Comparable Distribution of Minimal Residual Diesease between CD34 Positive Hematopoietic Progenitors, CD34 Negative Cells and Total White Cells in Patients with Chronic Myeloid Leukemia (CML) in Molecular Response.
Authors: Lange Thoralf , Thielsen Thomas , Leiblein Sabine , Alt Ruediger , Zschau Guido , Burckhardt Ralf , Kovacz Ines , Al-Ali Haifa K. , Franke Christina , Krahl Rainer , Niederwieser Dietger , Deininger Michael W. 
Published: October 2019

Objectives: Most patients with chronic phase CML attain a complete cytogenetic response (CCR) to imatinib (IM) but BCR-ABL remains detectable by RT-PCR, indicating persistence of residual disease. One small study (Bhatia et al. Blood101(12):4701–7, 2003) suggested that the levels of minimal residual disease (MRD) in CCR patients may be highest in the CD34+ cells but it is not clear whether the cohort under study was truly representative of patients with stable CCR. To address this question, we compared the level of MRD in total white cells (TWC), CD34-positive (CD34+), CD34-negative (CD34−) and CFU-GM colonies in patients with a molecular response (MR, defined as a BCR-ABL / ABL ratio below 0.12% as suggested by Paschka et al. Blood104 (11): #1013, 2004) to IM.

Patients and Methods: Twenty-two bone marrow samples were obtained from 13 CML patients with CCR [7 male/6 female, median age 57 (range 23–68) years]. At IM start all patients were in chronic phase (8 newly diagnosed, 5 refractory or intolerant to interferon alpha). The median time on IM at the date of the first analysis was 39 (range 10–50) month, the median duration of CCR and MR was 32 (range 4–49) and 20 (range 0–36) month, respectively. The frequency of CD34+ cells in unmanipulated BM as determined by FACS was compared to 33 patients with newly diagnosed CML and 9 healthy individuals. CD34+ cells were selected over immunomagnetic columns. At least 100 CD34+, CD34− and TWC were analyzed for BCR-ABL by FISH (Vysis LSI BCR-ABL ES probe, lab specific false positive rate of < 2%). CD34+ cells were plated in triplicate at 104 cells/ml and CFU-GM were counted on day 14. Quantitative PCR (qPCR) was performed on CD34+, CD34-, TWC and pools of 10 CFU-GM colonies. PCR-negative samples with less than 1000 copies of the ABL gene / μl cDNA were excluded.

Results: The median frequencies of CD34+ cells in patients with MR, newly diagnosed patients and healthy controls were 0.30 (range 0.10–1.56)%, 3.81 (range 0.04–17.77)% and 0.88 (range 0.46 to 1.47)%, respectively (p<0.0005, Kruskal-Wallis-Test). BCR-ABL was undetectable by interphase FISH in all three compartments. Eleven (50%) and 16 (72%) out of 22 samples derived from the CD34+ and CD34− cells contained sufficient c-DNA for PCR. No significant differences were observed between the levels of BCR-ABL mRNA in CD34+, CD34− and TWC [median 0.023 (range, 0.001 to 0.149)%, 0.020 (range 0,005– 0,134)% and 0.011 (range 0.0005 to 0.257)%], respectively (p= ns, Wilcoxon-test). In the patients with more than one sample analyzed, the distribution of MRD between the compartments did not change significantly over time. C-DNA from colonies was available for 14 patients (63%). Again, no significant difference in BCR-ABL mRNA in comparison to CD34+, CD34− and TWC was detected [median 0.001 (range 0,001 to 0.0425)%].

Conclusion:

In CML with MR to IM CD34+ cells are consistently BCR-ABL-negative. Comparable levels of BCR-ABL mRNA residual disease are present in total white cells, CD34+, CD34- and CFU-GM colonies, suggesting that residual disease is not limited to immature cells.