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Autologous Bone Marrow Transplantation in Acute Myelogenous Leukemia:
In Vitro Treatment With Myeloid-Specific Monoclonal Antibodies and Drugs
in Combination
By Roberto M. Lemoli, Christina Gasparetto, David A. Scheinberg, Malcom A S . Moore, Bayard D.Clarkson,
and Subhash C. Gulati
We report the results of a preclinical study comparing four
different purging protocols using a promyelocytic human cell
line HL-60 and myeloid leukemic progenitor cells (colonyforming unit-leukemic [CFU-L]) from acute myelogenous
leukemia (AML) patients assayed in semisolid culture. We
studied the antileukemic effect of (1) Single-cycle complement-mediated lysis by two different monoclonal antibodies
(MoAbs) (MI95 [CD33] and F23 [CD13] 40 pg/mL), reactive
with distinct antigens found on early myeloid cells and
monocytes, used alone and in combinations; (2) 4-Hydroperoxycyclophosphamide (4-HC) (80 kmol/L or 100 pmol/L)
alone; or (3) combined with VP-16 (5 pg/mL) and (4) a
cocktail of 1 through 3 as above (combined immunochemotherapy). More than 4 logs of HL-60 tumor cell elimination
were observed after 1 hour of incubation with both MoAbs
plus 4-HC VP-16 while the single treatment (immunotherapy or chemotherapy) provided 1.5 and 3.5 logs of colonyforming inhibition, respectively. When the same protocols
were tested on cryopreserved leukemic cells from eight
patients with AML, we observed a mean value of CFU-L
inhibition of 92.3% f 2.5% SD, 95.5% f 1.4% SD, and 99%
0.8% SD after MoAbs and complement lysis, 4-HC, and
4-HC + VP-16 treatment, respectively. The combined treatment of MoAbs and 4-HC VP-I6 produced more than 3-log
reduction of CFU-L colony formation. By comparison, the
mean recovery of committed normal bone marrow progenitors after incubation with MoAbs and complement was 12%
for CFU-granulocyte-macrophage(CFU-GM),22.9% for burstforming unit erythroid (BFU-E), and the recovery following
4-HC
VP-16 treatment was 4.4% for CFU-GM and 5.6%
BFU-E. In subsequent experiments, highly purified CD34’
blast cells, enriched by positive selection, and stimulated in
liquid culture by cytokines (interleukin-1 [IL-I], IL-3, and
combination of both) or MO-conditioned medium (MoCM),
demonstrated that immunochemotherapy spares hematopoietic colony-forming cells earlier than day 14 CFU-GM, in
vitro.
o 1991by TheAmerican Society of Hematology.
M
purified CD34’ blast cells stimulated by recombinant growth
factors (rGFs) or MO-T cell line conditioned medium
(MoCM) in liquid culture. In normal adults CD34’ cells
represent 1%to 4% of the mononuclear cells in BM. The
110- to 120-Kd glycoprotein referred to as CD34 antigen is
expressed on the cell surface of all the hematopoietic
committed progenitors (ie, CFU-GM, BFU-E, CFUmegakaryocyte [CFU-MK], CFU-granulocyte-eosinophilmacrophage-megakaryocyte [CFU-GEMM]) as well as more
immature progenitors (CFu-Bla~t).’.~
The latter undifferentiated cells are capable of self renewal and committment to
different hemopoietic lineages.’ CD34+ cells have been also
proven to restore hematopoiesis in lethally irradiated ba-
+
YELOABLATIVE CHEMOTHERAPY and radiotherapy followed by autologous bone marrow (BM)
rescue, may be curative for patients with acute myelogenous leukemia (AML) lacking a suitable HLA-matched
BM donor.’” However, tumor cell contamination of reinfused marrow could be a potential source of relapse in these
patients. Recent experiments and clinical trials demonstrated the feasibility of ex vivo treatment of human BM.’-4
Yeager et alz have reported encouraging results in patients
with AML who were transplanted in second or third
remission with 4-hydroperoxycyclophosphamide (4-HC)
purged marrow, the disease-free survival (43%) being
similar to that obtained with allogeneic transplantation.
