[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.

Prostate-specific membrane antigen expression in tumor-associated vasculature of breast cancers.

Prostate-specific membrane antigen (PSMA) has been found to be expressed in the tumor-associated neovasculature of multiple solid tumor types including breast cancers. However, thus far, the number of cases studied from some tumor types has been limited. In this study, we set out to assess PSMA expression in the tumor-associated vasculature associated with invasive breast carcinomas in a sizable cohort of patients. One hundred and six patients with AJCC stage 0-IV breast cancer were identified. Ninety-two of these patients had primary breast cancer [invasive breast carcinoma with or without co-existing ductal carcinoma in situ (DCIS) (74) or DCIS alone (18)]. In addition, 14 patients with breast cancer metastases to the brain were identified. Immunohistochemical staining for PSMA and CD31 was performed on parallel representative tumor sections in each case. Tumor-associated vascular endothelial cell PSMA immunoreactivity was semi-quantitatively assessed based on two parameters: overall percent of endothelial positivity and staining intensity. PSMA expression for tumor-associated vascular endothelial cells was scored 0 if there was no detectable PSMA expression, 1 if PSMA staining was detectable in 5-50%, and 2 if PSMA expression was positive in >50% of microvessels. CD 31 staining was concurrently reviewed to confirm the presence of vasculature in each case. Tumor-associated vasculature was PSMA-positive in 68/92 (74%) of primary breast cancers and in 14/14 (100%) of breast cancers metastatic to brain. PSMA was not detected in normal breast tissue or carcinoma cells. All but 2 cases (98%) showed absence of PSMA expression in normal breast tissue-associated vasculature. The 10-year overall survival was 88.7% (95% CI = 80.0%, 93.8%) in patients without brain metastases. When overall survival (OS) was stratified based on PSMA score group, patients with PSMA scores of 0, 1, and 2 had 10-year OS of 95.8%, 96.0%, and 79.7%, respectively (p = 0.12). When PSMA scores of 0 and 1 were compared with 2, there was a statistically significant difference in OS (96.0% vs 79.7%, respectively, p = 0.05). Patients with a PSMA score of 2 had a significantly higher median tumor size compared with patients in the lower PSMA score groups (p = 0.04). Patients with higher nuclear grade were more likely to have a PSMA score of 2 compared with patients with lower nuclear grade (p < 0.0001). Patients with a PSMA score of 2 had a significantly higher median Ki-67 proliferation index compared with patients in the lower PSMA score groups (p < 0.0001). Patients with estrogen receptor (ER)-negative tumors were more likely to have a PSMA score of 2 compared with patients with ER-positive tumors (p < 0.0001). Patients with progesterone receptor (PR)-negative tumors were more likely to have a PSMA score of 2 compared with patients with PR-positive tumors (p = 0.03). No significant association was observed between PSMA score group status and lymph node involvement (p = 0.95). Too little variability was present in Human epidermal growth factor receptor-2 (Her2/neu) amplified tumors to correlate with PSMA score group status. To date, this is the first detailed assessment of PSMA expression in the tumor-associated vasculature of primary and metastatic breast carcinomas. Further studies are needed to evaluate whether PSMA has diagnostic and/or potential therapeutic value.

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.

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.

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.

Immunohistochemical analysis of cancer stem cell markers in invasive breast carcinoma and associated ductal carcinoma in situ: relationships with markers of tumor hypoxia and microvascularity.

