Heiner Daus

MDM2 gene amplification and lack of p53 point mutations in Hodgkin and Reed–Sternberg cells: results from single‐cell polymerase chain reaction and molecular cytogenetic studies

Hodgkins disease (HD) is the most common haematological malignancy after chronic lymphocytic leukaemia, but very little is known about its pathogenesis or the genetic events that contribute to the malignant phenotype of the tumour cells. p53 is assumed to play an important role in the pathogenesis of HD, based on the observation that p53 protein is frequently accumulated in Hodgkin and Reed–Sternberg (H & RS) cells. We investigated single H & RS cells from five different HD patients for point mutations at the genomic level using multiplex polymerase chain reaction amplification and subsequent sequencing. No point mutations were detected in 50 single H & RS cells analysed. Hence, accumulation of p53 protein cannot be explained by mutations within the gene. A genome‐wide screening for genomic imbalances using comparative genomic hybridization revealed gain on chromosome 12q14, i.e. the mapping position of the MDM2 gene in several HD cases. Therefore, we assessed the copy number of the MDM2 gene using fluorescence in situ hybridization. In four out of six HD cases analysed, the copy number of the MDM2 gene was found to be increased. As gene amplification is frequently associated with protein overexpression, the observed accumulation of p53 in the nuclei of H & RS cells could be as a result of elevated MDM2 protein levels resulting in stabilization of p53 protein.

Diagnosis of pancreatic adenocarcinoma by polymerase chain reaction from pancreatic secretions.

As mutations at codon 12 of the Ki-ras oncogene have been shown to occur in 90% of pancreatic adenocarcinomas, a novel strategy for the detection of these mutations in pancreatic secretions obtained at routine endoscopies was developed. Ki-ras DNA was amplified and screened for the presence of mutations at codon 12 with a combination of different rapid, non-radioactive molecular biology techniques. Examination of DNA from cell lines and paraffin-embedded tumour samples was used to establish and test the strategy employed. Pancreatic secretions from 27 patients were examined for the presence of Ki-ras mutations. Mutations at codon 12 were detected in 16/16 secretions from patients with histologically confirmed carcinoma and from one patient with carcinoma of the bile duct. In six patients a mutation identical to the one found in the pancreatic secretions was also demonstrated in paraffin-embedded fine-needle biopsy or surgical samples. Of the remaining ten patients (who had pancreatitis or cholelithiasis) mutations were not found in nine. Ki-ras codon 12 mutation was identified in one of these patients however, and mucous cell hyperplasia of pancreatic ducts was found upon histological examination. These findings establish Ki-ras polymerase chain reaction from pancreatic secretions as a valuable new diagnostic procedure for the demonstration of malignant cells, possibly at an early stage of the disease.

The mechanism of the antiherpetic activity of zinc sulphate

The molecular mechanism of the effects of zinc ions against herpes simplex virus (HSV) infection was investigated. Zinc sulphate (100 microM) in the culture medium of an HSV-infected African green monkey kidney cell line did not block viral DNA synthesis and, at this concentration, only moderate cytotoxic effects were observed in uninfected cells. Nevertheless, virus yields were reduced to less than 1% of the control. Thus the long standing hypothesis that zinc might block multiplication of HSV by selective intranuclear inhibition of the viral DNA polymerase apparently has lost its validity. Inhibition of virus growth in the absence of severe cytotoxicity must therefore result from other effects of ZnSO4. Free virus is inactivated by 15 mM-ZnSO4 within a few hours of its addition. The inactivated virus is defective in the glycoprotein-dependent functions of penetration and, to some extent, adsorption. Electron micrographs show massive deposition of zinc onto virion components. In a virion, transmembrane transport of zinc ions is not expected and the established antiviral effect is therefore explained by an inhibition of virion glycoprotein function after non-specific accumulation of zinc into many virion membrane components.

