Prognosis in human melanoma: PAR-1 expression is superior to other coagulation components and VEGF

Aims: Two hundred and four accessible cases of malignant melanoma from the Grampian region of Scotland, collected over a period of 4 years, with minimum follow‐up of 2 years, were studied for coagulation factors and vascular endothelial growth factor (VEGF) expression as potential prognostic markers. The aim was to allow comparison with previous work using microvessel density on the same cases. Methods and results: Immunohistochemistry for VEGF, tissue factor (TF), fibrin and protease‐activated thrombin receptor (PAR)‐1 in 204 cases of melanoma was performed, and intensity of expression scored. Chalkley microvessel counts (MVD) were obtained for the tumour edge. TF expression and presence of fibrin correlated well with Breslow thickness and ulceration, reaching statistical significance, but surprisingly not for metastatic recurrence. Fibrin was variably present in over half the cases, located at the invasive edge, ulcerated surface and between tumour cell surfaces. In a few cases fibrin was within tumour cells, typically co‐located with melanin and confirmed by electron microscopy. In contrast, immunohistochemistry for PAR‐1 produced statistically significant results, correlating expression with Breslow thickness (P ≤ 0.001), ulceration (P = 0.001) and recurrence (P ≤ 0.005). Intensity of reactivity of VEGF correlated significantly with Breslow thickness, Clark level, ulceration and MVD, but not for metastatic recurrence. Conclusions: It appears paradoxical that VEGF expression is not more predictive of recurrence, but even low expression may be sufficient for tumour angiogenesis and other factors must govern tumour aggression. Antagonism of VEGF may still prove a successful adjunct in future therapeutic trials. Both MVD and PAR‐1 can be used as adjuncts to Breslow thickness and ulceration as prognostic indicators for melanoma, as they appear to give independent information for all thicknesses. PAR‐1 expression is the best antibody marker of recurrence risk from those studied. It remains to be seen whether this methodology can predict response to novel antiangiogenic therapies currently entering trial.     

Tumor angiogenesis as a prognostic factor in thick cutaneous malignant melanoma

The prognostic value of the extent of neovascularization in cutaneous melanoma is a highly controversial issue. The aim of the current study was to evaluate whether the morphometric analysis of tumor vascularity may be helpful in predicting the clinical outcome of patients with thick cutaneous melanomas. A series of 15 patients with melanoma (>3 mm in thickness) who did not experience disease progression after long-term follow-up (10 years) and 30 matched controls who underwent recurrence and/or metastases were selected for the study. Microvessels were immunohistochemically stained with anti-CD31 antibody. Several parameters, including vessel number, vascular density, vessel area, equivalent circle diameter, perimeter, shape factor, compactness, and the number of vascular ramifications per 100 vessel sections, were quantitatively assessed by a computer-aided semi-automatic image analysis system. Mean vessel area was 341.69 microm2 in cases without progression and 512.55 microm2 in the progressed melanomas (P=0.008, Mann-Whitney U test). The mean equivalent circle diameter was 18.95 microm in non-progressed melanomas and 22.57 microm in progressed melanomas (P=0.009). The mean number of ramifications was 0.8 in cases without progression and 1.9 in the controls (P=0.03). Microvessel count and vascular density were higher in progressed cases (17.37 vs. 11.73 and 28.94/mm2 vs. 19.55/mm2, respectively), but the difference did not reach statistical significance (P=0.06). Our results suggest that neovascularization is a critical event in the progression of thick melanoma. Its prognostic significance is better assessed by quantification of vessel area, equivalent circle diameter, and microvessel branching, whereas microvessel count and vascular density do not provide significant prognostic information.     

Microvessel density compared with the Chalkley count in a prognostic study of angiogenesis in breast cancer patients

