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Metastatic breast cancer is an extremely complex disease with limited treatment options due to the lack of information about the major characteristics of metastatic disease. There is an urgent need, therefore, to understand the changes in cellular complexity and dynamics that occur during metastatic progression. In the current study, we analyzed the cellular and molecular differences between primary tumors and paired lung metastases using a syngeneic p53-null mammary tumor model of basal-like breast cancer. Distinct subpopulations driven by the Wnt- and/or STAT3 signaling pathways were detected in vivo using a lentiviral Wnt- and STAT3 signaling reporter system. A significant increase in the overlapping populations driven by both the Wnt- and STAT3 signaling pathways was observed in the lung metastases as compared to the primary tumors. Furthermore, the overlapping populations showed a higher metastatic potential relative to the other populations and pharmacological inhibition of both signaling pathways was shown to markedly reduce the metastatic lesions in established lung metastases. An analysis of the unique molecular features of the lung metastases revealed a significant association with immune response signatures. Specifically, Foxp3 gene expression was markedly increased and elevated levels of Foxp3 + Treg cells were detected in close proximity to lung metastases. Collectively, these studies illustrate the importance of analyzing intratumoral heterogeneity, changes in population dynamics, and the immune microenvironment during metastatic progression.

Despite strong possibility that endothelial cells (ECs) of tumors and normal tissues may differ in various aspects, most previous studies on ECs have used normal cells. Here, we purified ECs from tumorous and normal human breast tissues, and studied the effect of radiation on angiogenesis and relevant molecular mechanisms in these cells. We found that in normal tissue-derived ECs (NECs), 4 Gy irradiation increased tube formation, matrix metalloproteinase 2 (MMP-2) expression and extracellular signal-regulated kinase (ERK) pathway activation. In cancer-derived ECs (CECs), however, 4 Gy irradiation significantly reduced tube formation, increased the production of angiostatin and interleukin-6 (IL-6), and upregulated AKT and c-Jun N-terminal kinase (JNK) pathway activation. Knockdown experiments showed that siMMP-2 efficiently inhibited tube formation by irradiated NECs, whereas siPlasminogen effectively attenuated the radiation-induced suppression of tube formation and the upregulation of angiostatin in CECs. Moreover, siIL-6 clearly inhibited the radiation-induced generation of angiostatin in CECs. Inhibition of ERK with a pharmacological inhibitor or small interfering RNAs (siRNAs) markedly suppressed the radiation-induced tube formation and MMP-2 upregulation in NECs, whereas the inhibition of either AKT or JNK with pharmacological inhibitor or siRNA treatment of CECs markedly attenuated the inhibition of tube formation and the upregulation of angiostatin and IL-6 caused by 4 Gy irradiation. These observations collectively demonstrate that there are distinct differences in the radiation responses of NECs and CECs, and might provide important clues for improving the efficacy of radiation therapy.

Background: The aim of this study was to construct a novel prediction model for the pathological complete response (pCR) to neoadjuvant chemotherapy (NAC) using immune-related gene expression data., Patients and Methods: DNA microarray data were used to perform a gene expression analysis of tumor samples obtained before NAC from 117 primary breast cancer patients. The samples were randomly divided into the training (n = 58) and the internal validation (n = 59) sets that were used to construct the prediction model for pCR. The model was further validated using an external validation set consisting of 901 patients treated with NAC from six public datasets., Results: The training set was used to construct an immune-related 23-gene signature for NAC (IRSN-23) that is capable of classifying the patients as either genomically predicted responders (Gp-R) or non-responders (Gp-NR). IRSN-23 was first validated using an internal validation set, and the results showed that the pCR rate for Gp-R was significantly higher than that obtained for Gp-NR (38 versus 0%, P = 1.04E-04). The model was then tested using an external validation set, and this analysis showed that the pCR rate for Gp-R was also significantly higher (40 versus 11%, P = 4.98E-23). IRSN-23 predicted pCR regardless of the intrinsic subtypes (PAM50) and chemotherapeutic regimens, and a multivariate analysis showed that IRSN-23 was the most important predictor of pCR (odds ratio = 4.6; 95% confidence interval = 2.7-7.7; P = 8.25E-09)., Conclusion: The novel prediction model (IRSN-23) constructed with immune-related genes can predict pCR independently of the intrinsic subtypes and chemotherapeutic regimens.

Casitas B-lineage lymphoma (CBL) protein family functions as multifunctional adaptor proteins and E3 ubiquitin ligases that are implicated as regulators of signaling in various cell types. Recent discovery revealed mutations of proto-oncogenic CBL in the linker region and RING finger domain in human acute myeloid neoplasm, and these transforming mutations induced carcinogenesis. However, the adaptor function of CBL mediated signaling pathway during tumorigenesis has not been well characterized. Here, we show that CBL is highly expressed in breast cancer cells and significantly inhibits transforming growth factor-β (TGF-β) tumor suppressive activity. Knockdown of CBL expression resulted in the increased expression of TGF-β target genes, PAI-I and CDK inhibitors such as p15(INK4b) and p21(Cip1). Furthermore, we demonstrate that CBL is frequently overexpressed in human breast cancer tissues, and the loss of CBL decreases the tumorigenic activity of breast cancer cells in vivo. CBL directly binds to Smad3 through its proline-rich motif, thereby preventing Smad3 from interacting with Smad4 and blocking nuclear translocation of Smad3. CBL-b, one of CBL protein family, also interacted with Smad3 and knockdown of both CBL and CBL-b further enhanced TGF-β transcriptional activity. Our findings provide evidence for a previously undescribed mechanism by which oncogenic CBL can block TGF-β tumor suppressor activity.

