Detection of MYCN gene amplification and deletions of chromosome 1p in neuroblastoma by in situ hybridization using routine histologic sections.
BACKGROUND
Amplification of the MYCN oncogene and partial deletion of chromosome 1p are genetic changes frequently seen in neuroblastoma that are indicators of poor prognosis. The identification techniques usually used--Southern blotting for MYCN gene amplification, and karyotypic analysis of viable tumor cells for large 1p deletions--are time-consuming and suited for specialized laboratories.


METHODS
We have developed an immunofluorescence in situ hybridization assay suitable for detection of MYCN amplification and 1p deletion in formalin-fixed, paraffin-embedded tissue sections. The technique is rapid, does not involve the use of radioactivity, and can be carried out in laboratories already familiar with conventional in situ hybridization and immunohistochemical detection methods.


RESULTS
MYCN gene amplification appeared as multiple signals per nucleus, corresponding to double minute chromosomes. Deletion of 1p was detected by loss of one of the normal signals present within the nucleus of a neuroblastoma cell line. Use of different fluorophores enabled the simultaneous detection of MYCN gene amplification and 1p deletion at the individual cell level. Detection was found to be enhanced by RNase and pepsin treatment of tissue sections before hybridization and by a novel microwave denaturation method.


CONCLUSIONS
Application of this methodology to formalin-fixed samples of neuroblastoma will permit comprehensive retrospective studies of these two genetic markers using archived tumors. In situ hybridization surveys will have widespread applications for studying known genetic aberrations in individual cells of a variety of solid tumors and for determining interrelationships and clinical significance in relation to tumor progression and patient outcome.
Leong
P K
PK
Department of Pathology, Hospital for Sick Children, Toronto, Canada.
Thorner
P
P
Yeger
H
H
Ng
K
K
Zhang
Z
Z
Squire
J
J
D002878:Chromosomes, Human, Pair 1; D003582:Cytogenetics; D005784:Gene Amplification; D017353:Gene Deletion; D016259:Genes, myc; D006801:Humans; D017403:In Situ Hybridization; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding