Association of angiogenesis in lymph node metastases with outcome of breast cancer.

BACKGROUND: Microvessel density (MVD) is a measure of the extent of new blood vessel growth or angiogenesis, which is required for tumor progression. Increased MVD in primary breast cancers appears to adversely affect disease-free survival and overall survival in patients with breast cancer. However, the clinical implications of angiogenesis in breast cancer metastases have not been well studied. The purpose of this study was to compare intratumoral MVD in primary breast cancer tissues with MVD in axillary lymph node metastases and to evaluate the relationships among primary- and metastatic-tumor MVD, disease-free survival, and overall survival in patients with lymph node-positive, stage II breast cancer who were treated with adjuvant chemotherapy in Cancer and Leukemia Group B Protocol 8082. METHODS: Immunostaining for factor VIII-related antigen was performed on tissue sections from 47 primary tumors and 91 axillary lymph nodes containing metastases from 110 patients with lymph node-positive breast cancer. Sections were examined for the presence or absence of focal areas of relatively intense neovascularization (vascular hot spots), and a quantitative assessment of intratumoral MVD was performed. RESULTS: The presence of vascular hot spots in axillary lymph node metastases, but not primary breast cancers, was associated with statistically significantly decreased disease-free survival (P =.006) and overall survival (P =.004) by univariate analysis. Similarly, increased MVD in metastases, but not in primary tumors, was statistically significantly associated with diminished overall survival in these patients (P =.02). In multivariate analysis, the number of positive axillary lymph nodes and the presence of vascular hot spots in axillary lymph node metastases predicted decreased disease-free survival (P =.0001 and.02, respectively) and overall survival (P =.0001 and.007, respectively). All P values were two-sided. CONCLUSION: This pilot study suggests that assessing neovascularization in axillary lymph node metastases may provide clinically useful information regarding survival in patients with primary 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.

Vascular permeability factor/vascular endothelial growth factor and vascular stroma formation in neoplasia. Insights from in situ hybridization studies.

The formation of vascular stroma plays an important role in the pathophysiology of malignancy. We describe the use of in situ hybridization in our laboratory as a tool to study the role of vascular permeability factor/vascular endothelial growth factor in the angiogenesis associated with malignancy.

Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in patients with ductal carcinoma in situ of the breast.

BACKGROUND: Prior studies have indicated that ductal carcinoma in situ (DCIS) lesions are capable of inducing a vascular stroma. However, the mechanisms responsible for angiogenesis in DCIS currently are not defined. The goal of this study was to determine the relationship between the expression of the angiogenic cytokine vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), and angiogenesis in patients with DCIS. METHODS: Forty-six breast biopsies with DCIS were characterized with regard to histologic features on hematoxylin and eosin stained sections, and microvessel density and distribution using sections immunostained for factor VIII-related antigen. In addition, in situ hybridization was performed on formalin fixed, paraffin embedded sections using 35S labeled riboprobes specific for VPF/VEGF. RESULTS: VPF/VEGF expression by tumor cells in DCIS was greater than that observed in adjacent benign ductal or lobular epithelial cells in 96% of the evaluable cases. Moreover, the degree of VPF/VEGF mRNA expression was significantly associated with the degree of angiogenesis in these lesions. Among 22 cases with strong VPF/VEGF mRNA expression, the median microvessel count was 100 +/- 30.6 vessels/field. In contrast, among 24 cases with low level VPF/VEGF mRNA expression, the median microvessel count was 71 +/- 48.6 vessels/field (P = 0.04). In addition, high grade DCIS lesions more commonly were associated with strong VPF/VEGF mRNA expression than low grade lesions, but the results were not statistically significant. CONCLUSIONS: These findings suggest that VPF/VEGF is an important angiogenic factor in patients with DCIS.

The relationship between shaved margin and inked margin status in breast excision specimens.

