The most common sub-variant of papillary thyroid carcinoma (PTC) is the so-called follicular variant (FVPTC), which is a particularly problematic lesion and can be challenging from a diagnostic viewpoint even in resected lesions. Although fine needle aspiration cytology is very useful in the diagnosis of PTC, its accuracy and utility would be greatly facilitated by the development of specific markers for PTC and its common variants. We used the recently developed Applied Biosystems 1700 microarray system to interrogate a series of 11 benign thyroid lesions and conditions and 14 samples of PTC (six with classic morphology and eight with follicular variant morphology). TaqMan® reverse transcriptase-polymerase chain reaction was used to validate the expression portfolios of 50 selected transcripts. Our data corroborates potential biomarkers previously identified in the literature, such as LGALS3, S100A11, LYN, BAX, and cluster of differentiation 44 (CD44). However, we have also identified numerous transcripts never previously implicated in thyroid carcinogenesis, and many of which are not represented on other microarray platforms. Diminished expression of metallothioneins featured strongly among these and suggests a possible role for this family as tumour suppressors in PTC. Fifteen transcripts were significantly associated with FVPTC morphology. Surprisingly, these genes were associated with an extremely narrow repertoire of functions, including the major histocompatibility complex and cathepsin families.

AIMS:Human herpesvirus 8 (HHV-8) has been identified in multicentric Castleman's disease and in angioimmunoblastic lymphadenopathies. However, the presence of the virus does not necessarily indicate an aetiological role in these conditions. This study investigates the cell types infected by HHV-8 in Castleman's disease and examines the correlation between HHV-8 and Castleman's disease lymph node angiogenesis.METHODS:Sixteen formalin fixed, paraffin wax embedded samples from patients with Castleman's disease (six multicentric, 10 solitary) were examined for the presence of HHV-8 using the polymerase chain reaction (PCR), non-isotopic in situ hybridisation, PCR in situ hybridisation (PCR-ISH), and real time quantitative TaqMan PCR to HHV-8 open reading frame 26 (ORF-26), and viral (v)-cyclin encoding regions. Vascularity was assessed using CD34, CD31, and factor VIII immunocytochemistry, and lymph nodes were scored as "low" or "high".RESULTS:Five multicentric Castleman's disease and two solitary Castleman's disease biopsies were positive for HHV-8. HHV-8 was identified in approximately 10% of intranodal B lymphocytes, in endothelial cells, and in subcapsular spindle cell proliferations. The copy number of HHV-8 was low at 10-50 copies/1000 cells. The highest copy number was in subcapsular spindle cells. There was no correlation between vascularity score and HHV-8 status.CONCLUSION:The preferential localisation of HHV-8 in subcapsular spindle cell proliferations (where early intranodal Kaposi's sarcoma initiates) and endothelial cells in Castleman's disease might finally explain the link between intranodal Kaposi's sarcoma and Castleman's disease.

In a period where the proportion of culture confirmed cases in the UK has been steadily declining, diagnosis by PCR has been used to increase the number of confirmed cases and provide additional epidemiological data. This report presents a comparative evaluation of the fluorogenic probe-based 5′ exonuclease assay (Taqman) using the Perkin-Elmer Applied Biosystems automated sequence detection system 7700 with previously reported polymerase chain reaction enzyme-linked immunosorbent (PCR ELISA) assays for the detection of meningococcal DNA in CSF, plasma and serum samples. Taqman assays developed were based on the detection of a meningococcal capsular transfer gene (ctrA), the insertion sequence IS1106 and the sialytransferase gene (siaD) for serogroup B and C determination and compared with similar assays in a PCR ELISA format. The Taqman ctrA assay was specific for Neisseria meningitidis, however the IS1106 assay gave false positive reactions with a number of non-meningococcal isolates. Sensitivity of the Taqman ctrA, IS1106 and siaD assays testing samples from culture-confirmed cases were 64, 69 and 50%, respectively, compared with 26, 67 and 43% for the corresponding PCR ELISA assays. Improvements to the DNA extraction procedure has increased the sensitivity to 93 and 91% for the TaqMan™ ctrA and siaD assays, respectively, compared to culture confirmed cases. Since the introduction of Taqman PCR a 56% increase in laboratory confirmed cases of meningococcal disease has been observed compared to culture only confirmed cases. The developed Taqman assays for the diagnosis of meningococcal disease enables a high throughput, rapid turnaround of samples with considerable reduced risk of contamination.

