Single-centre experience of the macrophage activation marker soluble (s)CD163 - associations with disease activity and treatment response in patients with autoimmune hepatitis.

BACKGROUND: Autoimmune hepatitis (AIH) is characterised by liver inflammation with reversibility upon anti-inflammatory treatment. Soluble (s)CD163, a specific macrophage activation marker, is associated with inflammation in other liver diseases, but never investigated in AIH. AIM: To investigate sCD163 in patients with acute AIH and in complete and incomplete responders to standard anti-inflammatory pharmacotherapy, and during follow-up in treatment naive patients. METHODS: In a cross-sectional design, we studied 121 AIH patients (female/male 89/32, median age 49 years); of these, we prospectively studied 10 treatment naïve AIH patients during prednisolone treatment and tapering. Twenty patients had variant syndromes of AIH and primary biliary cholangitis or primary sclerosing cholangitis. sCD163 was compared with markers of disease activity, severity and treatment response. RESULTS: In the patients with acute AIH (n = 21), sCD163 was sixfold increased compared with the normalised levels in patients (n = 32) with complete response to standard treatment [9.5 (3.3-28.8) vs. 1.6 (0.8-3.2) mg/L, P < 0.01)], while the patients (n = 27) with incomplete response had higher sCD163 [2.2 (1.3-7.9), P < 0.05] than the complete responders. sCD163 was positively associated with ALAT, IgG and bilirubin (rho: 0.45-0.59, P < 0.001, all), and negatively to external coagulation factors (rho:-0.34, P < 0.001). In the treatment naïve patients, sCD163 fell during high-dose prednisolone treatment and tapering. Immunohistochemical staining confirmed increased CD163 expression in liver biopsies from patients with acute AIH. CONCLUSIONS: sCD163 was markedly elevated in AIH in the acute phase, normalised by successful treatment in complete responders, but remained higher in the incompletely responding cases. Our results demonstrate macrophage activation in AIH paralleling disease activity, severity and treatment response, suggesting a role for macrophage activation in AIH.

E- to N-cadherin switch in melanoma is associated with decreased expression of phosphatase and tensin homolog and cancer progression.

BACKGROUND: Cadherin switch in melanoma, with loss of E-cadherin and upregulation of N-cadherin, is believed to underlie melanoma cell detachment from the epidermis and promotion of dermal and vascular melanoma invasion. The tumour suppressor phosphatase and tensin homolog (PTEN) has been suggested as a potential regulator of this cadherin switch. OBJECTIVES: To study the biological and clinical implications of cadherin switch and PTEN expression in melanoma progression. METHODS: We constructed tissue microarrays from primary tumour samples from 394 formalin-fixed paraffin-embedded melanomas diagnosed between 2001 and 2006. Median follow-up was 10 years. Tissue microarray sections were stained by immunohistochemistry for E-cadherin, N-cadherin and PTEN, and expression was analysed semiquantitatively. RESULTS: Breslow thickness correlated strongly with reduced/absent PTEN expression (P < 0·0001), low E-cadherin expression (P < 0·0001), high N-cadherin expression (P < 0·0001) and the combination of low E-cadherin and high N-cadherin expression (cadherin switch profile; P = 0·001). There was a significant association between reduced/absent PTEN and the presence of the cadherin switch profile (P = 0·03). In univariate analyses, low E-cadherin expression significantly predicted an adverse overall relapse-free (P = 0·04), melanoma-specific (P = 0·03) and distant-metastasis-free (P = 0·01) survival; reduced/absent PTEN predicted an adverse overall relapse-free survival (P = 0·006), and the cadherin switch profile predicted adverse melanoma-specific (P = 0·005) and distant-metastasis-free (P = 0·01) survival. In multivariate analysis, the cadherin switch profile was an independent prognostic marker of melanoma-specific (P = 0·04) and distant-metastasis-free survival (P = 0·02). CONCLUSIONS: Cadherin switch and reduced/absent PTEN expression are associated in melanoma, and both factors may play important roles in the progression of melanoma.

EBV-associated gastric carcinoma in high- and low-incidence areas for nasopharyngeal carcinoma.

