Relationship of WT1 Gene Expression to Disease Progression and Imatinib Responsiveness in Chronic Myelocytic Leukaemia Patients.
Authors: Varma Subhash , Anand Maninder Singh , Varma Neelam , Malhotra Pankaj 
Published: October 2019

Abstract
Abstract 3301
Poster Board III-189

Introduction
WT1 is an important regulatory molecule involved in cell growth and development. In adult tissues, WT1 expression is found in the urogenital system, the central nervous system and in tissues involved in hematopoiesis, including the bone marrow and lymph nodes. Because of the presence of zinc fingers in the C-terminal half of the protein, WT1 has been found to be a potent transcriptional regulator. Through transfection experiments, it has been found that WT1 can either enhance or repress the expression of specific target genes or constructs. Reported dual properties of WT1 as a tumor suppressor or oncogene are thought to be context / tissue specific. Most cases of chronic myelocytic leukemia (CML), acute myelogenous leukemia, acute lymphoblastic leukemia, and myelodysplastic syndrome (MDS) show WT1 overexpression. However, role of WT1 gene expression as a diagnostic and minimal residual disease (MRD) marker needs to be explored further. Tri-phasic nature of CML provides a unique opportunity to study the relationship of WT1 gene expression with various phases of disease and its responsiveness to imatinib mesylate (a tyrosine kinase inhibitor – TKI).

Patients and Methods
213 peripheral blood samples were obtained from 180 CML patients. 189 patient samples were from chronic phase (CP) patients, 6 from accelerated phase (AP) and 18 from blast crisis (BC) patients. Study population included imatinib naïve as well as imatinib treated CML patients. Total RNA was extracted using commercially available Qiagen kit. First strand cDNA was synthesized from 200 ng of total RNA using MBI Fermentas cDNA synthesis kit. RNA concentration, purity and amount were determined by UV spectrophotometry. Real-time-polymerase chain reaction (Real-time PCR) was performed for relative/comparative quantification of WT1 gene on the Stratagene Mx3005P QPCR system using SYBR Green chemistry. In comparative quantitation, the sample quantities were defined in terms of fold-change compared to a reference sample (calibrator). This approach eliminated determination of absolute template quantity and running standard curves with each experiment. Jurkat cell line was used as a calibrator. β-actin was used as a reference gene to check for sample-to-sample variation. All experiments were carried out in duplicate with appropriate negative controls. WT1 gene expression was compared (i) among 3 different phases of CML: CP (189 samples), AP (6 samples) and BC (18 samples), (ii) among 7 different sub-groups with respect to exposure and responsiveness to TKI; namely CML-CP (freshly diagnosed), 113 patient samples; CML-CP in complete haematological remission (CHR), 56 patient samples; CML-CP in not in CHR (NCHR), 20 patient samples; CML-AP, 6 patient samples; CML-BC (freshly diagnosed), 6 patient samples; CML-BC in CHR, 6 patient samples and CML-BC in NCHR, 6 patient samples. All 6 CML-AP patients were imatinib naïve. Log fold change in the values of WT1 gene expression was analyzed with respect to the 3 phases of CML (CP, AP & BC) and 7 different sub-groups with respect to exposure and responsiveness to TKI.

Results
WT1 gene expression was significantly higher among CML-AP and CML-BC, as compared to CML-CP (p=0.003). The expression of WT1 gene was significantly higher among CML-AP as compared to CML-CP. WT1 gene expression was significantly different among CML-CP (Fresh) vs. CML-AP (p=0.011), CML-CP (Fresh) vs. CML-BC (Fresh) (p=0.026), CML-CP (CHR) vs. CML-AP (p=0.050) and CML-CP (NCHR) vs. CML AP (p=0.036).

Conclusions
The level of expression of WT1 was directly proportional to disease progression (higher in CML-AP and BC compared to CML-CP), thus advocating its role as an important prognostic marker. In addition WT1 gene expression also changed significantly among groups with or without imatinib responsiveness, thereby indicating its possible usefulness as a surrogate marker for treatment monitoring.

Disclosures
No relevant conflicts of interest to declare.