Pergamon

01452126(95)00113-l

Leukemia Research Vol. 20, No. 4, pp. 327-332, ,996. Copyright 0 1996 Elsevier Science Ltd. All rights resewed

Printed in Great Britain 0145-2126196 $15.00 + 0 00

CLINICAL SIGNIFICANCE OF LEA-l EXPRESSION IN ADULT ACUTE MYELOID LEUKEMIA

Hiroshi Kawada*, Ryuki Fukuda*, Miyoko Yoshida Akira Masumoto*, Yoshiaki Ogawa*, Tamotsu Sasao

*, Mieko Takeij-, Nobumasa Kobayashi?, *, Nobuyuki Kubota*, Shigeki Watanabe*,

Yoshikatsu Umeda*, Kunihiko Yamauchi*, Shuji Yonekura* and Yukinobu Ichikawa* *The Fourth Department of Internal Medicine, Tokai University School of Medicine; and jCel1 Transfusion

Center of Tokai University Hospital, Bohseidai, Isehara, Kanagawa, 259-11, Japan

(Received 6 April 1995. Revision accepted 12 July 1995)

Abstract-h this study, we examined expressions of several adhesion molecules (AdMs), i.e. leukocyte function antigen-l (LFA-1: CD1 la/CD18), Hermes homing receptor (CD44) and intercellular adhesion molecule-l (ICAM-1: CD54), on leukemia cells from 51 adult patients with newly diagnosed acute myeloid leukemias (AMLs) to elucidate clinical significance of these AdM expressions. Those expressions in lymphoid malignancies have been correlated with tumor evolutions, but CD44 was detected in all the AML cases examined and CD54 expression did not associate with their clinical characteristics or outcomes. However, we found that LFA-1 expressions significantly correlated with splenomegaly, resistance to induction chemothera- pies and short survival periods in AML patients. Copyright G 1996 Elsevier Science Ltd.

Key words: Adhesion molecule, acute myeloid leukemia, flow cytometry, LFA-1 (CDlla/ CD18), Hermes homing receptor (CD44), ICAM-I (CD54), prognosis.

Introduction

A number of cell surface adhesion molecules (AdMs) are currently recognized and associated with multiple cellular functions [l-6]. These AdMs are also expressed on hematological malignancies and some have recently been shown to correlate with biological and clinical behaviors of the tumor cells [7-121.

Leukocyte function antigen-l (LFA-1) is an E/B heterodimeric (LFA-la: CDlla and LFA-lfi: CDlS) and noncovalently linked glycoprotein which belongs to /I2 integrins [13]. Hermes homing receptor (CD44)

Abbreviations: AdM, adhesion molecule; LFA, leukocyte function antigen; ICAM, intercellular adhesion molecule; AML, acute myeloid leukemia; FAB, French-American-British; BHAC, N‘t-behenoyl-fl-n-arabinofuranosylcytosine; Am-C, cy- tosine arabinoside; ACR, aclarubicin; ATRA, all-trans retinoic acid; CR, complete remission; MoAbs, monoclonal antibodies; FITC, fluorescein-isothiocyanate; PE, phycoerythrin; PBS, phosphate buffered saline; EDTA, ethylenediamine-tetraacetic acid; BSA, bovine serum albumin; NHL, non-Hodgkin lymphoma.

Correspondence to: Hiroshi Kawada, The Fourth Depart- ment of Internal Medicine, Tokai University School of Medicine, Bohseidai, Isehara, Kanagawa, 259-11, Japan (Tel: 0463 93 1121, Ext. 2234; Fax: 0463 92 4511).

consists of a transmembrane molecule with extensive O- linked sites of glycosylation [ 141. Intercellular adhesion molecule-l (ICAM-1: CD54), a member of the im- munoglobulin supergene family, is an integral mem- brane glycoprotein with seven potential N-linked glycosylation sites [15, 161. These three AdMs are crucial for a variety of cell interactions of leukocytes (granulocytes or lymphocytes) [3-6], and their possible roles in hematopoietic cell differentiation have been proposed [17, 181. The AdMs are also expressed in neoplastic counterparts of the progenitors [19]. How- ever, their clinical relevances in acute myeloid leukemia (AML) patients have not been sufficiently evaluated so far, while those in patients with lymphoid malignancies are relatively well characterized [7-lo].

In this study, we therefore determined the AdM expressions on AML cells by using flow cytometry and assessed their possible correlations with various clinical parameters of the patients.

Materials and Methods

Patients

Fifty-one newly diagnosed adult AML patients (31 males and 20 females, median age: 57 years, range:

327

328 H. Kawada et al.

Table 1. Expressions of adhesion molecules on AML cells

Adhesion molecule

FAB subtype Total

Positive cases MO Ml cases I tested (%I (n = 5) (n = 12) (Z8) $37) $45) $52) (nMp2)

CDlla 27 ; 51 (52.9) 315 4112 14118 O/7 515 112 Of2 CD44 50 50 WO) 315 12112 17117 717 515 212 212 CD54 15 I 50 (30.0) 315 2112 6118 l/7 314 012 012

Table 2. CR rates among patients with different FAB subtype and induction chemotherapy

Induction therapy

BHAC-DM BHAC-DMP BHAC-DOMP BHAC-AM Ara-C+ACR ATRA

Total CR rates (%I

FAB subtype Total CR

MO Ml M2 M3 M4 M5 M6 rates (%)

213 212 315 l/l 8111 (72.3) O/l 517 416 l/2 215 112 o/1 13124 (54.2)

212 Ill 313 (100) Ill Ill WO)

Ill 214 315 (60.0)

515 515 W)

315 9111 11117 617 215 112 112 33149 (67.3) (60.0) (81.8) (64.7) (85.7) (40.0) (50.0) (50.0)

16-94) who had been referred to Tokai University Hospital between July 1991 and February 1994, were included in the present study. Thirty (59%) of the AML patients were older than 59 years. They were classified according to the French-American-British (FAB) criteria [20-221 as shown in Table 1. After informed consent was obtained, the patients received one of the following six induction chemotherapies: daily p-behenoyl-fl-n-arabinofuranosylcytosine (BHAC; 170 mg/m2, 2-h infusions)tdaily 6-mercaptopurine (M; 70 mg/m2, orally, together with 300 mg/day of allopur- inol)tdaily aclarubicin (A, 14 mg/m2, bolus i.v.) (BHAC-AM) or intermittent daunorubicin (D; 25 mg/ m2, bolus i.v., on days 1, 2,5 and 6; and on days 8,9 and 10, if necessary) (BHAC-DM), BHAC-DMtdaily pre- dnisolone (P; 20 mg/m2, orally) (BHAC-DMP), BHAC- DMPtvincristine (0; 1 mg/m*, bolus i.v., on days 3 and 4) (BHAC-DOMP), continuous infusions of cytosine arabinoside (Ara-C; 15 mg/m2) and aclarubicin (ACR; 8 mg/m2) (Ara-CtACR) or daily all-trans retinoic acid (ATRA; 45 mg/m2 orally) (Table 2). We continued the chemotherapies until bone marrow nucleated cell count became fewer than lO,OOO/ul with less than 5% blasts. However, ATRA was administered until a complete remission (CR) was obtained. Two patients (a 90 year old male with Ml and a 94 year old female with M2) were excluded from present evaluations for clinical outcomes, since they did not receive induction che- motherapies because of their advanced ages. An

additional patient (a 49 year old male with M2), who did not attend our hospital after CR, was not included in the evaluations for CR durations and survival periods. CR was considered to be established when blast cells were less than 5% in normocellular marrow and peripheral neutrophils (15OO/yl) sand platelets (>lOO,OOO/ul) were recovered after one course of each chemotherapy. The overall CR rate was 67.3% (< 60 years old: 75.0% versus 360 years old: 57.1%, P > 0.05) in the 49 patients evaluated. Their median CR duration and survival period were 340 (range 57-840) days and 380 (range 2-1122t) days, respec- tively.

