Cloning efficiency and growth factor independence in subpopulations of cells in samples from patients with chronic myeloid leukemia and from normal bone marrow, assessed in 10-day serum-free liquid cultures of single cells
| Patient . | CD34+CD38−cells . | CD34+CD3+cells . | CD34−cells . | CD34+ G0cells . | CD34+G1/S/G2/M cells . | CD34+ TPO-resistant† cells . | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| +GF (% Ph−*) . | −GF (% Ph−) . | % GFI . | +GF . | −GF . | % GFI . | +GF . | −GF . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | |
| 1 | 51 (68) | 38 (0) | 74 | 39 | 2 | 5 | 5 | 2 | 47 | 26 (39) | 20 (0) | 76 | 28 (0) | 10 (0) | 37 | 30 (64) | 23 (0) | 77 |
| 2 | 57 (0) | 46 (0) | 82 | 19 | 6 | 31 | 1 | 0 | 0 | 53 (33) | 50 (0) | 94 | 34 (0) | 22 (0) | 64 | 28 (13) | 27 (0) | 95 |
| 3 | 39 (0) | 38 (0) | 96 | — | — | — | — | — | — | — (0) | — (0) | — | 32 (—) | 18 (—) | 55 | 13 (—) | 9 (—) | 75 |
| 4 | 50 (—) | 27 (0) | 54 | — | — | — | — | — | — | 48 (0) | 24 (0) | 50 | 34 (—) | 9 (—) | 26 | 43 (0) | 18 (0) | 42 |
| 5 | 90 (0) | 68 (0) | 76 | 42 | 21 | 50 | 20 | 7 | 32 | 21 (0) | 3 (—) | 15 | 24 (—) | 1.5 (—) | 6 | 40 (0) | 22 (—) | 55 |
| 6 | 74 (0) | 48 (0) | 65 | 22 | 6 | 29 | 1 | 0 | 0 | — (0) | — (0) | — | — | — | — | 15 (0) | 4 (0) | 29 |
| Mean ± SE | 62 ± 9 | 46 ± 7 | 75 ± 6 | 28 ± 7 | 11 ± 5 | 37 ± 7 | 7 ± 6 | 2 ± 2 | 11 ± 11 | 41 ± 10 | 26 ± 14 | 53 ± 23 | 31 ± 2.4 | 13 ± 4.6 | 38 ± 13 | 28 ± 6 | 16 ± 4 | 59 ± 12 |
| NBM 1 | — | — | — | — | — | — | — | — | — | 46 | 0 | 0 | 48 | 0 | 0 | 65 | 0 | 0 |
| NBM 2 | — | — | — | — | — | — | — | — | — | 59 | 0 | 0 | 53 | 0 | 0 | 71 | 0 | 0 |
| Patient . | CD34+CD38−cells . | CD34+CD3+cells . | CD34−cells . | CD34+ G0cells . | CD34+G1/S/G2/M cells . | CD34+ TPO-resistant† cells . | ||||||||||||
|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|---|
| +GF (% Ph−*) . | −GF (% Ph−) . | % GFI . | +GF . | −GF . | % GFI . | +GF . | −GF . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | +GF (% Ph−) . | −GF (% Ph−) . | % GFI . | |
| 1 | 51 (68) | 38 (0) | 74 | 39 | 2 | 5 | 5 | 2 | 47 | 26 (39) | 20 (0) | 76 | 28 (0) | 10 (0) | 37 | 30 (64) | 23 (0) | 77 |
| 2 | 57 (0) | 46 (0) | 82 | 19 | 6 | 31 | 1 | 0 | 0 | 53 (33) | 50 (0) | 94 | 34 (0) | 22 (0) | 64 | 28 (13) | 27 (0) | 95 |
| 3 | 39 (0) | 38 (0) | 96 | — | — | — | — | — | — | — (0) | — (0) | — | 32 (—) | 18 (—) | 55 | 13 (—) | 9 (—) | 75 |
| 4 | 50 (—) | 27 (0) | 54 | — | — | — | — | — | — | 48 (0) | 24 (0) | 50 | 34 (—) | 9 (—) | 26 | 43 (0) | 18 (0) | 42 |
| 5 | 90 (0) | 68 (0) | 76 | 42 | 21 | 50 | 20 | 7 | 32 | 21 (0) | 3 (—) | 15 | 24 (—) | 1.5 (—) | 6 | 40 (0) | 22 (—) | 55 |
| 6 | 74 (0) | 48 (0) | 65 | 22 | 6 | 29 | 1 | 0 | 0 | — (0) | — (0) | — | — | — | — | 15 (0) | 4 (0) | 29 |
| Mean ± SE | 62 ± 9 | 46 ± 7 | 75 ± 6 | 28 ± 7 | 11 ± 5 | 37 ± 7 | 7 ± 6 | 2 ± 2 | 11 ± 11 | 41 ± 10 | 26 ± 14 | 53 ± 23 | 31 ± 2.4 | 13 ± 4.6 | 38 ± 13 | 28 ± 6 | 16 ± 4 | 59 ± 12 |
| NBM 1 | — | — | — | — | — | — | — | — | — | 46 | 0 | 0 | 48 | 0 | 0 | 65 | 0 | 0 |
| NBM 2 | — | — | — | — | — | — | — | — | — | 59 | 0 | 0 | 53 | 0 | 0 | 71 | 0 | 0 |
Growth factor independence (GFI) was calculated as the cloning efficiency measured after 10 days in the absence of any added growth factors (GF) divided by the cloning efficiency obtained in the presence of 300 ng/mL Steel factor and Flt3-ligand plus 60 ng/mL interleukin 3, interleukin 6, and granulocyte colony-stimulating factor. Assays with and without growth factor (+GF and −GF, respectively) were set up with 1 to 3 96-well plates per condition. Differences between the CD34+CD38− cells and either the CD34+CD38+ cells or the CD34−cells, incubated either +GF or −GF, were significant (P = .02 and P < .001, respectively, for cultures +GF, and P = .002 and P < .001, respectively, for cultures −GF).
TPO indicates thrombopoietin; Ph−, Philadelphia chromosome negative; NBM, normal bone marrow; (—), analysis not done.
Cytogenetic or reverse transcriptase–polymerase chain reaction studies were performed on multiple individual clones (2-45 per condition) or, if clones were not available, samples from representative bulk cultures, to confirm Ph/BCR-ABL status.
TPO-resistant cells were those that did not divide during an initial 4-day culture in serum-free medium supplemented with TPO. These cells were isolated on the basis of their carboxyfluorescein diacetate succinimidyl ester fluorescence.