We performed immunohistochemical analysis of 3 cancer stem cell-related markers (CD44(+)/CD24(-/low), aldehyde dehydrogenase [ALDH]-1, CD133) in 94 invasive ductal carcinomas and assessed relationships with markers of hypoxia (carbonic anhydrase IX [CAIX]), tumor microvessel density (CD31), and clinicopathologic variables. Overall, 10% of tumors were CD44(+)/CD24(-/low), 13% were ALDH-1(+), 25% were CD133(+), 35% were immunonegative, and 1 tumor was immunopositive for all 3 markers. Associated ductal carcinoma in situ (DCIS) was present in 48% of tumors. Marker immunopositivity was detected in DCIS in 13% (CD44(+)/CD24(-/low)), 7% (ALDH-1(+)), and 32% (CD133(+)) of these tumors and was more likely present in DCIS when also detected in the invasive compartment (P = .03, P = .001, and P = .009, respectively). CD44(+)/CD24(-/low) cells were more common in progesterone receptor-negative tumors (P < .01), and ALDH-1(+) cells were more common in estrogen receptor-negative tumors (P < .01). CD133(+) cells were more common in patients younger than 50 years (P < .05) and in high grade (P < .01), localized (P < .05), and estrogen receptor-negative (P < .001), progesterone receptor-negative (P = .02), and triple-negative breast cancers (P < .001). CD44(+)/CD24(-/low) (P = .06) and CD133(+) (P = .02) tumor cells were more common in CAIX(+) versus CAIX(-) tumors, whereas ALDH-1(+) tumors had a higher mean microvessel density than did ALDH-1(-) tumors (P = .002). No significant relationships were observed between the markers studied and survival for 5 years. Our study demonstrated the presence of cancer stem cell marker-positive tumor cells in DCIS as well as invasive breast cancer and showed that CD44(+)/CD24(-/low) and CD133(+) cells were more frequently observed in hypoxic regions of tumor, whereas ALDH-1(+) cells more commonly colocalized to tumors with high microvessel density.

Correlation of the apparent diffusion coefficiency values on diffusion-weighted imaging with prognostic factors for breast cancer.

OBJECTIVE: The aim of this study was to correlate the apparent diffusion coefficient (ADC) value of breast cancer with prognostic factors. METHODS: 335 patients with invasive ductal carcinoma not otherwise specified (IDC NOS) and ductal carcinoma in situ (DCIS) who underwent breast MRI with diffusion-weighted imaging were included in this study. ADC of breast cancer was calculated using two b factors (0 and 1000 s mm(-2)). Mean ADCs of IDC NOS and DCIS were compared and evaluated. Among cases of IDC NOS, mean ADCs were compared with lymph node status, size and immunochemical prognostic factors using Student's t-test. ADC was also correlated with histological grade using the Kruskal-Wallis test. RESULTS: Mean ADC of IDC NOS was significantly lower than that of DCIS (p<0.001). However, the mean ADC of histological grade of IDC NOS was not significantly different (p=0.564). Mean ADC of oestrogen receptor (ER)-positive or progesterone receptor (PR)-positive cancer was significantly lower than that of ER-negative or PR-negative cancer (p=0.003 vs p=0.032). Mean ADC of Ki-67 index-positive cancer was significantly lower than that of Ki-67 index-negative cancer (p=0.028). Mean ADC values of cancers with increased microvascular density (MVD) were significantly lower than those of cancer with no MVD increase (p=0.009). No correlations were observed between mean ADC value and human growth factor receptor 2 expression, tumour size and lymph node metastasis. CONCLUSION: Low ADC value was correlated with positive expression of ER, PR, increased Ki-67 index, and increased MVD of breast cancer.

Imaging tumor vascularization for detection and diagnosis of breast cancer.

Breast cancer is one of the major causes of morbidity and mortality in western women. Current screening and diagnostic imaging modalities, like x-ray mammography and ultrasonography, focus on morphological changes of breast tissue. However, these techniques still miss some cancers and often falsely detect cancer. The sensitivity and specificity for detecting the disease can probably be improved by focusing on the consequences of tumor angiogenesis: the increased microvessel density with altered vascular characteristics. In this review, various techniques for imaging breast tumor vasculature are discussed. Dynamic contrast enhanced magnetic resonance imaging is the most-used imaging modality in this field. It has a proven high sensitivity, but a low specificity and cannot be applied in all women. Moreover, it has problems with detecting ductal carcinoma in situ (DCIS). On the contrary, contrast enhanced digital mammography can detect DCIS, but requires the use of ionizing radiation. Contrast enhanced ultrasound provides real-time information about true intravascular blood volume and flow. However, this technique still has difficulties with discriminating benign from malignant tissue. Moreover, these three imaging modalities all require the injection of contrast agents. Two relatively new techniques that do not use external contrast agents are diffuse optical imaging and photoacoustic imaging. Both visualize the increased concentration of hemoglobin in malignant tissue and thereby provide a high intrinsic contrast.

