Karl R. N. Baumforth

Novel markers for differentiation of lobular and ductal invasive breast carcinomas by laser microdissection and microarray analysis

BackgroundInvasive ductal and lobular carcinomas (IDC and ILC) are the most common histological types of breast cancer. Clinical follow-up data and metastatic patterns suggest that the development and progression of these tumors are different. The aim of our study was to identify gene expression profiles of IDC and ILC in relation to normal breast epithelial cells.MethodsWe examined 30 samples (normal ductal and lobular cells from 10 patients, IDC cells from 5 patients, ILC cells from 5 patients) microdissected from cryosections of ten mastectomy specimens from postmenopausal patients. Fifty nanograms of total RNA were amplified and labeled by PCR and in vitro transcription. Samples were analysed upon Affymetrix U133 Plus 2.0 Arrays. The expression of seven differentially expressed genes (CDH1, EMP1, DDR1, DVL1, KRT5, KRT6, KRT17) was verified by immunohistochemistry on tissue microarrays. Expression of ASPN mRNA was validated by in situ hybridization on frozen sections, and CTHRC1, ASPN and COL3A1 were tested by PCR.ResultsUsing GCOS pairwise comparison algorithm and rank products we have identified 84 named genes common to ILC versus normal cell types, 74 named genes common to IDC versus normal cell types, 78 named genes differentially expressed between normal ductal and lobular cells, and 28 named genes between IDC and ILC. Genes distinguishing between IDC and ILC are involved in epithelial-mesenchymal transition, TGF-beta and Wnt signaling. These changes were present in both tumor types but appeared to be more prominent in ILC. Immunohistochemistry for several novel markers (EMP1, DVL1, DDR1) distinguished large sets of IDC from ILC.ConclusionIDC and ILC can be differentiated both at the gene and protein levels. In this study we report two candidate genes, asporin (ASPN) and collagen triple helix repeat containing 1 (CTHRC1) which might be significant in breast carcinogenesis. Besides E-cadherin, the proteins validated on tissue microarrays (EMP1, DVL1, DDR1) may represent novel immunohistochemical markers helpful in distinguishing between IDC and ILC. Further studies with larger sets of patients are needed to verify the gene expression profiles of various histological types of breast cancer in order to determine molecular subclassifications, prognosis and the optimum treatment strategies.

The Epstein-Barr virus and its association with human cancers.

The Epstein-Barr virus (EBV) has been linked to the development of a variety of human malignancies, including Burkitts lymphoma, Hodgkins disease, nasopharyngeal carcinoma, some T cell lymphomas, post-transplant lymphoproliferative disease, and more recently, certain cancers of the stomach and smooth muscle. This review summarizes these associations and in particular the role of the viral latent genes in the transformation process.

Expression of the Epstein-Barr Virus-Encoded Epstein-Barr Virus Nuclear Antigen 1 in Hodgkin's Lymphoma Cells Mediates Up-Regulation of CCL20 and the Migration of Regulatory T Cells

In approximately 50% of patients with Hodgkins lymphoma (HL), the Epstein-Barr virus (EBV), an oncogenic herpesvirus, is present in tumor cells. After microarray profiling of both HL tumors and cell lines, we found that EBV infection increased the expression of the chemokine CCL20 in both primary Hodgkin and Reed-Sternberg cells and Hodgkin and Reed-Sternberg cell-derived cell lines. Additionally, this up-regulation could be mediated by the EBV nuclear antigen 1 protein. The higher levels of CCL20 in the supernatants of EBV-infected HL cell lines increased the migration of CD4(+) lymphocytes that expressed FOXP3, a marker of regulatory T cells (Tregs), which are specialized CD4(+) T cells that inhibit effector CD4(+) and CD8(+) T cells. In HL, an increased number of Tregs is associated with the loss of EBV-specific immunity. Our results identify a mechanism by which EBV can recruit Tregs to the microenvironment of HL by inducing the expression of CCL20 and, by doing so, prevent immune responses against the virus-infected tumor population. Further investigation of how EBV recruits and modifies Tregs will contribute not only to our understanding of the pathogenesis of virus-associated tumors but also to the development of therapeutic strategies designed to manipulate Treg activity.

Demystified ... the polymerase chain reaction.

