Radiobiological features of acute myeloblastic leukemia: comparison of self-renewal versus terminally differentiated populations

Int. J. Radiation

Oncology

Pergamon

Biol. Phys., Vol. 30, No. 5, pp. 1133-I 140, 1994 Copyright 0 1994 Elsevier Science Ltd Printed in the USA. All rights reserved 0360-3016/94 $6.00 + .OO

0360-3016(94)00338-6

l Biology Original Contribution

RADIOBIOLOGICAL FEATURES OF ACUTE MYELOBLASTIC LEUKEMIA: COMPARISON OF SELF-RENEWAL VERSUS TERMINALLY DIFFERENTIATED POPULATIONS DIDIER COWEN, M.D.,*

PIERRE RICHAUD, M.D.,+

PHILIPPE LAGARDE, M.D.,+

FFUN~OIS-XAVIER

JEAN-JACQUES BAUDET, M.D.,* NORBERT GUALDE, M.D.*

SERGE LANDRIAU,~ MAHON,

M.D.,#

FRANCOIS BELLOC, M.D.,”

AND JOSY REIFFERS, M.D.@

*Department of Radiotherapy, InstitutPaoli-Calmettes CancerCenter,232 Bd Sainte-Marguerite,

13009 Marseille; +Department of Radiotherapy and *Department of Immunology BergoniC Foundation South-West France Cancer Center, 180 rue de SaintGenes, 33076 Bordeaux Cedex; “Bone Marrow Transplantation Department Haut-Leveque Hospital, 33604 Pessac Cedex; ‘Bone Marrow Transplantation Laboratory URA CNRS 1456, C.H.R. Bordeaux, France

Purpose:To evaluate theradiosensitivity of self-renewing progenitor cellsin acute myeloblastic leukemia (AML), we have compared the radiosensitivity of the cells grown either in methylcellulose alone for 7 days, or first in suspension culture for 7 days before being plated in methylcellulose. Methylcellulose selects for terminal-dividing cells and suspension cultures have been developed because they allow self-renewal to occur: The exponential growth of the progenitors of AML cultured in suspension is due to self-renewal. Methods and Materials: Cells were harvested from previously untreated leukemic human bone marrows. The myeloblastic lineage of the colonies was assessed by morphological, cytochemical, and immunophenotypic analysis, and by the use of growth factors that did not stimulate the growth of T-lymphocytes. The cell-cycle distribution of the blasts was analyzed by flow cytometry and was comparable for all samples. The irradiation was performed with y-photons at a dose-rate of 0.05 Gy/min, similar to the clinical conditions used in our institution for total body irradiation (TBI). Results: The culture methods selected agressive leukemias. There were large variations of the individual radiosen-whatever culture method was used. The progenitor cells capable of self-renewal were more radiosensitive than terminal dividing cells. In two cases, a shoulder was found in the initial part of the cell-survival curves of cells capable of self-renewal. In these two cases, the best fit for the data was the linear quadratic model (survival = e -aD-BD2)with or/@ values of 1.49 Gy and 3.12 Gy, respectively. Conclusion: The very low values of c~/@suggest a reduced antileukemic effect in case of fractionated TBI, and may lead to more reliable screening methods to determine the most appropriate technique for radiation ablation of bone marrow prior to bone marrow transplantation (BMT). Total body irradiation, Bone marrow transplant, Acute myeloblastic

leukemia.

(29), and probablyalso

on the fractionation of total body irradiation (TBI) (3 1). Acute myeloblastic leukemia is one of the most frequent leukemias in France. Recurrent leukemia is a major cause of death after bone marrow transplantation (BMT), and more effective conditioning regimens appear to be necessary for better therapeutic efficacy. Available methods do not determine whether relapses occur because of radioresistance or chemoresistance of leukemic blasts.