BACKGROUND: The presence of tumor at the inked margins (IMs) of breast specimens is associated with an increased risk of local recurrence after breast-conserving therapy for invasive breast carcinoma and ductal carcinoma in situ (DCIS). Given the importance of margin status, some have advocated the use of shaved margins (SMs) as a means of conducting a more complete examination of the specimen margins than could be done with sections taken perpendicular to the IMs. However, it is not known whether these two methods of margin assessment provide comparable information. METHODS: To address this issue, the authors studied 22 consecutive breast reexcision specimens (10 DCIS, 6 infiltrating ductal carcinomas, and 6 infiltrating lobular carcinomas) in which the specimen surfaces were inked, the margins were shaved, and tumor was present in at least one of the SM sections. A total of 199 SMs were examined. The SMs were originally embedded in a way that permitted histologic sections to be cut opposite the inked surface. Sections of SM stained with hematoxylin and eosin (H & E) were reviewed and scored for the presence and extent (number of low-power fields) of cancer. The remaining tissue from the SM was then removed from the blocks, cut perpendicular to the IM, and reembedded to permit visualization of tumor in relation to the IM. Sections were then cut from two different levels of each reembedded block and stained with H & E. An SM was considered positive if tumor was present anywhere on the section. An IM was considered positive when tumor extended to the inked surface. RESULTS: Although all 22 excisions had at least 1 positive SM, tumor was present at an IM in only 12 specimens (55%). Among 69 positive SMs, the corresponding IM was positive in only 42 (61%). The likelihood of a positive IM increased with the number of low-power fields of involvement by invasive carcinoma or DCIS on the SM, as follows: 19% with 1 low power-field, 67% with 2 low-power fields, and 97% with > or = 3 low-power fields (all P < 0.02). When the SM was negative, the corresponding IM was negative in 98% of cases. CONCLUSIONS: Many patients with positive SMs do not have positive IMs. A positive SM more reliably predicts a positive IM when tumor involves > or = 3 low-power fields of the SM. The authors conclude that the clinical implications of a positive SM may not be the same as those of a positive IM. Clinical outcome studies are needed to define further the implications of positive SMs. [See editorial counterpoint on pages 1453-8 and reply to counterpoint on pages 1459-60, this issue.]

Vascular permeability factor/vascular endothelial growth factor and its receptors in oral and laryngeal squamous cell carcinoma and dysplasia.

Increased microvessel density has been described in squamous cell carcinoma of the head and neck and is related to patient prognosis. The factors responsible for the angiogenesis have not been identified. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional angiogenic cytokine expressed at high levels in many tumors. We examined 16 cases of squamous cell carcinoma, 10 cases of high-grade squamous dysplasia, and 19 cases of normal, reactive, or mildly dysplastic squamous mucosa by in situ hybridization for expression of VPF/VEGF and VPF/VEGF receptor mRNA. Strong expression of VPF/VEGF mRNA was seen in 12 of 16 squamous cell carcinomas and in 5 of 10 high-grade squamous dysplasias. In contrast, no cases of normal, reactive, or mild dysplastic squamous epithelium showed strong expression of VPF/VEGF mRNA. Furthermore, strong expression of VPF/VEGF receptor mRNA was seen in 5 of 9 cases of squamous cell carcinoma and 3 of 6 cases of high-grade squamous dysplasia but in only 2 of 14 cases of normal, reactive, or mild dysplastic squamous epithelium. Thus, expression of VPF/VEGF and its receptors is markedly increased in high-grade squamous dysplasia and invasive squamous cell carcinoma of the oral cavity and larynx and may play an important role in the angiogenesis associated with these lesions.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in endometrial carcinoma.

BACKGROUND: Solid tumors, including endometrial carcinomas, must induce a vascular stroma to grow beyond a minimal size. The mechanisms responsible for angiogenesis in endometrial carcinoma, however, are not well defined. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a multifunctional cytokine that is an important regulator of tumor angiogenesis. We evaluated VPF/VEGF mRNA and protein expression, as well as VPF/VEGF receptor mRNA expression, in endometrial carcinoma. METHODS: Fourteen examples of endometrial carcinoma were evaluated by in situ hybridization; in 7 cases, benign atrophic endometrium from the same patient was also examined. Histologic sections were subjected to in situ hybridization using 35S-labeled riboprobes specific for VPF/VEGF and, in a subset of cases, riboprobes specific for the VPF/VEGF receptors flt-1 and KDR. In addition, ten examples of endometrial carcinoma were evaluated for VPE/VEGF protein expression by immunohistochemistry. RESULTS: All 14 examples of endometrial carcinoma studied by in situ hybridization exhibited focal strong VPF/VEGF mRNA expression by tumor cells. In addition, the endothelial cells of surrounding microvessels strongly expressed flt-1 and KDR mRNAs in all ten cases examined. In contrast, no strong expression of VPF/VEGF, flt-1, or KDR mRNA was observed in the seven examples of benign atrophic endometrium studied. All ten cases of endometrial carcinoma studied by immunohistochemistry exhibited strong VPF/VEGF protein expression by tumor cells. CONCLUSIONS: These observations suggest that VPF/VEGF is an important angiogenic factor in endometrial carcinoma.