AIMS: Evaluation of angiogenesis by intratumoral vessel profiles can be performed by different methods. The aim of this study was to investigate the prognostic value of estimates obtained by the intratumoral microvessel density (MVD) method and then to compare with corresponding estimates obtained by the Chalkley method. METHODS AND RESULTS: A total of 330 patients treated for primary, unilateral, invasive breast carcinoma were included. The median follow-up time was 14 years and 4 months. The microvessels were immunohistochemically stained by antibodies to CD34. MVD was not significantly correlated with any clinicopathological variables. By univariate analysis, MVD showed no prognostic value with regard to recurrence-free survival (RFS) or overall survival (OS), while the Chalkley count had significant prognostic value (P < 0.0001; RFS and OS). In the Cox multivariate analysis, MVD had no prognostic impact [median HR [confidence interval (CI)] was 0.93 [0.66, 1.32] for RFS; and HR [CI] was 0.86 [0.62, 1.19] for OS], while the Chalkley count [median HR (CI) was 2.12 (1.48, 3.06) for RFS; and HR (CI) was 1.71 (1.23, 2.37) for OS] provided independent prognostic value when adjusted for age, menopausal status, axillary lymph node status, tumour size, histological grade, adjuvant systemic treatment and radiation therapy. In comparing the results obtained by MVD in our study with those from other published studies we find good agreement. CONCLUSIONS: The Chalkley count technique seems to be preferable for estimating angiogenesis with regard to the prognostic stratification of breast cancer patients, based on its strong prognostic impact, and acceptable reproducibility.     

Increased microvessel density in malignant and borderline mammary phyllodes tumours

AIMS: Tumour vascularity is considered a prognostic indicator in breast carcinoma, but its utility in mammary phyllodes tumour has not been explored. The authors report the correlation between intratumoral microvessel density and the histological grade of phyllodes tumour. METHODS AND RESULTS: Forty cases of phyllodes tumour were reviewed for stromal cellularity, overgrowth, cytological pleomorphism, mitotic count and margin pattern. Using established criteria, these were diagnosed as benign (n=28), borderline (n=10) and malignant (n=2). Microvessel density was counted on CD31-stained slides as the number of vessels per high power field. For benign phyllodes tumour, the range was 7-26.2 (mean 13.1); for borderline phyllodes tumour the range was 17.2-32.5 (mean 22.4); for malignant phyllodes tumour the range was 25.9-33.3 (mean 29.6). The difference between the benign and borderline groups was significant (P < 0.0001) but that between the borderline and malignant groups was not, due to the small number of malignant cases. CONCLUSIONS: There is a significant difference in stromal microvessel density between benign and borderline phyllodes tumour. Although the small number of cases of malignant phyllodes tumour limits further interpretation, we believe that microvessel density can be used as an additional objective histological parameter in the evaluation of phyllodes tumour.     

Prognostic Impact of Angiogenesis in Nonmuscle Invasive Bladder Cancer as Defined by Microvessel Density after Immunohistochemical Staining for CD34

Bladder cancer is the second most common malignancy of the urogenital region. The majority of bladder cancer deaths occur as a consequence of metastatic disease. Microvessel density (MVD), a surrogate marker for angiogenesis, has been shown to be predictive of progression and poor prognosis. The aim of this study was to evaluate the predictive value and prognostic significance of angiogenesis in human non muscle invasive bladder cancer (NMIBC) treated by BCG immunotherapy. The frozen sections of 28 non muscle invasive bladder cancer specimens were stained with CD34 antibody to label the vascular endothelium using the standard streptavidin-biotin immunoperoxidase method. Angiogenic activity was measured using microvessel count determined by the expression of vascular markers CD34.The prognostic significance of tumor stage, grade, loci number, tumor size, age and CD34 expression in determining the risk for recurrence was studied with both univariate and multivariate methods of analysis. According to univariate analysis of the prognostic significance for tumor stage, grade, tumor size, loci number, age and CD34 expression, in patients with NMIBC, the pT1 stage and high grade seem to be associated in a statistically significant manner with higher risk for recurrence (P=0.004, P=0.004, respectively). In the other hand, multivariate Cox regression’s analysis showed that microvessel density and multiplicity were independent predictor of recurrence after BCG immunotherapy (p=0.016, p=0.032, respectively). This study provides strong evidence that CD34 MVD is associated with recurrence after BCG immunotherapy. Independent studies, however, will be required on larger cohort to validate these findings.     

