Rabab A. A. Mohammed

Prognostic significance of vascular endothelial cell growth factors -A, -C and -D in breast cancer and their relationship with angio-and lymphangiogenesis

Vascular endothelial cell growth factors (VEGF)-A, -C and -D have potent angio and lymphangiogenic functions in experimental models, although their role in the progression of human breast cancer is unclear. The aims of the current study were to examine the relationship between the expression of the aforementioned growth factors with the angio and lymphangiogenic characteristics of breast cancer, and to assess their suitability as potential prognostic factors. Paraffin-embedded sections of 177 primary invasive breast cancer, with complete clinical follow-up information for 10 years, were stained for VEGF-A, -C, -D, podoplanin and CD34 using standard immunohistochemical approaches. The expression of the growth factors was correlated with clinicopathological criteria and patients’ survival. Lymph vessel density (LVD) and microvessel density (MVD) were assessed and correlated with expression of the growth factors. Vascular endothelial cell growth factor-A, -C and -D were highly expressed in 40, 37 and 42% of specimens, respectively. High expression of VEGF-A and - C, but not of -D, was associated with a higher LVD (P=0.013 and P=0.014, respectively), a higher MVD (P<0.001 and P=0.002, respectively), the presence of lymph node metastasis (P<0.001 and P<0.001, respectively), distant metastasis (P=0.010 and P=0.008, respectively) and a shorter Overall Survival (P=0.029 and 0.028, respectively). In conclusion, breast cancers that express high levels of VEGF-A and -C are characterised by a poor prognosis, likely through the induction of angio and lymphangiogenesis. Examination of expression of VEGF-A and -C in breast cancer may be beneficial in the identification of a subset of tumours that have a higher probability of recurrence and metastatic spread.

Improved Methods of Detection of Lymphovascular Invasion Demonstrate That It is the Predominant Method of Vascular Invasion in Breast Cancer and has Important Clinical Consequences

The presence of vascular invasion (VI), encompassing both lymphovascular invasion (LVI) and blood vascular invasion (BVI), in breast cancer has been found to be a poor prognostic factor. It is not clear, however, which type of VI plays the major role in metastasis. The aims of this study were to use an endothelial subtype specific immunohistochemical approach to distinguish between LVI and BVI by comparing the differential expression of blood vascular (CD34 and CD31) and lymphatic markers (podoplanin/D2-40) to determine their prognostic role in a well-characterized group of breast cancer patients with known long-term follow-up. Sections from177 consecutive paraffin-embedded archival specimens of primary invasive breast cancer were stained for expression of podoplanin, D2-40, CD31, and CD34. BVI and LVI were identified and results were correlated with clinicopathologic criteria and patient survival. VI was detected in 56/177 specimens (31.6%); 54 (96.4%) were LVI and 2 (3.5%) were BVI. The presence of LVI was significantly associated with the presence of lymph node metastasis, larger tumor size, development of distant metastasis, regional recurrence and worse disease-free interval and overall survival. In multivariate analysis, LVI retained significance association with decreased disease-free interval and overall survival. In conclusion, VI in breast cancer is predominantly of lymph vessels and is a powerful independent prognostic factor, which is associated with risk of recurrence and death from the disease. The use of immunohistochemical staining with a lymphendothelial specific marker such as podoplanin/D2-40 increases the accuracy of identification of patients with tumor associated LVI.

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.

Objective assessment of lymphatic and blood vascular invasion in lymph node‐negative breast carcinoma: findings from a large case series with long‐term follow‐up