Background: The aim of this study was to investigate the clinicopathological characteristics of GATA binding protein 3 (GATA3)-positive breast cancers as well as the association of GATA3 expression with response to chemotherapy., Patients and Methods: Tumor specimens obtained before neoadjuvant chemotherapy [paclitaxel followed by 5-fluorouracil/epirubicin/cyclophosphamide)] from breast cancer patients (n = 130) were subjected to immunohistochemical and mutational analysis of GATA3 and DNA microarray gene expression analysis for intrinsic subtyping., Results: Seventy-four tumors (57%) were immunohistochemically positive for GATA3. GATA3-positive tumors were significantly more likely to be lobular cancer, estrogen receptor (ER)-positive, progesterone receptor (PgR)-positive, Ki67-negative, and luminal A tumors. Somatic mutations were found in only three tumors. Pathological complete response (pCR) was observed in 8 (11%) GATA3-positive tumors and in 22 (39%) GATA3-negative tumors. multivariate analysis showed that tumor size, human epidermal growth factor receptor 2 (her2), and gata3 were independent predictors of pcr., Conclusions: GATA3-positive breast cancers showed luminal differentiation characterized by high ER expression and were mostly classified as luminal-type tumors following intrinsic subtyping. Interestingly, GATA3 was an independent predictor of response to chemotherapy, suggesting that GATA3 might be clinically useful as a predictor of a poor response to chemotherapy.

Background: We established the cell cycle profiling (C2P) assay for specific activity (SA; activity/expression) of cyclin-dependent kinases (CDKs). C2P risk score (C2P-RS) based on CDK1 and CDK2 SAs was significantly associated with relapse in breast cancer (BC). This study was conducted to investigate the predictive value of C2P-RS for neoadjuvant chemotherapy (NAC)., Patients and Methods: Among 124 eligible patients, 122 were treated with weekly paclitaxel followed by 5-fluorouracil, epirubicin and cyclophosphamide (P-FEC) and 2 were treated with paclitaxel monotherapy. C2P-RS was determined via C2P using frozen biopsy samples before NAC., Results: Negative estrogen receptor (ER), negative progesterone receptor (PR), positive human epidermal growth factor receptor 2 (HER2), high Ki-67 expression and intermediate + high C2P-RS were significantly associated with high pathological complete response (pCR) rates compared with positive ER (30% versus 9%), positive PR (25% versus 6%), negative HER2 (34% versus 11%), low Ki-67 expression (24% versus 7%) or low C2P-RS (24% versus 9%), respectively. The combination of C2P-RS and Ki-67 had a stronger impact on pCR than each parameter alone, and a multivariate analysis showed that the combination was an independent predictor of pCR (odds ratio 3.3, 95% confidence interval 1.1-9.5)., Conclusions: C2P-RS was significantly associated with pCR after P-FEC and may be a useful predictor for chemotherapy in BCs.

Background: In early-stage breast cancer, adjuvant chemotherapy is associated with significant systemic toxicity with only a modest survival benefit. Therefore, there is considerable interest in identifying predictive markers of response to therapy. Doxorubicin, one of the most common drugs used to treat breast cancer, is an anthracycline chemotherapeutic agent, a class of drugs known to be affected by hypoxia. Accordingly, we examined whether expression of the endogenous hypoxia marker carbonic anhydrase IX (CA IX) is predictive of outcome in early-stage breast cancer patients treated with doxorubicin., Methods: We obtained 209 early-stage pre-treatment surgically-resected breast tumours from patients, who received doxorubicin in their chemotherapeutic regimen and had >10 years of follow-up. Immunohistochemistry was used to detect CA IX, and we used fluorescence in situ hybridisation to detect both human epidermal growth factor receptor (HER2) and DNA topoisomerase II-alpha (TOP2A) gene amplification., Results: Carbonic anhydrase IX intensity was significantly correlated with progression-free survival (PFS) and overall survival (OS) in patients receiving 300 mg m(-2) of doxorubicin (HR=1.82 and 3.77; P=0.0014 and 0.010, respectively). There was a significant, inverse correlation between CA IX score and oestrogen receptor expression, but no significant correlations were seen with either HER2 or TOP2A ratio., Conclusion: We demonstrate that CA IX expression is correlated with worse PFS and OS for breast cancer patients treated with doxorubicin, independent of HER2 or TOP2A gene amplification. This study provides evidence that using CA IX to detect hypoxia in surgically-resected breast tumours may be of clinical use in choosing an appropriate chemotherapy regimen.

Association of estrogen receptor (ER), progesterone receptor (PR), HER2, Ki67 and 70-gene classifier (70-GC) with a response to paclitaxel (PAC) (n=79) or docetaxel (DOC) (n=55) was investigated in the neoadjuvant setting for breast cancer patients. Sensitivity of breast tumors to PAC, but not to DOC, was found to be significantly associated with ER negativity (P=0.003), PR negativity (P=0.007), and Ki67 positivity (P=0.007). Breast tumors classified into the responders by 70-GC showed a significantly (P=0.005) higher reduction rate to PAC and interestingly a significantly (P=0.009) lower reduction rate to DOC than those classified into the non-responders by 70-GC, suggesting that 70-GC might be useful for the differentiation of PAC-sensitive and DOC-sensitive breast tumors., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Purpose: Recently, Ki67 index (cell proliferation marker) has been attracting a considerable attention as a prognostic factor in breast cancer but the prognostic significance of Ki67 after neoadjuvant chemotherapy (NAC) has rarely been examined., Experimental Design: Primary breast cancer patients (n = 102) treated with NAC (sequential paclitaxel 12 cycles (q1w) and 5-FU/epirubicin/cyclophosphamide 4 cycles (q3w)) were recruited in the study. Ki67, estrogen receptor (ER) and progesterone receptor (PR) and breast cancer resistant protein (BCRP) and P-glycoprotein were determined by immunohistochemistry and HER2 was determined by FISH in tumor tissues obtained before and after NAC, and their association with patient prognosis (relapse-free survival) was examined., Results: Of the 102 patients, pCR was achieved in 30 (29.4%). In the 72 non-pCR patients, Ki67 index significantly (P < 0.001) decreased after NAC. Ki67 index after NAC, but not Ki67 index before NAC, was significantly associated with a patient prognosis (P = 0.022). Multivariate analysis has shown that Ki67 index after NAC is a marginally significant (P = 0.05) prognostic factor and that other biomarkers including ER, PR, BCRP, and P-glycoprotein before and after NAC are not significant., Conclusions: Ki67 after NAC, but not before NAC, is prognostic in breast cancer patients, and might be clinically useful in the prognosis prediction of patients who do not achieve pCR after NAC. On the other hand, BCRP and P-glycoprotein before and after NAC are unlikely to be useful as prognostic factors in these patients., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