Prognostic Impact of Angiogenesis in Nonmuscle Invasive Bladder Cancer as Defined by Microvessel Density after Immunohistochemical Staining for CD34

Bladder cancer is the second most common malignancy of the urogenital region. The majority of bladder cancer deaths occur as a consequence of metastatic disease. Microvessel density (MVD), a surrogate marker for angiogenesis, has been shown to be predictive of progression and poor prognosis. The aim of this study was to evaluate the predictive value and prognostic significance of angiogenesis in human non muscle invasive bladder cancer (NMIBC) treated by BCG immunotherapy. The frozen sections of 28 non muscle invasive bladder cancer specimens were stained with CD34 antibody to label the vascular endothelium using the standard streptavidin-biotin immunoperoxidase method. Angiogenic activity was measured using microvessel count determined by the expression of vascular markers CD34.The prognostic significance of tumor stage, grade, loci number, tumor size, age and CD34 expression in determining the risk for recurrence was studied with both univariate and multivariate methods of analysis. According to univariate analysis of the prognostic significance for tumor stage, grade, tumor size, loci number, age and CD34 expression, in patients with NMIBC, the pT1 stage and high grade seem to be associated in a statistically significant manner with higher risk for recurrence (P=0.004, P=0.004, respectively). In the other hand, multivariate Cox regression's analysis showed that microvessel density and multiplicity were independent predictor of recurrence after BCG immunotherapy (p=0.016, p=0.032, respectively). This study provides strong evidence that CD34 MVD is associated with recurrence after BCG immunotherapy. Independent studies, however, will be required on larger cohort to validate these findings.     

The correlation of angiogenesis with metastasis in primary cutaneous melanoma: a comparative analysis of microvessel density, expression of vascular endothelial growth factor and basic fibroblastic growth factor

AIM: To establish whether there is a correlation between angiogenesis and metastasis in primary cutaneous melanoma (PCMM). METHODS: We studied the microvessel density and the expression of vascular endothelial growth factor (VEGF) and basic fibroblastic growth factor (bFGF) in 22 cases of PCMM with metastasis at presentation (metastatic group) and 28 cases of PCMM without metastasis for 24 months or more (non-metastatic group). Microvessels were stained with CD31/PECAM-1 antibody and counted. We assessed the proportion of VEGF expression in tumour cells, lymphocytes infiltrating the tumour (TIL) and lymphocytes at the periphery of the tumour, as well as the proportion of bFGF expression in tumour cell cytoplasms, nuclei and intra- and peritumoral vessels. RESULTS: An increased microvessel density was detected in the metastatic group (15-33 [24.09 +/- 5.55] versus 2-24 [12.96 +/- 6.02]). Moreover, enhanced expression of VEGF in tumour cells and peritumoral lymphocytes (Chi-square p = 0.038 and p = 0.018) and bFGF in peritumoral vessels (chi(2) p = 0.013) correlated with the simultaneous presence of melanoma metastasis in PCMM. Furthermore, microvessel density was correlated with the expression of bFGF in peritumoral vessels (rs = 0.53, p = 0.049) and VEGF in tumour cells (rs = 0.37, p = 0.019). CONCLUSION: Microvessel density as well as the expression of both VEGF and bFGF might be informative concerning the progression of melanoma.     

Microvessel quantitation and prognosis in invasive breast carcinoma.

The prognostic significance of microvessel quantitation in invasive breast carcinoma was analyzed in a study group that comprised 88 patients with axillary node-negative carcinoma and 32 patients with axillary node-positive carcinoma who had a minimum follow-up period of 9 years. Microvessels were identified by immunohistochemistry using antibodies to endothelial markers, including factor VIII-related antigen and blood group isoantigens (ABH). Factor VIII-related antigen staining provided more consistent results for microvessel quantitation than did staining for ABH isoantigens. The three most vascular areas within a tumor were selected, and the microvessels within a x200 microscopic field of each area were counted by two investigators simultaneously. Node-positive carcinomas demonstrated significantly higher microvessel counts than did node-negative carcinomas (mean +/- SD, 99 +/- 42 and 73 +/- 22, respectively; P less than .001). In node-negative carcinomas, tumors from patients who experienced distant recurrence had higher microvessel counts than did tumors from patients who were disease-free (84 +/- 19 and 70 +/- 22; P = .01). Similarly, in patients with node-positive carcinoma, microvessel counts were considerably higher in tumors from patients who experienced distant recurrence than in patients who did not, although the difference did not reach statistical significance (113 +/- 44 and 93 +/- 34, respectively). Among patients with node-negative carcinoma, those with a microvessel count of less than 84 had a recurrence rate of 20% compared with 57% in patients with counts greater than 84 (P = .003). Microvessel counts were independent of histologic parameters, ploidy status, and S-phase fraction but correlated with peritumoral vascular invasion. Both microvessel counts and vascular invasion were independent prognostic parameters by multivariate analysis. High vessel counts may represent increased tumor angiogenesis and are correlated with tumor aggressiveness. Microvessel quantitation may be an additional prognostic factor that, when used in conjunction with more established parameters, can help in appropriate patient management.     