Background—Many lymph node abnormalities have been described in AIDS. These include opportunistic infections that sometimes result in spindle cell pseudotumours, Kaposi's sarcoma (KS), malignant lymphoma (Hodgkin's and nonHodgkin's), and florid reactive hyperplasia. Among these, reactive hyperplasia is the most common manifestation of AIDS related lymphadenopathy. Aim—To examine whether human herpesvirus 8 (HHV-8), the aetiological agent of KS, can be localised in AIDS related lymphadenopathy and whether its appearance in such nodes is predictive of Kaposi's sarcoma development. Methods—A series of human immunodeficiency virus (HIV) positive men (n = 21) with AIDS related lymphadenopathy who at the time of presentation had KS or subsequently developed KS (n = 5) were examined. The prevalence of HHV-8 was assessed in these patients using solution phase polymerase chain reaction (PCR), real time TaqMan quantitative PCR, and in cell amplification techniques (PCR in situ hybridisation (PCR-ISH) and labelled primer driven in cell amplification). Results—Using standard solution phase PCR in a nested format, only two of the 21 patients with AIDS related lymphadenopathy were positive for HHV-8. The lymph node of one of these patients contained KS lesions. Three HHV-8 positive patients were identified using TaqMan PCR (the original two positive patients and one additional patient). All of the positive patients either subsequently developed KS (n = 2) or had KS at the time of diagnosis (n = 1). Two additional patients subsequently developed KS, but were negative for HHV-8 by solution phase PCR and TaqMan PCR. Using PCR-ISH, HHV-8 amplicons were identified in some lymphoid cells (in one patient) and in spindle cells of the KS lesion in another. The positive lymphoid cells were predominantly concentrated in B cell areas of the affected lymph nodes, confirming the B cell tropism exhibited by HHV-8. Conclusions—The presence of HHV-8 in AIDS related lymphadenopathy is predictive of KS development and probably represents seeding of HHV-8 infected B cells from the peripheral blood. These findings support a role for HHV-8 in the pathobiology of KS.

AIMS: Human herpesvirus 8 (HHV-8) is now acknowledged as the infective cofactor in the pathogenesis of Kaposi's sarcoma. The mode by which HHV-8 causes Kaposi's sarcoma is unresolved and it is probable that it acts in conjunction with other factors including cytokines, anti-apoptosis proteins, and cell surface receptors. CD40, a cell membrane receptor belonging to the tumour necrosis factor receptor super family, promotes B cell survival and is expressed constitutively on endothelial cells. It is upregulated on cytokine treatment and has been documented recently in Kaposi's sarcoma. Because the HHV-8 genome contains cytokine homologues, this study investigated whether CD40 expression in Kaposi's sarcoma correlated with HHV-8 status, using a unique set of HHV-8 positive and negative specimens. METHODS: Twenty one paraffin wax embedded samples of Kaposi's sarcoma were selected, of which 18 were screened for the presence of HHV-8 using both conventional solution phase and TaqMan polymerase chain reaction (PCR). CD40 immunohistochemistry was assessed using a biotinylated amplification system. Staining was scored semiquantitatively. RESULTS: The results indicated that the expression of CD40 is independent of viral status, being present in both HHV-8 positive and negative specimens. CONCLUSIONS: This suggests that HHV-8 promotes Kaposi's sarcoma cell survival following infection by mechanisms other than those involving CD40.

BACKGROUND: Human herpesvirus 8 (HHV8) appears to be the agent responsible for Kaposi sarcoma. The mechanism remains undetermined but may involve cell cycle regulating genes including D type cyclins which are pivotal in cell cycle progression. Recent HHV8 genetic analysis has revealed the presence of a v-cyclin which is homologous to D type cyclins. AIMS: First, to assess whether there is an independent relation between endogenous cyclin D1 expression in Kaposi sarcoma and HHV8 status; second to determine whether v-cyclin mRNA expression varies with Kaposi sarcoma stage. METHODS: Cyclin D1 immunohistochemistry was performed on 17 paraffin embedded Kaposi sarcoma samples from 16 patients. HHV8 status was assessed in 15 of these using nested polymerase chain reaction (PCR) to ORF 26 and the newly described technique of TaqMan PCR. An additional 10 fresh Kaposi sarcoma samples (early and nodular) were examined for HHV8 v-cyclin RNA. RESULTS: One case, which did not contain amplifiable HHV8, showed strong cyclin D1 staining. The remaining cases were negative or weakly staining; v-cyclin transcript load was higher in early Kaposi sarcoma. CONCLUSIONS: While endogenous cyclin D1 expression is independent of HHV8 status, v-cyclin transcription is higher in early lesions, supporting the "viral hit" hypothesis.