Although 4-HC or 4-HC + VP-16 protocols have been
found to be effective for purging leukemia cells from the
marrow grafts,’r3 the combination of different purging
techniques might be more effective in eliminating tumor
cells. Based on this premise, we compared four purging
protocols using the promyelocytic human cell line HL-60
and myeloid leukemic progenitor cells (colony-forming
unit-leukemic [CFU-L]) from AML patients assayed in
semisolid culture. In particular, we studied the colonyforming inhibition activity of complement-mediated lysis by
two monoclonal antibodies (MoAbs) (directed toward CD33
and CD13 antigens); 4-HC used alone or combined with
VP-16; and a cocktail of MoAbs plus 4-HC
VP-16
treatment. We showed that purging was not complete after
MoAbs + C‘ procedure or drug treatment while combined
immunochemotherapy provided the highest tumor cell
killing. As no in vitro hemopoietic colonies (ie, CFUgranulocyte-macrophage [CFU-GM], burst-forming uniterythroid [BFU-E]) were observed in most of the experiments after treatment of normal light-density marrow cells
with the combined immunochemotherapy, we assessed the
recovery of colony-forming cells (CFU-GM) derived from
+
Blood, Vol77, No 8 (April 15). 1991: pp 1829-1836
*
+
+
From the Laboratories of Hematopoietic Cell Kinetics, Developmental Hematopoiesis and The Department of Medicine, Memorial
Sloan-KetteringCancer Center, New York, hY
Submitted October 9,1990; accepted December 12, 1990.
R.M.L. is supported by Italian Association for the Research against
the Cancer (A.I.R.C.),Milan, Italy. C.G. is a Fellow of the Leukemia
Society of America. D.A.S. is a Lucille P. Markey Scholar in
Biomedical Science. Research is supported by The United Leukemia
Fund, Lisa Bilotti Foundation, National Leukemia Association, and
the Morgan Murray Fund.
Presented in part at the American Society of Hematology Meeting,
Atlanta, GA, December 2-5, 1989.
Address reprint requests to S.C. Gulati, MD, PhD, FACP, ChieJ
Autologous Bone Marrow Transplant Team, Department of Medicine,
Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York,
NY 10021.
The publication costs of this article were defrayed in part by page
charge payment. This article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C.section 1734 solely to
indicate this fact.
0 1991 by The American Society of Hematology.
0006-4971191/7708-0018$3.00JO
1829
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1830
LEMOLI ET AL
boons and cancer patients treated with supralethal chemoradi~therapy.~.~
Our results demonstrated that the myeloid pre-CFU-GM
compartment is spared by immunochemotherapy protocols;
therefore, the use of 4-HC + VP-16 plus MoAbs treatment
may be suitable for clinical use in purging leukemic cells.
MATERIALS AND METHODS
Human cell line. HL-60 is a human acute promyelocytic leukemia cell line growing with a doubling time of 24 hours and a plating
efficiency (PE) of 7% to 12%.’’ The cell line was maintained in
exponential growth in RPMI-1640 supplemented with 10% fetal
calf serum (FCS; Hyclone, Logan, UT), 1%penicillin-streptomycinneomycin (PSN; GIBCO, Chagrin Falls, OH), and 1% L-glutamine
at 37°C in a humidified atmosphere of 5% CO, air. Cell viability
was always higher than 95% and cells were free of mycoplasma
contamination.
EM cells. BM was aspirated from the posterior iliac crest of
healthy volunteers after obtaining written informed consent and
BM mononuclear cells were collected after Ficoll-Hypaque gradient (1.077 g/cm’).
Leukemic samples. Cells were obtained from peripheral blood
(PB) of eight AML patients (Table 1). The diagnosis of AML was
established by morphologic criteria, cytochemical staining, and by
surface-marker analysis using a panel of MoAbs. Leukemic specimens were subclassifed according to the French-American-British
(FAB) classification system.” If the platelet count was sufficiently
high, the blood was first depleted of platelets by centrifugations
over Percoll (1.050 g/cm’) (Pharmacia Fine Chemicals Co, Piscataway, NJ), and light-density cells were subsequently obtained by
centrifugation over Percoll (1.075 picm’). The leukemic cells were
suspended in FCS and cryopreserved using a mixture containing
5% dimethyl sufoxide (DMSO) and 6% hydroxyethyl starch (HES)
as described.” Cells were frozen at -120°C in a Revco Freezer
(Revco Scientific, Asheville, NC). After thawing, the cells were
suspended in lscove’s modified Dulbecco’s medium (IMDM)
supplemented with 10% FCS (Hyclone) and 60 UimL of Deoxyribonuclease 1 (DNAse) (Cooper Biomedical, Malvern, PA) and
incubated for 30 minutes on ice. Viable cells were recovered by
Ficoll-Hypaque gradient. All samples contained more than 86% of
blasts and promyelocytes (Table 1). Content of residual T cells was
assessed by immunofluorescent staining with panel of MoAbs
(anti-T,, -Tl, and -Til) and was for all the patients less than 2%.
