High endothelial venule blood vessels for tumor-infiltrating lymphocytes are associated with lymphotoxin ß-producing dendritic cells in human breast cancer.

Blood vessels and tumor angiogenesis are generally associated with tumor growth and poor clinical outcome of cancer patients. However, we recently discovered that some blood vessels present within the tumor microenvironment can be associated with favorable prognosis. These vessels, designated tumor high endothelial venules (HEVs), appear to facilitate tumor destruction by allowing high levels of lymphocyte infiltration into tumors. In this study, we investigated the mechanisms regulating HEV blood vessels in human breast cancer. We found that lymphotoxin ß was overexpressed in tumors containing high densities of HEVs (HEV(high)) and correlated to DC-LAMP, a marker of mature DCs. DCs were the main producers of lymphotoxin ß in freshly resected HEV(high) breast tumor samples, and the density of DC-LAMP(+) DCs clusters was strongly correlated with the density of tumor HEVs, T and B cell infiltration, and favorable clinical outcome in a retrospective cohort of 146 primary invasive breast cancer patients. Densities of tumor HEVs and DC-LAMP(+) DCs were strongly reduced during breast cancer progression from in situ carcinoma to invasive carcinoma, suggesting that loss of tumor HEVs is a critical step during breast cancer progression. Finally, an increase in the infiltration of regulatory T cells was observed in HEV(high) breast tumors, indicating that tumor HEVs can develop in the presence of regulatory T cells. Together, our results support a key role for DCs and DC-derived lymphotoxin in the formation of tumor HEVs. These findings are important because novel therapeutic strategies based on the modulation of tumor HEVs could have a major impact on clinical outcome of cancer patients.

Dynamic contrast-enhanced MRI reveals the extent and the microvascular pattern of breast ductal carcinoma in situ.

To report the role of magnetic resonance imaging (MRI) in assessing the extent of breast ductal carcinoma in situ (DCIS). To assess whether the microvascularity pattern in DCIS correlates with magnetic resonance enhancement. Eighty-five histologically proven DCIS (77 pure DCIS, eight microinvasive DCIS) were prospectively studied with MRI. The morphology of magnetic resonance enhancement and the kinetic curve was recorded. Histopathologically, intraductal lesions were classified according to Van Nuys score. Tumor microvascularity was immunohistochemically assessed in a subset of 24 DCIS evaluating the number of microvessels, microvascularity area, and microvascularity pattern (diffuse or periductal). On the mammogram, 74% of DCIS appeared as microcalcifications. On MRI, 70% of DCIS showed enhancement. Non-mass-like uptake was observed in 78% of cases. The mean size of nonenhancing carcinomas was significantly lower than that of enhancing carcinomas (p = 0.033). The diffuse pattern was more frequent than the periductal pattern. A significant relationship between the morphology of MR enhancement and the microvascularity pattern was observed (p = 0.036); thus, 90% of DCIS showing segmental enhancement on MRI displayed a diffuse pattern while all DCIS with ductal enhancement showed a periductal pattern. There was a significant relationship between the maximum area of microvascularity and the vascular pattern (p = 0.015); periductal patterns showed larger areas than diffuse patterns. The lesion size was significantly larger as the Van Nuys score increased (p < 0.001) and was also related to the number of microvessels (p = 0.012). The mean area of microvascularity of DCIS was significantly larger as the Van Nuys score increased (p = 0.02). Breast MRI helps depict the extent of DCIS and reveals its microvascular pattern.

Breast cancer detection of large size to DCIS by hypoxia and angiogenesis using NIRS.

This investigation aimed to test all tumor-bearing patients who undergo biopsy to see if angiogenesis and hypoxia can detect cancer. We used continuous-wave near-infrared spectroscopy (NIRS) to measure blood hemoglobin concentration to obtain blood volume or total hemoglobin [Hbtot] and oxygen saturation for the angiogenesis and hypoxic biomarkers. The contralateral breast was used as a reference to derive the difference from breast tumor as a difference in total hemoglobin Δ[HBtot] and a difference in deoxygenation Δ([Hb]-[HbO2]). A total of 91 invasive cancers, 26 DCIS, 45 fibroblastomas, 96 benign tumors excluding cysts, and 67 normal breasts were examined from four hospitals. In larger-size tumors, there is significantly higher deoxygenation in invasive and ductal carcinoma in situ (DCIS) than in that of benign tumors, but no significant difference was seen in smaller tumors of ≤ 1 cm. With the two parameters of high total hemoglobin and hypoxia score, the sensitivity and specificity of cancer detection were 60.3 % and 85.3 %, respectively. In summary, smaller-size tumors are difficult to detect with NIRS, whereas DCIS can be detected by the same total hemoglobin and hypoxic score in our study.