BACKGROUND: Approximately 10% of gastric carcinomas are associated with Epstein-Barr virus (EBV). The Inuit in Greenland have a high incidence of EBV-associated nasopharyngeal carcinoma. METHODS: We conducted a population-based case-control study comparing gastric carcinomas in Greenland and in Denmark. RESULTS: The prevalence rate of EBV-associated gastric carcinomas was 8.5% in both populations. CONCLUSION: The findings of this study argue against a general susceptibility to EBV-associated carcinomas among the Inuit.

Tamoxifen's protection against breast cancer recurrence is not reduced by concurrent use of the SSRI citalopram.

Tamoxifen remains an important adjuvant therapy to reduce the rate of breast cancer recurrence among patients with oestrogen-receptor-positive tumours. Cytochrome P-450 2D6 metabolizes tamoxifen to metabolites that more readily bind the oestrogen receptor. This enzyme also metabolizes selective serotonin reuptake inhibitors (SSRI), so these widely used drugs - when taken concurrently - may reduce tamoxifen's prevention of breast cancer recurrence. We studied citalopram use in 184 cases of breast cancer recurrence and 184 matched controls without recurrence after equivalent follow-up. Cases and controls were nested in a population of female residents of Northern Denmark with stages I-III oestrogen-receptor-positive breast cancer 1985-2001 and who took tamoxifen for 1, 2, or most often for 5 years. We ascertained prescription histories by linking participants' central personal registry numbers to prescription databases from the National Health Service. Seventeen cases (9%) and 21 controls (11%) received at least one prescription for the SSRI citalopram while taking tamoxifen (adjusted conditional odds ratio=0.85, 95% confidence interval=0.42, 1.7). We also observed no reduction of tamoxifen effectiveness among regular citalopram users (>or=30% overlap with tamoxifen use). These results suggest that concurrent use of citalopram does not reduce tamoxifen's prevention of breast cancer recurrence.

High-throughput immunophenotyping of 43 ferret lymphomas using tissue microarray technology.

To validate the use of the tissue microarray (TMA) method for immunophenotyping of ferret lymphomas, a TMA was constructed containing duplicate 1-mm cores sampled from 112 paraffin-embedded lymphoma tissue specimens obtained from 43 ferret lymphoma cases. Immunohistochemical (IHC) expression of CD3, CD79alpha, and Ki-67 (MIB-1) was determined by TMA and whole mount (WM) staining of each individual case for result comparison. There was a high correlation between CD79alpha and CD3 results comparing ferret TMA and WM sections (kappa statistic 0.71-0.73 for single-core TMA and 0.79-0.95 for duplicate-core TMA) and between continuous data from Ki-67 staining of ferret TMA sections and WM sections (concordance correlation coefficients 0.77 for single cores and 0.87 for duplicate cores). Subsequently, a panel of commercially available antibodies was applied to the TMA for the analysis of expression in ferret lymphomas. The results of this study confirmed previously published results suggesting specific cross-reactivity of the applied IHC markers (CD3, CD79alpha, Ki67) with ferret lymphoma tissue. Other IHC markers (CD45Ro, bcl2, bcl10, MUM1, CD30, vimentin) were also expressed in subsets of the included ferret lymphomas. Further studies are necessary to determine the usefulness of these markers for diagnostic and prognostic evaluation of ferret lymphomas. In conclusion, the TMA technology was useful for rapid and accurate analysis of protein expression in large archival cohorts of ferret lymphoma cases.

[Undifferentiated salivary gland carcinomas]. / Undifferenzierte Karzinome der Speicheldrüsen.

Undifferentiated salivary gland carcinomas may be divided into small cell and large cell types. Among large cell undifferentiated carcinomas, lymphoepithelial carcinomas have to be distinguished, the latter of which are endemic in the Arctic regions and southern China where virtually all cases of these tumors are associated with the Epstein-Barr virus (EBV). Association with EBV may also be observed in sporadic cases, and detection of EBV gene products may aid their diagnosis. Immunohistology may be employed to resolve the differential diagnosis of undifferentiated salivary gland carcinomas, comprising malignant lymphomas, amelanotic melanomas, Merkel cell carcinomas, and adenoid cystic carcinomas, in particular in small biopsy materials. Because of the rarity of undifferentiated salivary gland carcinomas, the differential diagnosis should always include metastases of undifferentiated carcinomas arising at other primary sites, particularly when expressing the thyroid transcription factor-1 (TTF-1).

Is posttransplant lymphoproliferative disorder (PTLD) caused by any specific immunosuppressive drug or by the transplantation per se?

