Definition of the microvascular pattern of the normal human adult mammary gland

Abstract

The present report provides a detailed description of microvascularization in the normal human mammary gland, and defines two novel morphometric parameters to be used as a reference when angiogenesis in breast carcinoma is evaluated. Microvascularization was analysed by histology, immunohistochemistry and computer-assisted analysis in a set of breast tissue samples taken from nine women, during the pre-ovulatory phase of the menstrual cycle. The two parameters designed for image analysis were: vascular density (VD): [microvessel number/(microvessel area + residual stromal area)] × 10 000; and vascular area ratio (VAR): [microvessel area/(microvessel area + residual stromal area)]. In the lobules VD (mean value ± SE 2.48 ± 0.14) and VAR (0.33 ± 0.02) showed little variability and correlated significantly (P < 0.05). The areas occupied by microvessels, stroma and acini remained constant in all lobules (21.53 ± 1.87%, 42.65 ± 1.35% and 35.14 ± 1.57%, respectively). Microvascularization of the lobules was of a sinusoidal type, with large S-shaped capillaries. In the ducts VD (2.95 ± 0.16) and VAR (0.29 ± 0.03) showed little variability but did not correlate significantly. Microvascularization of the ducts was of a classic type, with capillaries normal in size and shape. The expression of oestrogen (ER) and progesterone (PR) receptors was analysed by immunohistochemistry and compared with the morphometric results. ER expression levels were in the range 20–25% (24.3 ± 2.1) and 14–18% (15.4 ± 1.5) in lobules and ducts, respectively. PR expression levels were in the range 10–13% (11.1 ± 1.6) and 14–17% (15.2 ± 1.4), respectively. No correlation was found between ER/PR expression and vascularization parameters.

Introduction

Neoangiogenesis is considered to be a crucial step in tumour growth and progression (Gasparini, 1996). The presence and the extension of angiogenesis may provide parameters for tumour prognosis (Weidner et al. 1993; Apolinario et al. 1997; Gasparini & Harris, 1999) and be used for developing new diagnostic (Pham et al. 1998; Miles, 1999; Young et al. 1999) and therapeutic strategies (Pluda, 1997; Gradishar, 1999).

Breast carcinoma represents a tumour model, in which angiogenesis has been extensively studied (Gasparini, 1999). The degree of vascularization can offer information relevant to the prognosis of invasive breast tumours (Gasparini, 1999), as well as to the characterization of pre-invasive lesions (Guidi et al. 1994; Engels et al. 1997).

However, methodological inconsistencies are perceived in the literature with respect to the analysis of vascularization, including vessel count, field size (ranging from ×100 to ×400) and class of antibody (factor VIII, CD31 or CD34). Moreover, a detailed description of microvascularization of the normal mammary gland is still missing. In particular, very little is known regarding the distribution and the number of capillaries in association with normal breast structures such as ducts and lobules. Furthermore, no data are available on the relationship between microvascularization and the expression of oestrogen (ER)/progesterone (PR) receptors.

We believe that an accurate definition of the microvascularization pattern in normal breast is essential in order to make a comparison with angiogenic alterations associated with pre-invasive and invasive breast lesions. With this in mind, immunohistochemistry can be successfully applied using antibodies against endothelial cell-associated proteins. In particular, antibodies against CD34 antigen, a 115-kDa molecular weight protein, have been shown to be very effective at highlighting vessels in routinely formalin-fixed and paraffin-embedded tissues (Ramani et al. 1990).

In this paper we analyse (1) the distribution and level of microvascularization in a set of normal human breast tissues by immunohistochemical analysis of CD34, and (2) the relationship between vascularization and ER/PR expression. We also describe a computer-assisted analysis developed for quantitative evaluation of the microvascularization pattern.

Materials and methods

Specimens

Breast tissue samples were obtained from breast reductions performed in nine young women between 18 and 28 years of age, during the pre-ovulatory phase (day 7 to day 10 from menstruation). Samples were formalin fixed and paraffin embedded. Eight ducts and eight lobules from each woman were selected at ×200 magnification (×20 objective lens and ×10 ocular lens). Five-micrometre adjacent sections were either stained with haematoxylin–eosin for histological analysis or immunostained to highlight the vessels and hormonal receptors. A total of 144 structures were selected and viewed using an image analyser.

