The latent human herpesvirus-6A genome specifically integrates in telomeres of human chromosomes in vivo and in vitro.

Previous research has suggested that human herpesvirus-6 (HHV-6) may integrate into host cell chromosomes and be vertically transmitted in the germ line, but the evidence--primarily fluorescence in situ hybridization (FISH)--is indirect. We sought, first, to definitively test these two hypotheses. Peripheral blood mononuclear cells (PBMCs) were isolated from families in which several members, including at least one parent and child, had unusually high copy numbers of HHV-6 DNA per milliliter of blood. FISH confirmed that HHV-6 DNA colocalized with telomeric regions of one allele on chromosomes 17p13.3, 18q23, and 22q13.3, and that the integration site was identical among members of the same family. Integration of the HHV-6 genome into TTAGGG telomere repeats was confirmed by additional methods and sequencing of the integration site. Partial sequencing of the viral genome identified the same integrated HHV-6A strain within members of families, confirming vertical transmission of the viral genome. We next asked whether HHV-6A infection of naïve cell lines could lead to integration. Following infection of naïve Jjhan and HEK-293 cell lines by HHV-6, the virus integrated into telomeres. Reactivation of integrated HHV-6A virus from individuals' PBMCs as well as cell lines was successfully accomplished by compounds known to induce latent herpesvirus replication. Finally, no circular episomal forms were detected even by PCR. Taken together, the data suggest that HHV-6 is unique among human herpesviruses: it specifically and efficiently integrates into telomeres of chromosomes during latency rather than forming episomes, and the integrated viral genome is capable of producing virions.

Fluorescent in situ hybridization on isolated tumor cell nuclei: a sensitive method for 1p and 19q deletion analysis in paraffin-embedded oligodendroglial tumor specimens.

In oligodendroglial neoplasms, losses of chromosomal material at 1p and 19q associate with chemosensitivity and prolonged survival. Thus, 1p/19q testing is increasingly proposed for use in brain tumor diagnosis and prognostic assessment. Fluorescent in situ hybridization (FISH) is a classic technique for investigation of 1p/19q status in paraffin-embedded tissues. A major limitation of this method is truncation of tumor cell nuclei complicating assessment of hybridization results. In our study, we analyzed 1p and 19q status in a series of 79 oligodendroglial neoplasms (49 oligodendrogliomas, 30 oligoastrocytomas, WHO: 57 Grade II, 22 Grade III tumors) and controls (gliotic brain tissue: n = 4, diffuse low-grade astrocytoma: n = 4) using FISH on isolated whole tumor cell nuclei, prepared as cytospin preparations, thus bypassing the problem of nuclear truncation. For interpretation of FISH results, we used consensus criteria as defined by the SIOP-Europe Neuroblastoma Study Group for analysis of peripheral neuroblastic tumors. FISH yielded interpretable results in 98.7% for 1p and 92.1% for 19q. Chromosome 1p/19q alterations comprised deletions (1p: 79.5%, 19q: 80%) and imbalances (1p: 11.5%, 19q: 12.9%). 1p aberrations were more frequent in oligodendroglioma than in oligoastrocytoma (100% versus 75.9%, P =.001). The frequency of 1p/19q alterations was not significantly different in WHO Grade II or Grade III tumors or in primary and recurrent tumors. We conclude that FISH on isolated cell nuclei, with application of the SIOP Europe Neuroblastoma consensus criteria, is a sensitive method for detection and interpretation of 1p and 19q aberrations in paraffin-embedded tissue specimens of oligodendroglial neoplasms.

Detection of disseminated tumor cells in neuroblastoma: 3 log improvement in sensitivity by automatic immunofluorescence plus FISH (AIPF) analysis compared with classical bone marrow cytology.