Vascular permeability factor (vascular endothelial growth factor) expression and angiogenesis in cervical neoplasia.

BACKGROUND: Angiogenesis is a critical factor in the progression of solid tumors, including cervical cancers. The mechanisms responsible for angiogenesis in cervical neoplasia, however, are not well defined. PURPOSE: Our goal was to determine the relationship between angiogenesis and the expression of the angiogenic cytokine vascular permeability factor (VPF), also known as vascular endothelial growth factor, and its receptors in cervical neoplasia. METHODS: Sixty-six cervical biopsy specimens were evaluated; among these, 16 samples were designated as benign, 17 as low-grade squamous intraepithelial lesions, 18 as high-grade squamous intraepithelial lesions, and 15 as invasive squamous cell carcinomas. Histologic sections immunostained for factor VIII-related antigen were evaluated quantitatively for microvessel density and for the presence of epithelial-stromal vascular cuffing. Sections were also evaluated for VPF messenger RNA (mRNA) expression by in situ hybridization. RESULTS: VPF mRNA expression, epithelial-stromal vascular cuffing, and microvessel density counts were significantly increased in invasive carcinoma and in high-grade intraepithelial lesions as compared with low-grade intraepithelial lesions and benign squamous epithelium. Vascular cuffing and increased microvessel density counts were also significantly associated with increased VPF mRNA expression. CONCLUSIONS: These observations suggest that VPF is an important angiogenic factor in cervical neoplasia.

Local recurrence after conservative surgery and radiation therapy for ductal carcinoma in situ: Possible importance of family history.

PURPOSE: The optimal treatment of ductal carcinoma in situ is controversial. Traditionally, women with this disease have been treated with mastectomy with excellent results, but recently the need for such extensive surgery has been questioned. Long-term data on the use of conservative surgery and radiation therapy for treatment are limited. A retrospective analysis was performed to assess treatment outcome and prognostic factors for patients with ductal carcinoma in situ treated with conservative surgery and radiotherapy. PATIENTS AND METHODS: From 1976 to 1990, 76 women with ductal carcinoma in situ were treated with conservative surgery followed by radiation therapy. The median age at diagnosis was 48 years. Seventeen patients had a positive family history of breast cancer in a first-degree (n=8) or second-degree (n=9) relative. Median follow-up interval was 74 months for the 71 survivors. In 54 patients, the carcinoma was detected by mammography alone; in 13 patients, by mammography and physical examination; and in 4 patients, by physical examination with a normal mammogram; and in 5 patients, by physical examination alone without mammography. Fifty patients had re-excision after initial biopsy. Final margins were positive in 11, close in 11, negative in 34, and unknown in 20. The median volume of excised tissue was 60 cm3. The axilla was surgically staged in 30 patients (39%) and all were negative. The whole breast was irradiated to a dose of 45 to 50 Gy in all patients. Seventy-two patients also received a boost to the primary site. The median total radiation dose to the primary site was 61 Gy (range, 46 to 71). RESULTS: Seven patients had a recurrence in the treated breast at 16, 18, 41, 63, 72, 83, and 104 months after treatment. The 5- and 10-year actuarial rates of local recurrence were 4% and 15%, respectively. Six of seven recurrences occurred in the vicinity of the original lesion. Four local recurrences were invasive, and three were ductal carcinoma in situ. Two patients developed a contralateral invasive carcinoma. The 5- and 10-year cause-specific survival rates were 100% and 96%, respectively. The 10-year actuarial rate of local recurrence was 25% in the group with a total excision volume less than 60 cm3, as compared with 0% in those with 60 cm3 or more excised (P=0.04). In patients with a positive family history, the 10-year actuarial rate of local recurrence was 37%, as compared with 9% in patients with a negative family history (P=0.008). Of the 17 patients with a positive family history, four developed either an ipsilateral or contralateral invasive breast cancer, whereas 1 of the 58 patients without a family history developed a subsequent invasive breast cancer (P=0.008). CONCLUSION: These results suggest that patients with ductal carcinoma in situ treated with conservative surgery and radiotherapy (including a boost to the primary site) appear to benefit from wide, rather than limited, resection. These results also suggest that family history may be an important prognostic factor for progression of disease.

Expression of vascular permeability factor (vascular endothelial growth factor) and its receptors in breast cancer.

