Pergamon

PII: SO145-2126(97)0012M

Leukemia Research Vol. 22, No. I, pp. 31-37. 1998. 0 1998 Elsevier Science Ltd. AU riehts reserved

Printed in -&at Britain 01452126/98 $19.00 + 0.00

CLINICAL SIGNIFICANCE OF GRANULOCYTE COLONY- STIMULATING FACTOR (G-CSF) RECEPTOR EXPRESSION IN

ACUTE MYELOID LEUKEMIA

Hiroshi Kawada, Tamotsu Sasao, Shuji Yonekura and Tomomitsu Hotta Fourth Department of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara,

Kanagawa 259-l 1, Japan

(Received 12 June 1997. Accepted 3 September 1997)

Abstract-We examined granulocyte colony-stimulating factor (G-CSF) receptor (GR) expres- sion on leukemic cells from 44 adults with newly-diagnosed acute myeloid leukemia (AML). GR expression was higher in female patients. G-CSF was administered to AML patients after initial induction therapy without significant acceleration of leukemia, irrespective of GR expression level. G-CSF administration after initial chemotherapy did not adversely influence clinical outcome of GR-positive patients. However, at first relapse, leukemia regrowth was accelerated in 3 of 15 GR-positive patients who received G-CSF after re-induction. It remains to be determined whether leukemia acceleration due to G-CSF contributes to re-induction failure and if G-CSF therapy is a significant risk in relapsed, GR-positive AML patients. 0 1998 Elsevier Science Ltd. All rights reserved.

Key words: Granulocyte colony-stimulating factor, granulocfle colony-stimulating factor receptor, acute myeloid leukemia, flow cytometry.

Introduction Materials and Methods

Acute myeloid leukemia (AML) cells express granulo- cyte colony-stimulating factor (G-CSF) receptor (GR) [l-6], and G-CSF occasionally accelerates the growth and/or differentiation of leukemic cells [7-111. How- ever, the clinical relevance of GR expression in AML patients has not been evaluated. Recently, it was noted that GR numbers measured by a radioisotopic binding assay correlated well with D values, the differences in fluorescence intensities of GR expression between negative controls and samples [4,12]. In this study, we determined the expression of GR on AML cells using flow cytometry to assess correlations with various clinical parameters.

Abbreviations: G-CSF, granulocyte colony-stimulating fac- tor; GR, G-CSF receptor; AhfL, acute myeloid leukemia; FAB, French-American-British; BHAC, N4-behenoyl-fi-n-arabino- furanosylcytosine; Ara-C, cytosine arabinoside; ACR, aclar- ubicin; ATRA, all-tram retinoic acid; CR, complete remission; BMT, bone marrow transplant; EDTA, ethylenediamine-tetra- acetic acid.

Correspondence fo: Hiroshi Kawada, M.D., Ph.D. Fourth Department of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-l 1, Japan (Tel.: 463-93-l 121, Ext. 2234; Fax: 463-92-4511).

Patients

Forty-four newly-diagnosed adult AML patients (28 males and 16 females; median age: 52 years, range: 16- 90 years) who were referred to Tokai University Hospital between January 1992 and February 1995 were included in the study. AML types were classified according to the French-American-British (FAB) cri- teria [13,14] (Table 1).

After informed consent was obtained, patients re- ceived one of the following induction chemotherapies: (a) BHAC-DM; daily N4-behenoyl-/?-u-arabinofurano- sylcytosine (BHAC; 170 mg/m2, 2-h infusion) + daily 6- mercaptopurine (M; 70 mg/m2, orally) + intermittent daunorubicin (D; 25 mg/m2, bolus infusion, on days 1, 2, 5, and 6; and on days 8, 9, and 10, if necessary); (b) Ara-C + ACR; continuous infusions of cytosine arabino- side (Ara-C; 15 mg/m2) and aclarubicin (ACR; 8 mg/ m2); or (c) daily all-trans retinoic acid (ATRA; 45 mg/ m2, orally). In patients who received the BHAC-DM regimen and whose leukemic cells expressed lymphoid antigens, daily prednisolone (20 mg/m2, orally) and intermittent vincristine (1 mg/m2, bolus infusion, on days 3 and 4) were added.

