S. Rumpler

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.

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.

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.

Demonstration of the translocation der(16)t(1;16)(q12;q11.2) in interphase nuclei of ewing tumors

The der(16)t(1;16) has been detected cytogenetically in a number of malignancies including Ewing tumors (ETs). To enable fast and reliable analysis of der(16) chromosomes, we established an interphase cytogenetic approach. By using two DNA probes hybridizing to the heterochromatic portions on the long arms of chromosomes 1 and 16, this technique allows the detection of this chromosomal aberration in nonproliferating cells. Formation of the der(16) leads to partial excess of 1 q material and partial loss of the long arm of chromosome 16. Double‐target fluorescence in situ hybridization (FISH) experiments were performed on cytospin slides of 13 ETs, near‐triploid tumor cells and normal cells to assess whether the FISH technique used permits the discrimination of nuclei harboring this aberration from nuclei without a der(16) chromosome. In five ETs, we found evidence for the presence of one or two der(16)t(1;16) chromosomes both by FISH and by conventional cytogenetics. Tumor cells displayed two signals for intact chromosomes 1, one or two additional fused signals for the der(16) chromosomes, and one signal for the intact chromosome 16. In one case without fused signals, the presence of a der(16) was demonstrated by hybridizing a painting probe for chromosome 16 simultaneously with the paracentromeric probe for chromosome 1. Our results suggest that double‐target FISH on interphase nuclei offers an ideal tool for analyzing tumors prospectively and retrospectively to assess the biological role and the possible prognostic impact of the der(16) in ETs and in other solid tumors. Genes Chromosom Cancer 17:141–150 (1996)

Visualization of episomal and integrated Epstein‐Barr virus DNA by fiber fluorescence in situ hybridization

For many Epstein‐Barr virus (EBV)‐associated malignancies, it is still a matter of controversy whether infected cells harbor episomal or chromosomally integrated EBV genomes or both. It is well established that the expression of EBV genes per se carries oncogenic potential, but the discrimination between episomal and integrated forms is of great relevance because integration events can contribute to the oncogenic properties of EBV, whereas host cells that exclusively harbor viral episomes may not carry the risks mediated by chromosomal integration. This notion prompted us to establish a reliable technique that not only allows to unequivocally discriminate episomal from integrated EBV DNA, but also provides detailed insights into the genomic organization of the virus. Here, we show that dynamic molecular combing of host cell DNA combined with fluorescence in situ hybridization (FISH) using EBV‐specific DNA probes facilitate unambiguous discrimination of episomal from integrated viral DNA. Furthermore, the detection of highly elongated internal repeat 1 (IR1) sequences provides evidence that this method permits detection of major genomic alterations within the EBV genome. Thus, fiber FISH may also provide valuable insights into the genomic organization of viral genomes other than EBV.

Fine-mapping of cytogenetically undetectable EWS/ERG fusions on DNA fibers of Ewing tumors

In contrast to the EWS/FLI1 fusion which is represented by a t(11;22)(q24;q12), EWS/ERG fusions are frequently cytogenetically not detectable. Three Ewing tumors (ET), two with apparently normal chromosomes 21 and 22, and one ET with a t(2;22)(p25;q12), were studied by FISH on interphase nuclei, metaphase chromosomes and on DNA fibers. EWS/ERG transcripts were detected by RT-PCR in all cases. FISH, using cosmids located proximally (F10, G9) and distally (F7) to the EWS breakpoint region, revealed no detectable separation of these probes in two cases. In contrast, co-hybridization of probe PT1526 containing the ERG breakpoint region with G9 revealed the juxtaposition of two signals per interphase nucleus in all three cases indicating the EWS/ERG fusions. Chromosome preparations displayed the juxtaposed signals on the der(22), and hybridization signals of the probes PT1526 and G9 on the non-rearranged chromosomes 21 and 22 in all cases, respectively. The PT1526 signal on the der(21) was seen only in cases 1 and 2. These results were confirmed by triple-target FISH on tumor DNA fibers. In all three cases, the hybridization pattern F10 – G9 – PT1526 indicates a centromere to telomere orientation. This finding suggests that EWS/ERG fusions in ETs may be generated by an inversion of the ERG gene or a part thereof followed by an insertion into the EWS gene on the der(22). Double-target FISH on interphase nuclei using probes flanking the EWS breakpoint region and probe PT1526 enables the detection of virtually all 22q12 rearrangements in ETs, thus providing a reliable diagnostic assay.