The sensitive detection of bone marrow involvement is crucial for tumor staging at diagnosis and for monitoring of the therapeutic response in the patient's follow-up. In neuroblastoma, only conventional cytomorphological techniques are presently accepted for the detection of bone marrow involvement, yet since the therapeutic consequences of the bone marrow findings may be far-reaching, the need for highly reliable detection methods has become evident. For this purpose, we developed an automatic immunofluorescence plus FISH (AIPF) device which allows the exact quantification of disseminated tumor cells and the genetic verification in critical cases. In this study, the power of the immunofluorescence technique is compared with conventional cytomorphology. 198 samples from 23 neuroblastoma patients (stages 4 and 4s) at diagnosis and during follow-up were investigated. At diagnosis, 45.6% of the samples (26 of 57) which were positive by AIPF investigation were negative by cytomorphology. During follow-up, 74.2% (49 of 66) of AIPF-positive samples showed no cytological signs of tumor cell involvement. False negative morphological results were found in up to 10% of tumor cell content. A tumor cell infiltrate below 0.1% was virtually not detectable by conventional cytomorphology. Using the sensitive immunofluorescence technique, the analysis of only two instead of four puncture sites did not lead to false negative results. Thus, the immunofluorescence technique offers an excellent tool for reliable detection and quantification of disseminated tumor cells at diagnosis and during the course of the disease.

Frequent low level expression in Ewing sarcoma family tumors and widespread absence of the metastasis suppressor KAI1/CD82 in neuroblastoma.

The transmembrane 4 superfamily member KAI1/CD82, a metastasis suppressor, is correlated inversely with the progression and invasion of several tumors. It is capable of inhibiting metastasis without affecting tumorigenicity per se. KAI1/CD82 expression is down-regulated in the progression of common solid epithelial tumors of adulthood. Mutation of p53 is suggested to be involved in the modulation of KAI1. As little is known about its expression and possible prognostic impact in pediatric tumors, we investigated KAI1/CD82 expression in cell lines and primary tumor samples from pediatric tumors of neuroectodermal origin, neuroblastoma and Ewing's sarcoma family tumor. Twenty-four of 29 Ewing's sarcoma family tumor cell lines, independent of p53 status, showed KAI1 mRNA positivity by reverse transcription-PCR analysis in contrast to zero of eight neuroblastoma cell lines. Among 13 primary Ewing's sarcoma family tumor samples from patients with different disease extension, KAI1 mRNA expression was low as detected by reverse transcription-PCR. Twenty of 30 primary neuroblastoma specimens were KAI1-negative by immunofluorescence analysis whereas the remaining 10 gave weak to moderate staining patterns. There was no apparent correlation of KAI1 expression with any clinical or genetic features of the patients whose tumor samples were studied. Consequently, KAI1 may not be of prognostic relevance in this group of tumors although there may be some role for KAI1 modulation in the biology of these neuroectodermal tumors.

[Detection, quantification and characterization of disseminated tumor cells]. / Erkennung, Quantifizierung und Charakterisierung von disseminierten Tumorzellen.

There are different reasons why the detection of disseminated tumor cells (DTCs) in the hematopoetic system is important. On the one hand the detection of disseminated tumor cells can provide vital information about a tumor's tendency to develop metastases. In some localized epithelial but also in embryonic tumors, for example a correlation between disseminated tumor cells and unfavorable outcome was observed (6, 14). These studies are based on the assumption that those tumor cells which appear in the hematopoetic system at a very early stage are responsible for the development of metastases. Another important aspect is the monitoring of the disease response to cytotoxic drugs by quantifying DTCs. During and after therapy there is no other possibility (except for an operation) to either directly analyze the effects the therapy has on the tumor cells or to determine their biological characteristics. The dissemination in the hematopoetic system, however, reveals the response to therapy and the biological features of the tumor cells. The prerequisites for such bone-marrow diagnosis, however, is the unequivocal identification of disseminated tumor cells. So in order to avoid false positive results (which are a risk in bone-marrow diagnostics), a system was developed to distinguish tumor cells from non-neoplastic cells and to facilitate insights into the biological make-up of tumor cells (2, 11).