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.

Lymphatic microvessel density, VEGF-C, and VEGFR-3 expression in different molecular types of breast cancer.

BACKGROUND: Breast cancer is a heterogeneous disease and five major distinct molecular types have been characterized by gene analysis and immunohistochemistry. The molecular types of breast cancer have different behavior, a particular profile of response to therapy, reflected in the differential survival of patients. Previous findings showed a particular preference for lymph node and distant metastases of different molecular types, but the specific lymphangiogenic profile of these types is lacking. PATIENTS AND METHODS: We investigated the differential expression vascular endothelial growth factor-C (VEGF-C), vascular endothelial growth factor receptor-3 (VEGFR-3) and D2-40 by immunohistochemistry, to evaluate lymphangiogenesis and the lymphatic microvessel density (LMVD), in patients with breast cancer, stratified according to the molecular classification. RESULTS: There was a differential expression of VEGF-C/VEGFR-3 and D2-40 in different molecular types of breast cancer, with highest level of expression for these markers being found in HER2 and luminal B types and the lowest in basal-like type. The lowest value of both intratumoral and peritumoral LMVD were found in normal-like type breast cancer. VEGF-C expression did not correlate with the grade of the tumor, but a significant correlation was found with lymph node metastasis. VEGFR-3 expression was found in 66.66% of the cases and correlated with the expression of VEGF-C in tumor cells. There was a positive correlation between VEGF-C, VEGFR-3 and LMVD only in the HER2 type, and a positive correlation in HER2 and normal-like types with VEGFR-3 expression in tumor cells. In addition, there was a correlation between HER2 type, VEGF-C and VEGFR-3 expression in tumor cells and lymphatic endothelium, respectively, and LMVD. CONCLUSION: Our results support a differential signature of lymphangiogenesis in different molecular types of breast cancer and these findings may have a direct impact on prognosis and therapeutic strategy of this disease.

D2-40: an additional marker for myoepithelial cells of breast and the precaution in interpreting tumor lymphovascular invasion.

D2-40 is a recently available mouse monoclonal antibody specific for human podoplanin and has been used in identifying lymphovascular invasion (LVI) of tumors. Although its expression has been evaluated in other tissues, its use as a marker for myoepithelial cells (MEC) of breast has not been studied. To explore its expression in the MEC of breast, paraffin-embedded tissue blocks from 48 patients with breast diseases were selected to include usual ductal hyperplasia (UDH, 41 cases), atypical ductal hyperplasia (ADH, 4 cases) and ductal carcinoma in situ (DCIS, 17 cases). Normal breast parenchyma and invasive carcinoma coexisting in the tissue sections were also included in the study. Immunohistochemistry for D2-40, calponin and p63 was performed and the staining patterns were reviewed and compared. D2-40 immunohistochemical staining is positive in the cytoplasm of MEC in UDH, ADH, and the majority of DCIS. The staining pattern of D2-40 is comparable with that of calponin, however D2-40 staining of MEC is weaker than that of calponin and with less background. In addition, myoepithelial cells and myofibroblasts at the edge of retraction spaces of DCIS are also stained by D2-40 that could be misinterpreted as tumor LVI. In conclusion, D2-40 immunohistochemistry reliably identifies the MEC of breast in a variety of lesions in a pattern similar to that of calponin and p63, and can be used as an additional MEC marker. Caution should be exercised when interpreting the staining of cells surrounding DCIS and carcinoma with retraction artifact.

Angiogenesis in the progression of breast ductal proliferations.