Expression of class 3 semaphorins and their receptors in human breast neoplasia.

AIMS: This study aimed to identify the involvement of class 3 semaphorins (Sema3) and receptors, neuropilins (Np1 and Np2) and plexins (A1-A4) in breast cancer development and angiogenesis. METHODS AND RESULTS: We quantified and correlated Sema3A, Sema3B, Sema3F and their known receptors and coreceptors Plexin-A1, Plexin-A3, Np1 and Np2 in sections of normal human breast, benign and pre-malignant hyperplastic tissue, pre-invasive and invasive cancer, and compared these findings with our previously published data on vascular endothelial growth factor (VEGF) and microvessel density (MVD) in the same samples. Histological analysis revealed that Sema3B was expressed more strongly and widely than Sema3A and 3F in normal breast tissue and all three semaphorins decreased with the transition from in situ to invasive cancer (P < 0.014). Plexin-A3 decreased significantly with progression towards invasive cancer (P < 0.045), whereas Plexin-A1 expression was only significantly reduced once invasion had occurred (P = 0.012). Np1 and Np2 were expressed in both endothelial and epithelial/tumour cells. Np2 expression did not change, but Np1 expression significantly increased in the spectrum from hyperplasia to ductal carcinoma in situ (P < 0.035), but decreased with invasive cancer. CONCLUSION: These data suggest that a decrease in class 3 semaphorin and their plexin receptors have some relationship with disease progression in ductal breast carcinoma.

Multivariate analysis of flow data in breast lesions and validation in a normal clinical setting.

PURPOSE: To improve differentiation between benign and malignant breast lesions by Doppler measurements and to validate results in a normal clinical setting in comparison to study conditions. MATERIALS AND METHODS: Doppler measurements of 458 patients were compared in benign and malignant tumors in a prospective study. In a multivariate analysis a diagnostic score was developed using a logistic regression model and stepwise selection. These results were compared with 272 patients who were examined under routine clinical conditions. RESULTS: Most measurements showed highly significant (p < 0.001) differences between benign and malignant tumors. For each measurement we considered two cut-points to define a diagnostic rule. Despite significant differences, none of the corresponding classification rules exceeded 90 % sensitivity and specificity. Multivariate analysis selected a model including age and the number of arteries and contralateral arteries. Although significant, the last factor barely improved diagnostic accuracy. Therefore, we deleted it from the multivariate model. Based on a simple model including age and the number of tumor arteries, we defined classification rules with high sensitivity and specificity. The RI measurement did not improve the discriminatory power of our score. In the validation study the sensitivity decreased from 89 - 98 % to 58 - 78 % with a specificity of 82 - 92 % vs. 83 - 86 %. CONCLUSION: Color Doppler can be used for breast cancer differentiation. However, in the clinical routine the sensitivity decreases considerably compared with optimized study conditions.

The prediction of lymph node metastasis in ductal carcinoma in situ with microinvasion by assessing lymphangiogenesis.

BACKGROUND AND OBJECTIVES: Lymph node (LN) metastasis of ductal carcinoma in situ with microinvasion (DCISM) is variable (0-14%). To ascertain the role of lymphangiogenesis in LN metastasis in DCISM, we compared the lymphatic vessel density with the presence of LN metastasis in a group of patients that underwent axillary dissection with breast surgery due to DCISM. METHODS: We identified 46 patients with a diagnosis of DCISM who underwent breast surgery with axillary dissection to evaluate LN status from June 1996 to March 2008. Microvessel density (MVD) and lymphatic vessel density (LVD) was measured by immunohistochemical staining with two markers, CD34 and D2-40. RESULTS: LVD of the patients with LN metastasis was significantly higher than that of the patients without LN metastasis (P = 0.04). Correlation in the total score of progesterone receptor and LN metastasis was also noted (P = 0.017). There was no statistically significant relation between LVD and clinicopathologic parameters such as size and type of underlying DCIS, nuclear grade, presence of lymphovascular invasion, hormone receptor, and HER-2 status. CONCLUSIONS: Lymphangiogenesis may be significantly associated with LN metastasis in DCISM. This is the first attempt to predict axillary LN metastasis in DCISM by quantifying the LVD.