In a Japanese study, cyclin-dependent kinase (CDK) based risk determined by CDK 1 and 2 activities was associated with risk of distance recurrence in early breast cancer patients. The aim of our study was to validate this risk categorization in European early breast cancer patients. We retrospectively analyzed frozen breast cancer specimens of 352 Dutch patients with histologically confirmed primary invasive early breast cancer. CDK-based risk was determined in tumour tissues by calculating a risk score (RS) according to kinases activity and protein mass concentration assay without the knowledge of outcome. Determination of CDK-based risk was feasible in 184 out of 352 (52%) tumours. Median follow-up of these patients was 15 years. In patients not receiving systemic treatment, the proportions of risk categories were 44% low, 16% intermediate, and 40% high CDK-based risk. These groups remained significant after univariate and multivariate Cox-regression analysis. Factors associated with a shorter distant recurrence-free period were positive lymph nodes, mastectomy with radiotherapy, and high CDK-based risk. There was no significant correlation with overall survival (OS). CDK-based risk is a prognostic marker of distance recurrence of patients with early breast cancer. More validation would be warranted to use of CDK-based risk into clinical practice.

Aims: It has been reported that glutathione S-transferase P1 (GSTP1) expression is implicated in resistance to taxanes (docetaxel and paclitaxel) in human breast cancer cells in vitro. In the study presented here, we examine whether GSTP1 expression is associated with resistance to docetaxel or paclitaxel in human breast cancers. We also investigated the relationship between GSTP1 methylation status and response to these taxanes., Material and Methods: Sixty two primary breast cancer patients were treated with docetaxel or paclitaxel as primary systemic treatment (PST). GSTP1 expression was detected immunohistochemically and the hypermethylation status GSTP1 gene was identified with a methylation specific primer assay., Results: The mean tumor reduction rate for all patients (n=62) was significantly (p<0.001) higher in GSTP1 negative (0.73+/-0.04; mean+/-standard error) than GSTP1 positive (0.31+/-0.09) tumors. The subset analysis showed that the mean reduction rate was significantly (p=0.005) higher in GSTP1 negative (0.59+/-0.06) than GSTP1 positive (0.11+/-0.13) tumors in the docetaxel group as well as in the paclitaxel group (p=0.006; GSTP1 negative tumors: 0.84+/-0.05; GSTP1 positive tumors: 0.56+/-0.08). On the other hand, GSTP1 methylation showed no significant association with the reduction rate., Conclusion: Our present study has suggested that GSTP1 protein expression, but not GSTP1 methylation status, might be associated with response to docetaxel and paclitaxel. This suggests that GSTP1 immunohistochemical expression might be a potentially clinically useful predictive factor for response to docetaxel and paclitaxel.

Background: We recently established a novel assay for specific activity (SA) of cyclin-dependent kinases (CDKs) using small tumor samples (>/=8 mm(3)). The aim of this study was to investigate the prognostic significance of CDK1SA and CDK2SA in human breast cancer., Methods: CDK1SA and CDK2SA were determined in 284 breast cancer patients and their prognostic significance was investigated., Results: Tumors with high CDK1SA and high CDK2SA showed significantly poorer 5-year relapse-free survival than those with low CDK1SA and low CDK2SA, respectively (66.9% vs 84.2% for CDK1SA; 43.6% vs 83.6% for CDK2SA). Moreover, combined analysis of CDK1SA and CDK2SA enabled the classification of breast tumors into high-risk and low-risk groups, where tumors in the high-risk group were strongly associated with unfavorable prognosis (5-year relapse-free survival 69.4% for the high-risk group and 91.5% for the low-risk group). Multivariate analysis showed that the risk determined by combined analysis of CDK1SA and CDK2SA is a significant (hazard ratio 3.09, P < 0.001) prognostic indicator for relapse, especially in node-negative patients (hazard ratio 6.73, P < 0.001)., Conclusion: Determination of CDK1SA and CDK2SA may be useful in the prediction of outcomes in breast cancer patients and has potential for use as a routine laboratory test.

The aim of the current study was to compare the diagnostic reliability of visual and quantitative indices of double phase Tc-99m MIBI scintimammography (M-SMM) and Tc-99m Tetrofosmin scintimammography (TF-SMM) for detection of breast cancer. Double phase M-SMM and TF-SMM (early; 10 minutes, delayed; hour) were performed after injection of 925 MBq of radiotracers in 75 highly suspected breast cancer patients (malignant:49, benign:26). For visual analysis, five scoring methods were used. For quantitative analysis, early, delayed lesions to non-lesion ratios (L/Ns), and washout rate (%, WR) were calculated. When over grade of visual grade was used as cut-off value in the detection of primary breast cancer, M-SMM and TF-SMM showed similar diagnostic accuracies. The optimal quantitative indices of M-SMM for the detection of breast cancer were 2.06 for early L/N and 1.72 for delayed L/N. Those of TF-SMM were 3.13 for early, and 2.56 for delayed image. Visual and quantitative analyses showed similar results. However, delayed L/N of M-SMM was superior to that of TF-SMM for the detection of breast cancer. In conclusion, the double phase M-SMM and TF-SMM showed favorable diagnostic accuracy in differentiating benign from malignant breast lesions, visually and quantitatively. The optimal visual interpretation grades for the detection of primary breast cancer of double phase M-SMM and TF-SMM were grade and 5. The optimal quantitative indices of M-SMM for the detection of breast cancer were 2.06 for early L/N and 1.72 for delayed L/N. Those of TF-SMM were 3.13 for early, and 2.56 for delayed image.