31

32 H. Kawada et al.

Table 1. G-CSF receptor expression according to FAB classification

G-CSF FAB subtype receptor expression MO Ml M2 M3 M4 M5 M6 Total

I 0 1 1 3 1 0 1 7 II 1 2 3 0 0 0 0 6 III 2 6 8 1 3 1 0 21 IV 0 4 0 3 2 1 0 10 Total 3 13 12 7 6 2 1 44

FAB, French-American-British; G-CSF, granulocyte col- ony-stimulating factor.

Chemotherapies were continued until the bone marrow nucleated cell count was less than 10 OOO/ul with < 20% blasts. However, ATRA was administered until a complete remission (CR) was obtained. A CR was defined as bone marrow blasts < 5% in a normocellular marrow and adequate peripheral neutro- phi1 and platelet counts (x1.5 x 109/1 and ~100 x 109/1, respectively). After achieving a CR, patients received cyclical post-remission chemotherapy including Ara-C and anthracyclines. At first relapse, patients received Ara-C + ACR or high-dose Ara-C containing che- motherapy.

Patients failing to achieve a CR after two induction courses were considered induction failures. Induction failures were excluded from the evaluation for CR duration but were included for CR rate, survival, and response to G-CSF. Patients who died during che- motherapy or aplasia were included in the evaluation for CR rate and survival. One patient (a 90-year-old man with Ml) was excluded from the evaluation for clinical outcomes because he did not receive intensive che- motherapy due to his advanced age. Patients who underwent bone marrow transplant (BMT) were cen- sored at the date of BMT.

After the initial induction chemotherapies, 24 of the 43 patients received daily G-CSF (filgrastim; Kirin Brewery Co Ltd., Tokyo, Japan) at a dose of 50 us/m2 subcutaneously or 100 ug/m2 in a 30-min intravenous infusion, starting from the first day after induction therapy and continuing until the peripheral neutrophil count exceeded 1.5 x 109/l. At that point, the dose was reduced to half. Treatment was discontinued if the neutrophil count remained above 1.5 x 109/l.

G-CSF receptor assay Heparinized bone marrow aspirate samples were

obtained prior to induction. Samples were lysed with hemolytic solution containing ammonium chloride (826 mg/dl), potassium bicarbonate (100 mg/dl), and tetrasodium ethylenediaminetetraacetic acid (EDTA;

3.7 mg/dl). After washing with a buffered saline-protein solution (RDF-1 wash buffer; R & D Systems, Minneapolis, MN), cells were resuspended in buffer. A 25 ul aliquot of the cell suspension (5 x lo4 cells) was mixed with 5 ul of biotinylated G-CSF (10 ug/ml) (R & D Systems) and reacted at 4°C for 1 h. After washing, cells were mixed with a streptavidin-phycoerythrin conjugate (Becton Dickinson Immunocytometry Sys- tems, Mountain View, CA) and incubated for 30 min at 4°C in the dark. Cells were washed and analyzed using a FACScan (Becton Dickinson) flow cytometer. Based on the morphological characteristics according to the FAB classification [13,14], appropriate gate settings were used to analyze the leukemic cell population, and more than 5000 cells were counted. As negative controls, cells were pre-incubated with 25 ul of excess unlabeled non- glycosylated G-CSF (filgrastim, 250 l.@nl) before mixing with biotinylated filgrastim, as previously described [6].

Overlay histograms in which the horizontal axis was fluorescence intensity and the vertical axis was the relative number of cells were obtained using Consort C- 30 software (Becton Dickinson). The D value, the maximum vertical displacement between the histogram of the sample and the histogram of the negative control, was calculated with the Kolmogorov-Smimov test as previously described [4,12]. GR expression was classi- fied into four grades: (I) D < 0.05; (II) 0.05 < D ~0.15; (III) 0.15 < Dc0.35; and (IV) 0.35 < D.

Expression of cell surface antigens and cell cycle distribution of the leukemic cells were analyzed by flow cytometry as previously described [ 15,161.