INTRODUCTION

Bone marrow transplantation has been succesfully used against a variety of hematological malignancies. Total body irradiation takes part in most of the conditioning regimens because of its immunosuppressive and antileukemic effects. While immunosuppression is easily achieved with the current preparative regimens, the antileukemic effect depends on the total dose of irradiation

This study was presented in part at ICRO 93’, 21-25 July 1993, Kyoto, Japan. Reprint requests to: Dr. Didier Cowen, Department of Radiotherapy, Institut Paoli-Calmettes Cancer Center, 232 Bd Sainte-Marguerite, 13009 Marseille, France. Acknowledgements-This study was supported by grants from

the Federation Nationale des Centres de Lutte Contre le Cancer (FNCLCC, Paris) and from the Ligue Regionale Anticancereuse d’Aquitaine. We also wish to thank E. A. McCulloch for his kind advice. Accepted for publication 3 June 1994.

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The efficacy of ionizing radiations against acute myeloblastic leukemia (AML) has been evaluated most often against established human or animal leukemia cell lines, and wide variations were reported (25, 36). However, up to now, none of the regimens of TBI under evaluation was tested directly against leukemic bone marrow progenitor cells (LPC) from AML patients before they underwent BMT. Another difficulty results from the fact that the LPC colony assays available for AML are based on plating efficiency in methylcellulose. This type of assay selects for terminal dividing cells and self-renewal (SR) is observed rarely (17, 18). McCulloch and others, have developed culture methods to explore SR in AML (5,22), and have found that patients whose blast cells had a high potential for SR also had a reduced probability of achieving remission (5, 7, 8, 16, 24). Efforts have been concentrated on finding drugs capable of reducing SR, and the dose-response curves for the effects of chemotherapy on AML blasts is dependent on the culture method used to measure it (22). To our knowledge, very little is known about the radiobiological properties of blast cells capable of self-renewal. We therefore compared the radiosensitivity of AML progenitor cells obtained with two different culture methods. The first, most usual method, consists in growing the cells directly in methylcellulose for 7 days after the irradiation. In this clonogenic assay, colony formation reflects primarily the terminal divisions. The second method consists in growing the cells after their irradiation in suspension for 7 days before the clonogenic assay. The exponential increase of the number of LPC in suspension is considered to reflect SR ( 17). We report large variations in the sensitivity of AML progenitor cells to ionizing radiation, a higher radiosensitivity of blast cells capable of SR, and shoulders at the initial part of some cell-survival curves exploring SR. METHODS

AND MATERIALS

Leukemic Cells Fresh leukemic bone marrow (BM) samples were procured by aspiration into heparinized syringes. We selected BM samples containing more than 60% blasts prior to further enrichment by density gradient separation on Fitoll-Hypaque (1.077 g/cm3) gradient. Blasts were then frozen in Iscove’s modified Dulbecco’s medium’ (IMDM) supplemented with 50% fetal calf serum’ and 10% dimethyl sulfoxyde3 (DMSO). The temperature of the cell suspension was subsequently brought to - 100°C at a rate of 2-5 “C/min and then stored at - 196°C in liquid nitrogen. On the day of the experiment, the cells were thawed ‘Sigma. 2Jacques Boy. 3Prolabo. 4Applig6ne.