There is increasing interest in the health-related quality of life (QOL) of patients with chronic lymphedema. The aim of this study was to ascertain whether complex decongestive therapy (CDT) for upper limb lymphedema results in long-term changes in lymphedema and QOL, and to determine whether the treatment-induced change in the percentage excess volume (PCEV) is correlated with any changes in QOL. Fifty-three patients who had lymphedema were treated with CDT. PCEV and QOL were recorded before and 1 month after CDT, and at a 6-month follow-up visit. PCEV was significantly (p<0.05) decreased at 1 month, but significantly (p<0.05) increased at 6 months compared to 1 month [but still significantly reduced (p<0.05) from baseline]. The QOL scores at 1 and 6 months were significantly higher than the score at baseline, indicating an improvement in the QOL. Significant changes were evident in the single domains of physical functioning, role-physical, mental health, and general health. The change in PCEV was associated with a change in physical functioning, vitality, bodily pain, and general health at 1 and 6 months (p<0.05). This study suggests that QOL significantly improved with upper limb lymphedema during the maintenance phase, which was necessarily correlated with the reduction in limb volume.

Purpose: To compare the diagnostic accuracy and incremental diagnostic role of quantitative indices of early and delayed lesion to non-lesion ratios (L/Ns) in the detection of primary breast cancer., Material and Methods: Double-phase (99m)Tc MIBI scintimammography (SMM) (early 10 min, delayed 3 h) was performed after injection of 750 MBq of (99m)Tc MIBI in 446 highly suspected breast cancer patients (malignant: 311, benign: 135). For visual analysis, five scoring methods were used, and, for quantitative analysis, early and delayed L/Ns were calculated. Receiver operating characteristic curve (ROC) analyses were performed to determine the optimal visual grade, and to calculate cut-off values of quantitative indices for differentiation of malignant and benign diseases and to investigate whether the quantitative indices could provide incremental diagnostic values in addition to visual analysis., Results: Optimal visual grades were above 4 and 5 in the detection of breast cancer. Sensitivity was 84.2% and specificity 79.3%; the area under the curve (AUC) was 0.832 (95% CI, 0.794-0.866) and standard error was 0.019. Early and delayed L/Ns of malignant breast disease were significantly higher than those of benign disease (early: 2.01 +/- 0.99 versus 1.13 +/- 0.26 (P < 0.001); delayed: 1.68 +/- 0.69 versus 1.11 +/- 0.23 (P < 0.001)). The optimal L/Ns for the detection of primary breast cancer were 1.27 for early and 1.12 for delayed imaging. When early L/N 1.27 was used as cut-off value for the detection of primary breast cancer, the sensitivity of SMM was 77.8% and specificity 85.2%. The AUC was 0.856 (95% CI, 0.820-0.888). When delayed L/N 1.12 was used, sensitivity and specificity were 81.4% and 78.5%, respectively. The AUC was 0.834 (95% CI, 0.796-0.867). The ROC comparison of early and delayed L/N showed no statistical difference in the detection of malignant breast disease (P=0.403). When the delayed L/N was added to the early one, early plus delayed quantitative analysis (E+D) showed 86.5% sensitivity and 74.8% specificity. However, the AUCs of E+D (0.854, 95% CI, 0.767-0.842) and early L/N (E) (0.856) showed no statistical difference (P=0.614). When grades 4 and 5 were used as cut-off visual grade, sensitivity and specificity were 84.2% and 79.3%, respectively. When the E was added to visual grade, visual plus early L/N (V+E) showed 89.4% sensitivity and 77% specificity. The AUC of V+E (0.867, 95% CI, 0.832 0.897) was significantly higher than that of visual analysis (V) (0.832, 95% CI, 0.794-0.866, P < 0.001). When the delayed L/N (D) was added to visual grade, visual plus delayed L/N (V+D) showed 89.4% sensitivity and 74.1% specificity. The AUCs of V+D (0.852, 95% CI, 0.816-0.884) and V revealed no statistical differences (P = 0.052)., Conclusion: From this study, the optimal visual grades for diagnosis of breast cancer were grades 4 and 5; the cut-off values of L/Ns were 1.27 for early and 1.12 for delayed imaging. It was also found that early L/Ns provide incremental value in addition to visual analysis. However, delayed L/N revealed no incremental value. Therefore, the delayed image should not be routinely performed for purposes of primary breast cancer detection