Since its initial description over twenty years ago the PCR has become one of the most valuable and flexible tools available to biomedical research. Subsequently, refinements and modifications to the basic approach, many of which have been described in this review, have enabled the application of the PCR to many areas of diagnostic medicine and have ensured its rapid acceptance as a routine test in many pathology disciplines. The growing importance of molecular approaches to the diagnosis of disease, particularly in histopathology, will continue to secure an ever expanding role for the PCR in diagnostic pathology.

Expression of the tumour necrosis factor receptor‐associated factors 1 and 2 in Hodgkin's disease

The tumour necrosis factor receptor‐associated factors (TRAFs) 1 and 2 participate in the signal transduction of various members of the tumour necrosis factor receptor (TNFR) family, including TNFR1, TNFR2, CD40, CD30, and the Epstein–Barr virus (EBV)‐encoded latent membrane protein‐1 (LMP1). Previous in situ hybridization studies have demonstrated TRAF1 transcripts in the malignant cells of the majority of Hodgkins disease (HD) tumours, where the expression of TRAF1 was higher in EBV‐associated tumours than in their EBV‐negative counterparts. In order to determine whether TRAF1 and also TRAF2 were expressed at the protein level in HD and whether there was any relationship to EBV status, immunohistochemistry has been used to detect these proteins in a series of HD specimens. TRAF1 protein was detected more frequently in Hodgkin/Reed–Sternberg (HRS) cells from EBV‐positive tumours than in their EBV‐negative counterparts. This difference was statistically significant (p=0.01). In contrast, TRAF2 expression by HRS cells appeared to be independent of EBV status. Using a sequential labelling approach, co‐localization of LMP1 with either TRAF1 or TRAF2 was also demonstrated in HRS cells from EBV‐positive tumours. Copyright

Three-dimensional culturing of the Hodgkin lymphoma cell-line L1236 induces a HL tissue-like gene expression pattern

To overcome some limitations of in vitro established cell-line tumor models for Hodgkin lymphoma (HL), we explored whether culturing in a three-dimensional (3D) matrix could improve the quality of the model. We used a novel designer peptide based self-organizing matrix. The gene expression profile of the 3D-cultured HL derived cell-line L1236 was compared with that of suspension-cultured (2D) L1236, as well as to the gene expression profile found in HL tumor samples. To validate our results we also included a gene expression data set of laser captured Hodgkin-Reed – Sternberg (H-RS) cells. The gene expression profiles were analyzed using Affymetrix™ technology. We found that the 3D culture affected gene expression of a HL derived cell-line inducing a more tumor-related expression profile. 3D culture affected the expression of 500 genes in L1236, upregulating genes involved in immune response and apoptosis and downregulating genes involved in cell division. It also affected genes involved in actin filament polymerization.

Expression of the matrix metalloproteinase 9 in Hodgkin's disease is independent of EBV status

Background—In vitro the Epstein-Barr virus (EBV) encoded latent membrane protein 1 (LMP-1) has been shown to upregulate expression of matrix metalloproteinase 9 (MMP-9), a member of a family of zinc dependent endopeptidases that is believed to facilitate tumour invasion and metastasis by degradation of the extracellular matrix. Aim—To test whether the expression of MMP-9 in Hodgkins disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkins disease cell line, L428. Methods—MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkins disease tumours and this expression was correlated with EBV status and survival. The influence of LMP-1 on MMP-9 expression was also investigated in the Hodgkins disease cell line, L428. Results—MMP-9 expression was demonstrated in the malignant Hodgkin and Reed-Sternberg cells of all (n = 86) formalin fixed, paraffin wax embedded Hodgkins disease tumours examined. Although the intensity of MMP-9 immunostaining varied between cases, there was no correlation between MMP-9 expression and EBV status or survival. MMP-9 expression was also detected in a variety of non-malignant cells, including fibroblasts. MMP-9 was detected by zymography in the L428 and KMH2 Hodgkins disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkins disease cell line. Induction of LMP-1 expression in the Hodgkins disease cell line L428 did not result in a detectable increase in the values of MMP-9 as measured by zymography. Conclusions—These results demonstrate that MMP-9 is consistently expressed by the Hodgkin and Reed-Sternberg cells of Hodgkins disease tumours and by the Hodgkins disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.

