Induction of lymphangiogenesis in and around axillary lymph node metastases of patients with breast cancer

We studied the presence of lymphangiogenesis in lymph node (LN) metastases of breast cancer. Lymph vessels were present in 52 of 61 (85.2%) metastatically involved LNs vs 26 of 104 (25.0%) uninvolved LNs (P<0.001). Furthermore, median intra- and perinodal lymphatic endothelial cell proliferation fractions were higher in metastatically involved LNs (P<0.001). This is the first report demonstrating lymphangiogenesis in LN metastases of cancer in general and breast cancer in particular

NF-κB activation in inflammatory breast cancer is associated with oestrogen receptor downregulation, secondary to EGFR and/or ErbB2 overexpression and MAPK hyperactivation

Activation of NF-κB in inflammatory breast cancer (IBC) is associated with loss of estrogen receptor (ER) expression, indicating a potential crosstalk between NF-κB and ER. In this study, we examined the activation of NF-κB in IBC and non-IBC with respect to ER and EGFR and/or ErbB2 expression and MAPK hyperactivation. A qRT–PCR based ER signature was evaluated in tumours with and without transcriptionally active NF-κB, as well as correlated with the expression of eight NF-κB target genes. Using a combined ER/NF-κB signature, hierarchical clustering was executed. Hyperactivation of MAPK was investigated using a recently described MAPK signature (Creighton et al, 2006), and was linked to tumour phenotype, ER and EGFR and/or ErbB2 overexpression. The expression of most ER-modulated genes was significantly elevated in breast tumours without transcriptionally active NF-κB. In addition, the expression of most ER-modulated genes was significantly anticorrelated with the expression of most NF-κB target genes, indicating an inverse correlation between ER and NF-κB activation. Clustering using the combined ER and NF-κB signature revealed one cluster mainly characterised by low NF-κB target gene expression and a second one with elevated NF-κB target gene expression. The first cluster was mainly characterised by non-IBC specimens and IHC ER+ breast tumours (13 out of 18 and 15 out of 18 respectively), whereas the second cluster was mainly characterised by IBC specimens and IHC ER− breast tumours (12 out of 19 and 15 out of 19 respectively) (Pearson χ2, P<0.0001 and P<0.0001 respectively). Hyperactivation of MAPK was associated with both ER status and tumour phenotype by unsupervised hierarchical clustering using the MAPK signature and was significantly reflected by overexpression of EGFR and/or ErbB2. NF-κB activation is linked to loss of ER expression and activation in IBC and in breast cancer in general. The inverse correlation between NF-κB activation and ER activation is due to EGFR and/or ErbB2 overexpression, resulting in NF-κB activation and ER downregulation

Tumor Cell Plasticity and Angiogenesis in Human Melanomas

Recent molecular studies provide evidence for a significant transcriptional plasticity of tumor cell subpopulations that facilitate an active contribution to tumor vasculature. This feature is accompanied by morphological changes both in vitro and in vivo. Herein, we investigated the morphological plasticity of tumor cells with special focus on vasculogenic mimicry and neovascularisation in human melanoma and mouse xenografts of human melanoma cell lines. In melanoma xenograft experiments, different vessel markers and green fluorescent protein expression were used to show how melanoma cells contribute to neovascularization. Additionally, we analyzed neovascularization in 49 primary melanomas and 175 melanoma metastases using immunostaining for blood (CD34) and lymphatic (D2–40) vessel-specific markers. We found significantly more lymphatic vessels in primary melanomas than in melanoma metastases (p<0.0001). In contrast to the near absence of lymphatic vessels within metastases, we found extensive blood micro-neovascularization. Blood micro-neovascularization was absent in micro metastases (less than 2 mm). A significant inverse correlation between Glut-1 expression (implying local hypoxia) and the presence of microvessels indicates their functional activity as blood vessels (p<0.0001). We suggest that the hypoxic microenvironment in metastases contributes to a phenotype switch allowing melanoma cells to physically contribute to blood vessel formation

Identification of cell-of-origin breast tumor subtypes in inflammatory breast cancer by gene expression profiling