Monoclonal antibodies (MoAbs) Fluorescein-isothiocyanate (FITC)- or phycoerythrin

(PE)-conjugated MoAbs identifying CDlla (LFA-la), CD18 (LFA-l/I), CD44 (Leu-44) and CD54 @u-54) were used in the present study. The other FITC- or PE- conjugated MoAbs employed were specific for CD2 (Leu-Sb), CD3 (Leu-4), CD4 (Leu3a), CD5 (Leu-1), CD7 (Leu-9), CDS (Leu-2a), CD10 (CALLA), CD13 (Leu-M7), CD14 (Leu-M3), CD16 (Leu-llc), CD19 (Leu-12) CD20 (Leu-16), CD33 (Leu-M9), CD34 (HPCA-2), CD38 (Leu-17), CD41b (TP-80), CD57 @u-7), HLA-DR and glycophorin-A. Anti-TP-80 and anti-glycophorin-A MoAbs were purchased from Ni- chirei Co. Ltd. (Tokyo, Japan) and Immunotech S.A. (Marseille, France), respectively. The remaining MoAbs

LFA-1 expression in AML 329

were obtained from Becton-Dickinson Immunocytome- try Systems (Mountain View, CA, U.S.A.).

Flow cytometric analysis Heparinized bone marrow aspiration samples were

obtained before the induction therapies. The median percentage of blast cells in the samples was 78.8% (range 32-97). Erythrocytes in the samples were lysed with hemolytic solution containing ammonium chloride (826 mg/dl), potassium bicarbonate (100 mg/dl) and tetrasodium ethylenediamine-tetraacetic acid (EDTA; 3.7 mg/dl). After washing with phosphate buffered saline (PBS) supplemented with 0.05 M disodium EDTA and 0.2% bovine serum albumin (BSA), cells were suspended in EDTA-BSA-PBS. Fifty microliters of the cell suspension (5 x lo5 cells) were mixed with the optimal concentration of FITC- and PE-MoAbs recom- mended by the manufacturers. The mixture was then reacted at 4°C for 30 min in the dark. Cells were also incubated with FITC- and PE-labeled isotype-matched mouse immunoglobulins and were used as negative controls in each test sample. After washing with EDTA-BSA-PBS, the cells were resuspended in 0.5 ml of EDTA-BSA-PBS, and were passed through a nylon mesh filter. More than 6000 cells were counted by a fluorescence activated cell sorter (FACSTAR, Becton-Dickinson). Measurement conditions were com- pensated by using CaliBRITETM beads (Becton-Dick- inson). Based on the morphological characteristics according to the FAB classification, appropriate settings of forward and 90” scatter gates were prepared to analyze the leukemia cell population from each patient. Under the considerations for unavoidable contamination of non-leukemic cells within the gated area, and the fact that normal lymphocytes generally express LFA-1 and CD44 [3,5], we determined the sum of normal T and B lymphocytes within the gated area (%CD3+ %CD20) in each test sample. The sum of normal T and B lymphocytes was 7.6+7.9% in the samples examined (n = 51). Based on this result, the leukemia cells were considered as positive for corresponding antigen, when the percentage of positively stained cells was higher than 20%.

Statistical analysis Correlations of the AdM expressions with various

patients’ clinical parameters were analyzed retrospec- tively. Relationships of the AdM expressions with other cell surface antigen expressions on AML cells and various patients’ clinical characteristics were deter- mined by chi-square test or Wilcoxon rank-sum test. Differences of survival periods and CR durations between corresponding antigen-positive and -negative cases were compared by generalized Wilcoxon test. Multivariate analysis for significant factors to predict

CR was performed by the stepwise logistic regression model.

Results

AML cells from 27 of the 51 cases (52.9%) were positive for CDlla. Of the 50 cases studied, CD44 was detected in all cases, and CD54 was expressed in 15 cases (30%) as shown in Table 1.

The CDlla-positive AML cases were frequently positive for CD18 (P < 0.01) CD34 (P < 0.05) and HLA-DR (P < 0.01) compared with the AdM-negative cases. The percentages of CDlla-positive cells also correlated with those of CD18-positive (r = 0.956, P < 0.001) and HLA-DR-positive cells (r = 0.685, P < 0.001) (Fig. 1). All CDlla-positive cases ex- pressed HLA-DR, while HLA-DR-negative cases did not express CDlla. The expressions of CD18 (P < 0.05) CD33 (P < 0.05) and CD34 (P < 0.01) were also frequent in the CD54-positive cases. However, the percentages of CD54- or CD4Cpositive cells did not correlate with those of any other antigen-positive cells.