Volume 30, Number 5, 1994

by immersion in a 37°C water bath, diluted stepwise to mitigate the osmotic stress in IMDM supplemented with 20% Fetal Calf Serum (FCS) and 10% DNAse I4 at a concentration of 1 mg/ml. Cells were then washed twice and resuspended in IMDM. Zmmunophenotyping Immunophenotyping was performed just before the irradiation, and at day 7 from pooled colonies as described previously (9). The surface antigen profiles of AML blasts were analyzed by indirect fluorescence and flow cytometry using a panel of monoclonal antibodies (MoAbs) that define human leukocyte differentiation antigens. Isotype controls were used for each experiment. Specifically, we used the MoAbs anti-CD, (T-lineage antigen), anti-CD9 (B-lineage antigen), anti-CD,,, (common antigen), antiCD, 3, anti-CD,s, anti-CD33, anti-CD34 (myeloid lineage antigens), which were purchased from IMMUNOTECH. Samples were considered to be positive if greater than 20% of cells bound the antibody used (9). Morphological Analysis Cells from Day 0 and from pooled Day 7 and Day 14 colonies, were deposited on slides by cytocentrifugation. The morphology of AML blasts was studied by MayGrtinwald Giemsa staining, and blasts were classified according to the French American British classification (2) revised and completed in 1980 and 1985 (3, 4). Cytochemical analyses was performed using Sudan Black B staining (30), characteristic of the myeloblastic lineage (15). Analysis of Cell Cycle Kinetics by Quantitative DNA Flow Cytometry After treatment with RNase A’, propidium iodide6 was used to quantify the DNA content of AML-LPC. Analysis was performed on an ATC flow cytometer’ equiped with an argon laser.8 The program used to calculate the percentage of cells in the GO/l, S, and G2/M phases of the cell cycle was as described by Baish (1). Flow cytometry data of at least 10,000 cells from each sample were acquired for analysis just before the irradiation. Irradiation AML blasts, 1 X lo6 cells/ml, in 5 ml IMDM supplemented with 22% fetal calf serum, were irradiated with 0.5 to 4 Gy y-rays in a single exposure using a 6oCo source, at room temperature. The dose rate (0.05 Gy/min) was chosen to be similar to the clinical conditions used for TBI in our institution (28). Preliminary data had shown that the cell-survival curves were not affected by hypoxia when the irradiation lasted less than 45 min: therefore. ‘AppligGne. 6Calbiochem. 70dam-Bracker, Wissenbourg, France. ‘2025 Spectra Physics, Les Ulis, France.

Acute myoblastic leukemia 0 D. COWENet a/.

the 4 Gy exposure was divided into two exposures of 40 min separated by a 15 min break, during which the cells were incubated at 37°C in a humidified atmosphere of 5% CO* in air before the second exposure. After the irradiation, lo6 AML blasts per sample were assayed in duplicate for the suspension culture, and lo5 AML blasts per sample were assayed in triplicate for the clonogenic assay, as described hereinafter. The surviving fraction after the clonogenic assay was determined using the following formula: survival after clonogenic assay (SCA) = plating efficiency in the irradiated samples/plating efficiency in the unirradiated control samples. The surviving fraction after the suspension culture was calculated using the formula: cell recovery after suspension culture (CRSC) = number of cells alive (Trypan-blue exclusion) in the irradiated samples/number of cells alive in the control samples. The radiation survival curves were constructed by plotting the results of the SCA for the samples cultured exclusively in methylcellulose, and by dividing the product CRSC X SCA (after suspension) (irradiated) by the CRSC X SCA (control-unirradiated) for the samples cultured first in suspension and then in methylcellulose. Common indicators of radiation sensitivity used to construct the cell-survival curves are presented in Table 1. The (Yand /3 values represent the slope of the initial and terminal parts of the survival curves. Survival data were fitted to the best predictive model between an exponential survival curve model (survival = e -“lD) or the linear quadratic model (survival = e-aD-BD2) by linear regression analysis. Computer programs used to fit the data were developed by the department of statistics of the Fondation Bergonie Cancer Centre.

pensions containing lo5 AML blasts from BM aspirates, suspended in 1 ml of IMDM supplemented with 30% FCS, 10% 5637-conditioned media, 0.9% methylcellulose (viscosity of a 2% aquous solution at 25°C: 4000 centipoises) and 10e4 M 2-mercaptoethanol, were cultured in 35-mm Petri culture dishes for 7 days at 37°C in a humidified atmosphere of 5% CO2 in air. Colonies with more than 30 cells were scored, and clusters with more than five cells were scored after 7 days of incubation. For each assay, three to five replicate plates were used. Liquid Suspension Culture Blast progenitors were cultured in suspension as described by Nara (23), modified by the substitution of 5637conditioned medium for PHA-LCM. Suspensions containing 5 X 1O6AML blasts suspended in 5 ml of IMDM supplemented with 20% FCS and 10% 5637-conditioned medium were cultured in cell culture flasks at 37°C in a humidified atmosphere of 5% CO2 in air. Cultured cells were harvested and counted at day 7, washed in IMDM, and then assayed for blast colony formation as described above, for another 7 days. RESULTS Plating Eficiency A linear relationship (r > 0.998) was found between the number of cells plated and colony formation when 5 X lo4 to 3 X lo5 cells per dish were plated (data not shown). Accordingly, lo5 cells were routinely plated. Among the first 28 BM samples tested, 8 cultures were lost either because 7 days were too long in suspension (3 cases) or because cells did not clone in methylcellulose (5 cases). In 20 cases, plating efficiency could be determined and was lower than 0.1% in 3 cases, between 0.1% and 1% in 3 cases, and higher than 1% in 14 cases. We therefore selected the 14 BM samples with the highest plating efficiencies to determine the radiosensitivity of the LPC. Among them, two cultures were lost, and in two other

