SCREENING OF FOUR COMMON LEUKAEMIA-ASSOCIATED TRANSCRIPTS IN NORMAL CORD BLOOD SAMPLES USING TAQMAN PROBE-BASED MULTIPLEX REAL-TIME PCR — ASN Events
  1. Schedule
  2. ASSCR Poster Presentations
  3. SCREENING OF FOUR COMMON LEUKAEMIA-ASSOCIATED TRANSCRIPTS IN NORMAL CORD BLOOD SAMPLES USING TAQMAN PROBE-BASED MULTIPLEX REAL-TIME PCR

SCREENING OF FOUR COMMON LEUKAEMIA-ASSOCIATED TRANSCRIPTS IN NORMAL CORD BLOOD SAMPLES USING TAQMAN PROBE-BASED MULTIPLEX REAL-TIME PCR (#386)

Pei Tian 1 , Elizabeth Algar 2 3 , Jeffrey Looi 1 3 , Ngaire J Elwood 1 3 4
  1. Director, BMDI Cord Blood Bank & Head, Cord Blood Stem Cell Research Program. Cell Biology, Murdoch Children's Research Institute, Melbourne, Victoria, Australia
  2. Childrens Cancer Centre, Murdoch Childrens Research Institute, Parkville, Victoria, Australia
  3. Department of Paediatrics, University of Melbourne, Parkville, Victoria, Australia
  4. BMDI Cord Blood Bank, Royal Childrens Hospital, Parkville, Vic , Australia

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.

  1. Mori H, Colman SM, Xiao Z, et al. Chromosome translocations and covert leukemic clones are generated during normal fetal development. PNAS 2002;99:8242-7.
  2. Lausten-Thomsen U, Madsen HO, Vestergaard TR, Hjalgrim H, Nersting J, Schmiegelow K. Prevalence of t(12;21)[ETV6-RUNX1] positive cells in healthy neonates. Blood 2011;117:186-9.