Clinical characteristics were compared between CDlla-positive and -negative patients (Table 3). The

(A)

(%)I00

y = -2.083 + 0.947x R = 0.956 (p<O.OOl)

(B)

0 20 40 60 80 100

HLA-DR (%)

Fig. 1. Correlations of CDlla expressions on AML cells with their expressions of CD18 (A) and HLA-DR (B).

330 H. Kawada et al.

Table 3. Clinical characteristics of AML cases according to the CDlla expressions

CDlla

Clinical characteristic

Age (years) Sex (M/F) RBC (x lO’*/lJ Platelet (x 10 /l) WBC (x 109/1) Blood blasts (%) Bone marrow blasts (%) Serum LDH (units/l) MPO positivity (%) Auer rod (y/n) Splenomegaly (y/n) Lymphadenopathy (y/n) Gum swelling (y/n) CNS involvement (y/n) Liver dysfunction and/or hepatomegaly (y/n) CR rate (%) CR duration (median day) Survival period (median day)

Positive Negative ( > 20%) (<20%)

59.9 f 19.0 53.1 f 12.7 15112 16/B

2.62 f 0.77 2.59 + 0.84 117.2f113.0t 44.8 f 36.3

35.7_+ 47.7 20.3 + 33.4 39.8 + 31.8* 58.2 & 30.7 67.4 + 20.2* 76.8 + 25.0 1492f2342 1018+678 47.6 k 40.2 69.5 f 35.3

12/15 1519 10/17* 2122 6121 3121 4123 6118 2125 3121 12115 12112

12/25 (48.0)t 21/24 (87.5) 321 334 292* 497

Abbreviations: M/F, males/females; RBC, red blood cells; WBC, white blood cells; LDH, lactate dehydrogenase; MPO, myeloperoxidase; y/n, yes/no; CNS, central nervous system. *P value less than 0.05, tP value less than 0.01 between CDlla- positive and -negative cases.

CD1 la-positive cases had significantly higher platelet counts (P < 0.01) and lower percentages of peripheral blood (P < 0.05) and bone marrow blasts (P < 0.05) than CDlla-negative cases. The incidence of spleno- megaly was high in the CDlla-positive cases (P < 0.05). When 30% positivity was used as the cut- off point, instead of 20%, splenomegaly became more prominent in the AdM-positive patients group (lo/15 versus 2/24, P < 0.01). Expressions of CD18, CD34 and HLA-DR also associated with high platelet count and/or low percentages of blast, but not with splenome- galy (data not shown).

Compared with CDlla-negative cases, the CR rate was significantly lower (P < 0.01) and the survival period was shorter (P < 0.05) in the antigen-positive cases. When 30% positivity was used as the cut-off threshold, the CR rate was still low in CDlla-positive cases (43.5% versus 88.5%, P < 0.01) and the differ- ence of survival periods became more significant (284 days versus 524 days, P < 0.01). However, CR durations were not different between the two groups (P > 0.05) when either threshold was used. Of the 13 CDlla-positive cases who were resistant to the first induction chemotherapy, 10 cases received further induction chemotherapies, but CR was obtained only in one of the cases. To assess the factors which can predict treatment outcome, we performed multivariate

analysis by using various clinical parameters described in Table 3, the cell surface antigen expressions examined, FAB subtypes and treatment regimens employed. The analysis also confirmed that only the CDlla expression was a predictive factor (P = 0.0039).

Although CD18-positive cases were also associated with low CR rate (P < 0.05) in the univariate analysis, any other clinical outcomes were not correlated with the CD18, CD34 and HLA-DR expressions.

We further analyzed the 35 cases who received almost similar regimens (BHAC-DM; 11 cases or BHAC-DMP; 24 cases). No significant differences in the CDlla expression, CR rates and survival periods were detected between the two patient groups. Of the 35 cases, the CDlla-positive cases still showed a low CR rate (41.2% versus 77.8%, P < 0.05). Short survival periods were also recognized in the antigen-positive cases (median survival periods: 123 days versus 501 days, P < 0.05), when the 30% threshold was used.

Clinical characteristics and outcomes were also compared between CD54-positive and -negative pa- tients, but no significant correlations were recognized.

Discussion

While the clinical significance of cell surface AdM expression on AML cells has not been extensively

LFA-1 expression in AML 331

evaluated so far, one report described that the expression of LFA-3 (CD58) correlated with a favorable prognosis of AML cases [23]. This previous study also examined CD54 expression on AML cells and showed that the AdM expression did not possess any clinical relevances in AML patients. In the present study, we also examined several other AdMs, including CD54, expressed on AML cells to clarify their clinical significance.

Of the AdMs examined, CD44 was expressed in all of the AML cases, although the AdM-positive patients with B-cell non-Hodgkin lymphoma (NHL) are reported to have a poor response to chemotherapies and signifi- cantly shorter survival periods [7]. Thus, CD44 was considered to be essential for AML cells as in their normal counterparts [17]. In addition, CD54 expression on AML cells was not associated with any clinical parameters as reported in the previous study [23], although various investigators recognized significant correlations of the AdM expression with tumor evolu- tions of NHL patients [8,9].

There have been no reports which have assessed the relationship of LFA-1 expressions with clinical variables in AML patients, whereas patients with NHL in remission have a significantly high frequency of LFA- l-positive tumor cells. In the present study, the tight correlation of CDlla expressions with those of CD18 were observed and the result indicates the known structural association between the two molecules of LFA-1 (CDlla/CD18) [13]. However, the correlations of CDlla and CD18 expressions with several clinical characteristics were not identical. The results might be explained by the fact that the expression of CD18 is also influenced by those of other AdMs, i.e. MAC-l (CDllb/ CD18) and ~150, 95 (CDllc/CD18) [13].

More importantly, we found that LFA-l-positive AML cases were less likely to enter CR. In addition, most of the LFA-l-positive AML patients who were resistant to the first induction chemotherapy could not achieve CR in spite of further induction chemotherapies. However, CR durations were not different between the AdM-positive and -negative patients. Thus, the resis- tance to induction chemotherapies was considered to reflect short survival periods observed in the AdM- positive patients. We further analyzed the correlations of LFA-1 expression with clinical outcomes in the patients who received almost identical regimens (BHAC-DM or BHAC-DMP) to exclude potential factors which influ- ence clinical outcomes according to different treatment protocols employed. The result confirmed that the prognostic values of the AdM expression still remained in the two patient groups.

Our study demonstrated that approximately one half of the AML cases had deficient expressions of LFA-1, although the AdM expression continues from very early stages of differentiation throughout further maturation in

normal myelomonocytic cell lineages [17]. We further disclosed significant positive correlations between the incidences of CDlla expression and those of CD34 and HLA-DR expressions on AML cells. These results suggest a different role of the LFA-1 molecule on AML cells from that on their normal counterparts and indicate immaturity of the AdM-positive AML cells [24-281.

In the present study, the percentages of CDlla- positive cells also positively correlated with those of HLA-DR-positive cells. A previous study reported that HLA-DR expressions correlated with functional imma- turity of AML cells in in vitro cell growth [29]. On the other hand, LFA-1 molecules have been suggested to play a role in AML cell adhesion to bone marrow fibroblasts [ll], and adhesion of AML cells to the stroma cells has been considered to support their growth and survival [ll, 12, 30,311. Thus, our present results concerning the LFA-1 expressions may be relevant to the stroma-dependent cell growth of AML cells, and suggest the possibility that AML cells closely binding to the stroma cells are more resistant to the treatments.

Finally, this study demonstrated frequent splenome- galy in the LFA-l-positive AML cases. A previous study of B-lymphoid malignancies (small lymphocytic lym- phoma and chronic lymphocytic leukemia) also sug- gested that LFA-1 expressions account for preferential tumor cell localization to the spleen [32]. These results may indicate an identical mechanism mediated by LFA- 1 in the splenic invasion of tumor cells.

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