Noninvasive In-Vivo Quantification of Mechanical Heterogeneity of Invasive Breast Carcinomas.

Heterogeneity is a hallmark of cancer whether one considers the genotype of cancerous cells, the composition of their microenvironment, the distribution of blood and lymphatic microvasculature, or the spatial distribution of the desmoplastic reaction. It is logical to expect that this heterogeneity in tumor microenvironment will lead to spatial heterogeneity in its mechanical properties. In this study we seek to quantify the mechanical heterogeneity within malignant and benign tumors using ultrasound based elasticity imaging. By creating in-vivo elastic modulus images for ten human subjects with breast tumors, we show that Young's modulus distribution in cancerous breast tumors is more heterogeneous when compared with tumors that are not malignant, and that this signature may be used to distinguish malignant breast tumors. Our results complement the view of cancer as a heterogeneous disease on multiple length scales by demonstrating that mechanical properties within cancerous tumors are also spatially heterogeneous.

PLASMINOGEN AND ANGIOSTATIN LEVELS IN FEMALE BENIGN BREAST LESIONS.

It is known that benign breast tissue exhibit relatively low angiogenic capacity. Activation of angiogenesis in mammary pre-malignant lesions could be associated with disease progression and high risk of transformation into the breast cancer. However, insight into the underlying molecular mechanisms involved in angiogenesis regulation in non-cancerous breast pathologies is still poorly defined. The purpose of the present study was to determine levels of plasminogen and its proteolytic fragments (angiostatins) in mammary dysplasia (mastopathy and breast cyst) and benign neoplasms (fibroadenomas). Plasminogen and angiostatins were analyzed using immunoblotting and quantified by densitometric scanning. The significant increase in plasminogen levels was found in fibrocystic, cysts, and non-proliferatious fibroadenoma masses (4.7-, 3.7-, and 3.5-fold, respectively) compared to healthy breast tissues (control). In the same benign lesions, 6.7-, 4-, and 3.7-fold increase in plasminogen 50 kDa fragment (angiostatin) levels as compared with control were also observed. Activation of matrix metalloproteinase-9, which was detected using gelatine zymography, could be responsible for plasminogen cleavage and abundance of angiostatin infibrocystic and cyst masses. In contrast, dramatic decrease of both plasminogen and angiostatin levels (3.8- and 5.3-folds, respectively) was shown in tissues of proliferatious form of fibroadenoma in comparison with that of the dormant type of this neoplasm. Based on the obtained results, we concluded that angiostatin, a potent vessel growth inhibitor and anti-inflammatory molecule, can play a crucial role in pathophysiology of non-cancerous breast diseases. Further studies are needed to evaluate potential diagnostic and clinical implications of these proteins for prediction and therapy of benign breast pathologies.

[Expression and clinical significance of MTDH and VEGF in triple-negative breast cancer].

OBJECTIVE: To study the expression and clinical significance of MTDH and VEGF in triple-negative breast cancer (TNBC). METHODS: Tissue samples of 168 breast cancers (including 112 TNBC tissue and 56 non-TNBC tissue), 10 breast fibroadenomas and 15 normal breast tissues were collected. Postoperative specimens were examined by immunohistochemistry for MTDH and VEGF expression. The correlation between the expression of MTDH and VEGF and clinicopathological features was analyzed. RESULTS: MTDH and VEGF were expressed in 57.1% and 49.4% of breast cancer patients, 64.3% and 56.3% in TNBC patients, respectively, significantly higher than that in the non-TNBC tissues, breast fibroadenomas and normal breast tissues (P<0.05 for all). Statistically significant correlation was found between the MTDH and VEGF expressions (r=0.356, P<0.001). Moreover, MTDH expression was correlated with tumor size, BMI index, lymph node metastasis, pathological stage, recurrence and metastasis, and the expression of p53 and Ki-67 proteins (P<0.05 for all). The VEGF protein expression was correlated with lymph node metastasis, pathological staging, recurrence and metastasis, and the expression of Ki-67 protein (P<0.05 for all). The patients with high expression of MTDH and VEGF showed a lower DFS and OS (P<0.05 for both). CONCLUSIONS: MTDH and VEGF expression may be correlated with tumor angiogenesis and progression and has the potential to be valuable prognostic factors in patients with TNBC.