Blast Colony Assay Blast colony formation was obtained as described by Minden (19) except that supernatants from cultures of the continuous line of bladder cancer cells (12), 5637, were used rather than media conditioned by leukocytes in the presence of phytohemagglutinin (PHA-LCM). Sus-

Table 1. Radiobiological

features of AML LPC according

Blast

1

2 3 4 5 6 7 8 9 10

to the population

(Pop”) explored

by the culture

Colony

Non-Self Patient

1135

Do

D,

n

1.31 2.08 0.79 1.25 0.74 0.74 1.51 1.71 0.49 1.23

0 0.63 0 0.22 0.55 0 0 0.07 0.58 1.01

1 1.4 1 1.2 2.1 1 1 1 3.2 2.3

Renewal

Ly 0.76 0.36 1.26 0.73 1.05 1.34 0.66 0.57 1.57 0.49

methods

Assay

Pop”

Self Renewal

P

4P

Do

Q

n

0 0.097 0 0.006 0.02 0 0 0.00 1 0.038 0.026

>lOO 37 >lOO >lOO 44 >lOO >lOO >lOO 42 18

0.73 2.03 0.38 0.71 0.5 1 0.74 0.92 0.71 1.09

0 0 1.15 0 0.52 0.45 0 1.11 0.41 0.72

1 1 19.9 1 2.8 3.2 1 3.3 5.1 1.9

O! 1.37 0.49 1.44 1.41 1.6 0.79 1.35 0.62 1.56 0.66

Pop”

B 0 0 0.096 0 0.033 0.53 0 0.039 0.5 0.02 1

fflP >lOO

>lOO 15 >lOO 48 1.49 >lOO 16 3.12 31

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1136

Table 2. Main characteristics

Patient 1 2 3 4 5 6 7 8 9 10

25 18 42 53 70 66 56 23 34 72

of the patients

F M M F F M F M M M

Patient Characteristics There were 4 females and 6 males; age ranged from 18 to 72 years, and mean white blood count at diagnosis was 142 X lo9 (range 2.3-470). The main characteristics of the 10 patients and the blast count at days 0, 7, and 14 are shown in Table 2. Cell Cycle The cell cycle distribution of leukemic blasts was evaluated for all patients and is shown in Table 3. The percentage of G2/M-phase cells in the leukemic bone marrow samples ranged from l-3%. Immunophenotypic Features Results of immunophenotyping are shown in Table 4. In two cases, immunophenotyping could not be performed. In patients number 2, 3, 6, and 10, enough cells were available at day 7 to confirm the myeloblastic lineage of the cultured cells. Radiobiological Features-Cell-Survival Curves The values of the plating efficiency after culture in methylcellulose and the clonogenic recovery after sus-

1 2 3 4 5 6 7 8 9 10

blasts S

86 95 98 87 92 94 91 95 95 95

13 4 1 10 6 4 7 4 3 4

Blasts D14 (%)

75 60 90 92 96 69 91 65 92 90

77 92 90 99 100 70 95 80 88 87

85 80 88 95 92 64 85 92 83 87

Cell Survival Without Suspension Culture. The cell-survival curves of the 10 samples cultured exclusively in methylcellulose are presented in Fig. 1. The best fit for the 10 survival curves was the exponential cell survival curve model and the (Yvalue, ranging from 0.36 Gy-’ to 1.57 Gy-‘. Cell-Survival Curves with Previous Suspension Culture. The cell-survival curves of 10 samples irradiated and then cultured, first in suspension and then in methylcellulose, are shown in Figs. 2 and 3. Figure 2 represents eight curves for which the best fit was the exponential cell survival curve model. The LYvalue ranged from 0.49 Gy-’ to 1.56 Gy-‘. Figure 3 represents two curves with an initial shoulder for which the linear quadratic model was the best fit. The a! value was 0.79 Gy-’ and 1.56 Gy-’ for case number 6 and number 9, and the 0 value was, respectively, 0.53 Gyp2 and 0.5 Gy-‘. The value of (Y/Pwas 1.49 Gy and 3.12 Gy for patients number 6 and number 9.

