Lymphatic and Blood Vessels in Basal and Triple-negative Breast Cancers

Characteristics And Prognostic Significance

Rabab AA Mohammed; Ian O Ellis; Ali M Mahmmod; E Claire Hawkes; Andrew R Green; Emad A Rakha; Stewart G Martin

Disclosures

Mod Pathol. 2011;24(6):774-785. 

In This Article

Abstract and Introduction

Abstract

Basal and triple-negative breast cancer phenotypes are characterised by unfavourable biological behaviour and outcome. Although certain studies have examined their pathological and molecular profile, the vascular characteristics of lymphatic and blood vessels have not been examined. Immunohistochemical staining with podoplanin, CD34 and CD31 was used to examine lymphatic and microvessel density, as well as vascular invasion in 197 basal-like and in 99 triple-negative breast tumours and compared against 200 non-basal and 334 non-triple-negative cases. All specimens were lymph node negative. Vascular invasion was identified as blood or lymphatic vascular invasion by the differential expression of markers. All measurements were correlated with clinicopathological features and prognosis. No significant difference was detected between the basal and triple-negative groups in terms of lymphatic or microvessel density or vascular invasion. However, both the basal and the triple-negative groups showed significantly higher microvessel density than did the non-basal and non-triple-negative groups (P=0.017 and P<0.001, respectively). Unlike microvessel density, no significant difference was detected in lymphatic density between the basal or triple-negative groups compared with their respective controls. Interestingly, vascular invasion, almost entirely lymphatic invasion, was detected in 27% of the basal and in 26% of the triple-negative groups with no significant difference in comparison with control groups. In both basal and triple negatives, vascular invasion was associated with poorer survival by univariate and multivariate analyses. The 20-year overall survival rate in basal-like tumours was 55% in vascular invasion-positive cases compared with 73% in vascular invasion-negative tumours (P=0.012), and 46% in triple-negative vascular invasion-positive compared with 79% in vascular invasion-negative tumours (P=0.001). Basal-like vs non-basal-like and triple-negative vs non-triple-negative tumours have similar vascular characteristics in terms of lymphatic vessel density and vascular invasion but higher microvessel density, suggesting that such groups may preferentially benefit from anti-angiogenic therapy. Vascular invasion was, in all phenotypes, almost entirely lymphatic vessel invasion and could stratify basal-like and triple-negative phenotypes into distinct prognostic groups.

Introduction

Breast cancer of the basal-like phenotype has been a focus of interest in breast cancer research over the last decade. This phenotype was initially described in a study by Perou and colleagues in which DNA microarray technology was used to examine gene expression patterns in primary breast cancers. Five groups were found to have distinct molecular, biological and clinical features. The existence of such types, namely luminal A, luminal B, ERBB2 (Her-2)-overexpressing, basal-like and normal-like types[1,2] have also been confirmed using immunohistochemical approaches.[3]

Basal-like breast cancer represents approximately 15–20% of invasive ductal carcinomas and displays specific molecular, epidemiological, phenotypic, morphological and mammographic features. It is characterised by high histological grade, high growth fraction and distinct genetic alterations. Such tumours are usually negative for hormone receptors and ERBB2 and show high expression of P53, EGFR and KIT. They are more likely to develop distant metastasis and have a generally poorer prognosis.[4] Their mammographic appearance is also different from that of non-basal tumours; they are more likely to manifest as an ill-defined mass or with comedo calcification, whereas non-basal-phenotype tumours are more likely to manifest as a spiculated mass.[5] There is no one 'gold' standard definition of basal-like breast cancer. It has been defined by negativity for ER, PR and HER2, with positive expression of either basal cytokeratins or EGFR.[6] In a different report, a positive expression of basal cytokeratins (CK5/6 and/or CK14) was used to identify the basal-like phenotype regardless of the expression of other markers.[7]

