Ann Hematol (1992) 64:78-82
Annals of
Hematology 9 Springer-Verlag 1992
Original article Phenotypic and clinical heterogeneity of CD56-positive acute nonlymphoblastic leukemia M.A. Reuss-Borst, B. Steinke, H.D. Waller, H.J. Biihring, and (2. A. Miiiler Medical University Clinic of Ttibingen, Second Department of Internal Medicine, Tt~bingen, Federal Republic of Germany Received August 19, 1991/Accepted December 18, 1991
Summary. The precise phenotype and clinical course are described of a subgroup of acute nonlymphoblastic leukemias (ANLL) expressing the NK-cell differentiation antigen CD56. As previously reported, C D 5 6 + leukemias occurred in a frequency of about 20% of A N L L cases showing clinical and immunophenotypical heterogeneity. Carrying various myelomonocytic markers, all cases were diagnosed to be of nonlymphoid origin. Positive or negative expression of CD34 allowed us to distinguish two major subtypes of C D 5 6 + leukemias representing immature and more differentiated cells carrying further differentiation antigens (CD14 and/or CD15) of the myelomonocytic lineages. These phenotypes correlated with the M0, M2, M4, and M5 leukemias of the FAB classification.
NK cells [5, 7]. CD56 has also been detected on small lung cell carcinomas as well as on the immature leukemia cell line KGla [ 7 - 9 ] . In 1983, Griffin et al. [4] reported that about 15% of acute nonlymphoblastic leukemias (ANLL) express CD56. However, aside from one case report on a C D 5 6 + acute monoblastic leukemia with phenotypical characteristics of immature monocytes [13], the lineage and differentiation specifity of these acute leukemias has so far not been analyzed in detail. In this study nine patients with CD56+ acute myeloid leukemia were investigated for their precise immunophenotype and individual hematological course to evaluate the clinical and lineage entity of this group of leukemias.
Key words: I m m u n o p h e n o t y p e - CD56 - Acute nonlymphoblastic leukemia
Material and methods Patients
Introduction During the Fourth International Conference on H u m a n Leukocyte Differentiation Antigens [6] the antigen CD56 was distinguished by six monoclonal antibodies (Leul9, N901, FP2-11.14, T-199, NKH1A, L185) and established to be identical with one (mol. wt. 140 kD) of the three isoforms of the neuronal cellular adhesion molecule (NCAM). This molecule, primarily expressed on normal and malignant neuroectodermal cells, is also referred to as a pan-natural-killer (NK) cell marker, since it is found on all cells mediating n o n - M H C - restricted cytotoxicity. In particular, in normal peripheral blood CD56 is expressed on a small subset of lymphocytes representing
Address for correspondence: C.A. MUller, Section for Transplan-
tation Immunology and Immunohematology, Second Department of Internal Medicine, Medical University Clinic, Otfried-MtillerStrasse 10, W-7400 Ttibingen, Federal Republic of Germany
Among 43 patients with acute nonlymphoblastic leukemia presenting at the University Medical Clinic, Dept. II, in Tt~bingen (Germany) between January 1989 and May 1991, nine patients (21%) were found to express the CD56 antigen on more than 20~ of their mononuclear cells in peripheral blood and bone marrow. Four patients were male and five were female, their ages ranging from 39 to 72 years (Table 1). Monoclonal antibodies (moabs)
For immunophenotyping the following monoclonal antibodies were used routinely: WT31 (a[3-T-cell-receptor), Leu19 (CD56), Leul2 (CD19), Leul5 (CDllb), LeuM9 (CD33), My10 (CD34) were purchased from Becton Dickinson (Sunnyvale, CA, USA); My7 (CD13) was derived from Coulter Immunology (Hialeah, FL, USA); OKTll (CD2), OKT3 (CD3) were purchased from Ortho Diagnostics (Raritan, N J, USA); and TA1061 (761-T-cell-receptor) was bought from T Cell Sciences (Cambridge, MA, USA). CD16 was derived from Dianova (Hamburg, FRG) and HNK-1 (CD57) was provided by the Fourth Workshop on Human Leukocyte Differentiation Antigens. TT1 (CD4), TT2 (CD8) [11], Tt~71 (CD5), Tt193 (CD7), 11C2 (CDllb), 46All (CD13), 6F3 (CD15), and TM1 (CD14) [3], as well as the HLA class-II antigen-specific antibody Tt~36 (anti-DR common) [15], were derived from our laboratory.
