically aggressive phenotype of breast cancer such as ER negativity. Their observation suggests a possibility that WT1 expression can serve as a risk factor for developing recurrences in breast cancer patients. Therefore, in the present study, we have studied the relationship between WT1 mRNA levels and various clinicopathological parameters as well as prognosis to clarify the prognostic significance of WT1 mRNA expression in breast cancer patients.
MATERIALS AND METHODS
Surgical Specimens and Patient Characteristics.
During the period from October 1995 to March 1998, 99 female patients with invasive breast cancer who underwent mastectomy or breast-conserving surgery were recruited in this study. Breast cancer tissues were obtained at surgery and snap frozen until use. Histological diagnosis was invasive ductal carcinoma for 97 patients and invasive lobular carcinoma for two patients. The details of patient characteristics are shown in Table 1⇓ . Histological grade was determined according to the modified Scarff-Bloom-Richardson criteria (17) . Normal breast tissues were obtained from 13 breast cancer patients who underwent mastectomy or breast-conserving surgery during the period from June 2000 to January 2001. Informed consent was obtained from each patient.
Patients’ characteristics
Patients had a physical examination every 3 months for 2 years postoperatively and every 6 months thereafter. Chest X-ray and abdominal ultrasonogram were obtained every 6 months postoperatively. Except five patients who received no adjuvant therapy, 94 patients received endocrine therapy (2 years administration of tamoxifen in 52 patients), chemotherapy (six cycles of cyclophosphamide + methotrexate + 5-fluorouracil in 17 patients), and chemoendocrine therapy (tamoxifen plus cyclophosphamide + methotrexate + 5-fluorouracil in 25 patients), essentially according to St. Gallen recommendations (18 , 19) . None of the patients had been treated with neoadjuvant therapy.
The median follow-up period of the 99 patients was 48 months (range, 39–60 months), and DFS of these patients was 73.1%. Twenty-one of 99 (21.2%) patients developed recurrences, i.e., 5 developed liver metastases, 4 developed lung metastases, 3 developed bone metastases, and 9 developed soft tissue metastases. Ipsilateral breast recurrences after breast-conserving surgery were not counted as recurrences.
RNA Extraction and Reverse Transcription.
Total cellular RNA was extracted from the frozen tumor specimens using TRIzol reagent according to the protocol provided by the manufacturer (Molecular Research Center, Cincinnati, OH). The 3 μg each of total RNA were used for synthesis of cDNA by Superscript II (Life Technologies, Inc., Rockville, MD) priming with oligo-(dT)15 primer under the condition at 42°C for 90 min, followed by heating at 70°C for 10 min.
Primers, Probes, and Real-Time PCR.
A real-time PCR amplification was carried out according to the method described previously (20) . Briefly, primer pairs for amplification were 5′-GATAACCACACAACGCCCATC-3′ and 5′-CACACGTCGCACATCCTGAAT-3′. The sequence of the probe, labeled with 6-carboxyfluorescein and 6-carboxy-N,N,N′,N′-tetramethylrhodamine at the 5′ and 3′ sides, respectively, was 5′-ACACCGTGCGTGTGTATTCTGTATTGG-3′. PCR reactions were carried out using ABI Prism 7700 Sequence Detection System (Perkin-Elmer Applied Biosystems, Foster City, CA) under the condition of 95°C 10 min, followed by 50 cycles of 95°C for 30 s and 63°C for 1 min. To quantify gene transcripts precisely, the β-glucuronidase transcripts were monitored as the quantitative control, and each sample was normalized on the basis of its β-glucuronidase transcript content. The primer probe mixture for β-glucuronidase was purchased from Perkin-Elmer Applied Biosystems, and the method of PCR was followed by the manufacturer’s protocol.
The standard curves for WT1 and β-glucuronidase mRNA were generated using the serially diluted solutions of plasmid clones with either WT1 (10−5 to 10−1 μg) or β-glucuronidase (10−8 to 10−4 μg) cDNA inserted by a part of each cDNA as templates. The amount of target gene expression was calculated from the standard curve, and quantitative normalization of cDNA in each sample was performed using the expression of β-glucuronidase gene as an internal control. Finally, WT1 mRNA levels were shown as ratios to β-glucuronidase mRNA levels when 10−5 μg and 10−8 μg of plasmid control defined as 1. Real-time PCR assays were conducted in duplicate for each sample, and the mean value was used for the calculation of mRNA expression levels.