Chemotherapeutic compounds. VP-16 (Bristol Labs, Syracuse,
NY)and 4-HC (kindly provided by Dr O.M. Colvin, John Hopkins
University, Baltimore, MD) were diluted immediately before use
with RPMI-1640 and phosphate-buffered saline (PBS) without
calcium and magnesium, respectively.
MoAbs. M195 is a mouse IgG2a MoAb that reacts with the
CD33 protein p67.” F23 is a mouse IgG2a MoAb (kindly provided
by Dr L.J. Old) whose target antigen (CD13) is expressed on early
and mature myeloid cells. HPCA-1 (MylO; Becton Dickinson,
Mountain View, CA) is a mouse IgGl MoAb directly toward CD34
antigen expressed on putative human hemopoietic progenitors.’
Incubation procedure and culture techniques. Tumor cells and
normal BM cells were adjusted to a concentration of 2 x 106/mL
and 20 x 106/mL,respectively. Drugs and MoAbs were added in a
single incubation: VP-16 was used at the concentration of 5 &mL;
4-HC, 80 Fmol/L or 100 kmol/L; M195 and F23, 40 pg/mL while
the optimal complement (baby rabbit complement; Pel Freez,
Brown Deer, WI) concentration was found to be 1:6 (volfvol). After
completion of 1 hour of incubation at 3 7 T , the cells were put on
ice for 5 minutes and washed twice with RPMI supplemented with
FCS. To simulate ex vivo BM purging conditions, the HL-60 cells
were also incubated together with irradiated (3,000 cGy) BM cells
(1:10 ratio). Tumor cells, after incubation alone or in mixture with
irradiated normal BM cells, were plated in 35-mm plastic Petri
dishes (Miles Lab, Naperville, IL) in quadruplicate. The culture
medium consisted of 1.1% methylcellulose (Methocel A4M; Dow
Chem Co, Midland, MI), in IMDM containing 20% FCS, 1%
antibiotics, and 1% L-glutamine. The number of cells plated in 1
mL of medium was 1,000 untreated and l@treated. Cells were
incubated at 37°C in humidifed atmosphere of 5% CO, in air and
scored for colonies ( > 50 cells) after 7 days of incubation using an
inverted microscope.
After treatment, normal BM cells were assayed for colonies
derived from CFU-GM, BFU-E, and CFU-GEM as already described.” Briefly, culture medium consisted of l mL of IMDM
(GIBCO), supplemented with 24% FCS, 0.8% bovine serum
mol/L of
albumin (BSA; Sigma Chemical Co,St Louis, MO)
2-mercaptoethanol (Sigma), 1 U of partially purified human
urinary erythropoietin (Epo; Toyobo Inc, New York, NY),10% of
a selected lot of MoCM prepared from the human MO-T lymphoblastic cell line14(kindly provided by Dr David Golde, UCLA, Los
Angeles, CA), and bovine hemin 0.2 mmolb (Sigma). Methylcellulose was added at a final concentration of 1.32%. Quadruplicate
cultures were incubated as described above and the colonies were
scored after 14 days of incubation. Clonogenic assay for CFW-L”
was the same as used for normal BM progenitors without the
addition of bovine serum and Epo. A total 10’ irradiated autologous cells (3,000 cGy with a ”’Cs source) were added per milliliter
of culture. The number of cells plated was adjusted to have about
100 colonies/mL of culture in the control samples; the treated
samples were cloned at higher cell concentration. Colonies ( > 20
cells) were scored after 10 days of incubation.
Enrichment of CD34’ cells and delta value. Purified progenitor
cells were obtained by immunopanning technique16 after 30 min-
Table 1. AML Cases
No.
Ageisex
Source of
Cells
FAB
Classification
% PE
% Blasts*
69lF
35lF
60lM
69lM
54lM
39lM
44lF
36lM
PB
PB
PB
PB
PB
PB
PB
PB
M1
M2
M4
M4
M5B
M5B
M2
M4
0.016
2.7
0.93
1.1
0.35
1.6
0.2
0.93
79
89
95
98
86
69
92
87
*The percentages of
after thawing.