Cytopathologic diagnosis of spontaneous infarction of fibroadenoma of the breast.

Infarction is an uncommon event in a fibroadenoma, which is the commonest benign tumor of the breast. Most often it occurs in pregnancy, lactation or is secondary to fine needle aspiration. Spontaneous infarction of a fibroadenoma in the absence of a predisposing condition is very rare. The cytopathologic features of infarction are necrosis and worrisome nuclear features, which are often misinterpreted as either inflammation or malignancy. We detail a report of accurate cytopathologic diagnosis of spontaneous infarction of fibroadenoma in a 17-year-old adolescent non pregnant girl. Careful attention to the cytopathologic clues like uniform thickness of the necrotic epithelial fragments, branching pattern reminiscent of the staghorn pattern despite atypical nuclear features and clinical details like young age of the patient and recent onset pain in a pre-existing lump helped arrive at the correct diagnosis and spared the patient of a radical excision. To the best of our knowledge, there are no earlier reports of correct cytopathologic diagnosis.

Fine needle aspiration diagnosis of a spontaneously infarcted fibroadenoma mimicking carcinoma: a case report.

BACKGROUND: Spontaneous infarction of fibroadenoma is an extremely rare complication in a nonpregnant/nonlactating female undergoing first-time aspiration. It can be misdiagnosed as carcinoma in all aspects of triple approach used for evaluation of patients with breast lesions. CASE: A 37-year-old woman presented to the outpatient surgical clinic with a 6-month history of a breast lump that was slowly increasing in size and had become painful during the past month. There was no history of any trauma or fine needle aspiration, and she was not pregnant or lactating. Mammogram and ultrasound revealed a 2.9-cm heterogenous hypoechoic suspicious lesion. No lymph nodes were detected in the axilla. Fine needle aspiration cytology was performed, and a diagnosis of benign breast lesion with features of infarction was rendered on cytology. The lump was excised surgically, and a histological diagnosis of infarcted fibroadenoma was made. CONCLUSION: Careful and diligent search for preserved benign epithelial cells on smears is the key to recognize this entity and avoid serious therapeutic implications.

Autoantibodies against muscarinic receptors in breast cancer: their role in tumor angiogenesis.

The presence of autoantibodies in cancer has become relevant in recent years. We demonstrated that autoantibodies purified from the sera of breast cancer patients activate muscarinic acetylcholine receptors in tumor cells. Immunoglobulin G (IgG) from breast cancer patients in T1N0Mx stage (tumor size≤2 cm, without lymph node metastasis) mimics the action of the muscarinic agonist carbachol stimulating MCF-7 cell proliferation, migration and invasion. Angiogenesis is a central step in tumor progression because it promotes tumor invasion and metastatic spread. Vascular endothelial growth factor-A (VEGF-A) is the main angiogenic mediator, and its levels have been correlated with poor prognosis in cancer. The aim of the present work was to investigate the effect of T1N0Mx-IgG on the expression of VEGF-A, and the in vivo neovascular response triggered by MCF-7 cells, via muscarinic receptor activation. We demonstrated that T1N0Mx-IgG (10(-8) M) and carbachol (10(-9) M) increased the constitutive expression of VEGF-A in tumor cells, effect that was reverted by the muscarinic antagonist atropine. We also observed that T1N0Mx-IgG and carbachol enhanced the neovascular response produced by MCF-7 cells in the skin of NUDE mice. The action of IgG or carbachol was reduced in the presence of atropine. In conclusion, T1N0Mx-IgG and carbachol may promote VEGF-A production and neovascularization induced by breast tumor cells via muscarinic receptors activation. These effects may be accelerating breast tumor progression.

Infarcted fibroadenoma of the breast: report of two new cases with review of the literature.

INTRODUCTION: Fibroadenomas are the most common benign breast tumors in young women. Infarction is rarely observed in fibroadenomas and when present, it is usually associated with pregnancy or lactation. Infarction can exceptionally occur as a complication of previous fine-needle aspiration biopsy or during lactation and pregnancy. MATERIALS AND METHODS: Retrospective review of 650 cases of fibroadenomas diagnosed at our institution during the 8-years period identified two cases of fibroadenomas with infarction (rate ~0.3%). RESULTS: Two partially infarcted fibroadenomas were diagnosed on core biopsy and frozen section in an adolescent girl (13 years old) and in a young woman (25 years old), respectively. No preceding fine-needle aspiration biopsy was performed in these cases, nor were the patients pregnant or lactating at the time of the diagnosis. CONCLUSION: Spontaneous infarction within fibroadenoma is a rare phenomenon in younger patients. The presence of necrosis on core biopsy or frozen section should be cautiously interpreted and is not a sign of malignancy. VIRTUAL SLIDES: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1556060549847356.