Inflammatory breast cancer (IBC) is an aggressive form of locally advanced breast cancer with high metastatic potential. Most patients have lymph node involvement at the time of diagnosis and 1/3 of the patients have distant metastases. In a previous study, we demonstrated that IBC is a distinct form of breast cancer in comparison with non-IBC. The aim of this study was to investigate the presence of the different molecular subtypes in our data set of 16 IBC and 18 non-IBC specimen. Therefore, we selected an ‘intrinsic gene set’ of 144 genes, present on our cDNA chips and common to the ‘intrinsic gene set’ described by Sorlie et al. [PNAS, 2003]. This set of genes was tested for performance in the Norway/Stanford data set by unsupervised hierarchical clustering. Expression centroids were then calculated for the core members of each of the five subclasses in the Norway/Stanford data set and used to classify our own specimens by calculating Spearman correlations between each sample and each centroid. We identified the same cell-of-origin subtypes in IBC as those already described in non-IBC. The classification was in good agreement with immunohistochemical data for estrogen receptor protein expression and cytokeratin 5/6 protein expression. Confirmation was done by an alternative unsupervised hierarchical clustering method. The robustness of this classification was assessed by an unsupervised hierarchical clustering with an alternative gene set of 141 genes related to the cell-of-origin subtypes, selected using a discriminating score and iterative random permutation testing. The contribution of the different cell-of-origin subtypes to the IBC phenotype was investigated by principal component analysis. Generally, the combined ErbB2-overexpressing and basal-like cluster was more expressed in IBC compared to non-IBC, whereas the combined luminal A, luminal B and normal-like cluster was more pronounced in non-IBC compared to IBC. The presence of the same molecular cell-of-origin subtypes in IBC as in non-IBC does not exclude the specific molecular nature of IBC, since gene lists that characterize IBC and non-IBC are entirely different from gene lists that define the different cell-of-origin subtypes, as evidenced by principal component analysis.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/44236/1/10549_2005_Article_9015.pd

Enrichment of the tumour immune microenvironment in patients with desmoplastic colorectal liver metastasis

Background Patients with resected colorectal liver metastasis (CRLM) who display only the desmoplastic histopathological growth pattern (dHGP) exhibit superior survival compared to patients with any non-desmoplastic growth (non-dHGP). The aim of this study was to compare the tumour microenvironment between dHGP and non-dHGP. Methods The tumour microenvironment was investigated in three cohorts of chemo-naive patients surgically treated for CRLM. In cohort A semi-quantitative immunohistochemistry was performed, in cohort B intratumoural and peritumoural T cells were counted using immunohistochemistry and digital image analysis, and in cohort C the relative proportions of individual T cell subsets were determined by flow cytometry. Results One hundred and seventeen, 34, and 79 patients were included in cohorts A, B, and C, with dHGP being observed in 27%, 29%, and 15% of patients, respectively. Cohorts A and B independently demonstrated peritumoural and intratumoural enrichment of cytotoxic CD8+ T cells in dHGP, as well as a higher CD8+/CD4+ ratio (cohort A). Flow cytometric analysis of fresh tumour tissues in cohort C confirmed these results; dHGP was associated with higher CD8+ and lower CD4+ T cell subsets, resulting in a higher CD8+/CD4+ ratio. Conclusion The tumour microenvironment of patients with dHGP is characterised by an increased and distinctly cytotoxic immune infiltrate, providing a potential explanation for their superior survival

Lymphatic marker podoplanin/D2-40 in human advanced cirrhotic liver- Re-evaluations of microlymphatic abnormalities

<p>Abstract</p> <p>Background</p> <p>From the morphological appearance, it was impossible to distinguish terminal portal venules from small lymphatic vessels in the portal tract even using histochemical microscopic techniques. Recently, D2-40 was found to be expressed at a high level in lymphatic endothelial cells (LECs). This study was undertaken to elucidate hepatic lymphatic vessels during progression of cirrhosis by examining the expression of D2-40 in LECs.</p> <p>Methods</p> <p>Surgical wedge biopsy specimens were obtained from non-cirrhotic portions of human livers (normal control) and from cirrhotic livers (LC) (Child A-LC and Child C-LC). Immunohistochemical (IHC), Western blot, and immunoelectron microscopic studies were conducted using D2-40 as markers for lymphatic vessels, as well as CD34 for capillary blood vessels.</p> <p>Results</p> <p>Imunostaining of D2-40 produced a strong reaction in lymphatic vessels only, especially in Child C-LC. It was possible to distinguish the portal venules from the small lymphatic vessels using D-40. Immunoelectron microscopy revealed strong D2-40 expression along the luminal and abluminal portions of the cell membrane of LECs in Child C-LC tissue.</p> <p>Conclusion</p> <p>It is possible to distinguish portal venules from small lymphatic vessels using D2-40 as marker. D2-40- labeling in lymphatic capillary endothelial cells is related to the degree of fibrosis in cirrhotic liver.</p

How useful is the assessment of lymphatic vascular density in oral carcinoma prognosis?

