We aimed to determine the frequency of t(12;21), t(1;19), t(4;11) and t(8;21) in a population of banked cord blood units (CBU). Four leukaemia cell lines, REH with t(12;21), 697 with t(1;19), MV4-11 with t(4;11) and Kasumi-1 with t(8;21), respectively expressing the fusion genes TEL-AML1, E2A-PBX1, MLL-AF4 and AML1-ETO, and a negative cell line, HL60, were used to establish a high-throughput Taqman probe-based multiplex real-time PCR protocol, which was then used to screen CB. RNA was extracted from CBU banked at the BMDI Cord Blood Bank and 500 CBU were screened for the 4 fusion transcripts.
The sensitivity of the assay could detect up to one mutant cell in 10,000 normal cells for all four fusion genes; the specificity of the assay was also validated. No transcripts were observed for E2A-PBX1, MLL-AF4, and AML1-ETO. At least 3 CBU displayed a positive result for TEL-AML1 transcript, with 1 or 2 out of 3 sample replicates being positive for each CBU. Conventional PCR and sequencing was subsequently used to confirm that 3 out of 500 CB units contained cells expressing the TEL-AML1 transcript at a frequency below 10-4.
In conclusion, we have developed a simple multiplex real time-PCR assay to detect the four most common rearrangements in childhood leukemia in a single reaction.
These findings are consistent with those of Mori et al1, whereby the frequency and level of TEL-AML1 positive cells in normal CB in the Australian population is in the order of 1%, and dispute the recent report of Lausten-Thomsen et al2 of a much lower frequency. The implication of this finding for the use of these CBU for transplant and their role in donor-cell-derived leukaemia remains to be determined. Greater numbers of CBU will be required to determine the frequency of the other 3 fusion transcripts.