BACKGROUND: An association between posttransplant lymphoproliferative disorder (PTLD) and cyclosporine A (CsA) and OKT3 has often been postulated on the basis of retrospective studies, although a randomized study with PTLD as the endpoint will probably never be performed. Because focus on PTLD coincided with the use of these drugs, a bias could be suspected. METHODS: In a retrospective, nonrandomized study, we reevaluated all lymphoma-like lesions arising in kidney-transplant patients grafted at our center during 1969 to 1998 and observed up to 2002. Case pathology was reviewed, and an association with Epstein-Barr virus (EBV) infection (and latency pattern) was assessed. RESULTS: We did not find any significant difference in the incidence of PTLDs when comparing the prednisolone/azathioprine, and CsA eras (P=0.89), the periods before or after OKT3 (P=0.61), and those before or after antilymphocyte globulin (ALG) (P=0.22). Occurrence time was shorter in the CsA (P=0.059), OKT3 (P=0.007), and ALG (P=0.007) eras. In the OKT3 era, 182 patients received, and 224 did not receive, OKT3; after the same observation time, there had been eight and five PTLDs, respectively (P=0.34). The use of mycophenolate mofetil (MMF) was associated with a reduction in the number of PTLDs (P=0.01). EBV was detected in 16 of 21 (76%) cases. CONCLUSIONS: We found no evidence to implicate any one drug regime preferentially in the development of PTLDs. The risk of developing PTLD seems to be a result of the whole transplantation process, which includes the antigenicity of the foreign graft, the immunosuppression resulting in inadequate cytotoxic T-cell activity, and the result of EBV infection. An important minority of cases are EBV negative.

Long-term follow-up of kidney transplant patients with posttransplant lymphoproliferative disorder: duration of posttransplant lymphoproliferative disorder-induced operational graft tolerance, interleukin-18 course, and results of retransplantation.

BACKGROUND: Posttransplant lymphoproliferative disorder (PTLD) can be resolved in many transplant patients by the reduction or cessation of immunosuppression, after which many grafts continue to function as the result of a form of operational tolerance. When graft function deteriorates, retransplantation may be an option. Cytokines such as interleukin (IL)-10 and IL-18 may play a role in PTLD tolerance induction and tumor regression. We report long-term follow-up on the duration of graft tolerance and the course of retransplantation in a series of patients who underwent kidney transplantation and demonstrated PTLD, and in whom we were able to perform IL-18 analyses. RESULTS: Patients were followed for up to 7 years after PTLD diagnosis. Treatment consisted of immunosuppression cessation with radiation therapy in cases with overt monomorphic lymphomas. All patients' PTLDs were resolved, and all patients but one (whose graft was removed) demonstrated a period of operational graft tolerance of up to 5 years. Five patients underwent retransplantation without sign of recurrence of the PTLD up to 3 years after transplantation. In the eight patients analyzed, IL-18 increased significantly during PTLD regression and follow-up in those with long-term operational tolerance. CONCLUSION: We report on a series of patients with resolved PTLDs demonstrating long-term recurrence-free survival, of whom most experienced a long period of operational graft tolerance. IL-18 seems to play a role in the resolution of the PTLDs. Five patients underwent retransplantation with standard immunosuppression without recurrence. A previous diagnosis of PTLD should not be regarded as a contraindication for later retransplantation.

Intense inflammation in bladder carcinoma is associated with angiogenesis and indicates good prognosis.

The aim of this study was to investigate the prognostic influence of microvessel density using the hot spot method in 107 patients diagnosed with transitional cell carcinoma of the bladder. In each case, inflammation was found in the invasive carcinoma, therefore we classified the degree of inflammation as minimal, moderate or intense. Microvessel density was then reevaluated in each tumour in areas corresponding to these three categories. Median microvessel density irrespective of degree of inflammation was 71. Areas of minimal, moderate and intense inflammation were found in 48, 92 and 32 tumours. Microvessel density increased significantly with increasing degree of inflammation. Disease-specific survival was improved if areas of intense inflammation were present in the carcinoma (P=0.004). High microvessel density, irrespective of the degree of inflammation, was associated with a significantly better disease-specific survival (P=0.01). Multivariate analysis using death of disease as endpoint demonstrated an independent prognostic value of N-classification (N0, hazard ratio (HR)=1 vs N1, HR=2.89 (range, 1.52-5.52) vs N2, HR=3.61 (range, 1.84-7.08)), and intense inflammation, HR=0.48 (range, 0.24-0.96). Malignancy grade, T classification and microvessel density were not independent significant markers of poor outcome. In conclusion, inflammation was significantly correlated to microvessel density, and areas of intense inflammation were an independent marker of good prognosis.