In a previous study on a small series of breast cancers, we developed objective methods for the assessment of vascular invasion (VI), using immunohistochemical staining. We found that VI was predominantly lymphovascular invasion (LVI), with minimal contribution of blood vascular invasion (BVI). The aims of the current study were: (a) to assess the frequency, extent and prognostic role of LVI and BVI in a large, well‐characterized series of LN‐negative breast cancers; and (b) to assess the ability of VI to stratify early breast cancer into different prognostic groups. Paraffin‐embedded sections from 1005 lymph‐node (LN)‐negative primary invasive breast cancers were stained for CD34, CD31 and podoplanin/D240 to detect BVI and LVI. VI lesions were assessed and the results were correlated with clinicopathological criteria and survival. VI was detected in 218 (22%); 211/218 (97%) were LVI, while BVI was detected in 7/218 (3%). The frequency of LVIs/section ranged from 1 to 79, with no significant difference between the frequency of LVI and outcome. The presence of LVI was significantly associated with adverse disease‐free interval (DFI) and poor overall survival (OS) in both univariate and multivariate analyses. The results from the study indicated that VI in early stage breast cancer is predominantly LVI and that its objective assessment is a powerful independent prognostic factor. Efforts to detect early metastatic activity, such as diligent pathological examination of sentinel LN biopsies would be complimented by the objective evaluation of VI status of the primary tumour. VI status should be included routinely in breast cancer staging systems. Copyright

Lymphatic and blood vessels in basal and triple-negative breast cancers: characteristics and prognostic significance

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.

Lymphatic and angiogenic characteristics in breast cancer: morphometric analysis and prognostic implications

Controversy exists regarding the topography of lymph vessels in breast cancer, their usefulness as prognostic factors, relationship with angiogenesis and whether active lymphangiogenesis occurs within the tumour. A series of 177 well-characterized breast cancers, with long term follow up, were stained with D2-40, CD31 and CD34. Distribution of lymphatics and lymph vessel density (LVD) were assessed in three areas, intratumoural, peripheral and peritumoural and correlated with clinicopathological criteria and patient prognosis. Microvessel density (MVD) was assessed and correlated with LVD. Double immunohistochemical staining with D2-40 and MIB-1 was carried out to assess the proliferative status of lymphatics and of the tumour emboli within. Peritumoural lymphatics were detected in all tumours whereas peripheral and intratumoural lymphatics were detected in 86 and 41% of specimens, respectively. Tumours with higher total LVD were significantly associated with the presence of lymph node (LN) metastasis and shorter overall survival (OS). In multivariate analysis, tumour grade, LN status and the presence of lymphovascular invasion, but not LVD, were independent poor prognostic factors. No association was found between LVD and MVD. Proliferating lymphatics were detected in 29% of specimens and were significantly associated with dense inflammatory infiltrate. In conclusion, lymphatics are located primarily in the peritumoural and peripheral areas in breast cancer and seem to play an important role in disease progression by being routes for tumour dissemination. The lack of correlation between lymphangiogenic and angiogenic characteristics suggests two distinct processes and the presence of active lymphangiogenesis, albeit in a small portion of specimens, may have important therapeutic implications.

Growth fraction as a predictor of response to chemotherapy in node-negative breast cancer

Accurate predictive markers of chemotherapeutic response in early breast cancer are still lacking. The role of tumour growth fraction as a predictor of response to chemotherapy was assessed in early breast cancer. In this study, immunohistochemical expression of MIB1 was studied in a well‐characterised series of early (Stages I and II) node‐negative breast carcinoma cases (n = 100) with long‐term follow‐up that have received adjuvant chemotherapy (cyclophosphamide/methotrexate/5‐fluorouracil regimen). In addition, 728 cases who did not receive adjuvant chemotherapy were used as a control group. Increased tumour growth fraction was associated with a better response to adjuvant chemotherapy in terms of longer breast cancer specific survival and disease‐free interval [hazard ratio (HR) = 0.354, 95% CI = 0.177–0.688, p = 0.003 and HR = 0.396, 95% CI = 0.205–0.768, p = 0.006, respectively]. In contrast to the control group, patients with high growth fraction tumour (>70%) showed an excellent outcome with infrequently reported events during the period of follow‐up. Importantly, patients with a low growth fraction (≤10%) showed frequent recurrences and shorter survival time with outcome comparable to those of high growth fraction who did not receive chemotherapy. Therefore, tumour growth fraction can be used to assign patients into distinct groups showing differential response to adjuvant chemotherapy. Patients with a high growth fraction appear to be ideal candidates for adjuvant chemotherapy while those with low growth fraction are less likely to benefit and are prone to the potential serious side effects of adjuvant chemotherapy.