Progranulin expression in breast cancer with different intrinsic subtypes.

Progranulin is a newly discovered 88-kDa glycoprotein originally purified from the highly tumorigenic mouse teratoma-derived cell line PC. We found that high progranulin expression was associated with higher breast carcinoma angiogenesis, reflected by increased vascular endothelial growth factor expression and higher microvessel density. However, no immunohistochemical evidence currently exists to correlate progranulin expression with clinicopathological features in different intrinsic subtypes of breast carcinoma biopsies. The aim of this study was to investigate the progranulin expression profiles in the intrinsic subtypes of breast carcinomas and their relevance to histopathological and clinicopathological features. Tissue blocks containing 264 cases of breast carcinomas from 2006 to 2009 were classified as different intrinsic subtypes. Tissues of four intrinsic subtypes were immunostained for progranulin, vascular endothelial growth factor and CD105. Their relevance to histopathological and clinicopathological features was also analyzed. Twenty tissue samples from breast fibroadenomas were included in this study. Progranulin expression showed no significant differences in different intrinsic subtypes, although an increasing tendency could be found in the triple-negative breast cancer (TNBC) subgroup (χ(2)=5.00, df=3, p=0.17). However, differences were significant when pathologically node metastasis-positive (pN(+)) TNBC were excluded (χ(2)=17.84, df=3, p<0.01). Some clinicopathological parameters, including CK5/6 (χ(2)=0.08, df=3, p=0.78), E-cadherin (χ(2)=0.71, df=3, p=0.40) and P53 (χ(2)=0.05, df=3, p=0.83), displayed no correlation with activity of progranulin in pathologically node metastasis-negative (pN(-)) TNBC. It was noted that the EGFR expression level of the pN(-) TNBC subtype was significantly higher in cases with strong progranulin expression than in cases with weak progranulin expression (χ(2)=11.26, df=1, p<0.01). A significantly higher expression level of progranulin in pN(-) TNBC suggests that progranulin is a promising new target for pN(-) TNBC treatment. Strong expression of progranulin correlates with positive EGFR expression in the pN(-) TNBC subtype. The close relationship between EGFR and progranulin/VEGF/CD105 expression may partly play a role in high angiogenesis levels in the pN(-) TNBC subtype.

The use of breast ultrasound color Doppler vascular pattern morphology improves diagnostic sensitivity with minimal change in specificity.

PURPOSE: The aim of this study was to evaluate the use of vascular morphology, around and within the B-mode region of abnormality, for improving the diagnostic accuracy of two of the most common solid breast pathologies. MATERIALS AND METHODS: The B-mode and Doppler images of 117 breast cancers and 366 fibroadenomas and lesions with a fibroadenoma-like appearance were reviewed retrospectively and the morphology of the vascular pattern was evaluated. The ratio of external to internal color Doppler, the external vascular pattern and the connecting vessels to internal vessels were assessed and differentiated into benign and malignant vascular patterns. These patterns were correlated with the histological diagnosis. RESULTS: Vascularity was demonstrated in 95 % of cancers and in 46 % of benign lesions with a trend to increasing vascularity in cancers. This provided poor specificity for excluding cancer in fibroadenomas. Variations in vascular pattern were recorded. The observed benign vascular patterns were avascularity, vascularity in the periphery and peripheral marginal vessels connecting with internal vascularity. The observed malignant vascular patterns were radially aligned external vessels with internal vessels being more numerous than external vessels which connected to radial vessels. (Fisher exact test p < 0.0001). Analysis of the vascular morphology improved the sensitivity for identifying cancers from 97 % (B-mode) to 99 % (B-mode and color Doppler) with a minimal reduction in specificity (93.7 to 92.6 %) or accuracy (94.6 to 94.2 %). CONCLUSION: The presence of vascularity within a lesion, by itself, is no longer a good predictor of malignancy because of the increase in Doppler sensitivity associated with improvements in ultrasound technology. The color Doppler ultrasound vascular pattern morphology improves the accuracy and sensitivity of B-mode image diagnosis, breast cancers and fibroadenomas with a minimal loss of specificity. Any breast lesion with radial rather than marginal connecting vessels should be regarded with suspicion.