Interobserver variation in interpretation of serial liver biopsies from patients with chronic hepatitis C.

Reliable and accurate assessment of liver histopathology in patients with chronic hepatitis C is important for decision regarding treatment and for evaluation of therapy. However, little data on interobserver variation have been published. In this study, five specialist histopathologists evaluated 46 liver biopsies from 20 patients treated with interferon-alpha. Knodell's and Ishak's scoring systems, De Groote's classification and a four level general necro-inflammatory activity score (GNAS) were applied. Besides kappa statistics, slide by slide analysis was performed. We defined an acceptable slide by slide agreement as eight of ten observer pairs agreed on 80% of the slides. The best agreement was seen for Knodell's and Ishak's fibrosis score, De Groote's classification and GNAS (mean weighted kappa (kappa(w)) = 0.49, 0.51, 0.50 and 0.44, respectively). By condensing data from Knodell's and Ishak's scores to presence or absence of cirrhosis and piecemeal necrosis respectively, concordance was substantial concerning cirrhosis (mean kappa = 0.69 and 0.72, respectively) but only moderate concerning piecemeal necrosis (mean kappa = 0.40 and 0.39, respectively). Slide by slide analysis showed the highest agreement on Knodell's fibrosis score and GNAS; only one point of difference in score was to be accepted to obtain 'eight of ten' agreement. In contrast, five points of difference were necessary to accept in order to reach the same agreement for Knodell's total activity score. Moreover, in serial biopsies the GNAS was sufficient to detect changes in disease activity following treatment. Thus, a simple scoring system with four category scales was reproducible and sufficient for detection of therapy induced changes.

Epstein-Barr virus (EBV) in Chinese pediatric Hodgkin disease: Hodgkin disease in young children is an EBV-related lymphoma.

BACKGROUND: The Epstein-Barr virus (EBV) is thought to be involved in the pathogenesis of some Hodgkin disease (HD) cases. EBV may be associated particularly with childhood HD, a disease rare in the West compared with developing countries. In this study, a large series of Chinese pediatric HD cases has been examined to determine the age-specific prevalence of EBV. METHODS: Paraffin sections from 104 pediatric and 52 adult Chinese HD cases were examined for EBV-RNA (EBERs) and EBV latent membrane protein-1. RESULTS: Most pediatric cases arose in boys and showed an histology of mixed cellularity. Prominent interfollicular involvement was seen frequently in the childhood cases. EBV was identified in tumor cells in 113 of 156 (72%) HD cases but was more frequent in pediatric cases (93 of 104; 89%) compared with adult cases (20 of 52; 38%) (P < 0.01; chi-square test). EBV was found in 86 out of 91 (95%) cases in children aged 3-10 years and in 7 out of 13 (54%) cases in children aged 11-14 years (P < 0.01; chi-square test). The virus was less frequent in cases in young adults than in old adults, although this trend was not significant (P > 0.05; chi-square test). Pediatric HD was associated with EBV irrespective of histologic subtype. In adults, EBV was associated more frequently with mixed cellularity than with other subtypes. CONCLUSION: To the authors' knowledge, this is to date the largest series of pediatric HD cases studied for EBV. Study findings provided further evidence that HD is etiologically heterogeneous. The authors believe that pediatric HD now should be regarded as a distinctive EBV-related lymphoma.

Laser-assisted microdissection of membrane-mounted paraffin sections for polymerase chain reaction analysis: identification of cell populations using immunohistochemistry and in situ hybridization.