% Promyelocytes’
blasts and promyelocytes were obtained by counting 200 cells from May-Grunwald-Giemsa-stained cytocentrifuge smears
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1831
IN VITRO IMMUNOCHEMOTHERAPY FOR AML
utes of incubation with HPCA-1 (MY10, 10 pg/mL, Becton
Dickinson) as reported elsewhere." The percentage of CD34+
blasts, evaluated by morphologic analysis and immunoflurescence
was 1% to 4% before purging procedures, 10% to 15% after
purging, and always higher than 90% after immunopanning.
CD34* cells, 5 x lo4, were plated in semisolid medium for
CFU-GM with recombinant granulocyte-macrophage colonystimulating factor (rGM-CSF) (1,000 U/mL; Amgen, Thousand
Oaks, CA), interleukin-3 (IL-3) (50 ng/mL; Genetics Institute,
Cambridge, MA), or MoCM (lo%, vol/vol) as colony-stimulating
factor. Colonies were counted after 14 days of incubation with an
inverted microscope and recorded as the Day 0 (DO) CFU-GM
frequency. Simultaneously, 1 x lo5cells were suspended in 1mL of
IMDM supplemented with 20% FCS containing IL-3 (50 ng/mL)
or IL-3 + IL-1 (100 U/mL; Syntex, Palo Alto, CA) or MoCM (10%
volhrol). On day 7, cells were counted and 1 x lo4cells from each
suspension were plated for CFU-GM in triplicate as described
above. Fourteen-day colonies were recorded as D7 CFU-GM
frequency, because 7 days elapsed from the beginning of the liquid
culture. The Delta value is determined by dividing the D7 CFU-GM
frequency by the DO CFU-GM frequency, and it is an indicator of
the number of progenitors of earlier stage than the 14-day
CFU-GM. When DO CFU-GM value was zero, the D7 CFU-GM
frequency was divided by 0.33 because the clonogenic assay was set
up in triplicate and one colony was the lower limit of detection.
Data ana&sk. All experiments were performed three or more
times and the mean ? standard deviation (SD) values of each
experiment were calculated. Colony counts after drug treatment
were expressed as a percent of the untreated control cells.
RESULTS
Effect of chemotherapy andlor immunotherapy on tumor
cells. Blood and/or marrow specimens from 50 AML
patients were screened for the development of 10-day
CFU-L-derived colonies induced by MoCM: 78% of AML
patients generated CFU-L present at 10 days." Eight
patients were chosen for the study on the basis of adequate
colony growth and their clinical characteristics are reported
in Table 1. The median age was 49 years (range 35 to 69),
the percent of leukemic blasts ranged from 69% to 98%,
and residual T cells were always less than 2%. Preliminary
experiments failed to demonstrate any advantage of sequential incubation of tumor cells with MoAbs (30 minutes at
4°C) and C' (30 minutes at 37°C) over simultaneous
treatment. Similarly, results of sequential treatment with
MoAbs +C' (30 minutes at 37°C) and 4-HC + VP-16 (1
hour at 37°C) did not significantly differ from the single
incubation. Therefore, only the data of the latter series of
experiments are reported. The results of HL-60 and CFU-L
clonogenic assay inhibition are shown in Figs 1 and 2. A
greater than 4 logs of HL-60 tumor cell elimination was
observed after immunochemotherapy treatment (ie, M195
plus 4-HC either used at 100 pmol/Lor 80 kmol/L + VP-16
and combination of M195 and F23 plus 4-HC + VP-16)
while the single procedure provided for a mean of 95% ?
0.9% SD of colony-forming inhibition (complementmediated lysis by M195 and F23 used together); 99.5% ?
0.8% SD 4-HC (100 pmol/L) used alone; and 99.95% 2
0.2% SD 4-HC and VP-16 mixture (Fig 1).
Eight AML patients were treated according to the four
protocols. The results are shown in Fig 2. No CFU-L (ie,
Lower limit of
-accurate
- - -detection
-- "."".
control
MoAb+C'
4HC
4HC+VP16
MoAb+C'+
4HC VP16
+
Fig 1. Cytotoxic activity of four protocols on HL-60 cell line mixed
C' treatment
(1:20) with an excess of irradiated BM cells. MoAb
represents one cycle of 40 pg/mL of M195 and F23 with complement
at % (vol/vol) dilution. 4-HC concentration is 100 pmol/L when used
alone. 4-HC at 80 pnol/L or 100 pmol/L gave the same result when
the drug was combined to VP-16 with and without MoAbs.
