Andrea Luegmayr

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.

Quality Assessment of Genetic Markers Used for Therapy Stratification

PURPOSE Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Regression and progression in neuroblastoma. Does genetics predict tumour behaviour

Neuroblastoma (NB) is a heterogeneous disease. The clinical course may range from spontaneous regression and maturation to very aggressive behaviour. Stage 4s is a unique subcategory of NB, generally associated with good prognosis, despite skin and/or liver involvement and the frequent presence of tumour cells in the bone marrow. Another type of NB is the locally invasive tumour without bone and bone marrow involvement which can also have a good prognosis, irrespective of lymph node involvement. Unfortunately, there is only limited biological information on such tumours which have not been treated with cytotoxic therapy despite lymph node involvement, residual tumour mass after surgery and/or bone marrow infiltration. In order to find specific genetic changes common to NBs with a benign clinical course, we studied the genetic abnormalities of these tumours and compared them with highly aggressive tumours. We analysed a series of 54 localised and stage 4s tumours by means of in situ hybridisation performed on fresh cells or on paraffin embedded tissues. In addition, we performed classical cytogenetics, Southern blotting and PCR analysis on fresh tumour tissue. The majority of patients had been treated with surgery alone, and in a number of patients tumour resection was incomplete. Deletions at 1p36 and amplifications of the MYCN oncogene were absent, and diploidy or tetraploidy were not seen in any case, with residual localised tumours possessing a favourable outcome. Unexpectedly, one patient with a tetraploid 4s tumour without any genetic structural changes not receiving any cytotoxic treatment, did well. Interestingly, this genetic spectrum contrasted with that of progressing tumours, in which most had genetic aberrations, the deletion at 1p36 being the most common event. These data, although limited, suggest that an intact 1p36 (recognised by D1Z2), the absence of MYCN amplification and near-triploidy (at least in localised tumours), represent prerequisites for spontaneous regression and/or maturation.

Intratumoural heterogeneity of 1p deletions and MYCN amplification in neuroblastomas.

BACKGROUND At least three genetic hallmarks identify aggressive tumour behaviour in neuroblastomas; amplification of the oncogene MYCN; deletion (loss of heterozygosity [LOH]) at the short arm of chromosome 1 (del1p36), seen in approximately 28% of the cases; and di-tetraploidy. The MYCN oncogene is amplified in approximately 23% of all neuroblastomas and becomes important for the stratification of therapy in localised and 4s tumours. Up to now, it has been believed that the genetic constellation of neuroblastic tumours is stable and does not alter during tumour evolution or during tumour progression. PROCEDURE Using fluorescence in situ hybridisation techniques (FISH) to investigate different tumour areas on touch preparations and histological sections, we show that genetic heterogeneity can be detected in neuroblastomas, especially in tumours detected by urinary mass screening. CONCLUSION The identification of such cell clones is important, because the MYCN amplification and/or the deletion at 1p36 appear to be responsible for aggressive local growth and development of metastases.

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.

Neuroblastoma cells can actively eliminate supernumerary MYCN gene copies by micronucleus formation—sign of tumour cell revertance?

Human neuroblastoma cell lines frequently exhibit MYCN amplification and many are characterised by the presence of morphologically distinct cell types. The neuronal cells (N-cells) and the so-called flat cells (F-cells) are thought to represent manifestations of different neural crest cell lineages and are considered to be the consequence of neuroblastoma cell pluripotency. In this study, various neuroblastoma cell lines were examined for micronuclei. In F-cells of neuroblastoma cell lines with extrachromosomally amplified MYCN, we observed the frequent occurrence of micronuclei. Using fluorescence in situ hybridisation (FISH) with a MYCN specific probe, we demonstrated that these micronuclei were packed with MYCN hybridisation signals. In addition, in a minor percentage of cells, MYCN signals occurred in clusters, adhered to the nuclear membrane and aggregated in nuclear protrusions. In F-cells, a substantial reduction or lack of amplified MYCN copies was observed. These observations let us conclude that extrachromosomally amplified genes can be actively eliminated from the nucleus resulting in a dramatic loss of amplified sequences in the F-cells. Moreover, reduction or loss of amplified sequences in F-cells was shown to be accompanied by downregulation of MYCN expression, by a decrease in proliferative activity and by upregulation of molecules of the major histocompatibility complex class I (MHC I). Interestingly, F-cells are not restricted to neuroblastoma cell cultures, but also occur in cell lines of other tissue origin. All F-cells share important biological features, interpreted as cell revertance, i.e. loss of the malignant phenotype and properties. This fact, together with the demonstration that neuroblastoma cells do not differentiate into Schwann cells in vivo [1] Ambros et al. NEJM 1996, 334, 1505-1511, do not support the hypothesis that F-cells represent Schwannian/glial differentiation in vitro. We therefore postulate that the elimination of amplified MYCN gene copies in cultivated neuroblastoma cells is in line with the phenomenon of tumour cell revertance.

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 patients 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.

Automatic detection and genetic profiling of disseminated neuroblastoma cells

BACKGROUND Rare tumor cells circulating in the hematopoietic system can escape identification. On the other hand, the nature of these cells, positive for an immunologiCal tumor marker, cannot be determined without any genetic information. PROCEDURE To overcome these problems a novel computer assisted scanning system for automatic cell search, analysis, and sequential repositioning was developed. This system allows an exact quantitative analysis of rare tumor cells in the bone marrow and peripheral blood by sequential immunological and molecular cytogenetic characterization. RESULTS AND CONCLUSIONS In that virtually all tumor cells in a mixing experiment could be recovered unambiguously, we can conclude that the sensitivity of this approach is set by the number of cells available for analysis. Sequential FISH analyses of immunologically positive cells improve both the specificity and the sensitivity of the microscopic minimal residual disease detection.

Unequivocal identification of disseminated tumor cells in the bone marrow by combining immunological and genetic approaches--functional and prognostic information.

Unequivocal identification of disseminated tumor cells in the bone marrow by combining immunological and genetic approaches n functional and prognastic information

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 Ewings sarcoma family tumor. Twenty-four of 29 Ewings 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 Ewings 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.