Exploratory study evaluating the association of polymorphisms of angiogenesis genes with hot flashes.

PURPOSE: Hot flashes are a common symptom and an important cause of decreased quality of life in women with breast cancer. Hot flashes involve vasodilatation and flushing, however, their complex etiology is not fully understood. We evaluated the association between germline polymorphisms in genes important to angiogenesis and subjective reporting of hot flashes. EXPERIMENTAL DESIGN: We recruited 1,244 subjects; 520 were breast cancer cases, 715 were documented healthy controls, and nine were of unknown status. Subjects were asked to provide a blood specimen and complete a questionnaire which included whether they had recently or had ever experienced hot flashes. We evaluated candidate polymorphisms in the following genes: hypoxia inducible factor-1 alpha (HIF1alpha), vascular endothelial growth factor (VEGF), VEGF-receptor 2 (VEGFR-2), endothelial nitric oxide synthase (eNOS), neuropilin-1 (NRP-1), and NRP-2. Testing for an association between polymorphisms and a history of current flashes or ever having hot flashes was performed. RESULTS: 441 premenopausal and 533 postmenopausal, Caucasian women were evaluable for hot flash analysis. For premenopausal women the eNOS-786 CT and TT genotypes were significantly associated with a greater likelihood of a subject reporting current hot flashes than the CC genotype (P = 0.03). After adjusting for clinical variables, the genotype association was no longer significant (P = 0.08). For postmenopausal women, the HIF1alpha 1744 CT and TT genotypes were significantly associated with a greater likelihood of a subject reporting current hot flashes (P = 0.05) and this remained significant after consideration of established clinical variables (P = 0.04). CONCLUSION: Hot flashes may be regulated by genes that control angiogenesis.

Significance of periductal lymphatic and blood vascular densities in intraductal carcinoma of the breast.

We investigated the significance of periductal lymphatic and blood vascular densities in intraductal carcinomas (IDC) of the breast. Thirty five cases of pure IDC treated by partial or total mastectomy were reviewed. Seven cases with normal breast tissue and 48 cases of invasive breast carcinoma were included as controls. All cases were immunostained with D2-40 and CD31. Positively stained microvessels were counted in densely vascular/lymphatic foci (hot spots) at 400x (=0.17 mm(2)) in the periductal areas. IDC without comedonecrosis showed a mean periductal D2-40 lymphatic microvessel density (LMD) of 5.8 +/- 5 (range 0-18), and a CD31 microvessel density (MD) of 14 +/- 8.9 (range 1-40). IDC with comedonecrosis showed periductal D2-40 LMD of 8.4 +/- 3.8 (range 4-18), and a CD31 MD of 24.3 +/- 7.6 (range 14-40). There was a significant difference between periductal D2-40 LMD and CD31 MD counts in IDC with and without comedonecrosis. There was a positive correlation of periductal D2-40 LMD and CD31 MD counts with high nuclear grade (r = 0.39 and 0.56) of IDC as well as with the presence of comedonecrosis (r = 0.49 and 0.59). Both D2-40 LMD and CD31 MD did not correlate significantly with tumor size, estrogen status, or progesterone status. As IDC with comedonecrosis and/or high nuclear grade has a worse prognosis than IDC without comedonecrosis and/or with low nuclear grade, it appears that lymphatic and blood vascular density evaluated by D2-40 and CD31, respectively, are independent prognostic indicators for patients with IDC of the breast and may be an indicator of early or unrecognized invasion or "regression."

Neovascularization in mucinous ductal carcinoma in situ suggests an alternative pathway for invasion.