+
more than 3 logs of tumor cells elimination) were found
after the combined treatment with MoAb M195 plus 4-HC
(100 kmol/L) and VP-16, whereas the mean colony-forming
inhibition was 92.3% 2 2.5% SD, 95.5% ? 1.4% SD, and
99% k 0.8% SD following antibody treatment and complement lysis, 4-HC and 4-HC + VP-16 treatment, respectively
(Fig 2).
Complement-dependent lysis by single MoAb (ie, M195
or F23) resulted in less than 90% of tumor cell elimination
when tested on both HL-60 cells and leukemic cells from
AML patients (data not reported).
Cytotoxic activity of four different purging protocols on
normal BMprogenitors. The mean recovery of committed
normal BM precursors after treatment with MoAbs with
and without 4-HC VP-16 is shown in Table 2. The low
number of CFU-GEM observed in our experiments did not
allow any statistical analysis; therefore, only the results of
CFU-GM and BFU-E recovery are reported. The single
treatments showed a mean recovery of 12% CFU-GM and
22.9% of BFU-E (immunotherapy), and 4.4% CFU-GM
and 5.6% BFU-E (chemotherapy). The colony-forming
efficiency of hematopoietic cells was completely abolished
by the combined protocol in most of the experiments. When
+
Lower limit of
-accurate
-- -detection
- ---
control
MoAb+C'
4HC
4HC+VPl6
MoAb+C'+
4HC+VPlS
Fig 2. Cytotoxic activity of four protocolson CFU-L. For details see
legend to Fig 1.
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LEMOLI ET AL
1832
Table 2. Recovery of Hematopoietic PrecursorsAfter Treatment
With MoAbs With and Without 4-HC VP-16
+
Colony-Forming Inhibition*
Treatment
CFU-GM
BFU-E
CTR + C’t
CTR MoAbs
M195 C’
F23 C’
M195 F23 C’
4-HC
4-HC + VP-16
M195 + C’ + 4-HC VP-16
F23 C‘ 4-HC VP-16
M195 + F23 C’ 4-HC + VP-16
11.8 i 4.6
10.4 f 7.1
62.7 i 7.2
48.3 f 10.2
88 c 7.4
84 f 10
96.4 3.5
99.6 f 1.4
99.4 f 2.8
99.5 f 3.2
9.6 f 3
12.3 f 2.4
46.8 2 5
43.7 i 6.8
67.1 c 9.6
80.3 f 12.5
97.2 i 2.3
100
99.8 f 1.6
100
+
+
+
+
+ +
____
+
+
+
+ +
____
~
*
~
*Aggregates (more than 50 cells) were scored at day 14. These
numbers are expressed as mean f SD of percentage of colony-forming
inhibition in comparison with control cultures. Mean numbers c SD of
CFU-GM and BFU-E colonies per l o 5 cells plated were 119 f 22 and
206 i 51, respectively.
tCTR represents control medium.
4-HC was tested at the concentration of 80 pmolL and
combined with VP-16 and MoAbs, the recovery of CFU-GM
and BFU-E was 2.7% 2 3% SD and 1% +- 2.5% SD,
respectively (data not shown in Table). Additional experiments were then designed to assess the recovery of progenitor cells from enriched population of CD34‘ cells after
immunopharmacologic treatment. Our assay consisted of
the stimulation of the pre-CFU-GM (CD34’) compartment by rCSFs or MoCM in liquid culture followed by a
clonogenic assay to evaluate the recovery of CFU-GM
derived from the enriched blast population. We also compared different culture conditions to investigate the optimal
combination of CSFs for stimulating early hematopoietic
progenitors. In this set of experiments, the addition of the
MoAb F23 to the combined treatment did not give different
results than the MoAb M195 used alone with drugs;
therefore, only the data of M195 plus 4-HC + VP-16
protocol are reported in Table 3.
