Molecular Profiling of CD34+ Cells from Diagnosis in CML Patients Identifies Low Expression of CD7 with High Expression of Proteinase 3 or Elastase as Predictors of Longer Survival.

Although most patients with chronic myeloid leukemia (CML) have the same initial molecular abnormality, the BCR-ABL fusion gene, the duration of chronic phase (CP) varies widely. To identify the possible molecular basis of this heterogeneity, we studied CD34⁺ cells collected at diagnosis from 68 CML-CP patients. In all cases the Philadelphia chromosome was the only cytogenetic abnormality identified. Cryopreserved cells had all been collected by leukapheresis within 3 months of diagnosis, when the patients still had high leukocyte counts prior to definitive treatment. Although the majority of patients were diagnosed in the pre-imatinib era and treated with hydroxyurea and/or interferon-α, 32 (47%) received imatinib during the course of their disease. The median age at diagnosis was 45.2 years (range 17.6–68.3), and the male: female ratio was 1.8: 1 (44 males, 24 females). Total RNA was extracted from CD34⁺ purified cells. Gene expression profiling using Affymetrix HG-U133A microarrays was performed on two subsets of patients, one comprising patients with an ‘aggressive disease’ who developed blastic transformation (BT) within 3 years of diagnosis (n=10) and, at the other extreme, patients with an ‘indolent disease’ whose BT occurred 7 or more years from diagnosis (n=9). There was no difference in the proportion of the CD34⁺ subsets of CD38⁺ Lin⁺, CD38⁺ Lin⁻ or CD38⁻ Lin⁻ cells between patients in the indolent as compared to the aggressive CML groups. Microarray data were analysed using a combination of MAS 5.0, dChip, RMA and GeneSpring 5.0 software, and revealed 20 genes differentially expressed in patients with aggressive and indolent disease, which were validated by quantitative real-time polymerase chain reaction (qPCR). The correlation between microarray and qPCR expression measures was highly significant for 18/20 genes (p<0.005, Spearman’s Rho). In the complete cohort of 68 patients, a multivariate Cox regression model identified the combination of low CD7 expression with high expression of proteinase 3 (PR-3) or elastase as associated with longer survival (p=0.0004 and p=0.0006, respectively). The levels of cytoplasmic PR-3 and surface CD7 protein expression by flowcytometry were also of prognostic value (p=0.029 and p=0.031, respectively). Patients who had more than 11% CD34⁺ cells expressing surface CD7 protein at diagnosis had a poor survival. Conversely, patients with more than 2% CD34⁺ cells expressing cytoplasmic PR-3 had a superior survival. This differential pattern of gene expression probably reflects the intrinsic heterogeneity of the disease; if so, assessing expression levels of selected genes at diagnosis may be valuable in predicting duration of survival in patients treated upfront with imatinib and the newer tyrosine kinase inhibitors.