CXCR3 chemokine receptor immunoreactivity in primary cutaneous malignant melanoma: correlation with clinicopathological prognostic factors

BACKGROUND: A role for CXCR3, the receptor for chemokines Mig, IP-10 and interferon-inducible T cell alpha-chemoattractant, in tumour cell migration during melanoma progression has been proposed. AIMS: To analyse CXCR3 expression in primary cutaneous malignant melanomas and its comparison with clinicopathological and prognostic factors. METHODS: A retrospective immunohistochemical study was carried out on formalin-fixed paraffin-wax-embedded sections from 82 patients with primary invasive cutaneous melanomas, with a monoclonal antibody to CXCR3 (clone 49801.111; R&D Systems). Immunoreactivity was semiquantitatively evaluated: labelling intensity (0, absent; 1, weak; 2, moderate; 3, strong) multiplied by the percentage of cells in each of the four intensity categories. A positive staining was considered when the score was >100. Melanomas were categorised by age, sex, primary site, tumour thickness, growth phase, ulceration, lymphocytic infiltration, recurrence, lymph node and distant metastasis, and survival. Univariate and multivariate statistical analyses were carried out. RESULTS: Of the 82 patients, a positive CXCR3 staining was found in 26 (31.7%) patients, whereas 56 (68.3%) were negative. In univariate analysis, a significant association of CXCR3-positive tumour cell immunostaining with tumour thickness >1 mm (p = 0.003), absence of lymphocytic infiltration (p = 0.04) and the presence of distant metastasis (p = 0.048) was found. Multivariate analysis found tumour thickness as the only independent factor with considerable association with distant metastases. CONCLUSIONS: Our findings of a positive correlation of CXCR3 tumour cell immunoreactivity in human primary cutaneous melanoma with tumour thickness >1 mm and absence of intratumoral lymphocytic infiltration support the biological implication of CXCR3 in the tumour progression of cutaneous malignant melanoma.     

Quantitation and prognostic value of breast cancer angiogenesis: Comparison of microvessel density,Chalkley count,and computer image analysis

In some studies of breast cancer, quantitation of immunohistochemically highlighted microvessel ‘hot spots’ has been shown to be a powerful prognostic tool. However, the antibody used, the number and size of the ‘hot spots’ assessed, and the stratification of patients into high and low vascular groups vary between studies. Furthermore, little is known about the relationship between microvessel density and other vascular parameters. These uncertainties and the laborious nature of the technique make it unsuitable for diagnostic practice. Both manual and computerized image analysis techniques were used in this study to examine the relationship between microvessel density and the vascular parameters in different sized microscopic fields in a pilot series of 30 invasive breast carcinomas. Automated pixel analysis of immunohistochemical staining, Chalkley point counting, and observer subjective vascular grading were also assessed as more rapid methods of measuring tumour vascularity. A Chalkley count was also performed on a further 211 invasive breast carcinomas. Significant correlations were observed between manual microvessel density and luminal perimeter (r=0·6, P=0·0004), luminal area (r=0·56, P=0·002), and microvessel number (r=0·57, P=0·0009) by computerized analysis. There were also significant correlations between the microscopic hot spots of 0·155 mm² and 0.848 mm² for microvessel number (r=0·81, P>0·00005), luminal perimeter (r=0·78, P<0·00005), and luminal area (r=0·65, P=0·0001). In addition, a significant correlation was observed between microvessel density and both subjective vascular grade (P=0·002) and Chalkley count (P=0·0001). A significant reduction in overall survival was observed between patients stratified by Chalkley count in both a univariate (P=0·02) and a multivariate (P=0·05) analysis in the 211 invasive breast carcinomas. These findings show that Chalkley counting is a rapid method of quantifying tumour angiogenesis and gives independent prognostic information which might be useful in diagnostic practice.     

