Assessment of NMYC amplification: a comparison of FISH, quantitative PCR monoplexing and traditional blotting methods used with formalin-fixed, paraffin-embedded neuroblastomas.
OBJECTIVE
To determine the degree of agreement between fluorescence in situ hybridization (FISH), Southern blot analysis and LightCycler monoplex polymerase chain reaction (PCR) analysis in the assessment of NMYC gene amplification status in neuroblastoma.


METHODS
We performed a retrospective analysis of NMYC amplification, using FISH, LightCycler monoplex PCR and Southern blot techniques to assess NMYC amplification in a series of 18 neuroblastomas and 20 histologically normal tissues (15 lymph nodes, 2 pancreas specimens, 1 section each of thyroid, prostate and uterus).


RESULTS
Nine neuroblastomas were NMYC amplified, and the remaining cases were nonamplified. All cases yielded interpretable results by Southern blotting and PCR monoplexing techniques. A single case of neuroblastoma was difficult to interpret by FISH due to high background debris. A single case demonstrated low-level NMYC amplification by LightCycler PCR monoplexing but was nonamplified by the other 2 techniques. FISH analysis in 1 case showed amplification, while the other 2 techniques demonstrated nonamplified status. The case in which FISH analysis incorrectly demonstrated amplification was the same one in which there was high background debris. The Southern blot results were reported as amplified or nonamplified, while numeric amplification ratios were obtained by both FISH and PCR LightCycler monoplex analysis. Comparison of these techniques demonstrated FISH to underestimate the degree of amplification in cases in which the amplification level was high by PCR. In fact, FISH appeared to saturate at amplification ratios > 10.


CONCLUSIONS
The study revealed a high level of concordance between the 3 techniques for assessment of NMYC amplification status. However, FISH analysis has the advantage of allowing concurrent assessment of NMYC amplification status and architecture. LightCycler PCR monoplexing appears to have the advantage of more accurately quantitating high levels of NMYC amplification, including those amplified 20-fold or higher. Both FISH and PCR LightCycler monoplexing have the advantage of being performable on formalin-fixed, paraffin-embedded tissue.
Layfield
Lester J
LJ
Department of Pathology, University of Utah School of Medicine, and ARUP Laboratories, Salt Lake City, Utah, USA. layfiel@aruplab.com
Willmore-Payne
Carlynn
C
Shimada
Hiroyuki
H
Holden
Joseph A
JA
D015139:Blotting, Southern / Q000379:methods; D045744:Cell Line, Tumor; D002648:Child; D005260:Female; D005557:Formaldehyde / Q000737:chemistry; D005784:Gene Amplification / Q000235:genetics*; D016259:Genes, myc / Q000235:genetics*; D005821:Genetic Techniques; D006801:Humans; D017404:In Situ Hybridization, Fluorescence / Q000379:methods; D008297:Male; D009447:Neuroblastoma / Q000235:genetics* / Q000473:pathology; D016612:Paraffin Embedding; D016133:Polymerase Chain Reaction / Q000379:methods; D039881:Prokaryotic Initiation Factor-2 / Q000235:genetics; D016707:Tissue Fixation
D039881:Prokaryotic Initiation Factor-2; D005557:Formaldehyde