Table 4. Immunophenotypes Patient

3

GO/G1

Blasts D7 (W)

pension culture are presented in Table 1. These values were used to construct the cell-survival curves presented in Figs. l-3.

1 2

Table 3. Percentage cell cycle distribution Patient

Blasts DO (%)

M2 M2 Ml M4 M5 M2 M5 M5 M3 Ml

25 470 212 119 180 85 212 98 2.3 17.5

cases the cells were so sensitive to ionizing radiation in the suspension culture that the clonogenic assay could not be performed. Finally, 10 complete results were available.

of the leukemic

and blast count at days 0, 7, and 14

FAB Classification

WBC X 10’

Sex

Age

Volume 30, Number 5, 1994

G2/M 1 1 1 3 2 2 2 1 2 1

4 5 6 7 8 9 10

CD: 7

9

10

13

18

* * *

*

N *

V t

-

-

_

_

Do D7 DO D7 D7

_

_

DO D7 D7 D7 D7 DO D7

_ * _ _ _

+ * * _ + _ _

_ N _ * _ _

33

34

R *

t

t

*

t

t t

$ * * V $ * z + * * t

*

+

*

*

t + R * 4 * + *-* +

DO = Day 0; D7 = Day 7; NVR = no valuable to 40%; 740 to 60%; $> 60%.

$

+

t 1: + z

* +

_ _

+ * *

results. *20

Acute myoblastic leukemia 0 D. COWEN et al.

0.001

1137

1 0

2

1

3

4

DOSE (Gy) . Patient 1

0 Patient 2

l

Patient 7

o Patient 8

x Patient 9

l

Patient3

o Patient 4

A Patient 5

n Patient 6

x Patient 10

Fig. 1. Cell survival curves with no previous suspension culture. DISCUSSION To our knowledge, this study is the first to compare the radiosensitivity of AML progenitor cells cultured with methods exploring the influence of self- renewal on radiation survival. The methods and the results should be discussed.

Methods The main problem arising from these techniques was the identification of the colonies observed in the culture dishes. The culture methods theoretically also allow the growth of normal bone marrow progenitor cells, which may be present even in highly blastic bone marrows, and T-lymphocytes, although we did not use phytohemaglutinin. Blast cells and T-lymphocytes were not separated as initially described by Minden (19) but the T-lineage of the colonies was unlikely for the following reasons: (a)

phytohemaglutinin was absent from the culture medium; (b) the morphological analysis at days 7 and 14 showed myeloid blast cells; and (c) immunophenotyping was in favor of a myeloid lineage. Furthermore, the colonies did not arise from normal bone marrow progenitors because the number and the appearance of the colonies is very different from that of normal marrow. In addition, the cytological analysis did not show mature granulocytic cells, unlike what is observed for normal bone marrow progenitor cells at day 7. Another problem arose when counting colonies and clusters. Because of the deferred cell death induced by ionizing radiation (33), the cell-survival curves are usually constructed after counting colonies of more than 64 cells corresponding to 6 cell divisions. Concerning AML-CFU assays, this is impossible because methylcellulose selects for terminal dividing cells (17) and inhibits the growth of the colonies (23). Therefore, in publications reporting cy-

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Volume 30, Number 5, 1994

0.1

a 5 f 0.01

0.001

DOSEGY) .

