Diagnostic histological and cytological specimens are routinely stored in pathology department archives. These biobanks are a valuable research resource for many diseases, particularly if they can be linked to high quality population-based health registries, allowing large retrospective epidemiological studies to be carried out. Such studies are of significant importance, for example in the search for novel prognostic and predictive biomarkers in the era of personalized medicine. Denmark has a wealth of highly-regarded population-based registries that are ideally suited to conduct this type of epidemiological research. We describe two recent additions to these databases: the Danish National Pathology Registry (DNPR) and its underlying national online registration database, the Danish Pathology Data Bank (DPDB). The DNPR and the DPDB contain detailed nationwide records of all pathology specimens analyzed in Denmark since 1997, and an incomplete but nonetheless valuable record of specimens from some pathology departments dating back to the 1970s. The data are of high quality and completeness and are sufficient to allow precise and efficient localization of the specimens. We describe the relatively uncomplicated procedures required to use these pathology databases in clinical research and to gain access to the archived specimens.

Sequence variations in the Epstein-Barr virus (EBV) encoded latent membrane protein-1 (LMP-1) gene have been described in a Chinese nasopharyngeal carcinoma-derived isolate (CAO), and in viral isolates from various EBV-associated tumors. It has been suggested that these genetic changes, which include loss of a Xho I restriction site (position 169425) and a C-terminal 30-base pair (bp) deletion (position 168287-168256), define EBV genotypes associated with increased tumorigenicity or with disease among particular geographic populations. To determine the frequency of LMP-1 variations in European wild-type virus isolates, we sequenced the LMP1 promoter and gene in EBV from lymphoblastoid cell lines from healthy carriers and patients without EBV-associated disease. Sequence changes were often present, and defined at least four main groups of viral isolates, which we designate Groups A through D. The widespread prevalence of LMP-1 sequence variations, particularly the Xho I polymorphism and the 30-bp deletion, indicate that they cannot be used as simple markers for oncogenic viruses related to particular forms of EBV-associated tumor. Several of the structural changes detected occur, however, at sites where they may affect transcription, translation, or function of LMP-1. Future in vitro studies should aim to establish the functional importance of variations at these sites.

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.

SJHD is in receipt of a research fellowship from the Danish Medical Research Council. Hamilton-Dutoit, S. J., Lou, H. & Pallesen, G. The expression of placental alkaline phosphatase (PLAP) and PLAP-like enzymes in normal and neoplastic human tissues. An immunohistological survey using monoclonal antibodies. APMIS 98: 797-8 1 1, 1990.The immunohistological expression of placental alkaline phosphatase (PLAP) and PLAP-like enzyme was studied in frozen sections from a wide variety (n = 254) of normal and malignant tissues using monoclonal antibodies reactive with PLAP (H3 17) and PLAP/PLAP-like enzyme (H I7E2; H3 15). PLAP/PLAP-like reactivity was seen in normal thymus, and foetal and neonatal testis, and in 2 1 out of 22 malignant germ cell tumours (GCTs), but was also found in normal endocervix, normal Fallopian tube and in 28 out of 167 non-GCTs (particularly in ovarian and proximal gastrointestinal tract tumours). Positivity for true PLAP (as demonstrated with H317) was seen in term placenta, in endocervix, and in Fallopian tube (but not in other normal tissues) and was commonly found in ovarian and proximal gastrointestinal tract tumours. Reactivity with H3 17 was unusual in malignant GCTs (2 out of 22 cases). These findings confirm that PLAP/PLAP-like positivity is a highly sensitive immunohistological marker for malignant GCTs, but one which by itself is of only moderate specificity. Furthermore, expression of true PLAP is rare in GCTs and favours instead an origin from the ovary or proximal gastrointestinal tract. The results also indicate that the predominant heat-stable alkaline phosphatase species in normal foetal and neonatal testis, and in thymus has a similar immunohistological profile to that found in malignant GCTs, and is a PLAP-like enzyme ("germ cell alkaline phosphatase") distinct from true PLAP. The occurrence of this marker in GCTs would appear to reflect increased eutopic production of an enzyme present in trace amount in corresponding normal tissues rather than a genuine example of ectopic expression.

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. Cancer 2001;91:1500–9. © 2001 American Cancer Society.

Traditional histological diagnosis of mycobacterial infection in formalin-fixed and paraffin-embedded (FFPE) tissues is insensitive and poorly specific. To improve this, we developed nested polymerase chain reaction (PCR) protocols for detecting a Mycobacterium genus-specific 65-kDa heat shock protein (HSP65) sequence and the M. tuberculosis complex-specific insertion sequence IS6110 in FFPE sections. Protocols were optimized on tissues from 20 patients with a final clinical diagnosis of mycobacterial infection. Amplicons were controlled by sequencing and restriction endonuclease digestion. PCR could detect as few as three mycobacterial genomes per reaction. Assays showed 100% sensitivity and specificity for both M. tuberculosis complex and M. avium complex infection. Paraffin blocks from a second group of 26 patients with histological evidence of necrotizing granulomas of unknown etiology were then analyzed as a surrogate group to test the assay under conditions similar to those applying during routine diagnosis. Twenty-three of these blocks contained amplifiable DNA; nine were positive for M. tuberculosis complex DNA and four for other types of mycobacterial DNA. Furthermore, digestion of HSP65 amplicons with NarI could distinguish M. tuberculosis from M. avium complex. In conclusion, our nested PCR assays can be used as reliable tools for the detection of mycobacterial infections in FFPE tissues. The assays are simple and rapid to perform and show improved sensitivity and specificity compared to previously reported protocols.

Epstein-Barr virus (EBV) infection in lymphoproliferative lesions has been assumed to be strictly latent. In order to investigate the possible occurrence of EBV replication in AIDS-related lymphoma (ARL) cells, we studied 13 cases by immunohistology using monoclonal antibodies to the EBV-encoded switch-protein BZLF1, early antigens (EAs), late replicative proteins [virus capsid antigens (VCAs) and membrane antigens (MAs)], and to the latent proteins EB nuclear antigen 2 (EBNA 2) and latent membrane protein (LMP). EBV genomes were detected by in situ hybridization. EBV genomes and/or gene products were demonstrated in ten cases, including all immunoblast-rich lymphomas, two Burkitts lymphomas, and a T-cell anaplastic large-cell lymphoma. The BZLF1 protein, which disrupts latency in B cells, was identified in six (60 per cent), and EAs in four (40 per cent) of the EBV-positive ARL. Only one lymphoma (10 per cent) expressed VCAs and MAs. EBNA 2 and LMP were detected in three (30 per cent) and eight (80 per cent) of EBV-positive cases, respectively. EBV DNA was detected in lymphoma cells in 7 of 12 (58 per cent) cases. The most important finding of this study was frequent spontaneous activation of latent EBV in ARL. Production of complete virus, however, was either aborted, or tumour cells expressing late productive cycle proteins (VCA, MA) were rapidly cleared from tissues. It is suggested that host factors that normally inhibit replication of EBV are deficient in AIDS patients.

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.