AIMS: Ductal carcinoma in situ (DCIS) associated with invasive mucinous carcinoma (IMC) has not been well characterized. The aim was to characterize mucinous DCIS (mDCIS) of the breast and to describe, to our knowledge for the first time, neovascularization in mucin. METHODS AND RESULTS: The pathology reports and slides were reviewed from 44 patients treated between 2003 and 2006 at The University of Texas M. D. Anderson Cancer Center, whose diagnosis fulfilled the criteria of IMC or DCIS with mucin production. The patients, all female, had a mean age of 62 years. DCIS was present in 93% of cases and the predominant histological types were solid, cribriform and micropapillary. The DCIS was grade 1 in 12 of 41 cases (29.3%), grade 2 in 25 of 41 cases (61%) and grade 3 in four of 41 cases (9.8%). Mucin was seen in the lumen of the ducts involved by DCIS in 88% of cases, mucin and vessels in 63.4% of cases and neither mucin nor vessels in 12.2%. The DCIS was vascular endothelial growth factor-positive, platelet-derived growth factor receptor-beta-positive and CDX-2-negative (100%). Occasional luminal cells within the DCIS were immunopositive for CD68. CONCLUSIONS: A significant number of mDCIS showed neovascularization in intraluminal mucin. When identified on core needle biopsy, the presence of vascularized mucin should not be used alone to discriminate between invasive and in situ carcinoma. A hypothesis proposed for the source of recruitment of vessels in the mucin is that mucin can promote neovascularization and that tumour cells invade not into the adjacent fibroconnective tissue, but rather into the mucinous, richly vascularized stroma that they have induced. Alternatively, it is possible that both cells and their secretory product invade together. To our knowledge, this is the first study to characterize neovascularization within the mucinous component of DCIS associated with and without IMC.

An expression signature of syndecan-1 (CD138), E-cadherin and c-met is associated with factors of angiogenesis and lymphangiogenesis in ductal breast carcinoma in situ.

INTRODUCTION: Heparan sulphate proteoglycan syndecan-1 modulates cell proliferation, adhesion, migration and angiogenesis. It is a coreceptor for the hepatocyte growth factor receptor c-met, and its coexpression with E-cadherin is synchronously regulated during epithelial-mesenchymal transition. In breast cancer, changes in the expression of syndecan-1, E-cadherin and c-met correlate with poor prognosis. In this study we evaluated whether coexpression of these functionally linked prognostic markers constitutes an expression signature in ductal carcinoma in situ (DCIS) of the breast that may promote cell proliferation and (lymph)angiogenesis. METHODS: Expression of syndecan-1, E-cadherin and c-met was detected immunohistochemically using a tissue microarray in tumour specimens from 200 DCIS patients. Results were correlated with the expression patterns of angiogenic and lymphangiogenic markers. Coexpression of the three prognostic markers was evaluated in human breast cancer cells by confocal immunofluorescence microscopy and RT-PCR. RESULTS: Coexpression and membrane colocalization of the three markers was confirmed in MCF-7 cells. E-cadherin expression decreased, and c-met expression increased progressively in more aggressive cell lines. Tissue microarray analysis revealed strong positive staining of tumour cells for syndecan-1 in 72%, E-cadherin in 67.8% and c-met in 48.6% of DCIS. E-cadherin expression was significantly associated with c-met and syndecan-1. Expression of c-met and syndecan-1 was significantly more frequent in the subgroup of patients with pure DCIS than in those with DCIS and a coexisting invasive carcinoma. Levels of c-met and syndecan-1 expression were associated with HER2 expression. Expression of c-met significantly correlated with expression of endothelin A and B receptors, vascular endothelial growth factor (VEGF)-A and fibroblast growth factor receptor-1, whereas E-cadherin expression correlated significantly with endothelin A receptor, VEGF-A and VEGF-C staining. CONCLUSION: Syndecan-1, E-cadherin and c-met constitute a marker signature associated with angiogenic and lymphangiogenic factors in DCIS. This coexpression may reflect a state of parallel activation of different signal transduction pathways, promoting tumour cell proliferation and angiogenesis. Our findings have implications for future therapeutic approaches in terms of a multiple target approach, which may be useful early in breast cancer progression.

A putative role for psoriasin in breast tumor progression.

