Feng X, Hui KM, Younes HM, Brickner AG. 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 (= 215) compared to healthy B-cell controls (= 12)(= 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. (6/11 B-ALL patients) with no detectable antigen expression in eight healthy volunteer samples (Figure ?(Figure1;1; Table ?Table2B).2B). All other genes studied (BCP-20, END, G250, HAGE, NY-ESO-1, p68 RNA helicase, SSX2IP and tyrosinase) were detectable in patient samples and healthy volunteers, except PAS domain-containing protein 1 (PASD1) and SSX2 which were not detected in either. Due to limited sample availability we choose six of the antigens, that were differentially expressed in patients compared with normal controls (Survivin, WT1 and END) or of particular interest to our group (PASD1, SSX2, SSX2IP), for further investigation by qPCR. Table 1A Patient information = 11) encompassing the analysis of 13 samples. qPCR analysis of antigen expression in B-ALL and healthy donor samples A two-way ANOVA test was used to determine whether there was a statistical difference between transcript expression of END, PASD1, SSX2, SSX2IP, Survivin and WT1, as determined by qPCR, in B-ALL patients (ALL001-8, 11 and 14) compared with healthy volunteers. Survivin had a significantly higher expression in seven of the ten B-ALL patients analysed, compared to healthy controls (= 0.015) (Figure ?(Figure2A).2A). Its median CT value (7.19) in patients was much lower compared to the median CT value (12.81) in normal controls. was expressed by three out of ten adult B-ALL patients (Figure ?(Figure2B)2B) however the median CT of B-ALL patients and normal controls, 12.88 and LY2452473 12.81 respectively, were almost equal. Therefore, there was no significant difference detected by the two-way ANOVA test between these two groups. Open in a separate window Figure 2 Relative expression of transcripts encoding each antigen in the B-ALL patients compared to the healthy volunteersDots indicate the CT values, whereby the CT of the endogenous control is subtracted from the CT of each gene. Genes that were not expressed were assigned a CT value of 40. The higher the CT value, the less antigen was expressed. All CT values for antigens Rabbit polyclonal to HIRIP3 were lower level than the reference gene and synovial sarcoma, X breakpoint 2 (expression were not detected in any of the adult B-ALL patients or healthy volunteers (Figures ?(Figures2C2C and ?and2D).2D). Nine out of ten patients expressed (Figure ?(Figure2E)2E) while seven out of ten expressed END (Figure ?(Figure2F).2F). Although the expression of these genes were high, their transcripts were also found in three of five healthy volunteers. Immunolabelling of antigen expression in B-ALL using immunocytochemistry The cell lines K562, OCI-LY3 and MDA-MB-231 were used LY2452473 to demonstrate the effectiveness of immunolabelling to detect the expression of END, PASD1, SSX2, SSX2IP, survivin, and WT1 (Table ?(Table3).3). Four out of five antigens had a cytoplasmic and nuclear localisation, while WT1 was only found in the cytoplasm of the K562 cells (Figure ?(Figure3).3). The immunoreactivity score of both survivin and WT1 was moderate, while SSX2 and SSX2IP showed a weak labelling in K562 (Table ?(Table3).3). END was not expressed in the K562 cell line. OCI-LY-3 cell line was used as an extra control for the expression of PASD1 and showed high levels of PASD1 in the cytoplasm and near the cell membrane. END was moderately expressed on the surface of MDA-MB-231 cells grown on coverslips, confirming the findings of previous studies . Table 3 Frequency of immunolabelling of the six antigens of interest in K562, OCI-LY-3 and MDA-MB-23 cells. = 10) expressed moderate to high levels of each antigen (SSX2, SSX2IP, Survivin and WT-1) while survivin expression was not detected LY2452473 in the healthy volunteer samples. In contrast, one of six healthy volunteers expressed SSX2 at High levels, two of six healthy volunteers had detectable SSX2IP expression detectable at moderate levels, and WT1 at moderate levels. The ICC experiments were performed twice, with controls, due to limited samples being available, but the results were reproducible. Table 4 Immunoreactivity score for the antigens SSX2, survivin, SSX2IP and WT1 LY2452473 as detected by ICC in B-ALL patient samples and healthy donors = 0.013) was significantly over-expressed in.