In the first three experiments, IL-3 alone (experiments 1
and 2) or combined with IL-1 (experiments 1,2, and 3) and
MoCM were chosen as stimulating factors in the suspension
culture while GM-CSF and IL-3 were used for inducing
CFU-GM growth in the semisolid assay. These three
experiments did not show any CFU-GM recovery after the
immunopharmacologic treatment before enrichment of
CD34+ cells by immunopanning. When CD34’ cells were
assayed, we observed few CFU-GM in two experiments (1
and 3) (DO value) and no granulocyte-macrophage progenitors were obtained in experiment 2. The number of
CFU-GM colonies scored after 1week of liquid culture (D7
value) was remarkably higher than the DO value specifically
following stimulation in suspension culture with MoCM
using either GM-CSF or IL-3 in semisolid assay (Delta
values of 361.3, >24, and 25.5 in experiments 1, 2, and 3,
respectively). Given the poor Delta value obtained after
incubation of enriched blast cells with IL-3 alone, we
decided to use only IL-1 + IL-3 and MoCM as CSFs in
liquid culture for the next experiments. In addition, MoCM
was also used as CSF in short-term assay because other
studies performed at the same time in our laboratory,
showed MoCM as the most effective stimulant for a highly
enriched blast cell population.” Experiments 4 and 5
reported the highest Delta value (which indicates the
proliferating capacity of CFU-GM derived from CD34’ cell
population) when IL-3 or GM-CSF were used in semisolid
assay after 1 week of stimulation with MoCM ( > 4 5 and
> 30, respectively, experiment 4).
Finally, experiment 6 compared the effect of two different immunochemotherapy protocols (4-HC used at 100
pmol/L [A] v 80 pmol/L [B]) on early CD34’ progenitors
cells. The pluripotent cell compartment seemed to be less
affected by using the lower dose of 4-HC (Delta value of 180
when MoCM was used after IL-1 and IL-3 stimulation and
> 144 when GM-CSF followed either MoCM or cytokines
combination stimulation) while the “contamination” of
committed myeloid progenitors was similar after immunopanning in both cases. Interestingly, in four experiments (2,
5, 6A, and 6B) BM progenitors lost their responsiveness to
IL-3 after exposure to the same GF, used alone or combined, in liquid culture.
DISCUSSION
The therapeutic benefit of various techniques for purging
BM in animal system is well established” and preliminary
studies using 4-HC or 4-HC
VP-16 purged BM for
patients with AML reported encouraging results.233Although there are no data available from randomized trials
to substantiate the role of BM purging for AML patients,
the multicenter registration data from the European Bone
Marrow Transplantation Group (EBMTG) in 1989 showed
superior results of mafosfamide-purged marrow versus
unpurged marrow in AML patients transplanted in first
complete remission after myeloblative conditioning regimen?”
Use of MoAbs for selective removal of tumor cells from
BM is another approach, and several groups have reported
the production of MoAbs reactive with antigens expressed
on myeloid cells that are also capable of recognizing
leukemic cells from patients with AML.2’-23
Some of those MoAbs have been also used in purging
protocol in the treatment of AML. Trials are currently in
progress involving MoAbs and complement-treated marrow for leukemic patient^.^^.'^
However, both of these approaches (ie, chemotherapy
and immunotherapy) did not show, when used alone,
convincing evidence of selective killing of clonogenic leukemic cells compared with normal BM progenitors. Purging
protocols using cyclophosphamide (CY)-active derivatives
(ie, 4-HC and mafosfamide) or other alkylating agents
alone or combined with different chemotherapeutics showed
a differential activity between myeloid cell lines and normal
BM precursor^^"^^ but the same results were not confirmed
when CFU-L from leukemic patients were
suggesting that the data obtained on continuously growing cell
lines may not be predictive in view of clinical studies.
