Leukemic engraftment of CD34+ cells from 10 NPM1-mutated AML in immunocompromised mice
| Patient code . | % CD34+ in original sample . | Purity of inoculum . | No./mice . | No. of cells/mice, ×106 . | No. of mice with AML/no. of mice evaluated . | Time of evaluation, w . | % hCD45 in mice BM . | % CD34+ in mice (gated on hCD45) . | % hCD45+/CD19+ (or CD20+) . | WB (NPMm) . | IHC (NPM) . |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 4 | 60 | 96.3 | 1/NOD | 5 | 1/1 | 4 | 27 | 50 | −‡‡ | + | c+ |
| 3/NOD | 2 | 2/3 | 8 | 60.4/80§ | ND/55 | −‡‡ | + | c+ | |||
| 10 | 27 | 98.5 | 2/NOD | 5 | 1/2 | 16 | 0.62 | ND | ND | ND | ND |
| 4/NOD | 2 | 3/4 | 16/16/20/20 | 12.8‖/9.7/0/17.4§ | 1.9/ND/—/2.4 | −‡‡ | + | c+ | |||
| 11 | 11.8 | 96.2 | 2/NOD | 2 | 2/2 | 20 | 39.8§/19.8 | 1 | −‡‡ | ND | c+ |
| 13 | 0.8 | 90 | 1/NOD | 2.5 | 1/1 | 19 | 32.3 | CD34−‡‡ | −‡‡ | ND | c+ |
| 22R* | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 |
| 17*† | 27 | 98.3 | 4/NOG | 2 | 4/4 | 9/9/12/20 | 4.5/15.8/52‖/80§ | ND/52/35/14 | ND/0/3.2**/0 | + | c+ |
| 4/NOG | 1 | 4/4 | 9/9/13/20 | 1.5/1.8/5.8/24.7 | ND/ND/21/23.7 | 0 | + | c+ | |||
| sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | |||
| 23† | |||||||||||
| CD34+ | 1.7 | 99 | 1/NOG | 0.2 | 1/1 | 15 | 21 | 0.57 | 0 | + | c+ |
| 2/NOG | 0.001 | 0/2 | 21 | 0 | — | — | — | — | |||
| CD34− | 99.9 | 1/NOG | 0.2 | 0/1 | 15 | 0 | — | — | — | — | |
| 2/NOG | 0.001 | 2/2 | 21 | 53.8¶/95.7§ | 0 | 0 | + | c+ | |||
| 30† | |||||||||||
| CD34+ | 2.93 | 79 | 4/NOG | 1 | 2/4 | 7/12/18/18 | 28#/27.3#/33**††/47.3**†† | 13/15.2/6.9/10.8 | 25/85/22/52.6 | − | c−/c+§§ |
| CD34− | 99.5 | 4/NOG | 1 | 0/4 | 7/12/18/18 | 0 | — | — | — | — | |
| 32† | |||||||||||
| CD34+ | 1.3 | 92.3 | 3/NOG | 0.5 | 2/3 | 12/18/20 | 8.2#/1.25**/1.54** | ND/0.42/1 | 81.5/15/1 | ND | c−/c+‖‖ |
| 3/NOG | 0.2 | 1/3 | 3/6/18 | 80.8#/32.8#/59.6**†† | 1.2/2.57/0 | 0/36/0.2 | −/+ | c−/c+‖‖ | |||
| 3/NOG | 0.1 | 1/3 | 17/19/20 | 0.72#/0.6††/0.14# | 0.1/2.7/0.3 | 50/5.2/0 | — | c−/c+‖‖ | |||
| 4/NOG | 0.01 | 0/4 | 3/3/15/20 | 0 | — | — | — | — | |||
| CD34− | 98.8 | 3/NOG | 0.5 | 0/3 | 13/18/20 | 0 | — | — | — | — | |
| 3/NOG | 0.2 | 0/3 | 3/6/15/20 | 0 | — | — | — | — | |||
| 4/NOG | 0.1 | 0/4 | 12/15/18/20 | 0 | — | — | — | — | |||
| 4/NOG | 0.01 | 0/4 | 6/7/20/20 | 0 | — | — | — | — | |||
| 34*†‡ | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 |
| Patient code . | % CD34+ in original sample . | Purity of inoculum . | No./mice . | No. of cells/mice, ×106 . | No. of mice with AML/no. of mice evaluated . | Time of evaluation, w . | % hCD45 in mice BM . | % CD34+ in mice (gated on hCD45) . | % hCD45+/CD19+ (or CD20+) . | WB (NPMm) . | IHC (NPM) . |
|---|---|---|---|---|---|---|---|---|---|---|---|
| 4 | 60 | 96.3 | 1/NOD | 5 | 1/1 | 4 | 27 | 50 | −‡‡ | + | c+ |
| 3/NOD | 2 | 2/3 | 8 | 60.4/80§ | ND/55 | −‡‡ | + | c+ | |||
| 10 | 27 | 98.5 | 2/NOD | 5 | 1/2 | 16 | 0.62 | ND | ND | ND | ND |