Laser microbeam microdissection (LMM) is an increasingly important method for obtaining pure cell samples for genetic and proteomic analysis. Immunohistochemistry (IHC) and in situ hybridization (ISH) are useful techniques for targeting specific cell populations for microdissection but are difficult to apply with the tissue support membranes often used during LMM. Using detection of cytokeratins and Epstein-Barr virus gene products in head and neck carcinoma as a model, we describe optimized protocols for membrane and section preparation and for low temperature antigen retrieval that allow IHC and ISH to be used reliably on membrane mounted paraffin tissue sections. Visualization of cellular targets was markedly improved by staining and this could be further improved using a variety of optical media before microdissection. Tissue fragments thus stained were suitable for subsequent polymerase chain reaction analysis of extracted DNA using standard techniques. These IHC and ISH procedures are generally applicable and will be useful for detecting a wide range of antigens and nucleic acids in paraffin sections in conjunction with LMM.

Epstein--Barr virus gene polymorphisms in Chinese Hodgkin's disease cases and healthy donors: identification of three distinct virus variants.

Epstein--Barr virus (EBV) is associated with several malignancies. Specific EBV gene variants, e.g. the BamHI f configuration, a C-terminal region 30 bp deletion in the latent membrane protein-1 (LMP1) gene (del-LMP) and the loss of an XhoI site in LMP1 (XhoI-loss), are found in Chinese cases of nasopharyngeal carcinoma (NPC), suggesting that EBV sequence variation may be involved in oncogenesis. In order to understand better the epidemiology of these EBV variants, they were studied in virus isolates from EBV-positive Chinese cases of Hodgkin's disease (HD; n=71) and donor throat washings from healthy CHINESE: Sequencing was performed of 15 representative EBV isolates, including the first analysis of the LMP1 promoter in Asian wild-type EBV isolates. The following observations were made. (i) Three EBV LMP1 variants were identified, designated Chinese groups (CG) 1--3. In both EBV-associated HD and in healthy Chinese, CG1-like viruses showing del-LMP1 and XhoI-loss were predominant. (ii) CG1viruses were distinct from European and African variants, suggesting that this profile is useful for epidemiological studies. (iii) Specific patterns of mutations were present in the LMP1 promoter in both CG1 and CG2. (iv) The BamHI f variant was not found in Chinese HD, in contrast to Chinese NPC and European HD. This study confirms that EBV isolates in Chinese HD and other tumours differ from those reported in Western cases. However, this reflects the predominant virus strain present in the healthy Chinese population, suggesting that these are geographically restricted polymorphisms rather than tumour-specific strains.

Patterns of angiogenesis in nonsmall-cell lung carcinoma.

BACKGROUND: Tumor angiogenesis plays a pivotal role in tumor growth, maintenance, and metastasis. The objective of the current study was to evaluate the prognostic value of estimates of tumor angiogenesis and vascular endothelial growth factor (VEGF) status in 143 primary tumors from patients who underwent radical surgery for nonsmall-cell lung carcinoma (NSCLC). METHODS: Tumor sections were stained by immunohistochemistry for CD34 and VEGF. Angiogenesis was estimated both by a modification of the method described by Weidner and by the use of a 25-point Chalkley eyepiece graticule. VEGF intensity was evaluated semiquantitatively in three groups of patients. The vascular data were correlated with histopathologic tumor type and grade, TNM classification, patient age, and the endpoint (death). RESULTS: The estimates of vascular score did not reveal any prognostic information. In 35 patients (24%), invasive tumor growth was identified with a highly ordered alveolar microvessel pattern. In parallel sections, the intensity of VEGF staining was weak in tumors that exhibited an alveolar microvessel pattern only, and it was more intense in tumors that demonstrated a mixed alveolar and diffuse angiogenic pattern. The 35 patients with alveolar microvessel pattern had a significantly better survival (P = 0.007). In a Cox multivariate analysis, the results demonstrated an independent bad prognostic value of high disease stage (P < 0.0001), adenocarcinoma (P = 0.004), greater age (P = 0.01), and angiogenic microvessel pattern (P = 0.01). CONCLUSIONS: The authors believe that the alveolar vascular pattern represented preexisting alveolar vessels, that is, the alveoli were filled up by tumor cells that exploited the existing highly vascular bed of the lungs. Therefore, this subgroup was characterized by tumor progression without the induction of angiogenesis. The current data do not support a significant prognostic role for tumor angiogenesis in patients who are diagnosed with NSCLC. This may have implications for therapy aimed at inhibiting tumor growth by the inhibition of angiogenesis.

Analysis of the Epstein-Barr virus (EBV) latent membrane protein 1 (LMP-1) gene and promoter in Hodgkin's disease isolates: selection against EBV variants with mutations in the LMP-1 promoter ATF-1/CREB-1 binding site.