FMkmt 1

OPlltkUltB

q Pamt2

*Patkant

QPathlt4

APattmt5

.Patht7

* Panent 10

Fig. 2. Cell survival curves with previous suspension culture (except patients number 6 and number 9). totoxicity assays against AML-CFU, the authors usually count smaller colonies and clusters (6, 14, 20). The last

problem lies in the use of supernatants from cultures of the bladder cancer cell line 5637, which contains at least G-CSF, GM-CSF, and IL-l (21). These growth factors have been shown to affect the shape of the cell-survival curves of human bone marrow progenitor cells, but only at high concentrations (35). However, the advantage of these supematants lies in the absence of IL-2, which could have stimulated the growth of eventual T-lymphocyte colonies. Nevertheless, despite the problems associated with this clonogenic assay, it remains the method of preference (26) because other methods are not helpful in AML. The MTT assay is not reproducible in AML (34), dye-exclusion tests cannot be used because of deferred cell-death, and the measure of the inhibition of incorporation of thymidine is not satisfying because of the contaminating population of normal progenitor cells. Results The samples used for the study were selected on the basis of selection of samples exhibiting the highest per-

centage of blast cells prior to Ficoll separation, selection of samples with the highest cloning efficiencies, and selection of samples growing under the defined conditions. Therefore, the results should be interpreted cautiously and could refer only to highly agressive leukemias. However, although the samples were selected, we found large differences in the radiosensitivity of AML progenitor cells, whatever culture methods were used. Furthermore, variations in cell-cycle distribution cannot account for the variation in radiosensitivity observed. Large variations of the radiosensitivity of leukemic cells have been reported (10, 13, 25, 27, 32, 37) with values of Do ranging from 0.6 Gy to 1.5 Gy. Although most of these studies concerned animal or human cell lines, values up to 5.8 Gy were reported for fresh acute lymphoblastic leukemia progenitor cells (36). The comparison of the survival curves obtained with both culture methods showed, for each individual sample, a higher radiosensitivity of the progenitor cells with selfrenewing abilities, and in two cases, a shoulder was found at the initial part of the cell-survival curves of the progenitor cells cultured to allow SR to occur. Uckun (36)

Acute myoblastic leukemia 0 D.

COWEN

1139

ef al.

0.1

0.01

0.001 0

1

2

3

4

DOSE(Gy) .

Patient6

0 Patient 9

Fig. 3. Cell survival curves with previous suspension culture (patients number 6 and number 9).

showed that better survivals could be observed after fractionation experiments, although the single dose cell-survival curves did not exhibit any shoulder. The same author emphasized that single dose cell survival curves may underestimate the repair capacities of the cell (11). Unfortunately, we could not perform fractionation experiments because the number of cells available was too low in each case. However, for cases 6 and 9, the CY//? ratio was very low, suggesting that the shoulders were broad

previously

enough to reduce significantly the antileukemic effect of the TBI regimen in case of fractionation. CONCLUSION Our data suggest that the radiosensitivity of the progenitor cells of AML is higher when culture methods allowing self-renewal to occur are used, and that the repair capacities of the progenitor cells capable of self-renewal could be underestimated with usual culture methods.

REFERENCES Baish, H.; Gohde, W.; Linden, W. A. Mathematical analysis of pulse-cytophotometric data to determine the fractions of cells in the various phases of cell-cycle. Radiat. Environ. Biophys. 12:31-39; 1975. Bennet, J. M.; Catovsky, D.; Daniel, M. T.; Flandrin, G.; Galton, D. A. G.; Gralnick, H. R.; Sultan, C. Proposals for the classification of the acute leukaemias. Br. J. Haematol. 33:45 l-458; 1976. Bennet, J. M.; Catovsky, D.; Daniel, M. T.; Flandrin, G.; Galton, D. A. G.; Gralnick, H. R.; Sultan, C. A variant form of hypergranular promyelocytic leukaemia (M3). Br. J. Haematol. 44: 169- 170; 1980.

4. Bennet, J. M.; Catovsky, D.; Daniel, M. T.; Flandrin, G.; Galton, D. A. G.; Gralnick, H. R.; Sultan, C. Proposed revised criteria for the classification of acute myeloid leukemia. Ann. Intern. Med. 103:626-629; 1985. Buick, R. N.; Minden, M. D.; McCulloch, E. A. Self-renewal in culture of proliferative blast progenitor cells in acute myeloblastic leukemia. Blood 54:95- 104; 1979. Buick, R. N.; Till, J. E.; McCulloch, E. A. Colony assay for proliferative blast cells circulating in myeloblastic leukaemia. Lancet i:862-863; 1977. Curtis, J. E.; Messner, H. A.; Hasselback, R.; Elhakim, T. M.; McCulloch, E. A. Contributions ofhost- and disease-

