Abstrakt
We conducted a large prospective study on pediatric ALL on behalf of the Czech Pediatric Hematology Working Group, and analyzed the frequency and clinical impact of various translocations, including t(4;11). We used the nested RT-PCR approach for the detection of the fusion gene MLL/AF4.
RNA was extracted using a method modified after the single-step technique described by Chomczynski and Sacchi.2 RNA integrity and quantity was evaluated in nondenaturing agarose gel electrophoresis. The single-round amplification of ABL exons A2 to A3 was then used for cDNA quality control.
As we know from our previous work (Divoky et al, manuscript submitted), exon 6 of the MLL gene is frequently absent in the alternatively spliced MLL/AF4 transcripts. Therefore, in contrast to the method used by Uckun et al, our RT-PCR used primers in MLL exon 5 for both the first and second round of our PCR.
The sensitivity of this approach, evaluated using limiting dilution of the MLL/AF4-positive cells into normal cells, is 10-4(0.01%), the sensitivity identical to the one described by Uckun et al. We started our prospective study in June 1994.
Since then, we found 7 infant patients positive for MLL/AF4 out of 11 tested (64%), contrasting with only 1 out of 206 patients aged 1 to 18 years (0.5%). Out of these 8 children, 7 were positive in the first round and 1, tested only after the induction therapy block and at subsequent relapse, in the second round.
Cytogenetic analysis was available in 5 of these patients, and in 4 cases t(4;11) was detected. In 1 remaining patient, cytogenetic analysis revealed deletion in 11q23 locus.
To test the hypothesis that MLL/AF4-expressing cells could be present among hematopoietic cells of healthy newborns, we applied the identical two-round RT-PCR approach to the cord blood samples from 103 full-term healthy newborns born in our hospital from February to March 1998 and found none of them to be MLL/AF4-positive.