Mobilization of CD34+CD38- hematopoietic stem cells after priming in acute myeloid leukemia

Table 1 Characteristics at diagnosis and outcome of the 8 acute myeloid leukemia patients

Pts	Age(yr)	Sex	Cytogenetics	CD34+(%)1	FAB	Molecular Biology2	WBC(x 109/l)2	Risk-group	G-CSFpriming	BM 15 d	CR	Consolidationand DFS
1	59	M	Normal	85	M5	Flt3-ITD neg CEBPA pos	55.1	Favorable intermediate	Yes	No blast	Yes	Chemotherapy 15+ mo
2	35	M	Normal	85	M2	Flt3-ITD neg CEBPA pos	4.8	Favorable intermediate	Yes	No blast	Yes	Chemotherapy 16+ mo
3	56	F	+8, del (20) (q11; q13)	93	M1	Flt3-ITD pos	12.6	Poor intermediate	Yes	ND	Yes	Chemotherapy 3 mo
4	19	M	Normal	67	M6	Flt3-ITD pos MLL pos	0.9	Unfavorable	Yes	ND	Yes	AlloSCT 6 mo
5	55	M	Normal	8	M1	Flt3-ITD pos NPM1 pos	147.3	Poor intermediate	Yes	ND	Yes	AlloSCT 14+ mo
6	55	F	Normal	3	M5	Flt3-ITD neg Evi-1 pos	3.7	Unfavorable	No	5% blasts	Yes	AlloSCT 14+ mo
7	44	M	Complex	94	M0	Flt3-ITD neg NPM1 neg	1.4	Unfavorable	No	> 5% blasts	No3	AlloSCT 11 mo
8	45	F	–7, +21	83	M4	Flt3-ITD neg NPM1 neg	2.4	Unfavorable	No	No blast	Yes	AlloSCT11+ mo

¹Expression of CD34 by leukemic cells;

²Molecular biology and WBC count at diagnosis;

³Morphological complete remission was subsequently achieved by salvage therapy combining idarubicin with high-dose cytarabine. Patients prognosis was defined according to the following classification based on cytogenetics and molecular marker analyses. Karyotype abnormalities that involved core binding factor (CBF) leukemias [t (16; 16) (p13; q22), inv (16) (p13; q22), or t (8; 21) (q22; q22)] with or without other cytogenetic abnormalities were considered favorable cytogenetics. Monosomies or deletions of chromosomes 5 and 7; abnormalities of the long arm of chromosome 3 (or Evi-1 gene mutation); 11q23 abnormality (or MLL gene mutation); or complex cytogenetic abnormalities (defined as at least five unrelated cytogenetic clones) were considered unfavorable risk factors. Other cytogenetic abnormalities and cytogenetically normal (CN)-AML were designated intermediate risk factors. Intermediate-risk cytogenetics was further subdivided into a favorable intermediate risk group [CN-AML with nucleophosmin (NPM1) or CCAAT/enhancer-binding protein-(CEBPA) mutations and no FLT3-ITD] and a poor intermediate risk group (other patients). Induction chemotherapy consisting of a TSC including a first sequence combining daunorubicin, 60 mg/m² per day IV on 1–3 d, and cytarabine, 500 mg/m² per day IV over the same period. The second sequence, administered after 4 d free interval, consisted of daunorubicin, 35 mg/m2 per day, IV on 8 d and 9 d, and cytarabine, 1000 mg/m² per 12 h on 8–10 d. Five patients received G-CSF (filgrastim) priming (5 μg/Kg per day) on 1 to 10 d. Three patients were not primed with G-CSF. AlloSCT: Allogeneic stem cell transplantation; BM: Bone marrow; CR: Morphological complete remission; DFS: Disease-free survival; F: Female; FAB: French-American-British morphological classification; M: Male; neg: Negative; pos: Positive; Pts: Patients; WBC: White blood cell; G-CSF: Granulocyte Colony-Stimulating Factor.