Immunohistochemical analysis

Adjacent sections were incubated with mouse anti-human CD34 (QB-END10, Novocastra Laboratory), ER (anti-ER, clone 6F11, Ventana Instruments) and PR (anti-PGR, clone 1A6, Ventana Instruments) antibodies, as previously reported (MacGrogan et al. 1996; Viacava et al. 2003). Microvessels were defined as discrete clusters or single cells stained for CD34; the presence of a lumen was not required for scoring. ER/PR expression was evaluated semiquantitetively as the percentage of positive nuclei in epithelial cells of lobules and ducts.

Computer-assisted analysis

Computer-assisted analysis was performed as previously described (Bocci et al. 1999, 2001). Images were digitized in a 512 × 512-pixel matrix, using a colour video camera (TK-1280E; JVC, Tokyo, Japan) and a microcomputer processor. Digitized pictures were visualized on a high-resolution colour display (SAMPO, Tao-Yuan-Hsien, Taiwan). The true colour image analysis software package KS300 v.1.2 (Kontron Elektronik GmbH, Eching, Germany) was run for manipulation, quantification of the images and data collection.

Morphometric analysis

Parameters: lobules (Fig. 1a,b)

Fig. 1.

(a) Normal lobule formed by CD34-positive microvessels (MV), acini (A) and stroma (S) (immunohistochemistry, ×100). (b) Computer image processing of the same lobule; LP, is the lobule perimeter drawn at the edge of loose lobular stroma and dense perilobular stroma. (c) Normal duct formed by lumen (L) and epithelial wall (E) surrounded by CD34-positive microvessels (MV) and stroma (S) (immunohistochemistry, ×100). (d) Computer image processing of the same duct. Microvessel analysis was performed in a stromal rime of 300 µm around the duct. Vascular area ratio (VAR) = [microvessel area/(microvessel area + residual stroma area)]; vascular density (VD) = [microvessel number/(microvessel area + residual stromal area)] × 10 000.

Total area:

the overall area occupied by the lobule, identified by the sharp margin between loose lobular stroma and dense interlobular stroma.

Acinar area:

the overall area occupied by the acini, including their lumen.

Vascular area:

the stromal area covered by microvessels, with or without a lumen.

Residual stromal area:

calculated by subtracting the acinar and the vascular areas from the total area.

Acimar index (AI):

acimar area/total area of the lobule.

Parameters: ducts (Fig. 1c,d)

Total area:

the overall area occupied by the duct, including lumen, epithelial wall and a 300-µm rime of stroma around the duct.

Ductal area:

the area occupied by the duct, including lumen and epithelial wall.

Vascular area:

the stromal area covered by microvessels, with or without lumen.

Residual stromal area:

calculated by subtracting the ductal and the vascular areas from the total area.

Values

Vascular density (VD):

number of microvessels present in a selected area, calculated as [microvessel number/(microvessel area + residual stromal area)] × 10 000.

Vascular area ratio (VAR):

the area occupied by microvessels within the selected area: [microvessel area/(microvessel area + residual stromal area)].

Furthermore, the area of each lobular component (acini with their lumen, microvessels and residual stroma) was also expressed as a percentage of the total lobular area.

Statistical analysis

The results of image analysis were reported as the mean ± SE of n observations and calculated using the GraphPad Prism™ software package (GraphPad Software Inc., San Diego, CA, USA). Statistical significance (P < 0.05) was calculated by Mann–Whitney test, with differences among patients estimated by anova, followed by Student–Newman–Keuls test. Correlation between the calculated parameters was analysed by linear regression analysis using GraphPad Prism™.

Results

Histology

Lobules showed variability in size, with some differences observed among different women and within the same mammary gland. Acini showed two types of cells: inner epithelial cells and outer myoepithelial cells. The intralobular stroma was moderately cellular. Medium- and large-sized ducts were lined with luminal and mioepithelial cells and surrounded by dense collagenous stroma.

Immunohistochemistry

A thin delicate framework of CD34 immunoreactive microvessels was observed in the stroma of both ducts and lobules. Microvessels in the intralobular stroma were relatively large with a well-defined lumen present in most cases. Microvessels in the periductal stroma were smaller and often without an identifiable lumen.

Computer-assisted analysis

Lobules

VD values ranged between 1.51 and 3.88 (mean value ± SE 2.48 ± 0.14) (Table 1), showing a high degree of homogeneity in all nine cases. VAR values ranged between 0.20 and 0.52 (0.33 ± 0.02) (Table 1), with only one case being significantly different from the others (P < 0.05). When the three components of the lobule were analysed, the percentage of the vascular area was 21.53 ± 1.87% of the total lobular area, the acinar area was 35.14 ± 1.57% and the residual stromal area was 42.65 ± 1.35%.

