Frequency of a Highly Polymorphic Variant, S36N GFI1, Is Increased in CML Patients

To date no single molecular biomarker, with proven clinical utility, that is predictive and identifies patients with more aggressive chronic myeloid leukaemia (CML) at diagnosis, has been reported. Such a molecular biomarker would enable stratification of patients according to aggressiveness of the malignancy and therefore at greater risk of progressing to blast crisis (BC). If these patients could be identified a more potent TKI could be prescribed at presentation or be considered for stem cell transplant up front. Recently a common polymorphic variant of growth factor independent 1 (GFI1), a zinc finger DNA binding transcriptional repressor, whereby serine is substituted by asparagine at residue 36 (S36N), was reported (Khandanpour et al. Blood. 2010; 115:22462) to be associated with a 1.6 fold increased risk of developing acute myeloid leukemia (AML). In addition, investigators reported a frequency of 11% of the S36N GFI1 allele among AML patients, compared with 3-7% among the European populations. Reports suggest S36N GFI1 variant fails to repress the target genes, including HOXA9, and as a consequence their expression levels are elevated. These observations are consistent with a critical role for GFI1 in myeloid differentiation. In view of these findings we hypothesized that the GFI1 S36N allele might influence CML progression, via up-regulation of HOXA9, from a relatively indolent chronic phase (CP) to invariably fatal BC, characterized by presence of leukaemic stem cells, resistant to tyrosine kinase inhibitors in peripheral blood. Therefore, we assessed if S36N allele frequency was increased in CML and quantified GFI1expression.

We retrospectively studied 106 cDNA samples, synthesized using 1 µg RNA extracted from nucleated cellular pellets isolated from peripheral blood or bone marrow aspirate. Total RNA was reverse transcribed using reverse transcriptase primed with random hexamers oligonucleotides. Of the 196 sample 89 were from highly heterogeneous CML patients and the remaining 17 comprised of randomly selected normal control samples from healthy adult volunteer blood donors. The 89 CML patients included 24 (accelerated phase: 2 ; BC:22) with 51 years median age (range 20-75) who had progressed to advanced disease (AD) of these 13 were males. The remaining 65 CML patients (M:43 ; F: 22) were in chronic phase (CP) with median age 74 years (range 7-90). Polymerase chain reaction products, containing GFI1 exons 2, 3 and 4 were subjected to restriction fragment length polymorphism using BfaI restriction enzyme to test for the S36N GFI1 allele. Samples with the S36N allele were confirmed by Sanger sequencing. Total GFI1 (i.e. serine and asparagine alleles), HOXA9 and GUSß (endogenous control gene) mRNA transcripts were quantified by quantitative real time using Taqman assay. Each Taqman assay included a standard curve constructed using 2 plasmids containing HOXA9 or GUSß insert. Serially diluted cDNA derived from MEG01 hematopoietic cell line was used to prepare the standard curve for GFI1quantification.

S36N GFI1 allele frequency was significantly increased (p<0.001) among the CML patients (11/89) compared with the normal control group (1/17), but we detected no significant difference between AD and CP samples. However, the total GFI1 mRNA expression was significantly increased in CP samples compared with the control group (p<0.0001). But its expression was significantly decreased in AD compared with those in CP (p=0.0096). Interestingly HOXA9 expression was significantly increased (p=0.018) among individuals expressing the asparagine allele (i.e. heterozygous and homozygous subjects). But we observed no correlation between HOXA9 and GFI1 expression in CML patients.

These data suggest an association of S36N GFI1 allele with CML. Furthermore, the study implies the variant allele is associated with increased HOXA9 expression, which may influence disease progression viaMusashi 2. HOXA9 is reported to upregulate Musashi 2, which we and others have shown is increased in CML BC patients. The observed inverse expression of GFI1 levels between CP and AD is consistent with its reported role in myeloid cellular differentiation. These observations imply two mechanisms by which GFI influences CML disease kinetics: decreased GFI1 expression and/or elevated HOXA9 levels due to the presence of the S36N GFI1 allele.