| 4/NOD | 2 | 3/4 | 16/16/20/20 | 12.8‖/9.7/0/17.4§ | 1.9/ND/—/2.4 | −‡‡ | + | c+ | |||
| 11 | 11.8 | 96.2 | 2/NOD | 2 | 2/2 | 20 | 39.8§/19.8 | 1 | −‡‡ | ND | c+ |
| 13 | 0.8 | 90 | 1/NOD | 2.5 | 1/1 | 19 | 32.3 | CD34−‡‡ | −‡‡ | ND | c+ |
| 22R* | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 |
| 17*† | 27 | 98.3 | 4/NOG | 2 | 4/4 | 9/9/12/20 | 4.5/15.8/52‖/80§ | ND/52/35/14 | ND/0/3.2**/0 | + | c+ |
| 4/NOG | 1 | 4/4 | 9/9/13/20 | 1.5/1.8/5.8/24.7 | ND/ND/21/23.7 | 0 | + | c+ | |||
| sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | |||
| 23† | |||||||||||
| CD34+ | 1.7 | 99 | 1/NOG | 0.2 | 1/1 | 15 | 21 | 0.57 | 0 | + | c+ |
| 2/NOG | 0.001 | 0/2 | 21 | 0 | — | — | — | — | |||
| CD34− | 99.9 | 1/NOG | 0.2 | 0/1 | 15 | 0 | — | — | — | — | |
| 2/NOG | 0.001 | 2/2 | 21 | 53.8¶/95.7§ | 0 | 0 | + | c+ | |||
| 30† | |||||||||||
| CD34+ | 2.93 | 79 | 4/NOG | 1 | 2/4 | 7/12/18/18 | 28#/27.3#/33**††/47.3**†† | 13/15.2/6.9/10.8 | 25/85/22/52.6 | − | c−/c+§§ |
| CD34− | 99.5 | 4/NOG | 1 | 0/4 | 7/12/18/18 | 0 | — | — | — | — | |
| 32† | |||||||||||
| CD34+ | 1.3 | 92.3 | 3/NOG | 0.5 | 2/3 | 12/18/20 | 8.2#/1.25**/1.54** | ND/0.42/1 | 81.5/15/1 | ND | c−/c+‖‖ |
| 3/NOG | 0.2 | 1/3 | 3/6/18 | 80.8#/32.8#/59.6**†† | 1.2/2.57/0 | 0/36/0.2 | −/+ | c−/c+‖‖ | |||
| 3/NOG | 0.1 | 1/3 | 17/19/20 | 0.72#/0.6††/0.14# | 0.1/2.7/0.3 | 50/5.2/0 | — | c−/c+‖‖ | |||
| 4/NOG | 0.01 | 0/4 | 3/3/15/20 | 0 | — | — | — | — | |||
| CD34− | 98.8 | 3/NOG | 0.5 | 0/3 | 13/18/20 | 0 | — | — | — | — | |
| 3/NOG | 0.2 | 0/3 | 3/6/15/20 | 0 | — | — | — | — | |||
| 4/NOG | 0.1 | 0/4 | 12/15/18/20 | 0 | — | — | — | — | |||
| 4/NOG | 0.01 | 0/4 | 6/7/20/20 | 0 | — | — | — | — | |||
| 34*†‡ | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 | sT4 |
BM indicates bone marrow; WB (NPMm), Western blot analysis with anti-NPM mutant specific antibody; IHC (NPM), immunohistochemical analysis for NPM1 subcellular localization on hCD45+ cells; —, not applicable; ND, not done; sT4, full details on the characteristics of the engraftment are provided in supplemental Table 4; NOD, NOD/SCID; and NOG, NOD/SCID/IL2Rγnull.
Experiments to calculate frequency of LIC were performed with these samples (see Table 5).
Paired experiments using corresponding doses of CD34+ versus CD34− cells were performed with these samples (Table 5, details of patients 17 and 34).
Higher cell doses of CD34− cells gave rise to engraftment as pattern 4 (see “Patterns of engraftment of CD34− cells from NPM1-mutated AML in immunocompromised mice” for description; Table 4).
Used for serial transplantation experiments.
FLT3-ITD+.
FLT3-ITD−.
Normal engraftment.
Mixed normal-leukemic engraftment.
Positive for NPM1 mutation by genomic DNA fragment analysis.
Evaluated only at immunohistochemistry.
In the same mice areas of leukemia (NPMc+) and normal engraftment (NPMc−) were detected.
In the same mice foci of NPMc+, cells were detected within areas of normal hemopoiesis.