Expression of glucose-regulated stress protein GRP78 is related to progression of melanoma

Aims:  Glucose-regulated protein 78 (GRP78) is a protein translated in response to endoplasmic reticulum (ER) stress that has been implicated in the pathogenesis and resistance to therapy of a variety of cancers. The aim of this study was to investigate its expression and role in the development and progression of human melanoma.
Methods and results:  The immunohistochemical expression of GRP78 in naevi, primary melanoma and melanoma metastases from 171 patients was correlated with clinicopathological factors and patient survival. The GRP78 immunoreactivity score (IRS) was 0.2 in compound naevi, 0.65 in dysplastic naevi, 4.65 in naevi adjacent to primary melanoma, 2.4 in melanoma in situ , 11.2 in thin (≤1.0 mm) and 18.1 in thick (>1.0 mm) primary melanoma. It was 18 and 17.3 in subcutaneous and lymph node metastases, respectively ( P  < 0.0001). GRP78 expression was positively correlated with increasing tumour thickness ( P  = 0.001) and with increasing dermal tumour mitotic index ( P  = 0.0004). Disease-free survival (χ² = 8.0703, P  = 0.0045) and overall survival (χ² = 6.2633, P  = 0.0123) in melanoma patients with IRS >25 were significantly lower than in melanoma patients with IRS <25.
Conclusions:  GRP78 expression appears to correlate with known correlates of melanoma progression and survival and requires further evaluation as a prognostic biomarker in melanoma.     

Tumoural vascularity as a prognostic factor in cancer patients: the evidence continues to grow

Accurate prognostic indicators would help in identifying patients at high risk for recurrence and death. Recent and previous studies indicate that intratumoural microvessel density (iMVD) of invasive breast carcinoma (a measure of tumour angiogenesis) is associated with aggressive tumour growth; iMVD may thus be a valuable prognostic indicator. Moreover, the bulk of accumulating data indicates that microvessel density in the area of most intense neovascularization in invasive breast carcinoma is an independent, significant and accurate prognostic indicator in predicting poorer survival. Such an indicator would be useful in the selection of high-risk patients with breast carcinoma for systemic adjuvant therapy. © 1998 John Wiley & Sons, Ltd.     

Prognostic relevance of P-cadherin expression in melanocytic skin tumours analysed by high-throughput tissue microarrays

AIM: To investigate whether protein expression or cellular localisation of P-cadherin is associated with clinicopathological characteristics in benign and malignant melanocytic skin tumours. EXPERIMENTAL DESIGN: P-cadherin expression and the Ki-67 labelling index were analysed immunohistochemically by using tissue microarrays (TMAs). Membranous and cytoplasmic expression was scored semiquantitatively (0 to 2+). RESULTS: P-cadherin protein expression of any intensity (1+ to 2+) was detected in the membrane in 41.5% (132/318) and in the cytoplasm in 64.2% (204/318) of patients. In general, P-cadherin expression was significantly reduced in malignant melanomas (p<0.001) and melanoma metastases (p<0.001), compared with benign nevi. Additionally, loss of membranous P-cadherin was associated with Clark level (p = 0.011) and tumour thickness (p<0.001). Interestingly, a significantly lower P-cadherin expression was shown by dermal nevi than by compound and junctional nevi (p = 0.005; p = 0.025). In primary melanomas, a Ki-67 labelling index <5% was not associated with P-cadherin protein expression, suggesting that loss of P-cadherin expression was not associated with proliferation. None of the other clinical and histological factors analysed was significantly related to P-cadherin expression. Low cytoplasmic P-cadherin expression was associated with tumour recurrence (p = 0.03) in all the patients who were analysed. After testing various multivariate Cox regression models, loss of cytoplasmic P-cadherin expression remained a highly significant adverse risk factor for tumour recurrence in patients with tumours <2 mm. CONCLUSIONS: Loss of cytoplasmic P-cadherin expression is common in advanced melanomas and can be a prognostic marker of progression in patients with melanoma, most useful in patients with primary tumours <2 mm in thickness.     

Objective assessment of blood and lymphatic vessel invasion and association with macrophage infiltration in cutaneous melanoma