Psoriasin (S100A7) was identifi;ed as a gene highly expressed in psoriatic keratinocytes and highly and more frequently expressed in ductal carcinoma in situ (DCIS) than in invasive breast carcinomas (IBC), suggesting a potential role in tumor progression. Psoriasin expression is associated with poor prognostic factors in both DCIS and IBC. Several putative functions have been proposed for psoriasin in various disease types, but none of these can fully explain its involvement in breast tumor progression. Here, we show that down-regulation of endogenous psoriasin expression via stable short hairpin RNAs in a human IBC cell line (MDA-MB-468) increases cell migration and invasion without influencing cell proliferation and survival in vitro but inhibits tumor growth in vivo. These seemingly paradoxical results are potentially explained by the dramatic up-regulation and down-regulation of matrix metalloproteinase-13 and vascular endothelial growth factor (VEGF), respectively, observed in cells with decreased psoriasin levels compared with controls. Correlating with this, high psoriasin expression in human IBC is associated with increased angiogenesis and worse clinical outcome, and psoriasin mRNA levels are coordinately regulated with VEGF and other genes related to hypoxia and mitochondrial reactive oxygen species (ROS). Based on these results, we propose that psoriasin may play a role in breast tumor progression by promoting angiogenesis and enhancing the selection for cells that overcome its anti-invasive function. This hypothesis may explain why psoriasin expression is highest in high-grade and/or estrogen receptor-negative tumors, as these are associated with increased hypoxia and ROS, a setting in which the angiogenic effects of psoriasin are most important.

[Development of an Innovative Cell Isolation Method for the Investigation of Breast Cancer Pathogenesis and Angiogenesis for Experimental In Vitro And In Vivo Assays]. / Entwicklung eines neuen Zellisolationsverfahrens zur Erforschung der Mammakarzinompathogenese und -angiogenese für experimentelle in vitro und in vivo Assays.

Background Breast cancer is the world's most common cancer among women. Autologous lipotransfer is increasingly used for breast reconstruction following surgical removal of the tumour. In cell-assisted lipotransfer, the transplant is enriched with stem cells from adipose tissue (ADSC). Despite positive clinical results, there are some concerns regarding oncological safety due to transplanted stem cells. To date there are only a few breast cancer studies using primary cells from the same patient to enable further investigation into the complexity of cell-cell interactions in breast cancer in an experimental setting. Materials and methods We performed literature research on the topic of autologous lipotransfer. 5 different cell types (epithelial, mesenchymal cells, ADSC, endothelial cells, endothelial progenitor cells) were isolated from mammary (carcinoma) tissue or blood and were subsequently characterised for gene and protein expression as well as functional properties. The arteriovenous (AV) loop model in the rat was evaluated as a possible in vivo model for breast cancer pathogenesis and angiogenesis in this study. Results The literature provided evidence for an in-vitro interaction between ADSC and cells of the mammary (carcinoma) tissue. In some clinical studies, certain subgroups of patients appeared to be exposed to an increased risk of tumour recurrence after lipotransfer, but in most studies no correlation between lipotransfer and tumour recurrence was found. Different cell populations, which differed significantly in terms of surface markers, gene expression and functional properties, were isolated from tissue of the same patient. Axial vascularised tissue was successfully generated in the AV loop model. Conclusion In this study we were able to isolate different cell populations from the same patient, which reflect the heterogeneity of the tumour tissue. This enables a precise analysis of cell-cell interactions and their effects on tumour angiogenesis and pathogenesis in breast cancer. In combination with the AV loop model, this offers new possibilities to generate vascularised mammary carcinoma tissue as well as healthy mammary gland tissue in vivo as an optimal model for the clinical setting.

Human breast carcinoma: fibrin deposits and desmoplasia. Inflammatory cell type and distribution. Microvasculature and infarction.

Study of 14 human infiltrating breast carcinomas revealed new features that shed light on the pathogenesis of tumor stroma formation and on host immunologic defense mechanisms. Fibrin deposits were observed in the stroma of all tumors, particularly at their growing edge. Fibrin may have contributed both to tumor angiogenesis and, with organization, to the formation of the fibrous stroma characteristic of these and other scirrhous carcinomas. We previously proposed similar mechanisms for several animal tumors. All breast carcinomas studied elicited some degree of lymphocytic response at the tumor periphery; lymphocytes penetrated the fibrous tumor stroma poorly, did not exit in significant numbers from central tumor vessels, and, even when greatly outnumbering tumor cells locally, appeared relatively ineffective at tumor cell killing. Microvascular endothelial cell damage was frequently observed and may have been responsible for zones of tumor infarction. Similar observations have been made in skin allografts and animal tumors where rejection was effected principally by microvascular damage and subsequent tissue infarction, not by lymphocyte contact with individual epithelial target cells.