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related attributes to the outcome of patients with acute myelogenous leukemia (AML). J. Clin. Oncol. 2:253-259; 1984. 8. Delmer, A.; Marie, J. P.; Thevenin, D.; Cadiou, M.; Viguie, F.; Zittoun, R. Multivariate analysis of prognostic factors in acute myeloid leukemia: Value of clonogenic leukemik cell properties. J. Clin. Oncol. 7:738-746; 1989. 9. Duboscq-Marchenay, N.; Lacombe, F.; Dumain, P.; Marit, G.; Montastruc, M.; Boisseau, M. R.; Reiffers, J. Role of blast cell immunophenotyping for the diagnosis and prognosis of acute myeloid leukemia. Hematol. Oncol. 10:235245; 1992. 10. Fitzgerald, T. J.; McKenna, M.; Kase, K.; Daugherty, C.; Rothstein, L.; Greenberger, J. S. Effects of x-irradiation on the clonogenic survival of experimental animal hematopoietic tumor cell lines: Evidence for heterogeneity. Int. J. Radiat. Oncol. Biol. Phys. 12:69-73; 1986. 11. Hendry, J. H.; Howard, A. The response of hematopoietic colony forming units to single and split doses of gamma rays or D-T neutrons. Int. J. Radiat. Biol. 19:5 l-64; 197 1. 12. Hoang, T.; McCulloch, E. A. Production of leukemic blast growth factor by a human bladder carcinoma cell line. Blood 66:748-75 1; 1985. 13. Laver, J.; Kwon, J. H.; Castro-Malaspina, H. Effects of low dose rate irradiation on human marrow hemopoietic and micro-environmental cells: Sparing effect upon survival of stromal and leukemic cells. Bone Marrow Transplant 2: 271-278; 1987. 14. Marie, J. P.; Zittoun, R.; Thevenin, D.; Mathieu, M.; Viguie, F. In vitro culture of clonogenic leukemia cells in acute myeloid leukemia: Growth pattern and drug sensitivity. Br. J. Haematol. 55:427-437; 1983. 15. Marmont, A. M.; Damasio, E.; Zucker-Franklin, D. Neutrophils. In: Zucker-Franklin, D., Marmont, A.M., eds. Atlas of Blood Cells. Function and Pathology. Vol. 1. Philadelphia, PA: Lea and Febiger; 1981:149-242. 16. McCulloch, E. A.; Curtis, J. E.; Messner, H. A.; Senn, J. S.; Germanson, T. P. The contribution of blast cell properties to outcome variation in acute myeloblastic leukemia (AML). Blood 59:60 l-608; 1982. 17. McCulloch, E. A.; Minden, M. D.; Curtis J. E. The influence of growth regulation on treatment strategy for acute myeloblastic leukemia. Cancer Surv. 9( 1): 169- 198; 1990. 18. McCulloch, E. A.; Till, J. E. Blast cells in acute myeloblastic leukemia: A model. Blood Cells 7:63-77; 198 1. 19. Minden, M. D.; Buick, R. N.; McCulloch, E. A. Separation of blast cell and T-lymphocyte progenitors in the blood of patients with acute myeloblastic leukemia. Blood 54: 186195; 1979. 20. Minden, M. D.; Till, J. E.; McCulloch, E. A. Proliferative state of blast cell progenitors in acute myeloblastic leukemia. Blood 52:592-600; 1979. 21. Miyauchi, J.; Kelleher, C. A.; Wong, G. G.; Yang, Y. C.; Clark, S. C.; Minkin, S.; Minden, M. D.; McCulloch, E. A. The effects of combinations of the recombinant growth factors GM-CSF, G-CSF, IL-3, and CSF-1 on leukemic blast cells in suspension culture. Leukemia 2(6):382-387; 1988. 22. Nara, N.; Curtis, J. E.; Senn, J. S.; Tritchler, D. L.; McCulloch, E. A. The sensitivity to cytosine arabinoside of the blast progenitors of acute myeloblastic leukemia. Blood 67: 762-769; 1986.