Table 1.

Vascular parameters of image analysis in normal breast ducts and lobules

Parameters	Lobules	Ducts
VD
range	1.51 ÷ 3.88	1.95 ÷ 4.07
mean ± SE	2.48 ± 0.14	2.95 ± 0.16
VAR
range	0.20 ÷ 0.52	0.12 ÷ 0.41
mean± SE	0.33± 0.02	0.29± 0.03

VD: vascular density; VAR: vascular area ratio; SE: standard error.

A significant correlation between VAR and VD was found in all cases (P = 0.007, r² = 0.94) (Fig. 2). No correlation was found between either VAR or VD and AI (P = 0.397, r² = 0.15 and P = 0.865, r² = 0.06, respectively).

Fig. 2.

Significant linear correlation between VAR and VD in lobular structures (P < 0.05).

Ducts

VD values ranged between 1.95 and 4.07 (2.95 ± 0.16) (Table 1). VAR values ranged between 0.12 and 0.41 (0.29 ± 0.03) (Table 1). No significant differences in either VD or VAR were observed. VAR and VD were not significantly correlated (P = 0.539, r² = 0.15) (Fig. 3).

Fig. 3.

Non-significant linear correlation between VAR and VD in ductal structures (P > 0.05).

When lobules and ducts were compared, VD was significantly higher in the stroma surrounding the ducts (P < 0.05), whereas no significant difference in VAR was found.

Hormonal receptor expression

ER was expressed in 20–25% (24.3 ± 2.1%) of the epithelial cells and 14–18% (15.4 ± 1.5%) of the epithelial cells in lobules and ducts, respectively. PR was expressed in 10–13% (11.1 ± 1.67%) of the epithelial cells and 14–17% (15.2 ± 1.4%) of the epithelial cells, in lobules and ducts, respectively. No correlation was found between ER/PR expression and vascularization parameters.

Discussion

In the present study we analysed the distribution and nature of microvascularization in a series of normal breast ducts and lobules. Several novel working criteria were adopted: (1) tissues were obtained from young nulliparous women, with no breast lesion detected; (2) tissues were all sampled during the same ovulatory phase (day 7 to day 10 of the menstrual cycle); (3) vascularization parameters were calculated by computer-assisted analysis; and (4) vascularization parameters were correlated with ER/PR expression.

An important anatomical feature of the mammary gland has been elucidated: ducts and lobules show two different patterns of microvascularization. Whereas ducts are surrounded by a high number of microvessels, with the appearance of typical small capillaries, lobules show fewer microvessels, which are larger and sinusoidal in shape. Such a difference could have functional implications. The sinusoidal capillary circulation of the lobules may be related to their role as a hormone target within the breast parenchyma. Large and tortuous capillaries would slow the blood flow, favouring a prolonged contact of the hormones with the lobular elements.

Further information on normal ducts and lobules was obtained by morphometric analysis, carried out by measuring a set of structural parameters that included total area, acinar or ductal area, vascular area and residual stromal area. All lobules showed similar ratios for each of their three components (vessels, acini and residual stroma), indicating that lobules are stable structures in the pre-ovulatory phase of the menstrual cycle. Such morphological stability is associated with functional stability as evidenced by similar levels of ER/PR expression.

In order to quantify microvascularization we defined two new parameters: vascular density (VD) and vascular area ratio (VAR). VD and VAR showed little variability within ducts and lobules, whereas VD was significantly higher in the stroma surrounding the ducts than in the lobules. VD positively correlated with VAR in the lobules, but no correlation was found in the ducts. This could be explained by the very small size of the capillaries, an increase in number of which would not significantly alter the vascular area surrounding the ducts. This set of results indicates that ducts and lobules are characterized by two different types of microvascularization, confirming the histological data.

The vascular pattern does not seem to be influenced by hormonal activity because we found no correlation between ER/PR expression and vascularization parameters.

We believe that our study offers a novel method of morphometric analysis, which allowed us to confirm and quantify a series of histological observations on the microvascularization of the mammary gland. We also defined several parameters that can be beneficially used as reference parameters for characterizing angiogenesis of both invasive and non-invasive phases of breast carcinoma.