Does thymidine phosphorylase correlate with angiogenesis in intraductal carcinoma of the breast?

OBJECTIVE: To determine the association between thymidine phosphorylase (TP) and angiogenesis, and other conventional prognostic markers. We also evaluated interobserver and intraobserver reliability for TP expression in ductal carcinoma, to achieve a more consistent results. METHODS: Our study included all cases diagnosed in Adnan Menderes University Medical Faculty Hospital, Aydin, Turkey as invasive ductal carcinoma or ductal carcinoma in situ (DCIS) with proven component of (>30%), between January 2003 and February 2005. The total number of the cases was 27 and their median age was 50 years. All sections were stained using monoclonal antibody-TP and examined at x40 magnification. Either nuclear or cytoplasmic staining was accepted as positive. The histoscore (H-score) was calculated for each specimen. The tumor stromal vascularity was assessed by monoclonal anti-CD34; and areas of intense vascularization were determined. Conventional immunohistochemical markers such as c-erb B2, Ki-67, estrogen and progesterone receptors and p53 were also applied to all slides. Three pathologists blindly examined each slide under 10 high-power fields (10 HPF) for 2 times in a 2 months period. RESULTS: There was no significant association between stromal vascularity and TP staining of cancer cells (p=0.1) and no correlation was determined between H-scores for TP staining in ductal carcinoma and DCIS components (p=0.5). CONCLUSION: There was no significant correlation noted between stromal and periductal vascularity with the anti-CD34 antibody was used. No significant correlation was identified between the TP H-score and stromal or periductal vascularity.

Overexpression of beta1-chain-containing laminins in capillary basement membranes of human breast cancer and its metastases.

INTRODUCTION: Laminins are the major components of vascular and parenchymal basement membranes. We previously documented a switch in the expression of vascular laminins containing the alpha4 chain from predominantly laminin-9 (alpha4beta2gamma1) to predominantly laminin-8 (alpha4beta1gamma1) during progression of human brain gliomas to high-grade glioblastoma multiforme. Here, differential expression of laminins was studied in blood vessels and ductal epithelium of the breast. METHOD: In the present study the expressions of laminin isoforms alpha1-alpha5, beta1-beta3, gamma1, and gamma2 were examined during progression of breast cancer. Forty-five clinical samples of breast tissues including normal breast, ductal carcinomas in situ, invasive ductal carcinomas, and their metastases to the brain were compared using Western blot analysis and immunohistochemistry for various chains of laminin, in particular laminin-8 and laminin-9. RESULTS: Laminin alpha4 chain was observed in vascular basement membranes of most studied tissues, with the highest expression in metastases. At the same time, the expression of laminin beta2 chain (a constituent of laminin-9) was mostly seen in normal breast and carcinomas in situ but not in invasive carcinomas or metastases. In contrast, laminin beta1 chain (a constituent of laminin-8) was typically found in vessel walls of carcinomas and their metastases but not in those of normal breast. The expression of laminin-8 increased in a progression-dependent manner. A similar change was observed from laminin-11 (alpha5beta2gamma1) to laminin-10 (alpha5beta1gamma1) during breast tumor progression. Additionally, laminin-2 (alpha2beta1gamma1) appeared in vascular basement membranes of invasive carcinomas and metastases. Chains of laminin-5 (alpha3beta3gamma2) were expressed in the ductal epithelium basement membranes of the breast and diminished with tumor progression. CONCLUSION: These results suggest that laminin-2, laminin-8, and laminin-10 are important components of tumor microvessels and may associate with breast tumor progression. Angiogenic switch from laminin-9 and laminin-11 to laminin-8 and laminin-10 first occurs in carcinomas in situ and becomes more pronounced with progression of carcinomas to the invasive stage. Similar to high-grade brain gliomas, the expression of laminin-8 (and laminin-10) in breast cancer tissue may be a predictive factor for tumor neovascularization and invasion.