Background: The aim of this prospective study was to evaluate the predictive value of [18F]fluorodeoxyglucose positron emission tomography (FDG PET) for the pathological response of breast cancer after completion of neo-adjuvant chemotherapy., Methods: Fifty patients with newly diagnosed, non-inflammatory, large or locally advanced breast cancer undergoing neo-adjuvant chemotherapy were eligible for this study. Clinical assessment was accomplished by comparing initial tumor size with preoperative tumor size. Pathological responses were classified into three groups: pathological non-response (pNR), pathological partial response (pPR) and pathological complete response (pCR). To determine the effect of reduction rate (RR) of peak standardized uptake values for tumor responses, logistic regression analyses were performed. To identify an optimal threshold value of RR for the prediction of pathological response, receiver operating characteristic analysis was performed., Results: Eight per cent (four of 50) of the patients had pCR and 46% had pPR. Ten per cent of patients had clinical CR and 52% had clinical PR. In clinical response, the RRs (+/- SD) of CR (-83.4 +/- 12), PR (-81.8 +/- 22.7) and NR (-79.7 +/- 31.9) showed no statistical differences (P > 0.05). However, for pathological responses, the RR of CR (-96.5 +/- 3.4) had a lower value than those of PR (-87.9 +/- 15.1) and NR (-56.2 +/- 29.6) (P = 0.0006; CR versus PR, P < 0.05; CR versus NR, P < 0.05; PR versus NR, P < 0.01). When -88% of RR was used as threshold value for differentiation between pCR and pPR, the area under the curve (AUC) was 0.788 [standard error (SE) 0.106; 95% confidence interval (CI) 0.589-0.920]. The sensitivity and specificity were 100% and 56.5%, respectively. When -79% of RR was used as threshold value for differentiation between pathological responders and non-responders, the AUC was 0.838 (SE 0.059; 95% CI 0.707-0.927). The sensitivity and specificity were 85.2% and 82.6%, respectively., Conclusions: Despite some limitations, this study suggests a possible predictive value of FDG PET for the assessment of the pathological response of primary breast cancer after neo-adjuvant chemotherapy. However, these findings deserve further investigation on a larger number of patients, and more frequent and earlier PET scans in each patient need to be performed to allow a better validation of the differentiation between the responder and non-responder groups.

The main reason for the recent interest in p53 is that almost 50% of human cancers contain p53 gene mutations. The majority of studies on p53 alterations in breast cancer have been limited to the isolated cases of ductal carcinoma in situ and infiltrating ductal carcinoma. The aims of this study were to determine the status and timing of p53 mutation in the progression from atypical ductal hyperplasia to invasive cancer, and to evaluate the patterns of p53 mutations in noninvasive and invasive lesions. Available lesions of invasive (n=88) and noninvasive (n=76) lesions were microdissected in 107 paraffin-embedded tissues (19 ductal carcinomas in situ, 57 invasive carcinomas with intraductal components, and 31 pure invasive carcinomas) and double-strand DNA sequencing was performed in exon 4-9 of the p53 gene. Among in situ cancers without invasive disease 36.8% had p53 mutations whereas in situ cancer with concurrent invasive disease showed p53 mutations in 33.3% of cases. In particular, two of seven atypical ductal hyperplasias harbored p53 alterations (one insertion and one missense mutation) in exon 8. The invasive component harbored p53 mutations in 30 of 88 cases (34.1%). We also discovered a novel deletion of 14 bp in exon 6 of two invasive lesions. The invasive component (1.33+/-0.13) carried a greater number of p53 mutations than its counterparts (1.19+/-0.10) and demonstrated more frequent multiple mutations (23.3% vs. 15.4%), but without statistical significance. Moreover, no statistical significance could be attached to the mutation frequency in the zinc-binding domains (26.7% vs. 15.4%), the directly DNA contact region (13.3% vs. 15.4%) and the missense mutation of p53 (50.0% vs. 57.7%) of the two groups. Based on our results, in spite of the small number of the lesions investigated, p53 mutation can occur at the stage of atypical ductal hyperplasia. The hypermutability and the specific p53 mutations involving the biologically functional domain (e.g., zinc binding domain or DNA contact region) have an insignificant influence on invasive progression in the breast cancer.

Objective: The purpose of this study was to assess whether the descriptive terminology and final assessment categories of the Breast Imaging Reporting and Data System (BI-RADS) lexicon can be used for breast carcinomas detected on MR imaging and to assess the inter- and intraobserver variabilities in the use of the descriptors and final assessment categories., Materials and Methods: In 82 patients, 101 masses, including 68 infiltrating carcinomas and 33 benign lesions, were interpreted independently by four radiologists and described by BI-RADS terminology with respect to mass shape and margin and BI-RADS final assessment categories. The enhancement pattern of the mass was also reported. In addition, two radiologists interpreted each case twice to evaluate intraobserver variability. The final case set for analysis was the 68 infiltrating carcinomas., Results: Most of the infiltrating carcinomas were described as irregular, spiculated, and heterogeneously enhancing masses. The final impression of the 68 carcinomas was BI-RADS category 5 (highly suggestive of malignancy) in 41 (61%), category 4 (suspicious abnormality) in 24 (35%), and category 3 (probably benign) in three (4%). Enhancement pattern was heterogeneous in 40 (59%), homogeneous in 14 (21%), and rim in 14 (21%). Interobserver agreement was moderate for mass margin, shape, enhancement, and final assessment category., Conclusion: This study suggests that the mammographic BI-RADS lexicon with some modifications may be applied to describe the features of infiltrating carcinoma seen on breast MR imaging.

Aberrant methylation in the CpG sites located in the promoter region of several tumor suppressor genes has been reported in various types of cancers. However, the methylation status of the p53 promoter has not been clearly determined and no information is available on its role in breast cancer. The aim of the study was to determine the presence and timing of the methylation of CpG sites in the p53 promoter, in the progression from ductal carcinoma in situ to invasive cancer. We also explored the correlation between the CpG methylation of the p53 promoter and p53 mutation during the progression of breast cancer. The corresponding lesions of both the invasive and noninvasive types were microdissected in paraffin-embedded tissue of 26 breast carcinomas. Bisulfite-modified DNA sequencing for methylation status in the p53 promoter was carried out, and double-strand DNA sequencing was performed in the promoter region and exons 4 to 9 of the p53 gene. CpG site methylation in the p53 promoter was detected in three cases (11.5%). Two noninvasive and three invasive lesions harbored CpG methylation in the p53 promoter. Methylations in more than one site were observed in three lesions, all of which contained methylation in two sites. The methylated CpG sites were located near the AP1 and YY-1 binding sites and at the YY-1 binding site. The p53 mutation was not found in the lesions where methylation in p53 promoter region was evident. In 16 cases (61.5%), neither methylation nor p53 mutation was detected. We conclude that the methylation in the p53 promoter region is found in the breast cancer irrespective of the status of invasion, and that the hypermethylation in the p53 promoter region is an alternative pathway to tumorigenesis where there is no p53 gene mutation.

