Detection of IL-3 and g-csf mRNA and protein in CD34+ progenitor cells and serum from cml patients

Our previous studies demonstrated that primitive subsets of leukaemic cells isolated from patients with chronic phase CML exhibit growth factor independent proliferation, in both single cell and bulk cultures, which is at least partially dependent on autocrine production of bioactive IL-3 and to a lesser degree G-CSF (PNAS 96; 12804, 1999). In those studies ∼85% of the cases investigated were CML patients in whom the stem cell compartment (LTC-IC) was already predominantly Ph + . Since such cases represent less than 10% of newly diagnosed patients, it was of interest to examine IL-3 and G-CSF mRNA expression in CD34 + progenitor cells and any corresponding protein production in serum in a larger series of CML patients independent of Ph status of their LTC-IC compartment. CD34-enriched lineage-negative cells were isolated using STEMSep TM from leucapheresis samples obtained at diagnosis from chronic phase patients and viable, propidium iodide (PI)-negative, annexin V-negative, CD34 + cells then isolated by FACS. Total RNA was extracted from ≥5 × 10 4 such cells and c-ABL, BCR-ABL, IL-3 and G-CSF expression examined using a 2-step RT-PCR procedure. 8 of the 10 CML samples tested to date are positive for IL-3 expression. Using sensitive ELISA kits, G-CSF was detected at significantly lower levels in the sera of CML patients (7.2 ± 2.9 pg/ml, n = 13) than in normal controls (24.5 ± 3.2 pg/ml, n = 9, p = 0.001), with levels correlating inversely to the WBC. In untreated patients with elevated WBC (45–166 × 10 9 /L) G-CSF levels proved to be undetectable. In patients on hydroxyurea, in whom the WBC was controlled (< 15 × 10 9 /L), levels were low but above the threshold for detection (2–11 pg/ml). Of interest, in patients who had achieved a complete cytogenetic remission following either allogeneic BMT or interferon therapy, levels were within the normal range. These findings support the proposed negative feedback mechanism between peripheral neutrophil count and serum G-CSF levels. IL-3 was detected in sera from 1 to 9 normal controls and 5 of 29 CMLs (range 1–35 pg/ml), 3 of whom were in accelerated phase. These results confirm that IL-3 mRNA expression in the progenitor compartment is typical of the majority of cases of CML. However, this does not necessarily lead to detectable levels of this growth factor in the circulation suggesting its effects in vivo may be largely confined to extravascular regions of the marrow and other tissues that are infiltrated by CD34 + leukaemic cells.