Ultrasound characteristics of breast fibroadenomas are related to clinical and histological parameters.

PURPOSE: We hypothesized that ultrasound characteristics of breast fibroadenomas (FA) vary in relation to the clinical and histological parameters: patient age, tumor size and histological classification. MATERIALS AND METHODS: Eleven ultrasound characteristics frequently observed in breast tumors were defined before the onset of our study. These characteristics, as well as a semi-quantitative score for vascularization on color-coded Doppler ultrasound, were analyzed in a retrospective study. Histology revealed adult type differentiation in all FA. They were divided into florid, regressive and mixed subtypes. The examiner was blinded for the histological classification during image analysis. RESULTS: Histological type: florid FA: more frequent in younger women (age group < 30 years; p < 0.001), and bigger than regressive FA (larger than 16 mm: p = 0.007). Statistically significant differences between florid and regressive FA regarding the ultrasound features: enhanced posterior ultrasound transmission (p < 0.001), homogenous echo pattern (p = 0.003) and lobulated margin contour (p = 0.042). Tumor size: patients with larger tumors (> 16 mm) were younger (mean age 35 vs. 43 years, p < 0.001). More often in bigger FA: enhanced dorsal ultrasound transmission (p < 0.001), hyperechoic spots (p < 0.001), strong vascularization (p < 0.001), inhomogeneous echo pattern (p = 0.001), horizontal axis (p = 0.009), lobulated margin contour (p = 0.009), lateral shadowing (p = 0.047). Age: more often in older patients (age group > 30 years): dorsal ultrasound shadowing (p = 0.008), irregular margin contour (p = 0.038), homogenous echo pattern (p = 0.047). CONCLUSION: Histological type, tumor size and patient age significantly influence ultrasound characteristics of breast FA. This might be helpful to consider when breast lesions are classified and decisions for biopsies are made.

[Hemodynamic changes in benign and malignant breast tumors and the mechanism].

OBJECTIVE: To compare the histological morphology, hemodynamics and angiogenesis-related molecules between benign and malignant breast tumor and investigate their variation in different perfusion regions in the same type of tumors. METHODS: Thirty patients with malignant breast carcinoma and 30 with breast fibroadenoma underwent contrast-enhanced ultrasound examination with time-intensity quantitative analysis. The perfusion indices including peak intensity (PI), area under the curve (AUC), time to peak (TTP) and wash-out time (WOT) were measured both inside and on the margin of the foci. The expressions of CD34, vascular endothelial growth factor (VEGF), and Flk-1/KDR in both groups were measured immuhistochemically. RESULTS: The time-intensity curve (TIC) of malignant tumor group was characterized by rapid ascent and slow descent, while that of the benign group presented with slow ascent and rapid descent. The AUC and WOT of the malignant tumor group were significantly higher than those of the benign group, while the PI and TTP showed no significant difference. In malignant tumor group, PI, AUC and WOT on the margin of the foci were significantly higher those of the inside region, while TTP showed a reverse pattern. No significant differences were found in the perfusion parameters between the inside and outside of the foci in the benign group. The distribution of CD34 was heterogeneous in breast carcinoma, and the micro-vessels were densely distributed especially on the margin of the cancer nest. The microvessel density of the malignant group (34.48-/+8.34) was significantly higher than that of the benign group (18.65-/+4.69). Diffuse or focal high VEGF expression was found on the margin of the cancer nest and necrotic tissue, but hardly detected in the benign group. Flk-1/KDR expressed diffusely or focally in breast carcinoma with especial high expression on the margin of the cancer nest and necrotic tissue, but was virtually undetectable in the benign group. CONCLUSION: The perfusion pattern, TIC, mean perfusion parameter and variation of the regional perfusion parameters provide valuable evidence for differential diagnoses between benign and malignant breast tumors. Molecular imaging targeting VEGF and Flk-1/KD shed light on new approaches to early diagnosis of breast carcinoma.