Transcriptional repression of the transforming growth factor (TGF)-1P type II receptor (TPRII) gene appears to be a major mechanism to inactivate TGF-beta responsiveness in many human cancers. Because histone acetylation/deacetylation plays a role in transcriptional regulation, we have examined the effect of MS-275, a synthetic inhibitor of histone deacetylase, in human breast cancer cell lines. MS-275 showed antiproliferative activity against all human breast cancer cell lines examined and induced TbetaRII mRNA, but not TGF-beta type I receptor mRNA. MS-275 caused an accumulation of acetylated histones H3 and H4 in total cellular chromatin. An increase in the accumulation of acetylated histones H3 and H4 was detected in the TbetaRII promoter after treatment with MS-275. However, the level of histone acetylation did not change in chromatin associated with the TGF-beta type I receptor gene. MS-275 treatment enhanced TGF-beta1-induced plasminogen activator inhibitor 1 expression. Thus, antitumor activity of MS-275 may be mediated in part through the induction of TbetaRII expression and consequent potentiation of TGF-beta signaling.

Background: Although there have been many reports on the immunocytochemical detection of bone marrow micrometastases in breast cancer patients, peripheral blood micrometastases (PBM) have rarely been studied by immunocytochemistry (ICC)., Methods: PBM in operable and metastatic breast cancer patients were studied using immunomagnetic separation of tumor cells followed by immunocytochemistry (IMS-ICC)., Results: PBM were not detected in any peripheral blood samples from 21 healthy women, six patients with benign disease, or in a 21 patients with primary operable breast cancer, of which there were 7 stage I (n=7), 9 stage II, 2 stage III, and 3 inflammatory tumors. On the other hand, PBM were detected in 8 of 29 patients with metastatic breast cancers (27.6%). The number of tumor cells per patient varied from 2 to 90 cancer cells (median: 8 cells). Positivity of PBM was not significantly associated with the first site of recurrence, number of involved organs, tumor marker status, performance status, or disease-free interval, but it was significantly (p<0.01) associated with progesterone receptor negativity., Conclusion: PBM are very rare in primary operable breast cancer patients but can be observed in a considerable number of metastatic breast cancer patients. The clinical significance of PBM still remains to be established.

The epithelium-specific transcription factor, ERT/ESX/ESE-1/ELF3, binds to the TGF-beta RII promoter in a sequence specific manner and regulates its expression. In this study, we investigated whether ERT could regulate endogenous TGF-beta RII expression in Hs578t breast cancer cells. Analyses of the Hs578t parental cell line revealed low RII mRNA expression and resistance to the growth inhibitory effects of TGF-beta. Infection of this cell line with a retroviral construct expressing ERT induced higher levels of endogenous RII mRNA expression and protein expression relative to cells infected with chloramphenicol acetyltransferase (CATneo) as a control. Relative to control cells, the ERTneo-expressing Hs578t cells show approximately a 50% reduction in cell growth in the presence of exogenous TGF-beta1, as well as a fourfold higher induction of activation in transient transfection assays using the 3TP-luciferase reporter construct. When transplanted into athymic mice, ERT-expressing Hs578t cells showed decreased and delayed tumorigenicity compared with control cells. This data strongly suggests that ERT plays an important role as a transcriptional activator of TGF-beta RII expression, and that deregulated ERT expression may play a critical role in rendering Hs578t human breast cancer cells insensitive to TGF-beta's growth inhibitory effects.

Background: it is well known that breast cancer patients with more than 10 axillary lymph nodes involved have poor prognosis even with extensive adjuvant chemotherapy. To improve this poor outcome, high-dose adjuvant chemotherapy has been applied to the these patients. This study was intended to clarify the efficacy and usefulness of high dose adjuvant chemotherapy (HDC) for high-risk breast cancer patients and its efficacy was compared with conventional adjuvant chemotherapy (non-HDC group)., Patients and Methods: Twelve patients with breast cancer involving more than 10 axillary nodes received high-dose chemotherapy with peripheral progenitor-stem cell transplantation (PBSCT). This regimen consists of BCNU (carmustine) 130 mg/m(2) x 3, CBDCA (carboplantin) 500 mg/m(2) x 3 and CPA (chyclophosphamide) 50 mg/kgx 2 after induction chemotherapy with 3 cycles of CE (chyclophosphamide 600 mg/m(2), epirubicin 60 mg/m(2))., Results: Twelve patients completed the high-dose chemotherapy regimen as planned, no patient died of chemotherapy related toxicity. After a median follow-up period of 44 months, disease-free and overall survival at 48 months after the operation for 12 patients determined by Kaplan-Meier methods was 63 % and 83 %, respectively. Disease-free survival was superior in the high-dose chemotherapy group compared with the control group but a statistical difference was not observed., Conclusion: High-dose chemotherapy seems to be an effective and feasible treatment for high-risk breast cancer patients. However, the usefulness of high-dose adjuvant chemotherapy for high-risk breast cancer patients should be confirmed by a large-scale randomized trial.

