WT1 Protein Expression in Myeloid Malignancies: An Immunohistochemical Study

Abstract 4886

The Wilms tumor 1 (WT1) gene encodes for a zinc-finger protein that may act both as a tumor suppressor and as an oncogene. It is widely expressed in normal tissues, especially in the urogenital system during fetal development. WT1 is a transcriptional regulator of genes involved in the cell growth and metabolism, such as c-Myc, bcl-2 and epidermal growth factor receptor (EGFR). Its abnormal mRNA overexpression in myeloid malignancies has been exploited as minimal residual disease marker. WT1 mutations have also been described in a percentage of acute myeloid leukemia (AML) with a normal karyotype. It has been reported that these patients had a bad outcome. Few data are available on WT1 protein expression in myeloid neoplasms.

WT1 expression was investigated in bone marrow samples obtained from 61 patients (54 AML, 1 MPN, 5 MDS and one case with myeloid sarcoma) and 5 controls. Blast cell immunophenotype and cytogenetics were available for each case. WT1 mutations were investigated in 14 AML cases. Immunohistochemistry procedures were performed on formalin-fixed paraffin embedded whole tissue sections with primary monoclonal antibody against WT1 (WT1 MxH 6FH2 clone, DAKO, Glostrup, DK). The staining intensity was graded on a 0 to 3 scale (0, negative; 1, weak; 2, moderate; and 3, intense staining) and the percentage of stained cells was scored. To obtain the final score, both values (staining intensity and percentage of positive cells) were multiplied. Cases with WT1 score under 10 were considered as negative. We also analyzed WT1 mRNA gene expression on the same cases by real-time PCR as previously described (Cilloni D et al, J Clin Oncol 2009).Relationship between quantitative variables was evaluated by non-parametric correlation analysis (Spearman correlation test). Two-sided p value under 0.05 was considered significative.

In normal bone marrow, WT1 immunohistochemical expression was restricted to the cytoplasm of occasional megakaryocytes and endothelial cells, whereas no nuclear staining was observed neither in hematopoietic cells nor in endothelial and circulating lymphoid cells. Immunohistochemical WT1 expression was found in 43 out of 61 (70.5%) cases. The mean value of mRNA WT1 expression was 3812.3 copies (standard deviation (SD):7163.9). We observed a positive correlation between WT1 score and mRNA WT1 expression (p<0.0001; Spearman's rho coefficient=0.611). Moreover, this correlation was maintained when we separated normal and complex karyotype groups (Normal: p<0.0001; Spearman's rho coefficient=0.755; Complex: p=0.023; Spearman's rho coefficient=0.318). Nevertheless, in four cases (3 AML and one CMML) with no WT1 protein expression, the WT1 mRNA levels were above 1800 copies (Normal<200). In the three cases with WT1 mutations the results were also discordant.

In myeloid malignancies, WT1 protein expression showed some degree of correlation with the WT1 mRNA levels. It remains to be investigated the mechanisms which can explain the discordances and if these findings could provide prognostically relevant information.