Low Sensitivity of the ki-ras Polymerase Chain Reaction for Diagnosing Pancreatic Cancer From Pancreatic Juice and Bile: A Multicenter Prospective Trial

PURPOSE Early detection of pancreatic cancer using molecular markers may improve outcome. Mutations of the ki-ras oncogene are detected in 70% to 90% of pancreatic adenocarcinomas. A prospective, partially blinded, multicenter diagnostic trial was performed to test the sensitivity and specificity of the ki-ras polymerase chain reaction (PCR) analysis of pancreatic juice and bile specimens. PATIENTS AND METHODS Specimens of pancreatic juice and bile were collected from 532 consecutive patients. Mutations in codon 12 of the ki-ras gene were identified by two independent enrichment PCRs and confirmed by direct sequencing. RESULTS One hundred seventy-four of 532 patients were excluded from the final analysis (reasons: no amplifiable DNA, no specimen or only duodenal juice sent, lost to follow-up). Sixty-three of 358 patients had ductal pancreatic cancer. In 24 (38.1%) of 63 patients, a mutated ki-ras gene was identified in pancreatic juice and/or bile. Ki-ras mutations were found in four (8%) of 50 cases of chronic pancreatitis, in 10 (18.7%) of 53 cases of other malignancies of the pancreaticobiliary tree, and in 14 (7.3%) of 192 cases of benign diseases or normal findings. Sensitivity and specificity of the ki-ras PCR analysis for the detection of pancreatic cancer was 38.1% and 90.5%, respectively. CONCLUSION In this prospective trial performed in nonselected patients, mutations of the ki-ras gene were detected in 38.1% of cases with pancreatic cancer. This test in its present form is not appropriate to confirm or screen for pancreatic cancer. More sensitive and/or quantitative PCR tests may improve the molecular diagnosis of pancreatic cancer.

LACK OF EVIDENCE FOR A PATHOGENIC ROLE OF CHLAMYDIA PNEUMONIAE AND CYTOMEGALOVIRUS INFECTION IN CORONARY ATHEROMA FORMATION

Atherosclerotic cardiovascular disease is generally accepted to be the result of metabolic disturbances. However, recent studies have suggested an infectious agent, especially Chlamydia pneumoniae or cytomegalovirus, to be involved in the pathogenesis of atherosclerosis. Atherosclerotic plaque specimens obtained from patients with coronary disease either by balloon dilatation catheter (13 cases) or atherectomy (16 patients) were examined for the presence of C. pneumoniae and cytomegalovirus. Using two primer pairs for C. pneumoniae, two primer pairs for the identification of unknown bacteria and primer pairs for the detection of immediate early gene E2 and the late genomic region of cytomegalovirus, we were unable to detect the suspected agents. The absence of C. pneumoniae, other bacteria and CMV in coronary atheromas is against the hypothesis of a pathogenetic role of these agents in coronary atheroma formation in the patients studied.

Improved method for the cyanogen bromide activation of agarose beads

The significant new feature of the procedure is the reaction control of the BrCN activation merely by the slow transit of BrCN from a dispersed organic phase to the aqueous phase containing agarose beads in concentrated buffer. The product thus obtained was applied in a model immunoaffinity chromatographic separation. Experimental conditions are given for the control of the degree of activation and of the multiplicity of attachment of the protein ligand, for optimizing the immunological reactivity of the immunosorbent and for minimizing leakage of covalently bound protein from the resin.

Detection of Epstein-Barr Virus DNA in Hodgkin- and Reed-Sternberg-Cells by Single Cell PCR

The Epstein-Barr virus (EBV) can be detected in the majority of lymph nodes involved by Hodgkins lymphoma using the highly sensitive polymerase chain reaction (PCR). However, the rate of EBV-DNA detection by in-situ hybridisation, which allows allocation of EBV to a defined cell population, i.e. the neoplastic H&RS-cells, is lower. In an attempt to combine the advantages of the high sensitivity of the PCR and the possibility of cellular allocation by in-situ hybridisation, we established a single-cell PCR of Hodgkin- and Reed-Sternberg (H&RS)-cells isolated by micromanipulation from biopsy tissues. We amplified EBV sequences from the BamW-region by single-cell PCR. Using this method we were able to detect EBV-DNA in the H&RS-cells from 4 of 6 patients. In EBV positive cases all H&RS-cells of a given patient were positive, proving the high sensitivity and reproducibility of the method. Other cells in the biopsy tissue involved by EBV-positive H&RS-cells were shown to be negative. This indicates that EBV may have a role in the pathogenesis of many but not all cases of Hodgkins disease.