The other recently recognised type of breast cancer are triple-negative tumours, which are characterised by being negative for ER, PR and HER2 and which account for ~16% of breast cancers.[7] They have distinguishable epidemiological[8,9] and imaging features.[10] It was initially believed that both Basal-like and triple-negative phenotypes were synonymous; however, recent results have suggested that both types may be distinct.[11] A comparative analysis of the clinicopathological characteristics and the expression of basal markers found that although triple-negative tumours were significantly associated with the expression of basal markers, 19% were negative for such markers and 7.3% of non-triple-negative tumours expressed them, suggesting that the triple-negative phenotype is not an ideal surrogate marker for basal-like breast cancers.[12] Recent studies have shown that each subtype, basal-like and triple-negative, is heterogeneous and consists of subgroups.[13]

Lymph vessels and blood vessels within and around malignant tumours have been known to have an important role in disease progression in many tumour types, including breast cancer. It has been appreciated for some time that angiogenesis (the process of formation of new blood vessels) has an important role in the progression of breast cancer.[14–16] Angiogenesis is commonly measured by counting blood vessels in tumour sections stained with one of the vascular markers such as CD34 or CD31, yielding microvessel density, and this been found to be associated with features of unfavourable outcome, such as larger size and poor differentiation. However, its role as a prognostic factor in breast cancer was found to be weak.[17] Much less is known regarding lymph vessels and their role in tumour progression and patient prognosis. The advent of robust markers that allow a clear delineation of lymphatic vessels from blood vessels has allowed lymphatic research to flourish over the last decade. Tumours with high lymph-vessel density have been found to be associated with a higher risk for axillary lymph-node metastasis;[18,19] however, its role in the prognostication of breast cancer is controversial. In a previous study, from the current authors, on a mixed group of breast cancers (various subtypes and a mix of lymph node-positive and lymph node-negative tumours), high lymph-vessel density was found to be associated with the presence of lymph-node metastasis and shorter overall survival. However, upon multivariate analysis, only tumour grade, lymph-node status and the presence of vascular invasion, but not lymph-vessel density, were found to be independent poor prognostic factors.[20] The presence of vascular invasion, as detected by standard H&E methodology, has been well characterised as an indicator of tumour types with higher metastatic potential, greater risk of recurrence and death from the disease,[21–23] and has been used to guide adjuvant systemic therapy for patients with node-negative breast cancer.[24] Vascular invasion includes both lymphatic vascular invasion and blood vascular invasion. As mentioned above, historically, it was difficult to distinguish between each type of invasion because of the lack of robust lymphatic-specific markers, but with the discovery of such markers, lymphatic research has expanded. Podoplanin (D2-40) expression can be used to specifically identify lymphatic vessels,[25–28] and through its use, it has become possible to identify and distinguish lymphatic from blood vascular invasion.[29] It has been previously reported that basal-like breast cancers are more likely to metastasise to the brain and lung (intra-pulmonary) and that this may be an indication for preferential haematogenous spreading.[5] Recent results by the current authors have suggested that breast cancer cells show an initial preference for lymphatic rather than for blood vessels.[29] Such work was conducted on a relatively small cohort of 177 patients, and hence it was not possible to examine whether different breast cancer subtypes showed different patterns of vascular invasion. The current study addresses this by examining vascular invasion patterns in primary basal-like and triple-negative phenotypes by studying a large cohort of lymph node-negative early-stage breast cancers.

The aims of this study were as follows: (1) to examine the vascular characteristics (lymphatic and microvessel density) in breast cancers of basal phenotypes and of triple-negative phenotypes and to compare these characteristics with non-basal tumour and non-triple-negative types; (2) to use immunohistochemical staining methods, with vascular markers, to distinguish between lymphatic and blood vascular invasion, as well as to examine the frequency of each type in both tumour groups; and (3) to examine the association between lymph-vessel density, microvessel density and vascular invasion with clinicopathological characteristics and patient prognosis.

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