Overexpression of PRDM16 in the presence and absence of the RUNX1/PRDM16 fusion gene in myeloid leukemias

In a recent study published in Genes, Chromosomes & Cancer, Sakai et al. (2005) reported the identification of a novel RUNX1 partner gene, MDS1/EVI1-like gene 1 (PRDM16), in a patient with acute myeloid leukemia (AML) M4 with the t(1;21) translocation. This fusion gene has also been described recently in a case of t(1;21) positive therapy-related AML (Stevens-Kroef et al., 2006). The t(1;21)(p36;q22) is a rare but recurrent translocation associated with de novo and therapy-related AML and with myelodysplastic syndromes (MDS). We, here, describe the first case of chronic myeloid leukemia (CML) blast crisis cells in which the RUNX1/PRDM16 fusion gene likely contributed to clonal evolution from CML to AML. A 59-year-old woman was referred in July 2000 for a CML. Cytogenetic analysis showed 46,XX,t(9;22)(q34.1;q11.2) in 20 metaphases (UPN 00-H59). She was treated initially with IFN for 20 months with a minor cytogenetic response and then with imatinib mesylate. After 6 months of imatinib, she developed a blastic transformation with 68% blast cells in the peripheral blood. Cells were obtained after an informed consent. Conventional G-banding karyotype showed a possible deletion at band 21q22 in addition to the t(9;22) in all metaphases. Spectral karyotyping of these cells revealed a t(1;21) translocation in addition to the t(9;22) translocation (UPN 02-H056)(Fig. 1A and 1B). The t(1;21) translocation was not present in chronic phase cells isolated from this patient. Fluorescence in situ hybridization (FISH) experiments were performed on peripheral blood metaphases of the blast phase cells using bacterial artificial chromosomes (BACs) and P1-derived artificial chromosomes (PACs) clones obtained from the BACPAC Resource Center (Children’s Hospital Oakland Research Institute in Oakland, California, http://bacpac.chori.org/ home.htm). Clone physical positions and covered genes are available at the UCSC Human Genome Browser (http://genome.ucsc.edu/). The RUNX1 gene located at band 21q22.12 was first studied using two selected BACs, RP11-771C10 (containing intron 6 to exon 8 and the 30 end of RUNX1) and RP11-299D9 (containing intron 1 to exon 8 and the 30 end of RUNX1). Split signals were observed with BAC RP11-299D9 (Fig. 1C). The candidate gene, PRDM16, located at band 1p36.32 was studied using two PACs, RP1-163G9 (containing the 50 end of PRDM16, exon 1 and intron 1) and RP4-785P20 (containing intron 3 to exon 17 of PRDM16) and the BAC RP11-659J6 (containing part of intron 1, exons 2 and 3, part of intron 3 of PRDM16). A split signal was detected using PAC RP1-163G9, indicating that the breakpoint was in intron 1 of PRDM16 (Fig. 1D). On the basis of the results of the FISH studies, we designed PCR primers covering most exons of RUNX1 in both directions (forward and reverse), and for PRDM16 in exon 1 (forward), exons 2–4 (reverse) to assess the expression of PRDM16/RUNX1 and RUNX1/ PRDM16 fusion products. RT-PCR analysis and sequencing of the amplicons revealed eight RUNX1/PRDM16 transcripts joining RUNX1 exons 5 or 6 and PRDM16 exons 2 or 3. Two RUNX1/ PRDM16 cDNAs represented an open reading frame encoding exons 1–5 or exons 1–6 of the RUNX1 gene containing the RUNT domain fused to exon 2 of PRDM16 that contain the PR domain (Fig. 2). However, the reciprocal PRDM16/RUNX1 fusion transcript was not detected. We analyzed the relative expression levels of PRDM16 using real-time PCR (ABI Prism 7900HT Sequence Detection System) in our CML case at the time of chronic phase (UPN 00-H59) and blast crisis (UPN 02-H056), and we compared PRDM16 transcript levels in this case to a series of 88 other myeloid malignancies without evident karyotypic abnormalities of chromosomal band 1p36.3 (including 4 CML-blast crisis derived cell lines, 6 CML blast crisis samples, 14 MDS and MDS transformed to AML samples, and 68 AML samples of different morphological and cytogenetic groups). The Ct (threshold cycle) values of PRDM16 were normalized to an endogenous control gene (GAPDH)