The phosphorylated form of connexin43 is up-regulated in breast hyperplasias and carcinomas and in their neoformed capillaries.

We applied an antiserum (SA226P) specifically recognizing the phosphorylated form of connexin43 (P-Cx43) to human breast samples including normal breast samples, with fibrocystic disease (FCD), fibroadenomas (FA), in situ and infiltrating carcinomas of all major types, and miscellaneous extramammary tumors. The findings were compared with those obtained with commercial antisera recognizing all Cx43 forms (pan-Cx43). A subset of samples was stained for Her2-neu and p44/42 to mitogen-activated protein kinase. Paraffin step sections were used. Immunoblots were performed on frozen samples of a representative subset of cases. In the normal breast, FCD, and FA, SA226P stained strongly and extensively most myoepithelial cells (MECs); luminal cells remained unstained. In proliferative FCD and some cellular FA, SA226P stained MEC and the capillary endothelium (CE). In ductal and lobular in situ carcinomas, SA226P reacted strongly and diffusely with the remaining MEC, the CE, and the transformed luminal cells. SA226P stained all infiltrating carcinomas except the tubular variant. In all breast carcinomas, the CE within and adjacent to tumors and some myofibroblasts stained with SA226P. By contrast, pan-Cx43 stained weakly and sporadically the MEC and rare samples of invasive carcinomas. Notably, Mab p44/42 reacted in parallel with the samples stained with SA226P, whereas reactions with Her2 were negative. Immunoblot findings paralleled those obtained immunohistochemically. We conclude that P-Cx43, restricted to MEC in the normal breast, is up-regulated in the same cells in hyperplasias and dysplasias and FA and is strongly up-regulated in invasive carcinomas. Notably, in some proliferative FCD and in most in situ and infiltrating carcinomas, P-Cx43 is strongly expressed in CE within and adjacent to the lesions but not away from them. These findings were paralleled by the strong nuclear reactions noted with Mab p44/42. These phenomena, although not exclusive to malignancy, are particularly conspicuous in breast carcinomas and seemingly reflect active proliferation associated with abnormal gap junctional intercellular communication.

Increased ipsilateral whole breast vascularity as measured by contrast-enhanced magnetic resonance imaging in patients with breast cancer.

BACKGROUND: Tumor-induced neovessel formation identified by gadolinium-enhanced magnetic resonance imaging (MRI) is a commonly used marker for breast malignancy. The purpose of this study was to assess possible differences in whole-breast vascularity as measured by contrast-enhanced MRI in the ipsilateral and contralateral breasts of patients with unilateral breast malignancies. METHODS: Gadolinium-enhanced MRI of the breast using a Siemens 1.0-T scanner with dedicated breast coil was performed on 22 consecutive patients with histologically confirmed unilateral breast carcinoma. Whole-breast vascularity of the breast containing the carcinoma was estimated as increased, decreased, or similar compared with the contralateral unaffected breast. Breast vascularity was then correlated to clinical factors including tumor size, histology, multifocality, nodal involvement, and patient age and menopausal status. RESULTS: Twenty patients had infiltrating carcinomas, and 2 patients had ductal carcinoma in situ. Four were multifocal. Fifteen of 22 patients demonstrated clear evidence of increased whole-breast vascularity in the ipsilateral breast containing the primary breast cancer compared with the contralateral breast. Although there was no clear correlation between the presence of increased whole-breast vascularity in the cancer-bearing breast with tumor size, histology, grade, mammographic appearance, or patient age and menopausal status, increased vascularity was present in 3 of 4 patients with multifocal disease and in 4 of 5 patients with metastatic disease in the axillary nodes. CONCLUSIONS: Measurable increases in whole-breast vascularity can be identified by contrast-enhanced MRI and appear with increased frequency in the cancer-bearing breast. These findings suggest that factors other than tumor size and histology may influence development of macroscopic vessels during tumor progression and may be indicative of angiogenic tumor biology.

Flt-4-positive vessel density correlates with vascular endothelial growth factor-d expression, nodal status, and prognosis in breast cancer.