ER Assay.
ER levels in breast cancers were measured by enzyme immunoassay using the kit provided by Abbott Research Laboratories (Chicago, IL). The cutoff value for ER was defined as 10 fmol/mg protein.
Statistical Methods.
The relationship between WT1 mRNA expression levels and clinicopathological characteristics was assessed by the Mann-Whitney test. DFS curves were calculated by the Kaplan-Meier method, and the log-rank test was used to evaluate the difference in DFS among the various patient subgroups. The proportional hazards model was used for the multivariate analysis. Statistical significance was assumed for P < 0.05.
RESULTS
Relationship between WT1 mRNA Levels and Various Clinicopathological Parameters.
WT1 mRNA expression levels in tumor tissues and normal breast tissues were quantified by a real-time PCR assay. WT-1 mRNA levels in breast tumors (3.92 ± 6.92, mean ± SD) were significantly (P < 0.0005) higher than those in the normal breast tissues (0.69 ± 1.27; n = 13; Fig. 1⇓ ).
WT-1 mRNA levels in breast tumors (n = 99) and normal breast tissues (n = 13). WT-1 mRNA levels are shown as ratios of WT-1:β-glucuronidase mRNA levels. Bars, mean ± SD.
The relationship between WT1 mRNA levels and the various clinicopathological parameters of breast tumors is shown in Table 2⇓ . Patients with disease recurrence showed a trend toward an increase (P = 0.09) in WT1 mRNA levels than those without disease recurrence. Tumors >2 cm also showed a trend toward an increase (P = 0.09) in WT1 mRNA levels as compared with tumors ≤2 cm. There was no statistically significant association between WT1 mRNA expression and any other clinicopathological parameters such as menopausal status, lymph node status, histological grade, and ER status.
Relationship between WT1 mRNA levels and clinicopathological parameters
Univariate and Multivariate Analysis of DFS according to WT1 mRNA Levels and Various Clinicopathological Parameters.
Breast tumors were dichotomized into the WT-1 mRNA high and low expression groups using the cutoff value of 3.3, which corresponded to the mean + 2 SD of the WT-1 mRNA levels in the normal breast tissues. According to this cutoff value, 66 tumors and 33 tumors were classified into the WT1 mRNA low and high groups, respectively. The high WT1 mRNA group showed a significantly (P < 0.05) lower 5-year DFS rate (62.6%) than the low WT1 mRNA group (77.2%; Fig. 2⇓ ). Lymph node metastasis, high histological grade, and ER negativity were also found to be significantly associated with a poor prognosis (Table 3)⇓ . Four parameters (lymph node status, histological grade, ER status, and WT1 mRNA levels), which were found to be significant by univariate analysis, were further analyzed by multivariate analysis (Table 4)⇓ . Multivariate analysis has revealed that the WT1 mRNA expression is a significant (P < 0.05) prognostic factor, independent of the other prognostic factors.
DFS rates of breast cancer patients according to WT1 mRNA levels.
Five-year DFS rates (%) according to various prognostic factors
Univariate and multivariate analysis of various prognostic factors
DISCUSSION
In the present study, we could demonstrate the WT1 mRNA expression in both breast tumors and normal breast tissues by a real-time RT-PCR method, and the WT1 mRNA levels were significantly higher in breast tumors than normal breast tissues. The fact that WT1 expression is up-regulated in tumors more than normal tissues is irreconcilable with the hypothesis that WT1 is a tumor suppressor gene and is rather suitable for the hypothesis that WT1 is an oncogene in breast cancer, as has been demonstrated in leukemia.