[Heterogeneity of angioarchitecture and their hemodynamic changes in benign and malignant breast tumors].

OBJECTIVE: To explore the differences between the angioarchitecture, hemodynamics, ultrastructure of neovasculr endothelial cells, and vascular distribution in different perfusion regions in benign and malignant breast tumors. METHODS: 30 cases of breast carcinoma (33 lesions) and 30 cases of breast fibroadenoma (34 lesions) were examined by contrast enhanced microvascular imaging (MVI), and perfusion indexes were collected both inside and at the margin of each focus according to time-intensity quantitative analysis, including peak intensity (PI), area under the curve (AUC), time to peak (TTP) and wash-out time (WOT). The ultrastructure of neovascular endothelial cells was examined by transmission electron microscopy. The expression of CD34, VEGF, Flk-1/KDR in both two groups were detected by immuhistochemistry. RESULTS: Significant differences were found between the two groups characterized with filling defect, vascular distortion, dilatation and uneven enhancement. Most of the curves of malignant group (87.9%, 29/33) ascended rapidly and dropped slowly while those of the benign group (79.4%, 27/34) ascended slowly and dropped rapidly. The AUC and WOT of malignant tumor group were significantly higher than those of benign group, while the PI and TTP had statistically no significant difference. In the malignant tumor group, PI, AUC and WOT collected from the margin of foci were significantly different from those collected inside the foci, however, there was no significant difference in the benign group. The margin of foci was characterized with dilated and distorted vessels, and the center of the foci was occupied by narrow or occluded blood vessels, sometimes with contracted endothelial cells and pericytes. Abundant microvascular areas located at the margin of foci. The ultrastructure of endothelial cells in the newly formed blood vessels of malignant group showed strong ability to divide, which was different from normal endothelium cells. CONCLUSION: The perfusion pattern, mode of time-intensity curve, mean perfusion parameter and variation of regional perfusion parameters provide a valuable diagnostic basis in distinguishing benign and malignant breast tumors. The density, morphology, distribution, structure and function of newly formed microvessels in tumor foci are also crucial factors when tumors are assessed by imaging examination.

[Relationship between serum VEGF level and VEGF, COX-2 and MVD expression in breast cancer tissues].

OBJECTIVE: To investigate the relationship between serum VEGF (sVEGF) level and VEGF, COX-2 and MVD expression in breast cancer, and to discuss their role in angiogensis of breast cancer. METHODS: sVEGF level was detected by ELISA in 68 preoperative breast cancer, 35 benign breast disease and 20 healthy women. The expression of VEGF, COX-2 and MVD was detected by immunohistochemical method in tissues of breast cancer and breast benign diseases, and to analyze the relationship of sVEGF, VEGF, COX-2 and MVD. RESULTS: (1) sVEGF level in preoperative breast cancers was 306.51 pg/ml (interquartile range from 190.44 to 442.04 pg/ml), in benign diseases was 150.82 pg/ml (interquartile range from 82.36 to 212.34 pg/ml), and in healthy control was 105.93 pg/ml (interquartile range from 78.54 to 157.77 pg/ml). The sVEGF level of preoperative breast cancer group was significantly higher than that of breast benign disease group and healthy women (P = 0.001). (2) The VEGF expression positive rate in breast cancer (67.65%) was significantly higher than that in breast benign disease (44.12%) (P = 0.015). The COX-2 expression positive rate in breast cancer (42.86%) was significantly higher than that in breast benign disease (11.43%) (P = 0.002). (3) the COX-2 expression positive rate in sVEGF high level patients (56.00%) was significantly higher than that in sVEGF normal level patients (11.11%) (P = 0.024), and MVD in sVEGF high level patients (27.32 +/- 3.40) was also higher than that in sVEGF normal level patients (15.31 +/- 6.16) (P = 0.011). (4) The sVEGF level (322.09 +/- 79.31) of 68 breast cancer patients whose VEGF was positive in breast cancer tissues was significantly higher than that in VEGF negative group (222.47 +/- 73.53) (P = 0.017). (5) The COX-2 expression positive rate in VEGF positive expression group (65.21%) was significantly higher than that in VEGF negative expression group (18.18%) (P = 0.017). The MVD expression in COX-2 positive expression group (22.94 +/- 5.51) was significantly higher than that in COX-2 negative expression group (10.30 +/- 4.42) (P = 0.027). CONCLUSION: sVEGF level in breast cancer is significantly higher than that in breast benign disease and healthy women, and is correlated with the expression of COX-2 and MVD in breast cancer tissues.