Vascular invasion in breast cancer; an overview of recent prognostic developments and molecular pathophysiological mechanisms

Vascular invasion (VI) is an essential step in breast cancer metastasis and the main cause of morbidity and mortality from the disease. Detection of VI in the primary tumour is a marker of metastatic potential. The prognostic value of VI in breast cancer has been known for more than four decades, but its application in clinical practice is still fraught with difficulties due to the limited number of studies conducted on large numbers of well‐characterized patients with long‐term follow‐up. Detection of VI in the primary tumour is currently assessed using sections stained with haematoxylin and eosin, which has some disadvantages. A number of vascular markers have been used to improve detection of VI; however, their sensitivity and specificity, as endothelial markers, vary considerably. In this review we describe the evolution of the prognostic importance of VI and the recent pathomolecular mechanisms that contribute to the ability of breast cancers to invade through vessels, in addition to the types, locations and methods of detection of vascular invasion.

Calpastatin is associated with lymphovascular invasion in breast cancer

Metastasis of breast cancer is a major contributor to mortality. Histological assessment of vascular invasion (VI) provides important prognostic information and demonstrates that VI occurs predominantly via lymphatics in breast cancer. We sought to examine genes and proteins involved in lymphovascular invasion (LVI) to understand the mechanisms of this key disease process. A gene expression array of 91 breast cancer patients was analysed by an Artificial Neural Network (ANN) approach using LVI to supervise the analysis. 89 transcripts were significantly associated (p<0.001) with the presence of LVI. Calpastatin, a specific calpain inhibitor, had the second lowest selection error and was investigated in breast cancer specimens using real-time PCR (n=56) and immunohistochemistry (n=53). Both calpastatin mRNA and protein levels were significantly associated with the presence of LVI (p=0.014 and p=0.025 respectively). The data supports the hypothesis that calpastatin may play a role in regulating the initial metastatic dissemination of breast cancer.

Lymphatic expression of CLEVER-1 in breast cancer and its relationship with lymph node metastasis

Background: Mechanisms regulating breast cancer lymph node metastasis are unclear. Staining of CLEVER-1 (common lymphatic endothelial and vascular endothelial receptor-1) in human breast tumors was used, along with in vitro techniques, to assess involvement in the metastatic process. Methods: 148 sections of primary invasive breast cancers, with 10 yr follow-up, were stained with anti-CLEVER-1. Leukocyte infiltration was assessed, along with involvement of specific subpopulations by staining with CD83 (mature dendritic cells, mDC), CD209 (immature DC, iDC) and CD68 (macrophage, M&phis;). in vitro expression of CLEVER-1 on lymphatic (LEC) and blood endothelial cells (BEC) was examined by flow cytometry. Results: in vitro results showed that although both endothelial cell types express CLEVER-1, surface expression was only evident on LEC. In tumour sections CLEVER-1 was expressed in blood vessels (BV, 61.4% of samples), lymphatic vessels (LV, 18.2% of samples) and in M&phis;/DCs (82.4% of samples). However, only CLEVER-1 expression in LV was associated with LN metastasis (p = 0.027) and with M&phis; indices (p = 0.021). Although LV CLEVER-1 was associated with LN positivity there was no significant correlation with recurrence or overall survival, BV CLEVER-1 expression was, however, associated with increased risk of recurrence (p = 0.049). The density of inflammatory infiltrate correlated with CLEVER-1 expression in BV (p < 0.001) and LV (p = 0.004). Conclusions: The associations between CLEVER-1 expression on endothelial vessels and macrophage/leukocyte infiltration is suggestive of its regulation by inflammatory conditions in breast cancer, most likely by macrophage-associated cytokines. Its upregulation on LV, related surface expression, and association with LN metastasis suggest that it may be an important mediator of tumor cell metastasis to LN.