Statistical analysis Relationships between GR expression and clinical

characteristics or results of therapy were determined by chi-square test or Wilcoxon rank-sum test. Differences in the time required to achieve CR, CR duration, or survival were compared by the generalized Wilcoxon test.

Results

Of the 44 cases examined at presentation, 37 were positive for GR (grade II: 6, III: 21, IV: 10, Table 1). The remaining seven were considered to be negative or faintly positive for GR (grade I). GR expression was heterogeneous within each FAB subtype, and there was no correlation between receptor expression and FAB classification. Although GR expression was higher in female patients (P < 0.05) no other correlations with the initial clinical characteristics were observed (Table 2).

In patients who received G-CSF after initial induction therapy, no significant regrowth associated with G-CSF

Clinical significance of granulocyte colony-stimulating factor (G-CSF) receptor expression in acute myeloid leukemia 33

Percent (A) CR duration

(1): G-CSF

(2): no G-CSF

Percent (B) Survival

(2) 60 - I (

50 -

40 - I I

30 - 30 - (1) 20 -

10 - (1)

20 -

10 - 0 I I I 0 I I I

500 1000 1500 500 1000 1500

Days Days

Fig. 1. Comparison of CR duration (A) and survival (B) according to G-CSF administration in patients with grade III or IV GR. No significant differences were observed using Kaplan-Meier curves comparing G-CSF with no G-CSF. G-CSF, granulocyte colony-

stimulating factor; GR, G-CSF receptor.

administration was observed, irrespective of the expres- sion level of GR.

We then divided patients whose leukemic cells were strongly positive for GR (grade III or IV) into two groups according to whether they received G-CSF after induction chemotherapy. Differences in the response to induction therapies, the time required to achieve CR,

Table 2. Clinical characteristics of AML patients according to G-CSF receptor expression

G-CSF receptor expression

Clinical characteristic I&II III & IV

Age (yews) 51.0 + 22.2 52.4 + 16.8 Sex (M/F) 11/2* 16/15 WBC (x 109/1) 29.9 + 44.8 47.6 f 76.7 Blood blasts (x 109/1) 23.2 + 38.5 37.5 f 73.6 Blood blasts (%) 58.3 + 29.5 50.1 + 35.8 Hb WW 8.7 +_ 2.3 8.2 +_ 2.1 Platelets (x 109/1) 67.5 + 53.8 88.3 + 127.3 Serum LDH (units/l) 1834 + 2835 1140 f 905 Bone marrow NCC (x l@/l.d) 32.1 + 23.8 28.1 + 28.4 Bone marrow blasts (%) 74.9 + 26.3 76.5 + 18.7 MPO positivity (%) 67.2 + 33.7 56.9 f 39.2 Auer rods (+/-) 1013 18/12 Karyotype abnormality (+/-) 4f7 12/14 Cell surface marker (+/-) CD7 517 9119 CD13 11/2 30/l CD33 12/l 2813 CD34 617 17/12 CD71 813 14/12 HLA-DR 914 2417 S phase (%) 5.6 + 3.7 6.0 f 4.8

AML, acute myeloid leukemia; G-CSF, gmnulocyte colony- stimulating factor; WBC, white blood cells; Hb, hemoglobin; LDH, lactate dehydrogenase; NCC, nucleated cell count; MPO, myeloperoxidase.

*The P-value is less than 0.05.

remission duration, or survival were not observed between these two groups (Table 3 and Fig. 1). No significant differences in clinical outcome were noted among patients with different GR expression (Table 4 and Fig. 2).

However, at first relapse, regrowth of leukemia was accelerated by G-CSF administration in 3 of 15 GR- positive patients who received G-CSF after re-induction chemotherapy. GR expression at diagnosis differed

Table 3. CR rates in patients with GR grade III/IV leukemic cells

G-CSF administration Induction Total therapy + - CR rates (%)

BHAC-DM 1 l/14 818 19/22 (86.4) Ara-C + ACR 314 l/l 415 (80.0) ATRA l/l 313 414 (1W Total CR rates 15/19 12112 2713 1 (87.1) (%I (78.9) VW

CR, complete remission; G-CSF, granulocyte colony- stimulating factor; GR, G-CSF receptor; BHAC-DM, N4- behenoyl-fl-o-arabinofuranosyl-cytosine + daunorubicin + 6- mercaptopurine; Ara-C, cytosine arabinoside; ACR, aclarubi- tin; ATRA, all-tram retinoic acid.