CD105/smooth muscle actin double immunostaining discriminate between immature and mature tumor blood vessels.

The aim of the study was to demonstrate the value of the double immunostaining for endothelial and perivascular cell to discriminate mature from immature tumor-associated blood vessels in mammary carcinoma. We used the specific endothelial marker CD105 to highlight the activated endothelial cells and antibodies against smooth muscle cell actin (SMA) for perivascular cells, applying Envision Doublestain system (HRP/DAB-APAAP/Fast Red). We found an inverse correlation between the immunoexpression of CD105 and SMA in normal vessels and a strong positive signal for CD105 in the intratumor single endothelial cells and immature vessels. Only few blood vessels were positive for both CD105 and SMA within the tumor area. The signal for the endothelial marker was weak and inconstant, and significantly diminished when the SMA immunoexpression was increased for the same vessel. The differentiation between vessels with and without perivascular cells coverage using double immunostaining for CD105/SMA may be an important step in the selection of the mammary tumors, which could have a high grade of responsiveness to antiangiogenic therapy with monoclonal antibodies against CD105 antigen.

Videodensitometry in the examination of focal breast lesions after injection of an ultrasound contrast agent.

BACKGROUND: The present investigation aimed at assessing the possibility of distinguishing between malignant and benign breast lesions by measuring the signal intensity in vessels of the suspect lesions over time after administration of the ultrasound contrast agent Levovist. MATERIALS AND METHODS: Levovist was administered intravenously to 21 patients with breast cancer and 12 patients with a benign breast lesion. In the subsequent ultrasound investigation (Color Power Angiography) the resulting increase in brightness over time in the vessels of the lesions was measured using the videodensitometry method. From the calculated time-brightness curves, the time to maximum brightness (T(max)), time to 90% of maximum brightness (T(90%)), maximum brightness and other time and brightness parameters were determined. The data were analyzed by means of the Mann-Whitney-Wilcoxon test. Additionally, the sensitivity and specificity were calculated for a sequence of cut-off levels for T(90%), T(max) maximum brightness and wash-in wash-out parameters. RESULTS: The differences between the benign and the malignant lesions for the parameters T(max) and T(90%) were statistically significant. The malignant foci showed a significantly more rapid in-flow of the contrast agent (p = 0.006) than the benign lesions. The wash-in wash-out time for Levovist was significantly shorter for the malignant lesions than for the benign lesions (p = 0.02). The time difference in attaining maximum brightness was not significant (p = 0.14). The specificity and sensitivity made a more precise differentiation between benign and malignant tumors possible. CONCLUSION: The use of a contrast agent in Doppler ultrasound enhances the diagnostic reliability in distinguishing between malignant and benign lesions, justifying the use of a contrast agent with a high specificity (92%) such as Levovist. However, invasive pre-operative methods such as punch biopsy are not, thereby, rendered unneccessary. It is possible that the combination of Levovist and videodensitometry will make it possible to increase the specificity of breast cancer diagnosis.

[Values of color Doppler flow imaging and imaging changes of breast fascia and ligament in differential diagnosis of small breast neoplasms].

BACKGROUND & OBJECTIVE: Color Doppler flow imaging (CDFI) has been widely applied to the diagnosis of breast neoplasms. But combined use of CDFI and imaging changes of breast fascia and ligament (superficial and deep fascia, and Cooper's ligament) on the differential diagnosis of small breast neoplasms (< or =2 cm) has rarely been reported. This study was to explore the value of combined use of CDFI and imaging changes of breast fascia and ligament in differential diagnosis of small breast neoplasms. METHODS: CDFI was performed on 110 patients with small breast neoplasms. Among them, 52 cases were diagnosed to be benign by pathologic examination after surgery, while the rest 58 were malignant. The blood flow and Doppler image inside and around the breast lesions were analyzed. Four grades were classified according to blood flow imaging. The infiltration of breast fascia and ligament was also considered. The CDFI result was compared with that of pathologic examination. RESULTS: The correct diagnosis rates of CDFI in diagnosing benign and malignant small breast neoplasms were 90.4% and 84.5%, respectively. The false-positive and false-negative rates of CDFI were 9.6% and 15.5%, respectively. There were obvious differences in blood flow grading, systolic maximum velocity (V(max)), resistance index (RI), and imaging changes of superficial, deep fascia and Cooper's ligament between benign and malignant small breast neoplasms. CONCLUSION: Combination of CDFI with imaging changes of breast fascia and ligament is valuable in clinical diagnosis and differential diagnosis of small breast neoplasms.