Volume 30, Number 5, 1994 23. Nara, N.; McCulloch, E. A. The proliferation in suspension of the progenitors of the blast cells in acute myeloblastic leukemia. Blood 65:1484-1493; 1985. 24. Nara, N.; Nagata, S. K.; Yamashita, Y.; Murohashi, I.; Adachi, Y. Relationship between in vitro sensitivity to cytosine arabinoside of blast progenitors and the outcome to treatment in acute myeloblastic leukemia. Br. J. Haematol. 70: 187-191; 1988. 25. O’Donoghue, J. A.; Wheldon, T. E.; Gregor, A. The implications of in vitro survival curves for the optimal scheduling of total body irradiation with bone marrow rescue in the treatment of leukemia. Br. J. Radiol. 60:279-283; 1987. 26. Puck, T. T.; Markus, P. I. Action of X-rays on mammalian cells. J. Exp. Med. 103:653-666; 1956. 27. Rhee, J. G.; Song, C. W.; Kim, T. H.; Levitt, S. H. Effect of fractionation and rate of radiation dose on human leukemic cells, HL 60. Radiat. Res. 101:519-527; 1985. 28. Richaud, P.; Chemin, A.; Cowen, D.; Marit, G.; Reiffers, J. Irradiation corporelle totale en arctherapie: Aspects techniques et dosimetriques. Bull. Cancer Radiother. 80:70-75; 1993. 29. Scarpati, D.; Frassoni, F.; Vitale, V.; Corvo, R.; Franzone, P.; Barra, S.; Guenzi, M.; Orsatti, M. Total body irradiation in acute myeloid leukemia and chronic myelogenous leukemia: Influence of dose and dose-rate on leukemia relapse. Int. J. Radiat. Oncol. Biol. Phys. 17:547-552; 1989. 30. Sheehan, H. L.; Storey, G. W. An improved method of staining leukocyte granules with Sudan black B. J. Pathol. Bacterial. 59:336; 1947. 31. Socie, G.; Devergie, A.; Girinsky, T.; Reiffers, J.; Vernant, J. P.; Le Bourgeois, J. P.; Hen&, P.; Guyotat, D.; Maraninchi, D.; Rio, B.; Michallet, M.; Jouet, J. P.; Milpied, N.; Leblond, V.; Pica, J.; Attal, M.; Belanger, C.; Gluckman, E.; Cosset, J. M. Influence of the fractionation of total body irradiation on complications and relapse rate for chronic myelogenous leukemia. Int. J. Radiat. Oncol. Biol. Phys. 20:397-404; 199 1. 32. Song, C. W.; Kim, T. H.; Khan, F. M.; Kersey, J. H.; Levitt, S. H. Radiobiological basis of total body irradiation with different dose rate and fractionation; repair capacity of hemopoietic cells. Int. J. Radiat. Oncol. Biol. Phys. 7:16951701; 1981. 33. Tubiana, M.; Dutreix, J.; Wambersie, A. Effets cellulaires des rayonnements ionisants. Les courbes de survie cellulaire. In: Hermann, ed. Radiobiologie. E. Mame: Paris; 1986:75103. 34. Twentyman, P. R.; Fox, N. E.; Rees, J. K. A. Chemosensitivity testing of fresh leukemia cells using the M.T.T. colorimetric assay. Br. J. Haematol. 7 1: 19-24; 1989. 35. Uckun, F. M.; Gillis, S.; Souza, L.; Song, C. W. Effects of recombinant growth factors on radiation survival of human bone marrow progenitor cells. Int. J. Radiat. Oncol. Biol. Phys. 16:415-435; 1989. 36. Uckun, F. M.; Song, C. W. Radiobiological features of fresh leukemic bone marrow progenitor cells in acute lymphoblastic leukemia. Cancer Res. 48:5788-5795; 1989. 37. Weichselbaum, R. R.; Greenberger, J. S.; Schmidt, A.; Karpas, A.; Moloney, W. C.; Little, J. B. In vitro radiosensitivity of human leukemia cell lines. Radiology 139:485-487; 198 1.