Table 4. CR rates among patients with different G-CSF receptor expression

G-CSF receptor expression Total

I II III IV CR rate

516 516 19/21 8/10 37143 (83.3) (83.3) (90.5) (80.0) (86.0)

Numbers in parentheses indicate percentages of CR patients. CR, complete remission; G-CSF, granulocyte colony-stimulating factor.

Tabl

e 5.

Clin

ical

ch

arac

teris

tics

befo

re r

egro

wth

of

leu

kem

ia

Clin

ical

char

acte

ristic

at

diag

nosi

s

1

at f

irst

rela

pse

afte

r at

ch

emot

hera

py

diag

nosi

s

Patie

nt n

o.

2 3

at f

irst

afte

r at

at

firs

t af

ter

rela

pse

chem

othe

rapy

di

agno

sis

rela

pse

chem

othe

rapy

Age

(yea

rs)

Sex

(M/F

) EA

B su

btyp

e W

BC

(x

109/

1)

Blo

od b

last

s (%

) I-%

W-W

Pl

atel

ets

(x

109/

1)

Seru

m L

DH

(u

nits

/l)

Bone

mar

row

NC

C (

x 10

4/p1

) Bo

ne m

arro

w b

last

s (%

) M

PO p

ositi

vity

(%

) Au

er r

ods

(+/-)

Ka

ryot

ype

abno

rmal

ity

(+I-)

C

ell

surfa

ce m

arke

r (+

I-)

CD

7 C

D13

C

D33

C

D34

C

D71

H

LA-D

R

S ph

ase

(%)

G-C

SF

rece

ptor

(gr

ade)

Th

erap

y to

indu

ce r

emis

sion

G

-CS

F ad

min

istra

tion

afte

r th

erap

y R

esul

t of

the

rapy

du

ratio

n of

firs

t C

R (

mon

ths)

60

F M2

2.8

4.4

15

18

7.4

12.1

89

23

94

6 16

04

47.2

9.

8 30

.2

56.4

75

53

+

+ -

-

- -

+ +

+ +

- +

- +

2.5

ND

II

II BH

AC-D

M

Ara-

C +

AC

R

+ +

CR

N

R

CR

N

R

CR

5.

5 9

5

51

M

Ml

0.6

62.1

0

97

6.0 15

949

0.6

62.8

2.

6 94

.6

92

+ - t + - ND

3:o

III

BHAC

-DM

-

5.0

63

8.7

98

558

9.2

77.2

58

+ - +

- +

+ +

+ +

+ -I-

+

ND

4:

o N

D

IV

Ara-

C +

AC

R

ADVP

* +

+ 20

F Ml

0.5

333.

5 0

96

8.1

537

2410

0.

6 58

.3

9.5

91.1

48

+ +

1.0

0.3

2 0

8.0

63

308

3.6

0.2

26.8

1.

2 32

+ + ND

N

D

ND

N

D

ND

N

D

ND

N

D

Ara-

C +

AC

R

+ PR

FAB,

Fr

ench

-Am

eric

an-B

ritis

h;

WB

C,

whi

te

bloo

d ce

lls;

Hb,

hem

oglo

bin;

LD

H,

lact

ate

dehy

drog

enas

e;

NC

C,

nucl

eate

d ce

ll co

unt;

MP

O,

mye

lope

roxi

dase

; C

R,

com

plet

e re

mis

sion

; N

R,

no r

espo

nse;

ND

, no

t do

ne.

*The

pat

ient

ach

ieve

d th

e fir

st C

R w

ith t

his

chem

othe

rapy

(A

DVP

; cy

tosi

ne a

rabi

nosi

de +

dau

noru

bici

n +

etop

osid

e +

pred

niso

lone

) fo

llow

ing

BHAC

-DM

.