Expression of hypoxia-inducible factor 1 alpha and its downstream targets in fibroepithelial tumors of the breast.

INTRODUCTION: Hypoxia-inducible factor 1 (HIF-1) alpha and its downstream targets carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) are key factors in the survival of proliferating tumor cells in a hypoxic microenvironment. We studied the expression and prognostic relevance of HIF-1alpha and its downstream targets in phyllodes tumors and fibroadenomas of the breast. METHODS: The expression of HIF-1alpha, CAIX, VEGF and p53 was investigated by immunohistochemistry in a group of 37 primary phyllodes tumors and 30 fibroadenomas with known clinical follow-up. The tumor microvasculature was visualized by immunohistochemistry for CD31. Proliferation was assessed by Ki67 immunostaining and mitotic counts. Being biphasic tumors, immunoquantification was performed in the stroma and epithelium. RESULTS: Only two fibroadenomas displayed low-level stromal HIF-1alpha reactivity in the absence of CAIX expression. Stromal HIF-1alpha expression was positively correlated with phyllodes tumor grade (P = 0.001), with proliferation as measured by Ki67 expression (P < 0.001) and number of mitoses (P < 0.001), with p53 accumulation (P = 0.003), and with global (P = 0.015) and hot-spot (P = 0.031) microvessel counts, but not with CAIX expression. Interestingly, concerted CAIX and HIF-1alpha expression was frequently found in morphologically normal epithelium of phyllodes tumors. The distance from the epithelium to the nearest microvessels was higher in phyllodes tumors as compared with in fibroadenomas. Microvessel counts as such did not differ between fibroadenomas and phyllodes tumors, however. High expression of VEGF was regularly found in both tumors, with only a positive relation between stromal VEGF and grade in phyllodes tumors (P = 0.016). Stromal HIF-1alpha overexpression in phyllodes tumors was predictive of disease-free survival (P = 0.032). CONCLUSION: These results indicate that HIF-1alpha expression is associated with diminished disease-free survival and may play an important role in stromal progression of breast phyllodes tumors. In view of the absence of stromal CAIX expression in phyllodes tumors, stromal upregulation of HIF-1alpha most probably arises from hypoxia-independent pathways, with p53 inactivation as one possible cause. In contrast, coexpression of HIF-1alpha and CAIX in the epithelium in phyllodes tumors points to epithelial hypoxia, most probably caused by relatively distant blood vessels. On the other hand, HIF-1alpha and CAIX seem to be of minor relevance in breast fibroadenomas.

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.

Color Doppler imaging of fibroadenomas of the breast with histopathologic correlation.

PURPOSE: This study was conducted to characterize the spatial distribution of blood vessels in breast fibroadenomas. METHODS: We performed a prospective study to map the anatomic distribution of the vessels in 29 fibroadenomas of the breast using color Doppler sonography. We categorized the detected vessels according to their location in or on the fibroadenoma, counted the different types of vessels, and tested for correlations between vessel distributions or numbers and histopathologic findings. RESULTS: Blood flow was demonstrated in 24/29 (83%) of fibroadenomas. We found 3 vessel types: feeding vessels, which are prominent vessels leading from the surrounding breast tissue into the fibroadenoma; capsular vessels, which are located within the tissue capsule; and segmental vessels, which are located within the fibrous septa of the fibroadenoma. Capsular and segmental vessels were demonstrated in 23/24 (96%) and 24/24 (100%) of the cases, respectively. Feeding vessels were seen in 12/24 (50%) of the cases. Histopathologic analysis revealed the same location and distribution of the vessels as color Doppler imaging. However, there was no correlation between numbers of vessels counted on sonograms and on histopathologic specimens. CONCLUSIONS: Examination of the vascularity demonstrated on color Doppler imaging helps in the diagnosis of benign breast neoplasms such as fibroadenomas.