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

BACKGROUND: Classical Hodgkin's 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 C1Qalpha, C1Qbeta 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.

Effect of all-trans retinoic acid on chemotherapy induced apoptosis and down-regulation of Bcl-2 in human myeloid leukaemia CD34 positive cells.

Acute myeloid leukaemia (AML) is a heterogeneous malignant disease in which disease progression at the level of CD34 positive cells has a major impact in drug resistance and relapse. The multi-drug resistance (MDR1) gene product, P-glycoprotein is expressed mainly in CD34 positive AML cells and Bcl-2 is expressed simultaneously with several putative drug resistance parameters in these cells. Bcl-2 over-expression is associated with CD34 positivity, poor response to chemotherapy and reduced overall survival in AML patients. Recently, all-trans retinoic acid (RA) has been reported to enhance cytarabine-induced apoptosis and downregulate Bcl-2 in several human myeloid leukaemia CD34 negative cells. The two CD34 positive human myeloid leukaemia cell lines: KG1 and KGla have the unique feature of expressing significant functional P-glycoprotein. Thus, the efficacy of RA in enhancing cytrabine- and fludarabine-induced apoptosis and overcoming the resistance was examined in both KG1 (CD34+CD7-) and KGla (CD34+CD7+) human myeloid leukaemia cells in the present study. Both cytarabine and fludarabine induced a dose dependent increase in the number of apoptotic cells in both CD34 positive cell types. Interestingly, the cytarabine-induced apoptosis was significantly more than fludarabine-induced apoptosis in both cell types. All-trans RA alone failed to induce apoptosis or inhibit proliferation of either of the two human CD34 positive leukaemia cell types. However, RA enhanced cytarabine- or fludarabine-induced apoptosis and inhibition of proliferation in KG1 CD34+CD7- but not in KGla CD34+CD7+ myeloid leukaemia cells. As single agents, RA, cytarabine and fludarabine reduced Bcl-2 expression in a dose dependent manner in both cell types. Using a quantitative ELISA assay, the Bcl-2 protein concentration was reduced by 86 or 100%, after 72 h of treatment with 10 microM cytarabine or fludarabine, respectively, in both CD34 positive leukaemia cell types. The addition of RA to cytarabine enhanced its induced reduction of Bcl-2 in KG1 CD34+CD7- but not in KGla CD34+CD7+ human myeloid leukaemia cells. Meanwhile, RA failed to augment fludarabine-induced reduction of Bcl-2 in both cell types. In conclusion, the present results suggest a potential role for the combination of RA and cytarabine in the treatment of refractory and/or relapsed AML patients with CD34+CD7- but not CD34+CD7+ blast cells.

Frequent expression of the tumor necrosis factor receptor-associated factor 1 in latent membrane protein 1-positive posttransplant lymphoproliferative disease and HIV-associated lymphomas.

The tumor necrosis factor receptor-associated factor 1 (TRAF1) participates in the signal transduction of various members of the tumor necrosis factor receptor (TNFR) family, including TNFR2, CD40, CD30, and the Epstein-Barr virus (EBV)-encoded latent membrane protein 1 (LMP1). In vitro, TRAF1 is induced by LMP1, and previous studies have suggested that expression of TRAF1 is higher in EBV-associated tumors than in their EBV-negative counterparts. To determine whether this was the case in posttransplant lymphoproliferative disease (PTLD) and related disorders, we used immunohistochemistry to analyze expression of TRAF1 in a total of 42 such lesions arising in a variety of immunosuppressive states. The specimens consisted of 22 PTLD lesions, 18 acquired immunodeficiency syndrome-associated lymphomas, including 6 primary central nervous system lymphomas, and 2 cases of Hodgkin disease. The presence of latent EBV infection was determined by EBER in situ hybridization, and expression of EBV-LMP1 was detected by immunohistochemistry. Latent EBV infection, as determined by a positive EBER signal, was detected in 36 of 42 tumors. Of the EBER-positive specimens, 30 of 36 also expressed LMP1. Twenty-four of 30 LMP1-positive tumors, including both Hodgkin disease specimens, expressed TRAF1, compared with only 3 of 12 LMP1-negative tumors. This difference was statistically significant (P <.005). These results show frequent expression of TRAF1 at the protein level in LMP1-positive PTLD and related disorders and suggest an important role for LMP1-mediated TRAF1 signaling in the pathogenesis of EBV-positive tumors arising in immunosuppressive states.

Down-regulation of ATM protein in HRS cells of nodular sclerosis Hodgkin's lymphoma in children occurs in the absence of ATM gene inactivation.

The tumour component of classical Hodgkin's lymphoma (cHL), Hodgkin Reed-Sternberg (HRS) cells, are believed to be derived from germinal centre (GC) B cells but intriguingly display a characteristic loss of B cell receptor (BCR) expression. The precise mechanisms by which BCR-negative HRS cell progenitors survive negative selection during the GC reaction remain obscure. Individuals with ataxia telangiectasia, caused by biallelic inactivation of the DNA damage response gene, ataxia telangiectasia mutated (ATM), have a higher risk of cHL development. Here we show that, in contrast to normal GC B cells that expressed low but detectable ATM protein, ATM protein was not detected in HRS cells of 17/18 cases of paediatric cHL, all but one with nodular sclerosis (NS) subtype. A comprehensive analysis of the ATM gene in microdissected HRS cells of nine representative tumours showed no evidence of either loss of heterozygosity or consistent pathogenic mutations. Furthermore, bisulphite sequencing of the ATM promoter from HRS cells of five tumours also revealed the absence of hypermethylation. Since our microarray data suggested significantly reduced ATM transcription in HRS cells compared to GC B cells, we conclude that loss of ATM expression could be the result of alterations in upstream regulators of ATM transcription. Importantly, ATM loss in paediatric cHLs has clinical implications and could be potentially exploited to guide future, less toxic, tumour-specific treatments.