PURPOSE: Metastasis to the regional lymph nodes through the lymphatic vessels is a common step in the progression of cancer and an important prognostic factor in many types of cancer. Recent evidence suggests that tumor lymphangiogenesis promotes lymphatic metastasis, and that the presence of Flt-4 on tumor blood and lymphatic vessels may play a important role in mediating lymphangiogenic factor-induced neovascularization. We assessed flt-4-positive vessel density (FVD) in breast cancer, and examined whether FVD associates with lymph node metastasis, VEGF-D expression, or prognosis. EXPERIMENTAL DESIGN: One hundred three invasive breast carcinomas with long-term follow-up were included in our study. Flt-4 was assessed using immunohistochemistry, then we analyzed the relationship between FVD and lymph node status, as well as VEGF-D expression and other established clinicopathological parameters. The relationship between FVD and prognosis was also investigated. RESULTS: Mean FVD of "hot spot" was 29.3 +/- 22.5 for each case. FVD was correlated significantly with lymph node metastasis (P < 0.0001), VEGF-D expression (P = 0.0019), tumor size (P = 0.0015), estrogen receptor (P = 0.0211), progesterone receptor (P = 0.0462), and c-erbB-2 (P = 0.0326). Survival curves determined by the Kaplan-Meier method and univariate analysis demonstrated that high FVD was associated with both worse disease-free survival (P = 0.0035) and overall survival (P = 0.0336). CONCLUSIONS: Increased FVD was correlated with lymph node metastasis and VEGF-D expression. High FVD may be a significant unfavorable prognostic factor for long-term survival in breast cancer. It is possible that Flt-4 becomes a target for antiangiogenic therapy to breast cancer.

Vascular stroma formation in carcinoma in situ, invasive carcinoma, and metastatic carcinoma of the breast.

The generation of vascular stroma is essential for solid tumor growth and involves stimulatory and inhibiting factors as well as stromal components that regulate functions such as cellular adhesion, migration, and gene expression. In an effort to obtain a more integrated understanding of vascular stroma formation in breast carcinoma, we examined expression of the angiogenic factor vascular permeability factor (VPF)/vascular endothelial growth factor (VEGF); the VPF/VEGF receptors flt-1 and KDR; thrombospondin-1, which has been reported to inhibit angiogenesis; and the stromal components collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin by mRNA in situ hybridization on frozen sections of 113 blocks of breast tissue from 68 patients including 28 sections of breast tissue without malignancy, 18 with in situ carcinomas, 56 with invasive carcinomas, and 8 with metastatic carcinomas. A characteristic expression profile emerged that was remarkably similar in invasive carcinoma, carcinoma in situ, and metastatic carcinoma, with the following characteristics: strong tumor cell expression of VPF/VEGF; strong endothelial cell expression of VPF/VEGF receptors; strong expression of thrombospondin-1 by stromal cells and occasionally by tumor cells; and strong stromal cell expression of collagen type I, total fibronectin, ED-A+ fibronectin, versican, and decorin. The formation of vascular stroma preceded invasion, raising the possibility that tumor cells invade not into normal breast stroma but rather into a richly vascular stroma that they have induced. Similarly, tumor cells at sites of metastasis appear to induce the vascular stroma in which they grow. We conclude that a distinct pattern of mRNA expression characterizes the generation of vascular stroma in breast cancer and that the formation of vascular stroma may play a role not only in growth of the primary tumor but also in invasion and metastasis.

Blood markers for vasculogenesis increase with tumor progression in patients with breast carcinoma.

Recent studies show that AC133-a hematopoietic stem cell antigen, when coex-pressed with endothelial markers, identifies a population of endothelial precursor cells (EPCs) in peripheral blood that function in tumor vasculogenesis in animals. Little is known about whether EPCs contribute to human tumor vasculogenesis. We attempted to determine if, through increased peripheral expression of AC133 or endothelial markers previously associated with EPCs,VEGFR-2 and Tie-2, we could detect an EPC response in the blood of patients with breast carcinoma. Thirty patients were segregated based on their breast biopsy histology into infiltrating carcinoma, DCIS and control groups. Using Real Time PCR, we measured the expression of the aforementioned markers in reverse transcribed RNA extracts from preoperative peripheral blood specimens. The cancer patients had significantly elevated Tie-2 expression with the highest levels associated with infiltrating carcinoma. Our data suggest increased circulating EPC markers in tumor patients, but further study of this cell population is needed to better define its role in tumor vasculogenesis.