Angiogenesis, the formation of blood vessels, is necessary for a tumor to grow, but when angiogenesis first appears in the progression of breast ductal carcinomas is unknown. To determine when this occurs, the authors examined microvessel density (MVD) by CD31 and CD105 immunostaining in normal ducts, 32 cases of usual hyperplasia, 19 cases of atypical hyperplasia, and 29 cases of ductal carcinoma in situ (DCIS). Simple hyperplasia had a 22-fold greater MVD than normal ducts (P < .0001). An increase during the progression of ductal changes was highly significant (P < .0001). To determine a possible mechanism, immunohistochemistry for vascular endothelial growth factor (VEGF) was evaluated. VEGF staining intensity of ductal epithelium increased during the progression from normal to hyperplastic to DCIS. This study shows that the first significant increase in angiogenesis occurs very early in the evolution of ductal proliferations as ductal cells become hyperplastic.

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.

Columnar cell lesions and pseudoangiomatous hyperplasia like stroma: is there an epithelial-stromal interaction?

The significance of association between cancer and its microenvironment has been increasingly recognized. It has been shown in animal models that interaction between neoplastic epithelial cells and adjacent stroma can modulate tumor behavior. Carcinoma associated stromal cells can transform normal epithelial cells into neoplastic cells. In breast, columnar cell lesions are non-obligate precursors of low grade ductal carcinoma in situ. Columnar cell lesions can be seen intimately associated with PASH-like-stroma, a lesion we termed as CCPLS. Our aim is to investigate epithelial-stromal interactions in CCPLS and compare them to PASH without columnar cell lesions in breast core needle biopsies. Normal terminal duct lobular unit (TDLU) epithelium was seen in association with columnar cell lesions as well as PASH. Eight (8) cases of each category were examined by a panel of immunostains: CD117 (C-kit), CD34, CD105, bFGF, AR, ER-beta, MIB-1. We observed a markedly decreased expression of c-kit in columnar cell lesions compared to TDLU-epithelium. CD105 showed a quantitative increase in activated vessels in CCPLS compared to PASH. A subset of CCPLS and PASH were androgen receptor positive. A strong nuclear positivity for ER-beta is observed in the epithelium and stroma of all CCPLS cases. We conclude that (1) activated blood vessels predominate in CCPLS; (2) A molecular alteration is signified by c-kit loss in columnar cell lesions; (3) ER-beta and androgen receptor positivity indicate CCPLS are hormonally responsive lesions. Our study suggests an intimate vascular and hormone dependent epithelial-stromal interaction exists in CCPLS lesions.

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."

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.

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.

Optical mammography: a new technique for visualizing breast lesions in women presenting non palpable BIRADS 4-5 imaging findings: preliminary results with radiologic-pathologic correlation.

The purpose of this prospective study is to determine the diagnostic accuracy of near-infrared breast optical absorption imaging in patients with Breast Imaging Reporting and Data System (BIRADS) 4-5 non-palpable lesions scheduled for biopsy, using pathology after core or excisional biopsy as a reference. The patient's breast was positioned onto a panel of red light-emitting diodes (640 nm). A soft membrane was inflated to exert a uniform pressure on the breast. Transmitted light was detected using a CCD camera. The entire acquisition sequence took 1 minute. Image processing generated dynamic images displayed in colour scale, to reveal time-dependent changes in the transmitted light intensity caused by the pressure change. Dynamic curves were classified in two categories: consistently decreasing intensity suspicious for malignancy, and sinusoidal increasing intensity considered as benign. Seventy-eight women consulting for non-palpable breast lesions were initially included in the study. An imaging-histology correlation was obtained for seventy-two patients, the remaining six patients were excluded for technical optical scan reasons. We experienced an overall sensitivity of 73% and specificity of 38%, the false negative results being mainly small size (<10 mm) infiltrating malignant lesions and ductal carcinoma in situ (DCIS). False positive results were seen in benign proliferative lesions. Dynamic optical breast imaging is a novel, low-cost, non-invasive technique yielding a new type of information about the physiology of breast lesions. Absorption is due to haemoglobin and its products, therefore reflecting the angiogenic status of breast tumours.

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.