Identification of survivin as a promising target for the immunotherapy of adult B-cell acute lymphoblastic leukemia

Abstract

B-cell acute lymphoblastic leukemia (B-ALL) is a rare heterogeneous disease characterized by a block in lymphoid differentiation and a rapid clonal expansion of immature, non-functioning B cells. Adult B-ALL patients have a poor prognosis with less than 50% chance of survival after five years and a high relapse rate after allogeneic haematopoietic stem cell transplantation. Novel treatment approaches are required to improve the outcome for patients and the identification of B-ALL specific antigens are essential for the development of targeted immunotherapeutic treatments. We examined twelve potential target antigens for the immunotherapy of adult B-ALL. RT-PCR indicated that only survivin and WT1 were expressed in B-ALL patient samples (7/11 and 6/11, respectively) but not normal donor control samples (0/8). Real-time quantitative (RQ)-PCR showed that survivin was the only antigen whose transcript exhibited significantly higher expression in the B-ALL samples (n = 10) compared with healthy controls (n = 4)(p = 0.015). Immunolabelling detected SSX2, SSX2IP, survivin and WT1 protein expression in all ten B-ALL samples examined, but survivin was not detectable in healthy volunteer samples. To determine whether these findings were supported by the analyses of a larger cohort of patient samples, we performed metadata analysis on an already published microarray dataset. We found that only survivin was significantly over-expressed in B-ALL patients (n = 215) compared to healthy B-cell controls (n = 12)(p = 0.013). We have shown that survivin is frequently transcribed and translated in adult B-ALL, but not healthy donor samples, suggesting this may be a promising target patient group for survivin-mediated immunotherapy.

Keywords: WT1; acute lymphocytic leukemia; antigen identification; immunotherapy; survivin.

Figure 1. Expression of transcripts encoding leukemia antigens in cell lines, primary adult B-ALL patients and normal donors

β-actin was used as a positive control for the ability of primers to amplify the cDNA. N was a no (cDNA) template control. Right hand side labels indicate expected product size and M indicates the location of the 1kB marker (HyperLadder I). RT-PCR data is representative of at least two independent experiments.

Figure 2. Relative expression of transcripts encoding each antigen in the B-ALL patients compared to the healthy volunteers

Dots indicate the ΔC_T values, whereby the C_T of the endogenous control GAPDH is subtracted from the C_T of each gene. Genes that were not expressed were assigned a C_T value of 40. The higher the ΔC_T value, the less antigen was expressed. All ΔC_T values for antigens were lower level than the reference gene GAPDH. Streaked dots, representing patient sample ALL004, were outliers that do not represent antigen expression. P-values were determined using a two-way ANOVA test. Ns, not significant.

Figure 3. Immunolabelling of the key antigens of interest in two primary adult B-ALL patient samples

Brown precipitate indicates the immunlabelling of test protein in the cell. Actin acted as the positive control for the assay. Two isotype control antibodies (Ms and Rb isotype) were used as negative controls to ensure there was minimal non-specific antibody binding to antigen. All pictures were taken at 400X magnification and were representative of at least two independent experiments.