CN-AML is the largest cytogenetic group of AMLs. Understanding the pattern of gene mutation in this patient group has an increasing influence on prognosis, treatment, and options for MRD [4]. WT1 is highly expressed in various types of leukemia, which has advanced its position as both a target for immunotherapy and a means of monitoring MRD [23].
The most important issue of the IPT in AML is the lack of constancy of antigen expression during the disease and that antigen densities for many antigens are identical on leukemic blasts and normal progenitors [24].
hMICL is a stable marker at diagnosis and during follow-up and is homogenously present on the CD34-positive patients, who can be otherwise poorly lineage-characterized IPT and difficult to monitor for MSD [25].
The current study investigated the relationship between WT1 genotypes and the promising marker hMICL receptor expression in 63 patients with CN-AML and determined the treatment outcome in these patients.
Regarding genotyping of WT1, the current study showed that the minor (mutant) allele of WT1 SNP rs16754 was found in 26.89% of patients with AML at the heterozygous state (AG), and in 23.8% of patients with homozygous state (GG), while the wild type (AA) was reported in 57.14% of patients with AML.
These frequencies are like those previously reported by Renneville et al. [26], who found that the minor allele of WT1 SNP rs16754 was expressed in 141 of 511 (27.6%) patients, at the heterozygous state (AG) in 123 patients (24%) and homozygous state (GG) in 18 patients (3.6%).
The same frequencies were reported by Hollink et al., Damm et al., and Ho et al. [27,28,29] However, in an old study by King-Underwood and Pritchard-Jones, they reported that mutations in WT1 occurred in only 10–15% of patients [30].
In the current study, CR was achieved in all AML cases with mutant AG plus GG WT1 genotypes. Also, the OS in mutant WT1 was significantly longer compared to that in wild type (AA). These findings suggest a favorable outcome for patients with mutant WT1 genotypes.
Our results agreed with those of Megías-Vericat et al. who reported in their meta-analysis that both 5 years survival and disease-free survival (DFS) were significantly higher in patients with the variant allele (G) although they did not find any significant effect of this variant on CR [1]. Similar results were reported by Long et al. [11] and Petiti et al. [31].
Similarly, two studies by Damm et al. and Ho et al., one performed in adult CN-AML and the other in pediatric AML, reported that the minor allele of WT1 SNP rs16754 significantly improved clinical outcome [28, 29].
However, a large cancer and leukemia group study reported by Becker et al. found that patients with CN-AML who had the rs16754 (WT1GG) genotype had a more favorable outcome among a subset of patients with FLT3-ITD [32].
Choi et al. in a Korean cohort study revealed that the different genotypes of rs16754 did not have any significant impact on clinical outcome in CN-AML [33].
Similarly, Rennevill et al., Ramzi et al., and Marcucci et al. found no significant difference between patients with wild and variant alleles based on CR and relapse as well as OS and DFS and adults and Hollink et al. found no prognostic impact of this SNP in pediatric AML [26, 27, 34, 35]. This difference can be explained by differences in sample size, laboratory methods racial, and age variations.
Niavarani et al. found good prognostic effect of another WT1 mutation, which is WT1 rs2234593 variant mutation. This supports our finding that exon 7 mutation of this gene carries good prognosis for CN-AML [21].
On the contrary, King-Underwood and Pritchard-Jones suggested that WT1 mutation may represent a poor prognostic indicator in AML [30]. In support of this study, Nyvold et al. noted the emergence of a WT1-mutated subclone following therapy, suggesting that mutation of WT1 could lead to progression of leukemia by conferring drug resistance [36]. Furthermore, Fitzgibbon et al. reported an association between acquired uniparental disomy of 11p and homozygous mutation of WT1 in patients with AML [37].
Owen et al. found a possible negative impact in AML with concurrent WT1 and FMS-like tyrosine kinase 3 (FLT3)-internal tandem duplication (ITD) mutations [38]. This discrepancy may be explained by three reasons: First, their AML cases had FLT3-ITD in addition to WT1 mutation and combined cytogenetic abnormality. Second, they used a clustering of mutations to exons in WT1 by fluorescence-based capillary electrophoresis analysis, which has provided different results. Lastly, their group included AML and ALL together in the survival analysis.
Of note, WT1 SNP rs16754 status did not correlate with the total WT1 messenger ribonucleic acid (mRNA) expression level in two previous studies [28, 29]. In addition, the SNP of WT1 (rs16754) consists of the replacement of a CGA by a CGG codon, which is used two times more often than the CGA to encode arginine.
Thus, the presence of the WT1 SNP rs16754 is increasing the rate of translation, which potentially affects protein folding. The location of WT1 rs16754 in exon 7 SNP rs16754 may not affect the splicing process [26].
The role of SNP rs16754 cannot exclude its possibility in linkage disequilibrium with another genetic aberration that affects drug metabolism and sensitivity. Confounding factors related to patient and disease characteristics, such as age, study restriction to CN-AML, and other accompanying genetic alterations may account for the discordant results recorded regarding the impact of WT1 SNP rs16754 genotype on prognosis.
Differences in treatment protocols between cooperative groups may also be responsible for these contradictory results. The various dosages of cytarabine in post-remission treatment for AML were shown to be related to somatic molecular abnormalities Rat Sarcoma Viral Oncogene (RAS) mutations [39]. One can hypothesize that leukemic cells harboring WT1 SNP rs16754 minor allele may be more sensitive to treatment by cytarabine, which was used in our current study.
Additionally, no significant relationship was detected between pretreatment patient parameters (age, sex, organomegaly, lymphadenopathy, AML phenotype, or other AML-IPT panels) and mutant or wild type. In agreement with our findings, Renneville et al. [26] and Schmid and his colleagues failed to correlate WT1 gene expression with other features of leukemia [40].
In the current study, we found that hMICL receptor expression was positive on leukemic cells in most patients with CN-AML. In agreement with our finding, van Rhenen et al. acknowledged hMICL as a surface antigen expressed on aCD34-positive AML and considered the antigen as a possible target in antibody-mediated therapy [41]. Several studies suggest that the routine use of hMICL can increase the value of FCM in diagnosis of AML [42].
Furthermore, Larsen et al. reported that hMICL was found in approximately 92% of studied patients with AML patients. Also, they reported that it was absent on lymphoid blast cells in all studied cases suggesting its specificity and potential value as a marker to discriminate AML from ALL in a routine FCM examination and revealed the validity of this antigen as a stable pan-AML marker that can increase both the diagnostic accuracy and MRD marker identification by FCM in AML cases during therapy and treatment follow-up [14].
To explore the prognostic impact of hMICL expression, response to treatment analysis revealed that all patients with negative hMICL expression achieved CR at day 28 of therapy and maintained CR till the end of follow-up period (24 months).
However, there was no statistically significant difference between hMICL expression and patients features, such as age, sex, hepatomegaly, lymphadenopathy, AML phenotype, and other IPT panels.
In agreement with our results, Bakker et al. recorded no significant difference between hMICL expression and different FAB subtypes studied, except for M3 cases, which recorded the highest expression of hMICL [43].
The OS was significantly longer in patients with negative hMICL expression compared to patients with positive hMICL expression. This suggested its important role in the selection of cases with good response to therapy.
In contrast to our results, Roug et al. found no relationship between disease remission and hMICL percentage expression [15]. Also, Eissa et al. found stable expression of hMICL through the disease and questioned the value of hMICL-based IPT in detection of treatment failure, which unfortunately happens in most patients with AML [44].
The current study had some limitations due to the small number of patients and short follow-up period so a larger study with longer follow-up period is recommended.