WT1 mRNA levels showed a trend toward an increase in patients with recurrence than those without it, suggesting that WT1 mRNA levels can be useful as a prognostic factor. In fact, we have been able to demonstrate that patients with high WT1 mRNA levels showed a significantly poorer prognosis than those with low WT1 mRNA levels. Furthermore, multivariate analysis has revealed that WT1 mRNA levels are a significant prognostic factor, independent of the conventional prognostic factors such as lymph node status, histological grade, and ER status. This is the first report that demonstrates the prognostic significance of WT1 mRNA levels in breast cancer patients. Although we used the mean + 2 SD of the WT1 mRNA levels in the normal breast tissues as the cutoff value for the WT1 mRNA high and low expression groups, an optimal cutoff value needs to be determined by a future study including a larger number patients.
It is suggested in acute leukemia that WT1 mRNA levels are useful in the prediction of chemosensitivity because it is based on the observation that patients with low WT1 mRNA levels achieve >90% CR rates, but those with intermediate and high WT1 mRNA levels achieve lower CR rates, i.e., 50 and 0%, respectively (11) . It is unknown, at present, whether there is a significant relationship between WT1 mRNA levels and response to chemotherapy or endocrine therapy in breast cancer. But if this is the case, the difference in treatment efficacy between the WT1 mRNA high and low expression groups could influence the prognosis, because all patients but five had been treated with adjuvant therapy in the present study. Ideally, a new prognostic factor is to be tested in the patients without adjuvant therapy. However, almost all breast cancer patients are treated with adjuvant therapy, according to the recommendation of the consensus meeting of St. Gallen (18 , 19 , 21) and NIH (22) . Thus, at present, it is nearly infeasible to evaluate a new prognostic factor among the patients without adjuvant therapy. Thus, strictly speaking, it is currently unknown whether the WT1 mRNA expression is a prognostic factor or a predictive factor or both. This problem, however, can be clarified, at least in part, by studying the significance of the WT1 mRNA expression as a predictive factor for endocrine treatment and/or chemotherapy in the neoadjuvant or metastatic setting.
The mechanism of up-regulation of WT1 mRNA expression has yet to be established. Although aberrant methylation of CpG islands in the promoter and the first intron of the WT1 gene has been reported in primary breast cancers (23) , Loeb et al. (15) have shown no association between the methylation status and WT1 mRNA expression in breast cancers. Thus, other transcriptional regulatory mechanisms seem to be implicated in the up-regulation of WT1 mRNA during carcinogenesis. Laux et al. (23) reported a novel 2.5-kb WT1 transcript lacking the first exon in the breast cancer cell line MDA-MB-231. Similarly, Dechsukhum et al. (24) detected a 2.1-kb transcript in prostate cancer cell line, leukemia cell line (K562), breast cancer cell line (MCF7), and acute leukemia. This 2.1-kb transcript consists of exons 6–10, together with a portion of intron 5 at the 5′ end of the transcript. The function of this short transcript is unknown at present, but the gene product possibly affects the function because it appears to contain the zinc-finger domain but lacks the transactivation region of WT1 (24) . Furthermore, the other two splicing variants of WT1, which lack the fifth exon or nine bases between exons 9 and 10, have been reported (1 , 25) . These results suggest that the WT1 function can be different according to the various isoforms. Because the real-time PCR method used in the present study could not differentiate the wild type WT1 mRNA from its splicing variants, the prognostic significance of these splicing variants still remains to be studied.
In conclusion, we have suggested that the WT1 mRNA levels can serve as a significant prognostic factor in breast cancer patients, independent of the conventional prognostic factors such as lymph node status, histological grade, and ER status. Our preliminary results need to be confirmed by a future study including a larger number of patients with a longer follow-up period.
Footnotes
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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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↵1 Supported in part by grants from Ministry of Education, Culture, Sports, Science and Technology, Japan.
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↵2 To whom requests for reprints should be addressed, at Department of Surgical Oncology, Osaka University Graduate School of Medicine, 2-2 Yamada-oka, Suita, Osaka 565-0871, Japan. Phone: 81-6-6879-3772; Fax: 81-6-6879-3779; E-mail: noguchi{at}onsurg.med.osaka-u.ac.jp
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↵3 The abbreviations used are: WT1, Wilms’ tumor suppressor 1; IGF, insulin-like growth factor; CR, complete remission; ER, estrogen receptor; DFS, disease-free survival.
- Received November 6, 2001.
- Revision received February 15, 2002.
- Accepted February 25, 2002.
References
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