Abstract Background Lymphatic vessels are major routes for metastasis in head and neck squamous cell carcinoma (HNSCC), but lymphatic endothelial cells (LECs) are difficult to recognize in tumor histological sections. D2-40 stains podoplanin, a molecule expressed in LECs, however, the potential prognostic usefulness of this molecule is not completely understood in HNSCC. We aimed to investigate the value of assessing peritumoral and intratumoral lymphatic vessel density (LVD) as prognostic marker for HNSCC. Methods Thirty-one cases of HNSCC were stained for D2-40 and CD31. LVD and blood vessel density (BVD) were assessed by counting positive reactions in 10 hotspot areas at ×200 magnification. Results D2-40 was specific for lymphatic vessels and did not stain blood vascular endothelial cells. LECs showed more tortuous and disorganized structure in intratumoral lymphatic vessels than in peritumoral ones. No statistical differences were observed between peritumoral-LVD and intratumoral-LVD or between peritumoral-BVD and intratumoral-BVD. Tumor D2-40 staining was positively associated with lymphatic vessel invasion (p = 0.011). Conclusion LVD is a powerful marker for HNSCC prognosis. We found significant differences in peritumoral and intratumoral D2-40 immunoreactivity, which could have important implications in future therapeutic strategies and outcome evaluation

Validation of tissue microarray technology in squamous cell carcinoma of the esophagus

Tissue microarray (TMA) technology has been developed to facilitate high-throughput immunohistochemical and in situ hybridization analysis of tissues by inserting small tissue biopsy cores into a single paraffin block. Several studies have revealed novel prognostic biomarkers in esophageal squamous cell carcinoma (ESCC) by means of TMA technology, although this technique has not yet been validated for these tumors. Because representativeness of the donor tissue cores may be a disadvantage compared to full sections, the aim of this study was to assess if TMA technology provides representative immunohistochemical results in ESCC. A TMA was constructed containing triplicate cores of 108 formalin-fixed, paraffin-embedded squamous cell carcinomas of the esophagus. The agreement in the differentiation grade and immunohistochemical staining scores of CK5/6, CK14, E-cadherin, Ki-67, and p53 between TMA cores and a subset of 64 randomly selected donor paraffin blocks was determined using kappa statistics. The concurrence between TMA cores and donor blocks was moderate for Ki-67 (κ = 0.42) and E-cadherin (κ = 0.47), substantial for differentiation grade (κ = 0.65) and CK14 (κ = 0.71), and almost perfect for p53 (κ = 0.86) and CK5/6 (κ = 0.93). TMA technology appears to be a valid method for immunohistochemical analysis of molecular markers in ESCC provided that the staining pattern in the tumor is homogeneous

Caveolin-1 expression in benign and malignant lesions of the breast

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Lymph vascular invasion in invasive mammary carcinomas identified by the endothelial lymphatic marker D2-40 is associated with other indicators of poor prognosis

<p>Abstract</p> <p>Background</p> <p>Immunohistochemical studies of lymphatic vessels have been limited by a lack of specific markers. Recently, the novel D2-40 antibody, which selectively marks endothelium of lymphatic vessels, was released. The aim of our study is to compare lymphatic and blood vessel invasion detected by hematoxylin and eosin (H&E) versus that detected by immunohistochemistry, relating them with morphologic and molecular prognostic factors.</p> <p>Methods</p> <p>We selected 123 cases of invasive mammary carcinomas stratified into three subgroups according to axillary lymph node status: macrometastases, micrometastases, and lymph node negative. Lymphatic (LVI) and blood (BVI) vessel invasion were evaluated by H&E and immunohistochemistry using the D2-40 and CD31 antibodies, and related to histologic tumor type and grade, estrogen and progesterone receptors, E-cadherin, Ki67, p53, and Her2/<it>neu </it>expression.</p> <p>Results</p> <p>LVI was detected in H&E-stained sections in 17/123 cases (13.8%), and in D2-40 sections in 35/123 cases (28.5%) (Kappa = 0.433). BVI was detected in H&E-stained sections in 5/123 cases (4.1%), and in CD31 stained sections in 19/123 cases (15.4%) (Kappa = 0.198). LVI is positively related to higher histologic grade (p = 0.013), higher Ki67 expression (p = 0.00013), and to the presence of macrometastases (p = 0.002), and inversely related to estrogen (p = 0.0016) and progesterone (p = 0.00017) receptors expression.</p> <p>Conclusion</p> <p>D2-40 is a reliable marker of lymphatic vessels and is a useful tool for lymphatic emboli identification in immunostained sections of breast carcinomas with higher identification rates than H&E. Lymphatic vessel invasion was related to other features (high combined histologic grade, high Ki67 score, negative hormone receptors expression) associated with worse prognosis, probable reflecting a potential for lymphatic metastatic spread and aggressive behavior.</p