Poster Board I-657
WT1 expression has proved to be of prognostic significance in AML, MDS and according to our unpublished data also in CML patients. There are four major WT1 isoforms with distinct functions in cells. Splicing variants encoding the WT1 isoforms differ from each other by the presence or absence of both the exon 5 and KTS region in the exon 9 of WT1 gene. It was shown that exon 5 containing isoforms appear to cause cell cycle arrest. Therefore, differences in WT1 splicing variant ratios might be associated with prognosis and response to therapy. All currently used primer/probe systems quantify all major splicing variants as expression of total WT1.
The aim of our study was to develop specific quantitative real-time PCRs (qRT-PCRs) for quantification of the four major WT1 splicing variants (+5+KTS; +5-KTS; -5+KTS; -5-KTS) at mRNA level and to apply these techniques for initial screening of WT1 splicing variants expression in AML and CML patients.
We designed several sequence variants of two forward (specific for +/-5) and two reverse primers (specific for +/-KTS) and one common TaqMan probe. After in silico analyses, selected primers were subjected for testing with plasmids each carrying target sequence of the splicing variant. The qRT-PCRs were performed on RotorGene (Corbett Research). GUS was used as housekeeping gene; primers, probes and protocols were adopted from Gabert et al. (Leukemia 2003, 17:2318). Absolute quantification was applied for data evaluation (reported in percentage).
Most of these tests focused on qRT-PCR specificity. The main test of quantification utility and specificity was performed by using 25 plasmid mixtures that simulate different ratios of particular variants (copies of each variant were in five different ratios to the remaining three variants; from 10/100000 to 100000/10 copies). Finally, total leukocytes from 45 AML and 13 CML patients at diagnosis were analysed to test system utility on real samples. The splicing ratio stability during the time was checked, because there were some specimens of peripheral blood not processed immediately but preserved at 4°C overnight.
Our results showed high specificity and utility of all four qRT-PCRs. We were able to quantify up to 100 copies of each variant. Even in case that one of the variants was in minority compared to others in the plasmid mixture, expected number of plasmid copies was assessed by the qRT-PCR. Splicing variant ratio was found to be conserved, when the blood was preserved overnight in refrigerator. Observed differences fall within the ranges of the technical variability of the four qRT-PCRs (12-50%).
In general, our preliminary data in patient samples showed the +5/-KTS variant as the most abundant in patients with CML and AML. Expression of the +5/-KTS and -5/+KTS highly varied among AML patients within the same FAB subtype. Low level of +5/+KTS variant (median 18% from total WT1) distinguished the high risk group patients (cytogenetic-based risk stratification) from the remaining groups (median 30% from total WT1). Our data also indicate that +KTS/-KTS variant expression levels independent of the presence of exon 5 may be of importance. While in low and intermediate risk groups +KTS and –KTS variants were expressed without significant differences, in the high risk group of patients the expression of –KTS variants was significantly higher. At the same time, the high risk group showed significantly decreased +5 variants levels (independent of the presence of KTS sequence).
In conclusion, we have developed the specific qRT-PCRs detecting the four WT1 splicing variants on mRNA level. Further research is ongoing to confirm the aforementioned significance of different expression of the particular WT1 isoforms.
Supported by MZOUHKT2005.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.