BMI-1 expression and outcome. (A) OS according to BMI-1 expression as assessed by Q-RT-PCR in the whole cohort of 84 patients. (B) LFS according to BMI-1 expression. (C) Cumulative incidence of grades 2 to 4 acute GVHD according to BMI-1 expression. In multivariate analysis, when comparing grades 0 to 2 or grade 2 acute GVHD versus the severe acute GVHD group (grades 3-4), the statistical association with BMI-1 remains significant (P = .01 and P = .03, respectively). In terms of chronic GVHD, 74 patients survived to day 100 and were evaluable for chronic GVHD: 41 did not develop any form of chronic GVHD and 33 had a limited (n = 4) or an extensive (n = 29) form. In univariate analysis, no statistically significant associations were found between the 4 analyzed genes and chronic GVHD (BMI-1, P = .07; CD7, P = .12; ELA-2, P = .93; PR3, P = .12). When added to a multivariate analysis, none of these genes was found to be significant. The median gene expression level is used to segregate the patients into a “low BMI-1” group (BMI-1 expression less than median) and a “high BMI-1” group (BMI-1 expression greater than median). Values of BMI-1 represent the Q-RT-PCR expression as a ratio to the GAPDH control gene. For establishment of the Q-RT-PCR assay, the Jurkat cell line was used as a positive control for BMI-1 expression with a standard curve being produced for the amplification of logarithmic dilutions (10−1 to 10−5) of its cDNA. An average of the duplicates of each data point was taken and plotted against the cycle threshold (Ct). The technical variability between duplicate samples in our RT-PCR assays has been established for a number of different genes as less than 1.3-fold at the 95% level of confidence.