Solid tumors must induce a vascular stroma to grow beyond a minimal size, and the intensity of the angiogenic response has been correlated with prognosis in breast cancer patients. Vascular permeability factor (VPF), also known as vascular endothelial growth factor (VEGF), is a secreted protein that has been implicated in tumor-associated angiogenesis. Vascular permeability factor directly stimulates endothelial cell growth and also increases microvascular permeability, leading to the extravasation of plasma proteins, which alter the extracellular matrix in a manner that promotes angiogenesis. To determine whether VPF has a role in breast cancer, we used in situ hybridization to study VPF mRNA expression in normal breast tissue (13 specimens), comedo-type ductal carcinoma in situ (DCIS) (four specimens), infiltrating ductal carcinoma (12 specimens), infiltrating lobular carcinoma (two specimens), metastatic ductal carcinoma (three specimens) and metastatic lobular carcinoma (one specimen). Vascular permeability factor mRNA was expressed at a low level by normal duct epithelium but was expressed at high levels in tumor cells in all cases of comedo-type DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, VPF mRNA was not expressed at high levels in infiltrating lobular carcinoma. We also used in situ hybridization to study the expression of two recently described endothelial cell surface VPF receptors, flt-1 and kdr. Vascular permeability factor receptor mRNA was strongly expressed in endothelial cells of small vessels adjacent to malignant tumor cells in DCIS, infiltrating ductal carcinoma, and metastatic ductal carcinoma. In contrast, no definite labeling for receptor mRNA was detected in infiltrating lobular carcinoma or nonmalignant breast tissue. The intense expression of VPF mRNA by breast carcinoma cells and of VPF receptor mRNA by endothelial cells of adjacent small blood vessels provides strong evidence linking VPF expression to the angiogenesis associated with comedo-type DCIS, infiltrating ductal, and metastatic ductal breast carcinoma.

Microvessel density and distribution in ductal carcinoma in situ of the breast.

BACKGROUND: Prior studies have suggested that microvessel density is an important prognostic factor in invasive breast cancer. However, the extent and distribution of microvessels in association with ductal carcinoma in situ (DCIS) have not been well defined. PURPOSE: Our goal was to determine the density and distribution of stromal microvessels in DCIS and to investigate the relationships among microvessel density, histopathologic features, HER2/neu oncogene expression, and tumor proliferation rate. METHODS: Of 61 consecutive cases of DCIS identified from hospital pathology reports, 55 cases were evaluated. Breast biopsy specimens had been preserved in paraffin blocks for each DCIS case. Histologic sections of formalin-fixed, paraffin-embedded tissue were stained with hematoxylin-eosin and immunostained for factor VIII-related antigen, the HER2/neu oncoprotein, and the proliferative-associated antigen detected by the Ki-S1 antibody. Factor VIII-stained sections from each case were independently examined by two pathologists and overall tumor-associated stromal microvessel density was scored semiquantitatively on a 1+ to 3+ scale by each observer. Quantitative microvessel counts of DCIS-associated stromal microvessel density were performed. The presence or absence of a cuff of microvessels in immediate apposition to the basement membrane of involved spaces was also evaluated. RESULTS: A variable number of microvessels were found to be present in a diffuse pattern surrounding spaces involved with DCIS. Semiquantitative microvessel scores were 2+ in the majority of cases (53%); 22% of cases were 1+, and 25% were 3+. Quantitative microvessel counts ranged from 17 to 80 vessels per 100x field (0.45 mm2), with a mean +/- SD of 42.9 +/- 16.6. Comedo-type lesions were significantly (P = .004) more often associated with 3+ microvessel density than non-comedo-type lesions by semiquantitative assessment. As determined by both semiquantitative and quantitative analysis, respectively, the presence of prominent microvessel density was significantly associated with marked stromal desmoplasia (P = .05 and P = .04), HER2/neu expression (P = .03 and P = .0002), and high Ki-S1 proliferation index (P = .05 and P = .01). Vascular cuffing around involved spaces was identified in 21 of the 55 cases (38%) and was not significantly associated with histologic features, HER2/neu expression, or Ki-S1 proliferation index. CONCLUSIONS: DCIS of the breast is characterized by two patterns of stromal microvessels. The first pattern is a diffuse increase in stromal microvessels surrounding involved spaces. This pattern is particularly prominent in comedo-type lesions with marked stromal desmoplasia. The second pattern is microvessel cuffing of involved spaces that is present in only a minority of cases and appears unrelated to histologic features evaluated, including DCIS subtype.