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

Materials and Methods

Patients and Tissue Samples

Clinicopathological data of early-stage 1000 specimens of primary invasive breast cancer were retrieved from the Department of Histopathology (Nottingham University Hospitals, City Hospital Campus). The basal phenotype was identified as specimens expressing CK5/6 and/or CK14 as described previously.[7] Overall, 197 of 1000 (19.7%) specimens were found of the basal-like phenotype for which paraffin-embedded archival specimens were retrieved from the Department of Histopathology. A total of 82 patients (42%) were younger than 50 years. In all, 58 (29%) of the specimens were <1.5 cm. All specimens were lymph node negative. Overall, 74 (38%) patients had developed a recurrence by the time of the last follow-up and 56 (31%) had died from breast cancer. To compare the vascular characteristics with non-basal types, 200 consecutive non-basal lymph node-negative primary invasive breast cancer specimens were identified and retrieved from the 1000 cohort. This gave 397 specimens for the current study. More details about the clinicopathological characteristics of the basal and the non-basal groups are summarised in Table 1. From the 397 cohort, 99 tumour specimens were of the triple-negative phenotype (negative ER, negative PR and negative HER2) and 298 were of the non-triple-negative phenotype. A total of 44 (44%) of the triple negatives were from postmenopausal women, 80 (80%) were >1.5 cm in size, 88 (88%) were grade III and 8 (8%) were grade II. In all, 26 patients died from the disease, 34 developed a recurrence and 21 (21%) developed distant metastases. This project was approved by the Nottingham Ethics Committee 2 with 20-year clinical follow-up information available for analysis. The Nottingham Local Research Ethics Committee waived the need for written informed consent.

Immunohistochemistry

For identification of blood vessels and lymph vessels, representative paraffin-embedded sections from each specimen were stained with CD34 (Serotec, MCAP547, 1:500 dilution) and with podoplanin (AngioBio, 11-003, 1:100 dilution), respectively. In 97 sections, there was CD34 reactivity around tumour cell clusters suggesting the possibility of blood vascular invasion. This was confirmed, or otherwise, by staining parallel sections with CD31 (monoclonal, 0823, DakoCytomation, Denmark, 1:100 dilution). In brief, 4-μm-thick sections were deparaffinised and rehydrated. Antigen retrieval for CD31 and CD34 was achieved by incubating sections in 0.01 mol/l sodium citrate buffer (pH 6.0) in an 800-W microwave for 20 min. Podoplanin did not require antigen retrieval. After blocking of endogenous hydrogen peroxidase and non-specific reactions, sections were incubated for an hour at room temperature with primary antibodies. Visualisation of staining was conducted using streptABC kit (StreptABComplex/HRP Duet, Mouse/Rabbit kit, Dako Corporation, K0492) according to the manufacturer's instructions. Immunohistochemical reactions were developed with 3,3′-diaminobenzidine as the chromogenic peroxidase substrate (Dako Corporation, K3468), counterstained with Myer's haematoxylin and mounted.

Assessment of Lymph-vessel Density and Microvessel Density Lymph-vessel density was assessed in podoplanin-stained sections as described previously.[20] All lymph vessels in the whole tumour section were counted using × 100 magnification with a surface area of 3.46 mm2. The sum of lymph vessels was divided by the sum of the surface area of all counted fields to adjust lymph-vessel density to variation in tumour size. The lymph-vessel density was presented as the number of lymph vessels/mm2. Microvessel density was assessed in CD34-stained sections using the Chalkley counting method, at × 200 magnification, as described previously.[20] In brief, the microvessel density count is the average of the count of three vascular hot-spot fields using the Chalkley grid. This method was found to be reliable and reproducible in the assessment of tumour angiogenesis.[30,31]

Assessment of Vascular Invasion Parallel CD34- and podoplanin-stained sections were examined. Where tumour cell clusters were detected within a CD34-positive structure, a further parallel section was stained with CD31 for confirmation of the vascular nature of the lesion (97 section). Vascular invasion lesions were categorised as blood vascular invasion or lymphatic vascular invasion according to the differential expression of the three markers. Tumour clusters within podoplanin+ were identified as lymphatic vascular invasion regardless of the expression of CD34 or CD31, the expression of which is variable in tumoural lymph vessels (Figure 1).[32–34] Blood vascular invasion was identified if tumour cells were detected within podoplanin-negative and both CD34- and CD31-positive vessels.

Figure 1.

The upper panel shows parallel sections of a breast cancer specimen stained with podoplanin, CD34 and CD31 showing a tumour embolus within a podoplanin-positive, CD34-positive and CD31-positive vessel indicating the lymphatic nature of the vessel, scale bar=50 μm. The lower panel shows parallel sections of a breast cancer specimen section stained with podoplanin (showing low lymphatic vessel density, arrows point at lymph vessels) and CD34 (showing high microvessel density, red arrows point to blood vessels), scale bar=100 μm. The colour reproduction of this figure is available on the html full text version of the manuscript.

Statistical Analysis

The following four levels of statistics were performed using SPSS for windows, version 17: (1) Mann–Whitney tests were conducted to compare the means of lymph-vessel density and microvessel density (as continuous data) between the basal and non-basal groups and between the triple-negative and non-triple-negative groups; (2) specimens were divided into two categories according to the median values of lymph-vessel density and microvessel density. The association between lymph-vessel density, microvessel density and clinicopathological criteria was evaluated in univariate analysis using a 2 × 2 table and χ 2 test. (3) Survival analysis of disease-free interval and overall survival was accomplished using the Kaplan–Meier method, and the statistical significance of differences in the cumulative survival curves between groups was evaluated by the long-rank test. Multivariate analysis, using a Cox proportional hazards model, was conducted using patient age, tumour size, tumour grade and vascular invasion. All statistical analyses were two sided with significance defined as P <0.05.

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