Purpose: To evaluate the ability to use breast magnetic resonance (MR) imaging to assess disease extent in patients with posterior breast masses who are suspected to have tumor invasion into underlying muscle., Materials and Methods: Nineteen patients with posterior breast masses underwent three-dimensional, gradient-echo, 1.5-T MR imaging before and after the administration of gadopentetate dimeglumine. Thirteen had deep palpable masses that were clinically determined to be fixed to the underlying chest wall. Twelve had mammographic findings that caused muscle involvement to be suspected, and seven had normal mammograms. All patients underwent surgery. MR images were reviewed and were correlated with histologic findings., Results: Enhancing masses were identified on MR images in all 19 patients. Five (26%) of the 19 patients had masses that abutted the muscles, with obliteration of the fat plane and muscle enhancement. All five had muscle involvement at surgery. In the remaining 14 (74%) patients, no enhancement of muscle was seen; none of these had invasion of the muscle at surgery., Conclusion: Extension of adjacent tumor into underlying musculature was indicated by abnormal enhancement within these structures. Violation of the fat plane between tumor and muscle, without other findings, did not indicate tumor involvement of these deep structures.

We experienced a case of hepatic arterial infusion chemotherapy using docetaxel for liver metastasis, which showed no response to CEF therapy, from breast cancer. A 63-year-old woman had undergone modified radical mastectomy for right breast cancer (T2aN1bM0: Stage II) in October, 1995. Six-cycle CMF therapy and toremifene citrate (40 mg/day) were administered as adjuvant therapy, but multiple recurrent tumors in liver, lung, and local site were detected in February 1997. Six-cycle CEF therapy was given for recurrent disease and there was a complete response for lung and local recurrence, but no change in liver metastasis. Chemoendocrine therapies using 5'-DFUR or CMitF in addition to TAM and fadrozole hydrochloride hydrate had developed progressive disease for liver metastasis. A catheter and port kit were operatively inserted and implanted in March 1998. Hepatic arterial infusion of docetaxel (30-40 mg/body/month, one hour administration) was repeated 4 times, once in our clinic. Leukopenia, general fatigue and fever, which were mild and did not require any treatment, appeared as side effects. This treatment reduced multiple liver metastatic sites on abdominal CT finding and was thought to be a partial response. However, the patient had multiple brain metastasis and died on August 2, 1998. While docetaxel, even by systemic administration, has a 36-77% response rate for liver metastasis, arterial infusion might have a good response and mild side effect with a lower dose than by intravenous administration.

Previous studies suggest that estrogen receptor-positive (ER+) breast cancer cells acquire resistance to transforming growth factor-beta (TGF-beta) because of reduced expression levels of TGF-beta receptor type II (RII). We now report that treatment of ER+ breast cancer cells with the DNA methyltransferase inhibitor 5-aza-2'-deoxycytidine (5-aza-2'-dC) leads to accumulation of RII transcript and protein in three different cell lines. RII induction restored TGF-beta response in MCF-7L breast cancer cells as indicated by the enhanced activity of a TGF-beta responsive promoter-reporter construct (p3TP-Lux). A transiently transfected RII promoter-reporter element (RII-chloramphenicol acetyltransferase) showed an increase in activity in the 5-aza-2'-dC-treated MCF-7L cells compared with untreated cells, suggesting the activation of a transactivator of RII transcription. Using electrophoretic mobility shift assays, the enhanced binding of proteins from 5-aza-2'-dC-treated MCF-7L nuclear extracts to radiolabeled Sp1 oligonucleotides was demonstrated. An RII promoter-chloramphenicol acetyltransferase construct containing a mutation in the Sp1 site was not expressed in the 5-aza-2'-dC-treated MCF-7L cells, further demonstrating that induction of Sp1 activity by 5-aza-2'-dC in the MCF-7L cells was critical to RII expression. Northern analysis indicated that 5-aza-2'-dC treatment did not affect the Sp1 transcript levels. Western blot analysis revealed an increase of Sp1 protein in the 5-aza-2'-dC-treated MCF-7L cells, but there was no change in the c-Jun levels. Studies after cyclohexamide treatment suggested an increase in the Sp1 protein stability from the 5-aza-2'-dC-treated MCF-7L extracts compared with untreated control extracts. These results indicate that the transcriptional repression of RII in the ER+ breast cancer cells is caused by suboptimal activity of Sp1, whereas treatment with 5-aza-2'-dC stabilizes the protein thus increasing steady-state Sp1 levels and thereby leads to enhanced RII transcription and subsequent restoration of TGF-beta sensitivity.

Urokinase-type plasminogen activator (u-PA) is a key protease in cancer invasion and metastasis. Recent studies demonstrated that u-PA, plasminogen activator inhibitor type-1 (PAI-1), and tissue-type plasminogen activator (t-PA) are prognostic factors in breast cancer. However, there have been no prospective studies of node-negative breast cancer on a multicenter basis. On the other hand, some patients, even those with node-negative breast cancer, developed recurrence, and only tumor size is available as a predicting factor in this group. Therefore, it is necessary to find other prognostic factors in node-negative breast cancer to determine suitable adjuvant therapies. Tissue samples in this prospective study were obtained from 130 patients with node-negative invasive breast cancer who underwent radical operation at four hospitals. The median follow-up was 52.6 months. u-PA, PAI-1, and t-PA antigen levels were assayed by ELISA kits using the cytosolic fractions of tumors. Patients with high u-PA, high PAI-1, or low t-PA had significantly higher relapse rates than did those with low u-PA, low PAI-1, or high t-PA, respectively, by the Kaplan-Meier method (P = 0.006, 0.032, and 0.028, respectively). Analyses of the combinations of both u-PA and PAI-1 or both u-PA and t-PA showed that the differences in relapse rate between the high- and low-risk groups were statistically very significant. In the univariate analysis, u-PA, PAI-1, t-PA, progesterone receptor, and tumor size (T3 versus T1) were significantly correlated with relapse. However, the multivariate analysis revealed that only u-PA (P = 0.023) was an independent prognostic factor. This study showed that u-PA was a new significant independent prognostic factor in node-negative breast cancer.

