Pattern of HER-2 Gene Amplification and Protein Expression in Benign, Borderline, and Malignant Ovarian Serous and Mucinous Neoplasms.

Amplification of HER-2 gene and overexpression of HER-2 receptor play a significant role in the progression of a number of malignancies such as breast cancer. Trastuzumab (anti-HER-2 therapeutic agent) has been used successfully in treatment of breast cancer. The aim of this study was to assess the pattern of HER-2 gene amplification and of HER-2 receptor expression in a spectrum of serous and mucinous ovarian tumors to determine whether HER-2 is altered in these neoplasms similar to that occurring in breast cancer. Formalin-fixed paraffin-embedded microarray tissue sections from 212 specimens were stained with HER-2 antibody using immunohistochemistry and with anti-HER-2 DNA probe using chromogenic in situ hybridization. Specimens consisted of 65 benign tumors (50 serous and 15 mucinous), 26 borderline (13 serous and 13 mucinous), 73 malignant tumors (53 serous carcinoma and 20 mucinous carcinoma), 18 metastatic deposits (13 serous and 5 mucinous), in addition to 30 normal tissues (16 ovarian surface and 14 normal fallopian tube). HER-2 protein-positive expression was not detected in the normal or the benign tissues. Borderline neoplasms showed positive staining, but no overexpression. HER-2 overexpression was seen only in 4 carcinoma specimens: 1/53 (1.8%) primary serous carcinomas and 3/20 (15%) primary mucinous carcinomas. HER-2 gene amplification was seen in 4 specimens: 2 primary mucinous carcinomas and 2 malignant deposits of these 2 mucinous carcinomas. In conclusion, alteration of HER-2 was not detected in ovarian serous neoplasms; however, in mucinous carcinoma, HER-2 amplification and overexpression occur.

Prognostic significance of lymphatic invasion in lymph node-positive breast carcinoma: findings from a large case series with long-term follow-up using immunohistochemical endothelial marker.

The poor prognostic significance of lymphatic invasion (LI) in breast carcinoma (BC) as a whole and in lymph node (LN)-negative patients in particular has been recognized in several studies; however, its prognostic role in LN-positive patients is still questionable. Aim of the current study was to assess prognostic role of LI in LN-positive BC specimens. Sections from non-selected 557 LN-positive BC specimens were stained with antibody to podoplanin/D2-40. LI was identified and correlated with clinicopathological features and patients' outcome. Twenty-year overall survival (OS), disease-free interval (DFI), and development of distant metastasis (DM) or recurrence were known for all patients. LI was detected in 262/557 (47%) of specimens ranging from 1 to 350 lesion per tumor section. Its presence was associated with higher grade tumors (P<0.0001), negative hormonal receptors (P<0.0001), high HER-2 expression (P=0.006), and with increased number of positive LNs (P=0.019). In the whole LN-positive BC, presence of LI was a poor prognostic factor for OS, DFI, and development of DM both in univariate and in multivariate analysis. In further stratification of patients, LI was associated with poorer prognosis in patients with single positive LN and not in patients with >1 positive LN. In T1N1 stage, LI was highly associated with poor OS (P=0.002), DFI (P<0.0001), and DM (P<0.0001). In T2N1 patients, LI was associated only with poorer DFI (P=0.037) but not with death or DM. In the two former patient groups, LI lost significance in multivariate analysis. In conclusion, LI is a poor prognostic factor in LN-positive BC particularly for patients having single positive LN. LI therefore would add further prognostic significance when considered in treatment in those patients. We recommend incorporation of LI in breast carcinoma staging and in prognostic indices.

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.

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, Mϕ). 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 Mϕ/DCs (82.4% of samples). However, only CLEVER-1 expression in LV was associated with LN metastasis (p = 0.027) and with Mϕ 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.

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.

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.

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.

IL-17 expression by breast-cancer-associated macrophages: IL-17 promotes invasiveness of breast cancer cell lines.

INTRODUCTION: IL-17 plays an important role in autoimmunity, promoting autoimmunity, inflammation and invasion in multiple sclerosis, rheumatoid arthritis and type I diabetes. The role of IL-17 in cancer is unclear, however, as there are few studies examining IL-17 protein expression in cancer. We therefore examined IL-17 protein expression in human breast cancer and modelled its potential biological significance in vitro. METHODS: Immunohistochemistry was used to determine IL-17 expression in breast cancers. Matrigel invasion assays were employed to examine the effect of IL-17 on cancer cell invasion by a panel of breast cancer cell lines. The role of matrix metalloproteinases (MMPs) was investigated with selective antagonists and immunoassays for MMP-2, MMP-3, MMP-9 and tissue inhibitor of MMP. RESULTS: IL-17-expressing cells with macrophage morphology were identified in the peritumoural area of a proportion of patients (8/19 patients). Macrophages were confirmed by CD68 staining on serial sections. With the exception of occasional lymphocytes, one patient with rare multinucleate giant cells and one patient with occasional expression of IL-17 in tumour cells, no other IL-17-positive cells were detected. Addition of IL-17 to cell lines in vitro stimulated marked invasion of Matrigel. In contrast, IL-17 did not promote the invasion of MCF7 or T47D cell lines. Invasion was initially thought to be dependent on MMPs, as evidenced by the broad-spectrum MMP inhibitor GM6001 and selective antagonists of MMP-2/MMP-9 and MMP-3. Measurement of MMP-2, MMP-3 and MMP-9, and tissue inhibitor of MMP 1 secretion, failed to reveal any changes in expression following IL-17 exposure. In contrast, TNF promoted secretion of MMPs but IL-17 did not augment TNF, indicating that IL-17 acts via an independent mechanism. CONCLUSIONS: The present study is the first to describe in situ expression of IL-17 protein in human breast tumours and to propose a direct association between IL-17 and breast cancer invasion. The precise effectors of IL-17-dependent invasion remain to be characterised but could include a range of proteases such as a disintegrin and metalloproteinase protein or astacins. Nevertheless, this work identifies a novel potential mechanism for breast cancer invasion and tumour progression, the prognostic implication of which is currently under investigation.

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 from 177 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.