Isolation and analysis of two strongly transforming isoforms of the Epstein‐Barr‐virus(EBV)‐encoded latent membrane protein‐1 (LMP1) from a single Hodgkin's lymphoma

Two genes encoding the latent membrane protein 1 (LMP1) of the Epstein‐Barr virus (EBV) were isolated from a single case of Hodgkins disease (HD) and were tested for their biological activities. The LMP1 gene from the Reed‐Sternberg cells contained point mutations relative to the prototype LMP1 gene, leading to amino‐acid exchanges. The LMP1 gene from passenger lymphocytes showed identical point mutations, but also had an in‐frame insertion of 132 base pairs within the 33‐bp repeat region. This insert encoding 44 amino acids contained the sequence PSQQS, corresponding to the potential TRAF‐binding motif PXQXT/S. When compared to the B95.8 gene, both HD‐derived LMP1 genes showed an increase in the transformation of Rat‐1 rodent fibroblasts. The transforming ability of the LMP1 gene with the insertion was greater than that of the other HD‐derived LMP1, and was comparable with the highly transforming LMP1‐Cao gene derived from a nasopharyngeal carcinoma. The HD‐derived genes stimulated expression of the cell‐surface markers, CD40 and CD54, similarly to the LMP1‐B95.8 gene, while the LMP1‐Cao gene had a significantly reduced ability to induce these proteins. In contrast, the LMP1‐Cao transactivated an NF‐κB‐response element more efficiently than did the HD‐derived genes. Transfer of the 132‐bp insert alone into the B95.8 gene did not increase its transforming activity to the LMP1‐Cao level, indicating that additional mutations in the LMP1 gene are necessary for modulating this function. Int. J. Cancer 76:194–200, 1998.© 1998 Wiley‐Liss, Inc.

Enrichment polymerase chain reaction for the detection of Ki-ras mutations: relevance of Taq polymerase error rate, initial DNA copy number, and reaction conditions on the emergence of false-positive mutant bands

Abstract Screening for oncogene mutations as a marker for malignancy can be a powerful tool for the early diagnosis of cancer. The enrichment polymerase chain reaction (PCR) is a sensitive method for the detection of low-frequency mutations in small samples. However, false-positive results, caused by methodological errors, may have severe clinical implications. When applied to the detection of Ki-ras mutations in pancreatic secretions, the assay sensitivity is limited to approximately 1:1400. Our investigation of Ki-ras mutations in blood samples from patients with pancreatic carcinoma revealed PCR bands presumably derived from mutant Ki-ras in samples from healthy volunteers, while all blood samples of the patients with pancreatic carcinomas showed a wild-type band pattern. Mathematical modeling of the PCR reaction reveals that the rate of false positive PCR results depends on the initial amount of DNA, the Taq polymerase error rate, the number of PCR reaction cycles, reaction efficiency and the restriction endonuclease chosen. The overall error rate of false positive results of the enrichment PCR can be reduced to the square of the rate of a single-step analysis if repeated amplifications of the same DNA specimen show an identical result.

Assessment of clonality of rosetting T lymphocytes in Hodgkin's disease by single-cell polymerase chain reaction: detection of clonality in a polyclonal background in a case of lymphocyte predominance Hodgkin's disease

Abstract. Rosetting of CD4+ T cells around the neoplastic Hodgkin and Reed-Sternberg (H&RS) cells is a characteristic feature of Hodgkins disease (HD). To answer the question whether this phenomenon is solely due to chemokine-mediated attraction of T cells or whether the rosetting T cells in addition recognize antigens presented by the H&RS cells, we examined the T cells adherent to H&RS cells. Cells from five cases of HD [four classic HD and one lymphocyte-predominant (LP) HD] were examined by single-cell analysis for the T-cell receptor (TCR) γ gene. Between 5 and 17 rosettes containing one to ten rosetting lymphocytes and the corresponding H&RS cells were amplified in separate plastic tubes. Of the resulting 119 TCRγ polymerase chain reaction (PCR) products, 87 were sequenced. While no evidence of a clonal expansion was obtained in the lymph nodes from four of five patients with classic HD, clonal TCRγ sequences were found in the lymph node from the patient within LPHD in two independent experiments analyzing seven and ten different rosetting complexes, respectively. Of 13 products, 11 showed identical Vγ9 sequences. Unrelated products were found in all other TCRγ family subgroups in this case. Single H&RS cells picked as controls were negative for TCRγ rearrangements. Our results demonstrate that clonal proliferations on a polyclonal background can occur among the T cells forming rosettes with Hodgkin cells and lend support to the view that Hodgkin cells may also function as cells presenting antigens to the adhering T cells.