Clinical significance of granulocyte colony-stimulating factor (G-CSF) receptor expression in acute myeloid leukemia 35

Percent (A) CR duration

(2): III & IV

500 1000 1500

Days

Percent (B) Survival

(2): III & IV

60 -

50 - ’ ’ 4 (2) 40 - 30 - (1) 20 - 10 -

0 I I I

500 1000 1500

Days

Fig. 2. Comparison of CR duration (A) and survival (B) among patients with different GR expression. No significant differences were found between grade I and II cases, and grade III and IV cases. CR, granulocyte colony-stimulating factor receptor.

among these three patients (Table 5). In patient 1, the level of GR expression was also determined at relapse and was again determined as grade II. In all three patients with regrowth, severe marrow aplasia with a marked decrease in the number of blasts was noted before G-CSF administration. The clinical course of patient 1 from Table 5 is summarized in Fig. 3.

G-CSF 75 p g SC -

ACR 10 mg Cont. DIV ////////

Ara-C 20 mg Cont. DIV

NCC (x 104/pI) 9.8 5.3 0.6 33.0

,,i i 0 0 Blast (9%) 56.4 44.9 2.6 86.6

Discussion

Leukemic cells from women showed a higher expression of GR, but there were no other correlations with GR expression, including the major prognostic factors in AML, i.e., age, WBC count, serum level of LDH, FAB subtype, karyotypic abnormality, or Auer rods [17].

38.0 7.0 0.4 2.9

87.6 12.7 1.6 34.6 0

hn - (x 109/l) 5;

c 40

30

20

10 1 9- 8-

September October November December 1993

Fig. 3. Clinical course of patient 1 at the time of first relapse. The patient received re-induction chemotherapy with continuous infusions of Ara-C and ACR. Chemotherapy finished on day 14, and G-CSF was started. On day 23, blood blasts suddenly appeared, and G-CSF was stopped. After informed consent was obtained, G-CSFkombined chemotherapy [16] was begun. G-CSF was resumed, and blasts increased. Ara-C and ACR were then added and continued with G-CSF for 10 days. Although the patient did not achieve a CR, the percentage of blasts declined after this therapy. Ara-C, cytosine arabinoside; ACR, aclarubicin; G-CSF,

granulocyte colony-stimulating factor; NCC, nucleated cell count; BM, bone marrow; CR, complete remission.

36 H. Kawada et al.

We found that G-CSF could be safely administered to AML patients after initial induction therapy without significant acceleration of leukemia, irrespective of the expression level of GR. Moreover, although this preliminary study included several different induction regimens, G-CSF administration after initial therapy did not adversely influence the clinical outcome of GR- positive patients. The absolute level of GR expression also did not affect clinical outcome. These results may be relevant to the following in vitro findings: (i) AML cells showing an abnormality of the signaling pathway induced by G-CSF have been detected [18], suggesting that AML cells do not always respond to G-CSF; (ii) GR numbers on AML cells are less than in normal myeloid cells [l]; and (iii) although G-CSF can accelerate clonogenic leukemic cell growth, high concentrations are necessary to stimulate leukemic cells [7,11,19].

However, regrowth of GR-positive AML cells can be accelerated by G-CSF at re-induction; regrowth could occur even if the level of GR expression at relapse was considered to be low. Usuki et al. reported that Ah4L resurgence after chemotherapy was likely to occur in patients whose leukemic cells had low numbers of G- CSF receptors [20]. In contrast, a previous in vitro study showed no correlation between colony-forming abilities of leukemic cells which respond to G-CSF and the level of GR [2].

Although we could not clarify why regrowth was significantly accelerated at relapse, G-CSF clearly should be used carefully in relapsed, GR-positive AML patients. Use of G-CSF at a higher dose may stimulate AML cells preferentially. However, a large- scale study will be necessary to determine whether G- CSF administration primarily contributes to re-induction failure, especially since relapsed AML tends to have resistance to chemotherapy and a poor prognosis [21].

Acknowledgements-We thank Mieko Takei, Shiho Kura- shima, and Nobumasa Kobayashi for their technical assistance.

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