The aims of this study were to investigate the role of vascular invasion (blood and lymphatic), vessel density and the presence of tumour-associated macrophages as prognostic markers in 202 cutaneous melanoma patients. Sections of primary melanoma were stained with lymphatic-specific antibody D2-40 to assess lymphatic vessel invasion and density in intratumoural and peritumoural areas; an antibody against endothelial marker CD34 was used to determine blood vessel invasion and density, and an antibody against CD68 was used to determine macrophage counts. Immunohistochemically determined vascular invasion (combined blood and lymphatic) was compared with that determined using haematoxylin and eosin (H&E) staining. The use of immunohistochemistry increased detection of vascular invasion from 8-30% of patients, and histological exam of H&E-stained tissue was associated with a false positive rate of 64%. Lymphatic vessel invasion occurred at a much higher frequency than blood vessel invasion (27 and 4% of patients, respectively). Although immunohistochemically detected vessel invasion was significantly associated with histological markers of adverse prognosis, such as increased Breslow thickness, ulceration and mitotic rate (all P<0.001), no associations with relapse-free or overall survival were observed. High macrophage counts were significantly associated with markers of aggressive disease, such as Breslow thickness, ulceration and mitotic rate (P<0.001, P<0.001, P=0.005, respectively), and lymphatic vessel invasion and high microvessel density (P=0.002 and P=0.003, respectively). These results suggest that vascular invasion is more accurately detected using immunohistochemistry and occurs predominantly via lymphatic vessels. The association of vessel characteristics with histological characteristics of the primary melanoma provides evidence for their biological importance in melanoma, but that they were not associated with clinical outcome attests to the value of existing histological prognostic biomarkers. We note that a high macrophage count may be associated with neovascularisation and primary tumour growth, and may also promote invasion through lymphatic vessels.     

Tenascin-C in primary malignant melanoma of the skin

AIMS: To investigate the expression and the prognostic role of glycoprotein Tenascin-C (Tn-C) in primary melanoma of the skin. METHODS AND RESULTS: The immunohistochemical expression of Tn-C was studied in 98 primary melanomas and related to inflammation, invasion, and patient outcome. Patients were followed up for disease recurrence for 0.04-7.4 years (median 3.9) and for survival for 0.5 to 12.1 years (median 9.3). The expression of Tn-C was evaluated for each tumour invasion border; the stromal and intracytoplasmic Tn-C of the melanoma islets were also recorded. Tn-C is widely expressed in primary melanoma samples, the staining pattern varying from focal to diffuse in different parts of the tumour. No correlation existed between intensity of Tn-C staining and inflammation. No stromal Tn-C was detected at the upper dermal lateral border in 12 patients, nor at the deep, dermal or subcutaneous border in 14 patients. These patients showed better disease-free survival (DFS) than did those cases with focal or diffuse staining (P = 0.06, P = 0.05). Also, absence of intracytoplasmic Tn-C was a beneficial prognostic factor for DFS (P = 0.04). In multivariate analysis, tumour ulceration and intracytoplasmic Tn-C expression of melanoma cells were independent adverse prognostic factors for DFS. CONCLUSIONS: In primary melanoma of the skin, absence of Tn-C in the stroma of invasion fronts and within tumour cells seems to be related to a more benign disease behaviour with a lower risk of developing metastases.     

Prognostic significance of angiogenesis by Chalkley counting in node negative cancer of the ampulla of Vater

Angiogenesis is essential for tumor growth and metastasis. Currently, the Chalkley assay with CD34 immunostaining is the proposed standard method for angiogenesis quantification in solid tumor sections. The purpose of this study was to evaluate the expression of CD34 and its prognostic significance using the Chalkley method in node negative carcinoma of the ampulla of Vater. Between January 1997 and December 2006, 56 node negative patients who had curative resection for carcinoma of the ampulla of Vater were retrospectively reviewed. The Chalkley count was expressed as the mean value of the three counts for each tumor and further divided into two groups according to the mean value of the Chalkley count: low < 4 or high ≥ 4. The mean Chalkley count value was 4.0 (± 3.1). In the low Chalkley group, the 1- and 3-yr recurrence rates were 18.3%, 47.6% respectively; in the high Chalkley group, the 1- and 3-yr recurrence rates were 26.5% and 60.6% respectively. Only high Chalkley count had statistical significance as a factor in recurrence of node negative ampulla of Vater carcinoma. Assessment of angiogenesis may have an important role in the prognostic evaluation of node negative cancer of the ampulla of Vater.     