Vitamin E succinate (VES) inhibited the proliferation of the estrogen receptor-negative human breast cancer cell line, BT-20, in the G1 phase of the cell cycle. The E2F proteins are integral transcriptional components in the regulation of cell growth. Overexpression of E2F-1 blocked the ability of VES to inhibit BT-20 cell growth, suggesting that VES regulation of E2F-1 activity leads to growth arrest of BT-20 cells. VES, although having little effect on E2F-1 steady-state protein levels, decreased E2F-1 phosphorylation and transactivation activity and increased cyclin A binding to E2F-1. GAL4-E2F-1 deletion mutant studies indicated that cyclin A negatively regulates E2F function. In VES-treated BT-20 cells, the cyclin A protein exhibited reduced kinase activity, which correlated with decreased steady-state levels and binding of cyclin-dependent kinase-2 to cyclin A and increased steady-state levels and binding of p21cip1 to cyclin A and cyclin-dependent kinase-2. The functional consequence of the negative regulatory effect of VES on E2F-1 function was shown by the ability of VES to inhibit the transcriptional activation of an E2F-1 responsive gene, c-myc. These studies show that VES induces growth inhibition of BT-20 cells through a mechanism that involves cyclin A-negative regulation of E2F-mediated transcription.

Background: Thrombomodulin (TM) is a natural anticoagulant which inhibits thrombin. Recent studies have reported that TM is correlated with vascular diseases and a few cancers. The aim of this study was to evaluate the role and the prognostic value of TM in breast cancer., Patients and Methods: TM expression in samples from 60 invasive breast cancer patients was examined immunohistochemically with a polyclonal antibody against TM., Results: TM staining was observed mainly on both the cytoplasm and cell surface in cancer cells and on endothelial cells around or in cancer tissue. TM expression in cancer cells was not correlated with the clinicopathological features. However, low TM expression was significantly correlated with a high relapse rate (p = 0.047 by the chi 2 test and 0.05 by the Kaplan-Meier method)., Conclusions: TM might play an active role in cancer invasion and metastasis, and serve as a new prognostic factor in invasive breast cancer.

Background: There have been many reports that the u-PA system plays an important role in cancer invasion and metastasis. The binding of u-PA to its specific cell-surface receptor, u-PAR, is necessary for the activation of u-PA system. The aim of this study is to evaluate the role and the prognostic value of u-PAR in cancer invasion and metastasis., Patients and Methods: u-PAR expression in 104 breast cancers was investigated immunohistochemically using a monoclonal antibody against u-PAR., Results: u-PAR expression was mainly observed both on cancer cells and stromal cells. Patients with high u-PAR expression in cancer cells or stromal cells had a high relapse rate compared with patients with low u-PAR expression by the Kaplan-Meier method (p = 0.035 and 0.011, respectively). In uni- and multivariate analysis, u-PAR expression in stromal cells was significantly correlated with relapse (p = 0.017 and 0.043, respectively)., Conclusions: This study has shown that not only cancer cells but also stromal cells have an important roles in breast cancer invasion and metastasis, and that u-PAR expression in cancer cells and stromal cells might be a novel prognostic factor in breast cancer.

Urokinase-type plasminogen activator (u-PA), which cleaves plasminogen to yield plasmin, is a serine protease of fibrinolysis and is presumed to play a key role in extracellular proteolysis and facilitate the migration of cancer cells. This study was conducted prospectively to evaluate the prognostic significance of u-PA antigen level in breast cancer tissues. u-PA concentrations in the cytosol of 226 breast cancer tissues were determined prospectively by enzyme-linked immunosorbent assay using cytosol fractions prepared for steroid hormone assay. The median follow-up period of the patients was 60 months. Various prognostic factors were evaluated by univariate analysis or multivariate analysis using the Cox proportional-hazards method. Patients with primary breast cancer containing high levels of u-PA had a significantly shorter disease-free survival than patients with low levels of u-PA antigens. In multivariate analysis, a high level of u-PA was an independent risk factor for disease-free survival, being independent of age, axillary node status, and estrogen receptor status. Among the major prognostic factors, a high u-PA antigen level, lymph node involvement, and a positive estrogen receptor status were the most important for predicting relapse-free survival (P = 0.044, P < 0.0001, P = 0.0039). This first prospective study confirmed the prognostic significance of the u-PA antigen level in association with other major prognostic factors. The results of our present study suggest that u-PA in breast cancer tissue might be involved in breast cancer invasion and metastasis.

The proteolytic processes are thought to be the critical point in tumor invasion and metastasis, mainly by matrix metalloproteinases (MMPs) and serine proteases. We measured the activity of MMP-2 from 28 normal, 12 benign and 126 breast cancer tissues using gelatin zymography. Inactive MMP-2 (72 kD) was expressed in 53.6% of the normal and 66.6% of the cancer tissues, respectively (P = 0.77), while active MMP-2 (62 kD) was expressed in 28.6% and 73.0%, respectively (P = 0.003). The enzymatic activity of active MMP-2 (62 kD) measured in the gel band area was 4.0 +/- 7.2 mm2 in normal breasts, 7.7 +/- 9.8 mm2 in benign breast diseases, 9.5 +/- 8.5 mm2 in ductal carcinoma in situ (DCIS), and 12.0 +/- 13.7 mm2 in invasive cancers. The MMP-2 activation ratio (62 kD/62 kD + 72 kD) was 0.12 +/- 0.18 in normal tissues, 0.10 +/- 0.20 in benign diseases, 0.61 +/- 0.22 in DCIS, and 0.50 +/- 0.28 in invasive cancers. In conclusion, MMP-2 activation was the main cause of the increased 62 kD MMP-2 activity during the early phase of breast cancer, while production of MMP-2 supplemented the increased 62 kD activity in the late phase. We suggest, therefore, that these differential expressions of MMP-2 activation and production during the different stages of breast cancer progression are potential therapeutic targets for biological or gene therapy under the concept of stage-oriented cancer treatment.