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Article Figures & Data

Figures

  • Figure 1.

    Purification strategy for isolating cells stained with CD34/CD10 or CD19. ALL blast cells were gated on the basis of low forward and side scatter (A) and subsequently gated for expression of CD34-PE and CD10- or CD19-FITC, R1 to R4 (B). Sort gates R1 to R4 were separated by at least 10 channels, and quadrants K1, K2, and K4 in panel B were set up to exclude at least 99.9% of cells in isotype controls. (C-D) Reanalysis of CD34+/CD19+ (C) and the CD34+/CD19- subfractions (D) after sorting.

  • Figure 2.

    Growth of ALL cells in different long-term culture systems. Growth of ALL cells from 9 patients were evaluated in suspension culture supplemented with F36GMGS or with IL-3, IL-7, and SCF and compared with growth in standard LTBMC and LTBMC supplemented with IL-3, IL-7, and SCF over a 6-week period. Cultures were maintained with weekly half-media changes, and the number of viable cells present at each week was determined using flow cytometry. Results from 8 patients where cell expansion was observed in culture are shown. Data are expressed as means ± standard error of measurement.

  • Figure 3.

    ALL cell expansion in suspension culture. The proliferative capacity of ALL cells from 47 patients was evaluated in suspension culture (SC). Cells from 40 patients were maintained in the culture system for at least 4 weeks, and expansion of cell numbers was observed in 28 of these 40 cases. The graph depicts the proliferation observed in the 28 cases and that observed using cells from 10 NBM donors. Proliferation of cells from both pre-B and c-ALL subtypes was observed, and there was no correlation between proliferation, prognostic indicators, or diagnostic and relapse samples. Data are expressed as means ± standard error.

  • Figure 4.

    Expression of CD34 and CD10 or CD19 on ALL progenitor cells. ALL cells from 19 patients were sorted for expression of CD34 and CD10 (A), and cells from 16 patients were sorted for coexpression of CD34 and CD19 (B). The proliferative capacity of the sorted subfractions and unsorted controls was evaluated in SC. Absolute cell counts, derived from each sorted population, and unsorted controls were determined by flow cytometry at weeks 1, 3, 5, and 6. These values were then used to calculate the proportion of the total viable cells represented by each sorted population. The proportion of the total cells derived from the each sorted subfraction is presented as the mean ± SE; n indicates number of patient samples evaluated at each time point.

  • Figure 5.

    Phenotype of NOD/SCID-engrafting ALL cells. ALL cells were sorted for expression of CD34 and CD10 (n = 6) (A) or CD34 and CD19 (n = 5) (B) and evaluated in the NOD/SCID assay. Each patient is represented by a specific symbol, and each symbol depicts the engraftment obtained as measured by CD45+ cells present in the bone marrow of the NOD/SCID recipients.

Tables

  • Table 1.

    Engraftment characteristics of unsorted ALL cells in NOD/SCID mice

    % CD45+ in NOD/SCID*, by no. of unsorted cells injected
    Patient no.SubtypeKaryotype5 × 1051065 × 106107% FISH-positive
    2 c-ALL Complex 1.9 3 100
    9 pre-B 46,XX 13
    10 c-ALL t(12;21) 0 0.12 0.6 80
    11 c-ALL 46,XX 0.16 0.3 8
    18 c-ALL 46,XY 0.12 0.14
    19 c-ALL 58,XY 0.15 80
    21 pre-B 11q23 5 44 100
    25 pre-B 46,XX 7 57 80
    28 pre-B 46,XY 4 37 82 99
    31 c-ALL del(9p), del(17p) 59 80 92 98 > 90
    32 pre-B 46,XY 0 0.13
    43 pre-B 46,XX 0.6
    47 c-ALL del(17p) 9.2 24 100
    53 pre-B del(6p) 2.23 60 89 > 83
    56 c-ALL t(12;21) 3 11 > 70
    69 c-ALL t(12;21) 2 29 > 87
    • — indicates not tested.

    • * Each value represents the mean level of CD45+ cells in the femoral marrow of at least 2 NOD/SCID mice

    • Relapse sample

  • Table 2.

    Expression of lineage-specific and differentiation markers on CD45+ cells harvested from NOD/SCID bone marrow

    Patient no./recipient% human leukocytes CD45% B lineage% progenitors CD34% FISH-positive
    CD10CD19CD22
    CD34+/CD10-
          9/1° 35 83 87 56 88
          9/2° 46 85 90 58 83
       10/1° 28 88 92 80 72 89
       10/2° 25 92 90 77 80 90
       43/1° 6 87 96 60 93
       43/2° 2 91 92 58 89
       47/1° 20 84 88 80 100
       47/2° 28 87 80 73 100
    CD34+/CD19-
          9/1° 12 86 91 63 85
          9/2° 15 80 83 55 83
       10/1° 79 92 95 84 90 91
       10/2° 82 90 97 80 86 100
       25/1° 95 78 80 75 79
       25/2° 95 82 85 80 75
    • 1° indicates primary NOD/SCID recipient; 2°, secondary NOD/SCID recipient; and —, not tested.