Growing interest now focuses on improvements of in situ polymerase chain reaction (PCR) technology for the detection of DNA and RNA cellular sequences. In this study, reverse transcription PCR in situ hybridisation (RT PCR-ISH) was developed and used to determine gene expression of pyruvate dehydrogenase in a cell model system, using human peripheral blood lymphocytes (PBLs). The success of in cell RNA amplification depends on the type of cell/tissue fixation, cell permeabilisation, and the efficiency of reverse transcription and cDNA amplification. This paper presents new approaches to overcome the critical aspects of fixation, permeabilisation, and reverse transcription when performing in cell RNA amplification. A novel fixative, "Permeafix", possessing fixative and permeabilisation properties, was used for cell fixation procedures. "Permeafix" obviated the need for pre-amplification proteolysis, facilitating entry of PCR reagents to target sequences within the cell. In addition, a simple on step RNA in cell amplification protocol using recombinant Thermus thermophilus (rTth) DNA polymerase, which reverse transcribes mRNA efficiently to cDNA and then catalyses cDNA amplification, was used. The value of a semi-junctional primer system for in cell gene expression studies, without the need to perform DNase digestion, is demonstrated.

AIMS: Human herpes virus 8 (HHV-8) is the infectious agent implicated in the pathogenesis of Kaposi's sarcoma, although its mode of action is unclear. Recent work indicates that the HHV-8 genome encodes a viral Bcl-2 homologue (v-Bcl-2). The aim of this study was to explore Bcl-2 expression in Kaposi's sarcoma using a unique set of HHV-8 positive and negative cases, and to determine whether there is a relation with p53 expression. METHODS: Up to 18 specimens from 17 patients were selected. HHV-8 status was determined using nested polymerase chain reaction (PCR) to the open reading frame (ORF) 26, with further confirmation by TaqMan PCR. In addition, Bcl-2 and p53 immunohistochemistry were performed using standard protocols. RESULTS: The results suggest that Bcl-2 and p53 expression is independent of HHV-8 status. In addition, there does not appear to be a direct correlation with disease stage. CONCLUSIONS: HHV8 histopathogenesis is likely to be a multifactorial complex process, which may be mediated in part by viral genes and apoptosis regulating homologues.

AIMS:Kaposi's sarcoma is a vascular tumour of uncertain pathogenesis possibly caused by an infectious agent, identified in high risk groups. Accumulating solution phase polymerase chain reaction (PCR) and seroepidemiological data suggest that a previously undescribed herpes DNA virus (human herpesvirus 8 (HHV8)) is the causative agent. Using a unique cohort of early Kaposi's sarcoma, the precise cell type infected with HHV8 in such lesions was identified to elucidate further the role of HHV8 in the pathobiology of Kaposi's sarcoma.METHODS:Sixteen cases of early Kaposi's sarcoma (derived from skin and lymph node) were assessed for the presence of HHV8 using both standard solution phase PCR and TaqMan PCR to the KS330 Bam region of HHV8. In situ amplification was also performed on a selected group in an attempt to identify the candidate infected cells.RESULTS:Using both conventional solution phase and TaqMan PCR, 87% of cases were positive. In addition, HHV8 amplicons were localised in situ to endothelial and spindle cell proliferations in early Kaposi's sarcoma. The HHV8 viral load varied from lesion to lesion.CONCLUSIONS:The presence of HHV8 in early lesions supports a role for HHV8 in the pathogenesis of Kaposi's sarcoma. Coupled with recent seroepidemiological studies, these results suggest that HHV8 is the aetiological agent of Kaposi's sarcoma. Its precise interaction with other factors known to be involved in the development of Kaposi's sarcoma, including cytokines and anti-apoptosis genes, requires elucidation.