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 Burkitt's lymphoma, Hodgkin's 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.

Zonal rotor purification and characterization of "mannosomes": a tubular membrane system in gastropod mollusc digestive gland.

Mannitol oxidase (manox) is an H2O2-generating oxidase apparently unique to molluscs and especially abundant in alimentary tissues. In the digestive gland it is localized to an organelle ("mannosome") that forms an unusual tubular membrane system. We have developed a novel centrifugation procedure for >100-fold purification of these membranes in 20% yield from approximately 30 g of digestive gland of the slug Arion ater. Mannosomes from several other gastropod species are also substantially purified by the procedure. Four successive density gradient separations are employed which minimize structural damage by exploiting near isosmotic conditions early on and by completely avoiding traumatic pelleting and resuspension. Plasma membrane contamination is reduced by digitonin-induced density perturbation. The purified preparation is characterized by a predominant 68-kDa integral membrane protein and retains the in situ appearance of hexagonally arranged tubules with an enveloping outer membrane.

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 Hodgkin's 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.

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 Hodgkin's disease correlates with EBV status and survival and to investigate whether LMP-1 expression affects MMP-9 concentrations in the Hodgkin's disease cell line, L428. METHODS: MMP-9 expression was measured by means of immunohistochemistry in a series of Hodgkin's 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 Hodgkin's 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 Hodgkin's 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 Hodgkin's disease cell lines, whereas low or undetectable amounts of MMP-9 were found in the L591 Hodgkin's disease cell line. Induction of LMP-1 expression in the Hodgkin's 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 Hodgkin's disease tumours and by the Hodgkin's disease cell lines, L428 and KMH2. However, this expression does not appear to be related either to LMP-1 values or to survival.

Reactivity and isotype profiling of monoclonal antibodies using multiple antigenic peptides.

The characterisation of monoclonal antibodies (MAbs) is essential for the development of assay systems particularly where antigens have been developed using synthetic peptides. Indeed some peptide-carrier conjugates fail to induce immune responses and may not generate antibodies that bind to native protein. As an alternative to peptide-carrier conjugates, multiple antigenic peptides (MAPs) have been used for immunization strategies, but with little regard to the characteristics of the MAbs produced. In this study, we used 3 MAPs of Epstein-Barr virus (EBV) latent membrane protein 1 (LMP1) to immunise BALB/c mice. Overall, the polyclonal antibody responses from tail bleeds showed that MAPs evoked B-cell responses. However, on screening 144 hybridomas, 24 MAb supernatants exhibited weak to moderate reactivity in enzyme-linked immunosorbant assay (ELISA) and against cell cytospin preparations (B95.8 and AG876 LCL), respectively. Isotype profiling of hybridoma supernatants also showed that 11 out of 24 were IgM. Further characterization of 6 MAbs in Western blotting showed reactivity to recombinant LMP1 and only one MAb (B28D) showed weak reactivity to the malignant cells (Hodgkin/Reed-Sternberg; HRS cells) of an EBV+ Hodgkin's lymphoma using paraffin-embedded tissue. It is probable that these MAPs failed to augment T-cell help and contributed to the production of low affinity (IgM) antibodies. These observations may be of importance to future immunization strategies, where MAPs are used in the production of monoclonal reagents.

Absence of prolactin gene expression in colorectal cancer.

AIMS: Previous studies documenting hyperprolactinaemia in patients with colorectal cancer have suggested that the tumour is the source of hormone production. The aim of this study was to determine the frequency of hyperprolactinaemia in patients with colorectal cancer before, during, and after surgery, and also to determine whether prolactin is produced by these tumours. METHODS: Serum prolactin concentrations were measured in 20 patients with colorectal cancer before, during, and after surgical resection of their tumours. Samples taken during surgery included peripheral venous blood and blood taken from the main veins draining the tumour. To determine whether the tumour was responsible for the production of prolactin in these patients, paraffin wax embedded sections of tumour specimens were subjected to immunohistochemistry and western blotting using a monoclonal antibody to prolactin. RESULTS: Five patients (three women, two men) had preoperative prolactin concentrations above the normal reference range, although this increase was of clinical importance in only two. After surgical resection of their tumours, prolactin concentrations remained high in both patients. All 20 patients had greatly raised prolactin values at the time of surgery, irrespective of whether this was measured in peripheral blood or in blood taken from veins draining the tumour. All 20 colorectal cancer tissue samples, including those with raised preoperative and/or postoperative prolactin concentrations, were negative for prolactin staining. Frozen tissue was also available in four cases. The absence of prolactin gene expression in these four tumours was confirmed both by repeat immunohistochemistry and by western blotting. A further 50 colorectal cancer cases examined by immunohistochemistry alone were also unreactive for prolactin. CONCLUSIONS: The results of this study suggest that serum prolactin concentrations may occasionally be raised in colorectal cancer patients, but that the tumour is not the source of hormone production.