Table 2 Circulating CD34⁺ cell subsets before, during and after intensive chemotherapy

Time	CD34 vs CD38
	CD34+CD38-	CD34+CD38low	CD34+CD38+
At diagnosis (T0)
Median	7.32%	35.45%	56.74%
Mean ± SD	15.40% ± 19.21%	32.35% ± 20.41%	53.81% ± 35.37%
At 4 d (T4)
Median	12.82%	39.15%	37.30%
Mean ± SD	21.17% ± 22.68%	34.01% ± 23.08%	45.11% ± 37.48%
At 8 d (T8)
Median	15.99%	26.21%	36.81%
Mean ± SD	30.14% ± 29.51%	30.28% ± 20.87%	39.06% ± 32.98%
At 10 d (T10)
Median	41.35%	22.99%	18.90%
Mean ± SD	48.18% ± 26.79%	27.86% ± 18.65%	24.29% ± 22.73%
At 15 d (T15)
Median	73.56%	26.18%	3.93%
Mean ± SD	66.42% ± 20.18%	27.33% ± 9.26%	8.05% ± 12.24%
At recovery (TR)
Median	5.37%	32.76%	63.42%
Mean ± SD	10.63% ± 13.81%	29.53% ± 11.23%	61.79% ± 18.39%

TR: Time of cell recovery.

Table 3 Mean percentage of viable, apoptotic and necrotic cells in peripheral blood and bone marrow samples at T0, T15 and time of cell recovery

Cell population	T0 (BM)	T0 (PB)	T15 (BM)	T15 (PB)	TR (BM)	TR (PB)
Immature cells
Viable cells	75.5 ± 17.6	52.0 ± 21.2	18.5 ± 3.5	14.5 ± 12.0	37.0 ± 16.9	34.0 ± 14.1
Apoptotic cells	17.5 ± 17.6	19.5 ± 21.9	33.0 ± 7.0	27.5 ± 23.3	34.5 ± 13.4	24.5 ± 7.7
Necrotic cells	3.0 ± 1.4	28.0 ± 0.0	45.0 ± 11.3	54.0 ± 38.1	25.0 ± 2.8	38.5 ± 24.7
CD34+ cells
Viable cells	77.5 ± 20.5	59.5 ± 33.2	34.0 ± 19.7	24.5 ± 3.5	65.0 ± 25.4	29.0 ± 19.7
Apoptotic cells	20.5 ± 20.5	34.5 ± 28.9	31.0 ± 29.6	38.0 ± 15.5	32.0 ± 22.6	44.0 ± 1.4
Necrotic cells	2.5 ± 0.7	5.0 ± 2.8	35.0 ± 8.4	34.0 ± 21.2	3.0 ± 2.8	24.0 ± 19.7
CD34+CD38- population
Viable cells	68.0 ± 19.7	59.5 ± 34.6	52.5 ± 24.7	21.5 ± 10.6	51	32.5 ± 30.4
Apoptotic cells	27.5 ± 24.7	38.0 ± 33.9	33.5 ± 23.3	60.0 ± 2.8	47	52.5 ± 16.2
Necrotic cells	0.5 ± 0.7	2.0 ± 1.4	13.5 ± 2.1	18.0 ± 14.1	1	22.5 ± 0.7
CD34+CD38low population
Viable cells	72.5 ± 26.1	48.0 ± 43.8	15.5 ± 6.3	15.0 ± 7.0	33.5 ± 2.1	30
Apoptotic cells	25.5 ± 26.1	45.0 ± 38.1	34.5 ± 28.9	38.5 ± 20.5	54.0 ± 5.6	49
Necrotic cells	2.0 ± 1.4	7.0 ± 5.6	49.5 ± 33.2	46.5 ± 27.5	12.5 ± 3.5	18
CD34+CD38+ population
Viable cells	78.0 ± 19.7	70.5 ± 30.4	21.5 ± 7.7	27.5 ± 6.3	67.0 ± 25.4	44.5 ± 36.0
Apoptotic cells	19.0 ± 19.7	23.5 ± 27.5	26.0 ± 26.8	20.5 ± 20.5	31.0 ± 24.0	30.5 ± 0.7
Necrotic cells	2.0 ± 0.0	5.0 ± 2.8	53.0 ± 18.3	42.0 ± 43.8	1.5 ± 0.7	28.0 ± 39.5

BM: Bone marrow; PB: Peripheral blood; TR: Time of cell recovery.