AIMS: To study the distribution of Epstein-Barr virus (EBV) variants containing mutations in the latent membrane protein 1 (LMP-1) oncogene and promoter in EBV associated Hodgkin's disease and infectious mononucleosis compared with previous findings in asymptomatic EBV carriers. METHODS: Sequence analysis of the EBV LMP-1 promoter and gene in isolates from Danish patients with Hodgkin's disease (n = 61) and infectious mononucleosis (n = 10). RESULTS: Viruses (previously designated group D) that contain two mutations in the activating transcription factor/cAMP response element (ATF/CRE) in the LMP-1 promoter, which are known to decrease promoter activity greatly, were significantly less frequent in Hodgkin's disease than in both infectious mononucleosis (p = 0.0081) and asymptomatic EBV carriers (p = 0.0084). In some cases, the LMP-1 gene contained mutations in a recently identified cytotoxic T cell (CTL) epitope. Most viral isolates contained mutations shown to increase nuclear factor kappa B (NF-kappa B) activation and had one of two newly identified C-terminal activation regions 3 (CTAR-3) deleted. The exon 1 Xho-I restriction site in the LMP-1 gene could be lost through a range of different mutations. CONCLUSIONS: These findings indicate selection pressure against EBV strains with weak LMP-1 promoter activity in Hodgkin's disease and thus provide further strong circumstantial evidence for the pathogenic role of EBV (and LMP-1) in this disease. Mutation of the CTL epitope suggests immune selection of EBV strains. Many EBV isolates contain functionally important mutations in the LMP-1 gene. Loss of the Xho-I restriction site should not be used as a marker of specific LMP-1 variants.

Risk of Hodgkin's disease and other cancers after infectious mononucleosis.

BACKGROUND: Infectious mononucleosis, which is caused by the Epstein-Barr virus, has been associated with an increased risk for Hodgkin's disease. Little is known, however, about how infectious mononucleosis affects long-term risk of Hodgkin's disease, how this risk varies with age at infectious mononucleosis diagnosis, or how the risk for Hodgkin's disease varies in different age groups. In addition, the general cancer profile among patients who have had infectious mononucleosis has been sparsely studied. METHODS: Population-based cohorts of infectious mononucleosis patients in Denmark and Sweden were followed for cancer occurrence. The ratio of observed-to-expected numbers of cancers (standardized incidence ratio [SIR]) served as a measure of the relative risk for cancer. SIRs of Hodgkin's disease in different subsets of patients were compared with the use of Poisson regression analysis. All statistical tests including the trend tests were two-sided. RESULTS: A total of 1381 cancers were observed during 689 619 person-years of follow-up among 38 562 infectious mononucleosis patients (SIR = 1. 03; 95% confidence interval [CI] = 0.98-1.09). Apart from Hodgkin's disease (SIR = 2.55; 95% CI = 1.87-3.40; n = 46), only skin cancers (SIR = 1.27; 95% CI = 1.13-1.43; n = 291) occurred in statistically significant excess. In contrast, the SIR for lung cancer was reduced (SIR = 0.71; 95% CI = 0.58-0.86; n = 102). The SIR for Hodgkin's disease remained elevated for up to two decades after the occurrence of infectious mononucleosis but decreased with time since diagnosis of infectious mononucleosis (P: for trend <.001). The SIR for Hodgkin's disease tended to increase with age at diagnosis of infectious mononucleosis (P: for trend =.05). Following infectious mononucleosis, the SIR for Hodgkin's disease at ages 15-34 years was 3.49 (95% CI = 2.46-4.81; n = 37), which was statistically significantly higher than the SIR for any other age group (P: for difference =.001). CONCLUSION: The increased risk of Hodgkin's disease after the occurrence of infectious mononucleosis appears to be a specific phenomenon.

Human immunodeficiency virus-associated malignant lymphoma in eastern Denmark diagnosed from 1990-1996: clinical features, histopathology, and association with Epstein-Barr virus and human herpesvirus-8.

