Rapid detection of MYCN gene amplification and telomerase expression in neuroblastoma

Amplification of the MYCN gene and high telomerase activity predict a poor prognosis for the patients with neuroblastoma, We used PCR techniques for r apid detection of MYCN gene amplification and human telomerase reverse tran scriptase (hTERT) expression in neuroblastoma specimens. The detection of M YCN gene amplification is based on differential PCR in which three primer p airs were used to coamplify a 178-bp fragment of target MYCN gene with two reference gene fragments, a 237-bp of p53 exon 7 and a 120-bp of beta-globi n exon 3, in a single tube of 40 surgically resected tumor samples. MYCN am plification was identified by this differential PCR in all 10 samples carry ing more than 10 copies (already known to have MYCN gene amplification by S outhern blot analysis). There were no false-negative or false-positive case s, and the relative intensity of MYCN bands in the differential PCR correla ted significantly with the copy number determined by Southern blot analysis (gamma = 0.99, P < 0.0001). This protocol was also applicable in the biops y or aspirated samples, as well as the parafiin-embedded tissues, and in de tecting intratumoral heterogeneity. Using RT-PCR procedures, hTERT mRNA exp ression was detectable in all 13 tumors with high telomerase activity, Thes e nonradioisotopic PCR-based protocols for detecting,MYCN gene amplificatio n and hTERT mRNA expression are rapid and reliable and are likely to be use ful to determine the biological behavior of neuroblastoma.