Assessment of the Wilms' Tumor Gene (WT1) Expression in High-Risk Neuroblastoma (NB) Patients WHO Underwent Autologous Hematopoietic Stem Cell Transplantation (HSCT)

In spite of intensive multimodal therapy including HSCT, it is difficult to assess remission in NB. We have previously demonstrated that WT1is a surrogate marker of proliferation in leukemia.  To assess the status of NB cell proliferation post HSCT, WT1 gene expression was evaluated. 42 random bone marrow (BM) samples (from 34 patients) were obtained, at diagnosis (n=24) and post HSCT (n=18; 13 in remission and 5 in clinical relapse). Quantitative RT-PCR was used for the evaluation of the level of gene expression as positive (> 1 x 10⁰ ng/µl), weakly positive (9.9 x 10^-1 to 6.5 x 10^-1 ng/µl) and negative (< 6.5 x 10^-1 ng/µl). Results were extrapolated from a ten fold serial dilution standard curve using a NB cell line (NGP). The levels of WT1 gene expression in NGP were arbitrarily defined as 1.0. The integrity of RNA was confirmed by amplification of the housekeeping gene GAPDH. At diagnosis 83% (18/24 positive, 2/24 weakly positive) (95.9 + 74.2 (0.890- 1500) ng/µl) patients expressed the WT1 gene, where 17% (4/24) (0.199 + 0.0946 (0.007- 0.440) ng/µl) were negative. Post HSCT in remission 92% (12/13) were negative (0.0822 + 0.0192 (0.0026-0.200) ng/µl) in comparison to the time of clinical relapse where 60% (3/5) (1.57 + 0.418 (1.10 – 2.40) ng/µl) were positive for WT1 gene expression. A comparison t-test was performed between the initial gene expression at diagnosis to remission and only positive WT1 gene expression at diagnosis to clinical relapse. Significant difference (p<0.0002) was found when comparing WT1 transcription at diagnosis to remission, whereas insignificance (p<0.5) was found when comparing positive WT1 expression at diagnosis to clinical relapse. This signifies that the WT1 gene is indicative of cellular proliferation further illustrating the status of BM involvement in NB patients post HSCT.