Thymidine phosphorylase expression and stromal vascularity in ductal carcinoma in situ of the breast

AIMS: Periductal angiogenesis in ductal carcinoma in situ is associated with an increased risk of subsequently developing a recurrence. This study aimed to (1) identify the relation between periductal and stromal vascularity and recurrence and (2) determine whether thymidine phosphorylase (TP) is associated with angiogenesis or recurrence in ductal carcinoma in situ (DCIS). METHODS: Twenty cases of DCIS that did not subsequently recur, 20 that developed a subsequent in situ recurrence, and 12 that developed a subsequent invasive recurrence were investigated. Periductal and stromal (hotspot) microvessel density were determined quantitatively using antibodies to CD34 and von Willebrandt factor (vWF). TP expression by DCIS was assessed semiquantitatively using the H score method. RESULTS: Stromal and periductal microvessel density assessed by anti-vWF gave similar mean values, and showed a strong positive correlation. When angiogenesis was assessed with anti-CD34 this association was lost. Not only were the mean values for both types of microvessel density higher than those obtained with anti-vWF, but the periductal microvessel density was significantly greater than the stromal microvessel density. TP expression was associated with stromal microvessel density assessed with anti-vWF, but not with anti-CD34. TP expression was not related to recurrence. No significant difference was identified in TP expression or stromal vascularity in DCIS between cases that recurred as DCIS and those that recurred as invasive carcinoma. CONCLUSIONS: Recurrent in situ or invasive disease after excision of DCIS does not appear to be related to stromal microvessel density or to TP expression by DCIS cells.     

Microvessel area using automated image analysis is reproducible and is associated with prognosis in breast cancer

Microvessel density may be one measure of tumor associated angiogenesis but is methodologically difficult to standardize and reproduce. We used our automated quantitative image analysis system, AQUA, to more objectively assess microvessel area. Cytokeratin and CD31 were used to create tumor and vessel compartments respectively with AQUA. Microvessel area was defined as CD31 compartment area normalized to the tissue spot area (CD31 area/area of entire tissue spot). Consecutive breast cancer whole sections were stained with CD31 to compare pathologist-based microvessel density with AQUA microvessel area. Microvessel areas of 3-fold redundant tissue microarrays of 652 primary breast cancers were also assessed. CD34 and factor VIII-related antigen were also tested. There was nearly linear correlation between pathologist's microvessel density and AQUA microvessel area with regression coefficient R = 0.846. On the redundant arrays, of the 67% evaluable cases, 52% were microvessel area high and 48% low with good reproducibility of scores (Spearman rho 0.551). AQUA microvessel area was associated with larger tumors, node positivity, and estrogen receptor negativity, with 20 year survival at the univariate and multivariate levels (P < .0001 and P = .0121, respectively). CD34 or factor VIII-related antigen were more heterogenous, had poor association with CD31, and did not correlate with outcome. AQUA-based microvessel area was significantly correlated with both standard breast cancer prognostic parameters as well as with clinical outcome. In the future, it may also allow the use of the AQUA-based algorithms to quantify the expression of angiogenic biomarkers to either tumor or microvessel area-specific compartments.     

Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression.

Angiogenesis and lymphangiogenesis are essential for breast cancer progression and are regulated by vascular endothelial growth factors (VEGF). To determine clinical and molecular correlates of these processes, we measured blood and lymphatic vascular microvessel density in 29 invasive carcinomas (22 ductal, six lobular, one papillary), using the vascular marker CD31 and the novel lymphatic marker D2-40. Microvessel density was assessed microscopically and by image cytometry, and was compared with tumor histology, grade, stage, lymph node metastasis, hormone receptors, HER2/neu status, and expression of VEGF, VEGF-C and VEGF-D by immunohistochemistry or quantitative RT-PCR. Strong correlation was observed between visual and image cytometric microvessel density using D2-40 but not CD31 (P=0.016 and 0.1521, respectively). Image cytometric CD31 microvessel density correlated with tumor size, grade, stage and lymph node metastasis (P=0.0001, 0.0107, 0.0035 and 0.0395, respectively). D2-40 microvessel density correlated with tumor stage (P=0.0123 by image cytometry) and lymph node metastasis (P=0.0558 by microscopy). Immunohistochemical VEGF signal in peritumoral blood vessels correlated with image cytometric CD31 and D2-40 microvessel density (P=0.022 and 0.0012, respectively), consistent with the role of VEGF in blood and lymphatic vascular growth. Intratumoral VEGF-C and VEGF-D expression by quantitative RT-PCR correlated with D2-40 (P=0.0291 by image cytometry) but not with CD31 microvessel density, which could suggest a selective role of VEGF-C and VEGF-D in lymphangiogenesis. CD31 and D2-40 microvessel density correlated significantly with several prognostic factors, including lymph node metastasis. Thus, measurements of angiogenesis and lymphangiogenesis may have utility for breast cancer pathology, particularly for estimation of metastatic risk.