Similarly, studies of the expression of cell surface antigens
+
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IN VITRO IMMUNOCHEMOTHERAPY FOR AML
1833
Table 3. Pre-CFU-GM Assay In Vitro. Ficoll/lmmunochemotherapy/CD34+Population
Stimulus in
SuspensionCulture
Exp 1
IL-3
IL-1
+ IL-3
MoCM
Exp 2
IL-3
IL-1
+ IL-3
MoCM
Exp 3
IL-1
+ IL-3
MoCM
Exp 4
IL-1
+ IL-3
MoCM
Exp 5
IL-1
+ IL-3
MoCM
Exp 6A
IL-1 IL-3
+
MoCM
Exp 6BS
IL-1 IL-3
+
MoCM
Conditioned Medium in
Semisolid Assay
CFU-GMt From CD34' Cells
CFU-GMX
DO
D7
Delta
Value
GM
IL-3
0
0
3
7
168
51
56
7.3
GM
IL-3
0
0
3
7
615.6
92
205.3
13.1
GM
IL-3
0
0
3
7
1,084
367.2
361.3
52.4
GM
IL-3
0
0
0
0
GM
IL-3
0
0
0
GM
IL-3
4
0
> 12
> 24
0
8
0
0
0
0
0
8
8
> 24
GM
IL-3
0
4
4
36
6
8
1.5
GM
IL-3
0
0
4
4
72
102
18
25.5
GM
IL-3
MoCM
1
1.5
2.5
0
0
2.5
3
5
6
GM
IL-3
MoCM
1
1.5
2.5
0
0
2.5
10
15
18
GM
IL-3
MoCM
0
1
1.5
1
2.5
4
24.5
56
24.5
0
14
GM
IL-3
MoCM
0
1
1.5
1
2.5
4
17.5
17.5
49
17.5
7
12.2
GM
IL-3
MoCM
0
1.5
0.5
0
0
1
9
0
9
GM
IL-3
MoCM
0
1.5
0.5
0
0
1
6
12
3
GM
IL-3
MoCM
0.5
2
5.5
0
1
0.5
48
12
90
> 144
GM
IL-3
MoCM
0.5
2
5.5
0
1
0.5
48
15
36
> 144
0
*Number of colonies (more than 50 cells) before enrichment of CD34' cells. The value is expressed for 1 x
tNumber of colonies before (DO) and after (D7) 1 week of liquid culture expressed for 1 x l o 5cells plated.
*4HC concentration in this experiment was 80 bmol/L.
0
lo5cells plated.
0
0
> 24
>9
> 15
2.4
> 30
> 45
7.2
> 27
0
9
> 18
> 36
3
12
180
15
72
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LEMOLI ET AL
1034
tion capacity. Therefore, a more sensitive assay of early
on AML cells demonstrated that phenotypic features are
stem cell viability and capability to give rise to more mature
acquired in an analogous manner to their normal counterprogenitors is needed. Recent experimental evidences sugp a r t ~ . ~Therefore,
’,~~
the lack of a specific MoAb that reacts
gest that early hematopoietic progenitors with self-renewal
on the leukemic cells is the major obstacle for this proceproperty expressed CD34 antigen and that they are distindure. In addition, the heterogeneity in antigen expression
on CFU-L could allow some leukemic cells to escape lysis.34 guishable from more differentiated myeloid cells?-’
Furthermore, CD34+ cell fraction can be divided into
To improve current purging methods, we attempted to
remove occult leukemic cells from autologous marrow by
distinct precursors populations according to the coexpression of HLA-DR and CD33 antigens.’ Andrews et a1 have
combining two different purging methods. In particular, a
multiagent chemotherapeutic protocol (4-HC VP-16) was
recently demonstrated that only CD34+,CD33- cell population was able to sustain CFU cells (CFU-C) production for
compared and combined with complement-mediated lysis
5 weeks in long-term BM culture (LTMC) system while
by two MoAbs broadly reactive with myeloid cells (antiCD33 and anti-CD-13).
cells that expressed both the antigens generated few CFUC.39The importance of CD34’ cells is also underlined by
A similar approach (ie, immunochemotherapy) has been
studies showing that these cells, isolated by avidin-biotin
already proposed for purging of T-cell acute lymphocytic
immunoadsorption technique, can restore lymphoheleukemia (T-ALL) and Burkitt’s lymphoma cells providing
matopoiesis in lethally irradiated baboons and humans.8r9
a significant increase in tumor cell killing when compared
More recently, Siena et al demonstrated that many
with the single treatment although the results of normal
BM recovery have not been reported in the first s t ~ d y . ~ ’ . ~ ~ CD34’ cells are induced to circulate in the PB of cancer
patients after high-dose chemotherapy and intravenous
The 4-HC VP-16 (100 Fmol/L and 5 pglmL, respecadministration of rhuGM-CSF, and that the enrichment of
tively) purging protocol has proven to have a synergistic
CD34’ cell fraction is a crucial point to ensure recovery of
activity on HL-60 cells and antagonistic on normal BM
normal hematopoiesis after myeloablative chemotherapy.40
precursors,” and it is being used as a purging treatment for
Therefore, we tried to assess the ability of the immunoAML and non-Hodgkin lymphoma patients at Memorial
chemotherapeutic treatment to spare the putative heM195 MoAb recognizes CD33 antigen (p67)
matopoietic precursors collected after positive selection by
and it was found expressed on most of the myeloblastic
panning with monoclonal anti-MY10 antibody. A shortleukemias studied. M195 and rabbit complement were able
term suspension culture system, which was shown specific
to eliminate almost all of the committed BM progenitors in
to self renewal of pluripotent myeloid precursors in mua clonogenic assay (ie, 14-day CFU-GM and BFU-E) while
rine41,42 and human mode1,I7 was then set up and the
no binding of 1251 M195 was found on early blast cells
increase in number and clonogenic efficiency of blast cells
collected by negative selection.2LThese observations could
was assessed following stimulation with IL-3, IL-1 + IL-3,
suggest a therapeutic advantage for a protocol involving the
and MoCM. Our results demonstrated the recovery of the
use of M195 (alone or combined with an anti-CD13 M o m ,
hematopoietic progenitors earlier than day-14 CFU-GM
namely F23) that is positive on the majority of Ah4L cells
after immunochemotherapy. In particular, cultures containbut not on the earliest normal colony-forming cells. A
ing both IL-1 and IL-3 or MoCM, in the absence of any
similar MoAb, MY9, also reactive with CD33 antigen has
preformed feeder layer, showed a marked increase in
been used to purge AML patients marrows from residual
pluripotent precursors, whereas IL-3 used alone was less
leukemic cells.’6
effective.
In our experimental system HL-60 leukemic cell line
Finally, an experiment was addressed to compare the
clonogenic efficiency was reduced more than 4 logs, in
effect of two different combined protocols (4-HC used at 80
presence of marrow cell excess, after 1 hour of incubation
pmol/L v 100 pmol/L, which both gave the same results in
VP-16 while the treatment with
with M195 plus 4-HC
terms of HL-60 clonogenic efficiency inhibition) on pluripochemotherapy or immunotherapy alone was less effective
tent stem cells. The lower dose of 4-HC allowed a better
on clonogenic tumor cells. The same combined protocol
recovery of the hematopoietic progenitor compartment and
produced, at a sensitivity level of 3 logs, a complete
the Delta value obtained was comparable with the values
eradication of CFU-L from eight patients with AML. The
observed in a large series of experiments on normal BM
addition of F23 MoAb did not further enhance the eliminasamples where different techniques were used to ensure the
tion of both HL-60 and CFU-L cells while a lower concenoptimal recovery and stimulation of early blast ~e1ls.l~
tration of 4-HC (ie, 80 pmol/L) showed the same results on
Further experiments are currently in progress to investigate
HL-60 of the optimal 4-HC concentration (100 pmolL)
the recovery of leukemic cells after immunochemotherapy
found in a previous
and panning with anti-CD34 MoAb. In addition, the
After incubation of normal marrow with the immunocomparison between the LTMC system and our short-term
chemotherapy protocol, CFU-GM and BFU-E were undeliquid culture is underway to accurately quantitate pretectable in most of the experiments by direct assay in
CFU-GM precursors.
semisolid medium. However, because infusion of chemopuIn summary, our results showed that the combination of
rified marrow containing no detectable CFU-GM produced
single cycle complement-mediated lysis by MoAbs and
hematologic recovery in transplanted patients3*this assay
pharmacologic protocols can produce additive tumor cell
does not adequately predict the hematopoietic reconstitu-
+
+
+
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IN VITRO IMMUNOCHEMOTHERAPY FOR AML
killing on leukemic cells while, under the same experimental conditions, early hematopoietic precursor are substantially spared. Conversely, in our experiments the depletion
of the committed hematopoietic progenitors was nearly
complete, suggesting, according to Rowley et al,” the
efficacy of our immunopharmacologic purging method in
view of its clinical application. The responsiveness of early
BM progenitors to IL-1 and IL-3 in combination also
suggests that preincubation of autologous marrow with
these cytokines may enhance the delayed hematopoietic
reconstitution following the reinfusion of purged marrow.
1835
Recent data from our laboratory support the use of IL-1
and IL-3 combination, which has been shown to expand the
committed hematopoietic progenitor compartment without
affecting the stem cell pool in the LTMC system.MThus, the
use of 4-HC VP-16 and MoAbs would seem a reasonable
approach to purge minimal residual leukemia from autologous graft.
+
ACKNOWLEDGMENT
The authors thank Judy Reid and Claudette Bryant for their
assistance in preparing this manuscript.
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1991 77: 1829-1836
Autologous bone marrow transplantation in acute myelogenous
leukemia: in vitro treatment with myeloid-specific monoclonal
antibodies and drugs in combination
RM Lemoli, C Gasparetto, DA Scheinberg, MA Moore, BD Clarkson and SC Gulati
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