The clinicopathological features of human immunodeficiency virus (HIV)-associated lymphoma were investigated in a retrospective study of 85 adult patients in eastern Denmark diagnosed during the period 1990-1996. The possible pathogenetic role of Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) in these tumours was also studied. Seventy patients (82%) presented with extranodal disease and 26 (31%) had CNS involvement at diagnosis. Diffuse large cell B-cell lymphoma was the most frequent histological subtype, comprising 65 of 79 cases available for microscopic re-evaluation (82%) and including 20 of 23 evaluable patients with CNS lymphoma (87%). EBV RNA was demonstrated by in situ hybridization in 51 of 65 evaluable tumours (79%) and in 14 of 16 cases (88%) with CNS-lymphoma. Three cases showed a T-cell phenotype. The presence of HHV-8 DNA was analysed by PCR in 32 cases. A strong band consistent with tumour cell infection was detected in only one case, weaker bands being seen in 4 cases. None of these patients had primary effusion lymphomas. In conclusion, Danish AIDS-related lymphomas are of predominantly high-grade B-cell type with extranodal localization and atypical presentation. Our results provide further evidence that EBV plays a major role in the pathogenesis of large cell AIDS-related lymphoma, whereas HHV-8 does not appear to contribute significantly to the development of solid lymphomas in this group of patients.

EBNA-1 sequence variation in Danish and Chinese EBV-associated tumours: evidence for geographical polymorphism but not for tumour-specific subtype restriction.

The Epstein-Barr virus (EBV) nuclear antigen (EBNA)-1 is consistently expressed in EBV-associated tumours. Recently, EBNA-1 carboxy (C)-terminal sequence variants have been described based on the amino acid signature at codon 487, and designated prototype (P)-ala (identical to prototype B95.8 strain), P-thr, variant (V)-val, V-leu, and V-pro. These studies suggest that certain EBNA-1 variants show selective cell tropism and may be preferentially associated with different EBV-positive malignancies; for example, in contrast to P-ala subtypes, V-val appeared to be restricted to the oral compartment and to be associated with undifferentiated nasopharyngeal carcinoma (NPC). To test the hypothesis that V-val subtypes are restricted in distribution, EBNA-1 variants were investigated in NPC and throat washings (TWs) from a low (Denmark) and a high (China) NPC risk area. For comparison, cases of Hodgkin's disease (HD) were also studied. V-val was found to be the dominant EBNA-1 subtype, not only in Chinese TWs and NPC biopsies, but also in Chinese HD. Furthermore, V-val was not detected in any of the Danish NPC biopsies or TW samples. These findings show that V-val is not associated with NPC, nor is it restricted to the oral compartment, but rather that it represents a dominant Asian EBNA-1 subtype, both in EBV-associated malignancies and in the general population.

Detection of clonal B cells in microdissected reactive lymphoproliferations: possible diagnostic pitfalls in PCR analysis of immunoglobulin heavy chain gene rearrangement.

AIMS: To evaluate the specificity of standard and fluorescence based (Genescan) polymerase chain reaction (PCR) immunoglobulin heavy chain (IgH) gene rearrangement analysis in complete and microdissected paraffin wax embedded sections from lymphoid proliferations. METHODS: PCR IgH gene rearrangement analysis of whole sections and microdissected fragments (n = 62) from paraffin wax embedded reactive lymph nodes (n = 6) and tonsils (n = 3). Amplificant analysis used both standard methods and automated high resolution fluorescence based quantification and size determination using GENESCAN software. RESULTS: Whole tissue sections were consistently polyclonal in control experiments. IgH gene amplification was successful in 59 of 62 microdissected fragments; only two of 59 showed a polyclonal rearrangement pattern, the remainder being oligoclonal or monoclonal. Reanalysis was possible in 33 samples; six showed reproducible bands on gel analysis and satisfied accepted criteria for monoclonality. Use of high resolution gels with Genescan analysis improved sensitivity and band definition; however, three samples still appeared to be monoclonal. CONCLUSIONS: These results confirm that PCR based IgH gene rearrangement analysis is a sensitive and specific method for demonstrating B cell clonality in whole paraffin wax embedded sections. However, oligoclonal and monoclonal rearrangement patterns are regularly encountered in small tissue fragments from otherwise unremarkable reactive lymphoproliferations, possibly because of preferential priming or detection of local B cell clones. Data from clonal analysis of small, microdissected or lymphocyte poor samples must be evaluated critically. It is recommended that analyses should be run in parallel on at least two tissue specimens. Only reproducible bands present in more than one sample should be considered to be suggestive of neoplasia.