Inflammation and tissue repair markers distinguish the nodular sclerosis and mixed cellularity subtypes of classical Hodgkin's lymphoma.

Background:Classical Hodgkins lymphoma (cHL), although a malignant disease, has many features in common with an inflammatory condition. The aim of this study was to establish the molecular characteristics of the two most common cHL subtypes, nodular sclerosis (NS) and mixed cellularity (MC), based on molecular profiling and immunohistochemistry, with special reference to the inflammatory microenvironment.Methods:We analysed 44 gene expression profiles of cHL whole tumour tissues, 25 cases of NS and 19 cases of MC, using Affymetrix chip technology and immunohistochemistry.Results:In the NS subtype, 152 genes showed a significantly higher expression, including genes involved in extracellular matrix (ECM) remodelling and ECM deposition similar to wound healing. Among these were SPARC, CTSK and COLI. Immunohistochemistry revealed that the NS-related genes were mainly expressed by macrophages and fibroblasts. Fifty-three genes had a higher expression in the MC subtype, including several inflammation-related genes, such as C1Qα, C1Qβ and CXCL9. In MC tissues, the C1Q subunits were mainly expressed by infiltrating macrophages.Conclusions and interpretations:We suggest that the identified subtype-specific genes could reflect different phases of wound healing. Our study underlines the potential function of infiltrating macrophages in shaping the cHL tumour microenvironment.

Evidence for a pathophysiological role of cysteinyl leukotrienes in classical Hodgkin lymphoma

Classical Hodgkin lymphoma (cHL) is characterized histologically by a minority of malignant Hodgkin Reed‐Sternberg cells surrounded by abundant inflammatory cells, generally believed to be of major importance in the pathophysiology of the disease. Here, we present data that link inflammatory cell‐derived arachidonic acid metabolites, the cysteinyl leukotrienes (CysLT), to the pathogenesis of cHL. Two HL cell lines, L1236 and KMH2, were shown to express functional CysLT1 receptors, responding with a robust calcium signal upon leukotriene (LT) D4 challenge. LTD4 stimulated protein release of tumor necrosis factor‐α, interleukin‐6 and ‐8 by L1236 cells and interleukin‐8 by KMH2 cells. Importantly, all these LTD4‐induced effects were blocked by the CysLT1 receptor‐specific antagonist zafirlukast. Immunohistochemical studies of cHL biopsies and microarray analysis of microdissected cells revealed that the CysLT1 receptor is expressed also by primary Hodgkin Reed‐Sternberg cells. As these cells are surrounded by CysLT‐producing eosinophils, macrophages and mast cells, our results suggest the CysLTs as mediators in the pathogenesis of cHL, contributing to the aberrant cytokine network of this lymphoma.

Microarray analysis of bicalutamide action on telomerase activity, p53 pathway and viability of prostate carcinoma cell lines.

Bicalutamide is a non‐steroidal anti‐androgen commonly used in the treatment of prostate carcinoma. We analysed the transcriptional response to bicalutamide treatment with the aim of explaining the inhibition of telomerase in the androgen‐sensitive cell line LNCaP and the effects of bicalutamide on the androgen‐insensitive cell line DU145. Cells treated with 80 μm bicalutamide in steroid‐depleted medium for 1 day were analysed in duplicate by Affymetrix Human Genome Focus Arrays. Response to bicalutamide in LNCaP cells was represented by downregulation of androgen‐regulated genes, activation of the p53 pathway and inhibition of telomerase, which was associated with downregulation of v‐myc avian myelocytomatosis viral oncogene homologue (MYC) and telomerase reverse transcriptase subunit. In DU145 cells we observed the influence of cell density on bicalutamide effectivity such that highly confluent cells showed lesser sensitivity than low confluent ones. In conclusion, we provide an explanation for telomerase inhibition after androgen receptor blockade in LNCaP cells and we also report activation of the p53 pathway in LNCaP cells and in‐vitro sensitivity to bicalutamide of low confluent androgen‐insensitive DU145 cells. These findings might have implications for both experimental and clinical research into prostate cancer. In particular, activation of the p53 pathway after treatment with 80 μm bicalutamide could justify usage of bicalutamide dosages higher than 150 mg daily in androgen‐sensitive carcinoma therapy.