79 Table l. Clinical and hematological characteristics of the patients Patients 1
2
3
4
5
6
7
8
9
Clinical data Sex (m/f) Age (years) Skin infiltrates MDS 1 Remission2 Survival (months)
f 72 2
f 72 + _ + 6
m 39 _ + 6
m 67 + _+ 9
m 63
f 67
f 54
m 54
m 75
--
+
+
--
+
5
6
114
1
1
Peripheral blood WBC ( x 109/1) Blasts (%) Hb (g/100 ml) Platelets ( x 109/1)
28 72 10.2 129
39.4 49 9.2 39
19.4 60 7.9 29
129 88 10.6 21
34 20 9 189
26.7 72 10.1 195
4.6 7 6 105
10 78 10 90
10.8 22 4.8 98
Bone marrow Blasts (%) FAB subtype 3 Auer rods
75 M0 -
60 M4 -
72 M2 +
70 M2 -
58 M2
85 M4
50 M4
95 M5a
65 M4
] Myelodysplastic syndrome 2 Remission: -, none; _+, partial; +, complete 3 French-American-British classification 4 Still alive
Indirect immunofluorescence analysis Mononuclear cells obtained by Fieoll-hypaque density gradient centrifugation from heparinized peripheral blood and bone marrow aspirates were labeled by the indirect immunofluorescence technique with the use of moabs as first, and F(ab)2 fragments of fluoresceinisothiocyanate (FITC)-conjugated goat-anti-mouse immunoglobulins (Dianova, Hamburg, FRG) as second antibody layer. Double staining experiments were performed with a mixture of the specific moabs as first, and a mixture of FITC and phycoerythrin (PE)-conjugated goat-anti-mouse immunoglobulin isotype specific antibodies (Southern Biotechnology, Birmingham, USA) as second antibody layer. Fluorescence was evaluated on a FACS IV cell sorter (Becton Dickinson, San Jose, CA, USA), as previously described [10]. Dual scatter gates (forward x 90 ~ scatter) were set to electronically select subpopulations, of which the fluorescence distribution was analyzed. The green fluorescence of the FITC-labeled cells was measured through a 530-nm band pass filter and the yellow fluorescence of PE-stained compounds through a 570-nm band pass filter. After proper compensation with control beads, the fluorescence intensities (log scale, 4 decades) and the scatter signals (linear scale) were analyzed by a data lister and evaluated on an IBM-AT using inhouse programs.
Results
Clinical presentation and hematological course T h r e e (nos. 2, 3, a n d 5) o f nine p a t i e n t s evaluated presented at the h e m a t o l o g y d e p a r t m e n t with unspecific s y m p t o m s o f fever, night sweat, a n d weakness. Patients 2 a n d 5 showed leukemic infiltrates o f the skin at the t i m e o f first clinical a d m i s s i o n (Table 1). F o u r o t h e r p a t i e n t s (nos. 4, 6, 7, a n d 9) were p r i m a r i l y d i a g n o s e d as suffering
f r o m a m y e l o d y s p l a s t i c s y n d r o m e ( M D S ) a n d developed acute l e u k e m i a a few m o n t h s later. O n e p a t i e n t (no. 1) was f o u n d to have a leukocytosis with myeloblasts in the differential c o u n t o f p e r i p h e r a l b l o o d cells on a r o u t i n e check u p b u t h a d no clinical s y m p t o m s . I n a n o t h e r p a tient (no. 8) acute m y e l o i d l e u k e m i a was f o u n d to cause a c h l o r o m a with s u b s e q u e n t p a r a p l e g i a . Petechiae a n d h e m a t o m a s due to t h r o m b o c y t o p e n i a were n o t i c e d in three p a t i e n t s (nos. 2, 3, a n d 4). In all except one p a t i e n t (no. 7) p e r i p h e r a l leukocytosis with blast cell counts between 7~ a n d 88O7o was observed. I n four cases (nos. 2, 6, 7, a n d 9) b o n e m a r r o w smears stained with May-Grt~nwald-Giemsa revealed p o l y m o r p h i c m o n o c y t o i d blasts beside t y p i c a l myeloblasts. Since on c y t o c h e m i c a l stains m y e l o p e r o x i d a s e ( M P O ) a n d ~ - n a p h thyl-acetate-esterase ( a - N A E ) were positive, these cases were d i a g n o s e d as M 4 leukemias, a c c o r d i n g to the FAB classification. I n three p a t i e n t s (nos. 3, 4, a n d 5) b o n e m a r r o w aspirates revealed p o l y m o r p h i c m y e l o i d blast p o p u l a t i o n s with granules, A u e r rods (no. 3), a n d an increased n u m b e r o f promyelocytes. A c c o r d i n g to their m o r p h o l o g y a n d c y t o c h e m i c a l staining, these cases were classified as M 2 leukemias. Case 8, d i a g n o s e d as suffering f r o m M 5 a leukemia, showed large M P O - n e g a t i v e , b u t a - N A E strongly positive blasts with slightly basophilic, wide c y t o p l a s m as the d o m i n a n t cell p o p u l a t i o n . I n p a t i e n t 1, 75~ o f the cells on the b o n e m a r r o w s m e a r represented large u n d i f f e r e n t i a t e d blast cells. Cytoc h e m i c a l stainings p r o v i d e d n o f u r t h e r characteristics o f the l e u k e m i a s w h i c h was therefore d e f i n e d as A M L - M O
80 Table 2. Immunophenotype of peripheral blood or bone marrow cells (% positive cells) Patients 1 BM
2 BM
3 PB
4 BM
5 BM
6 PB
7 BM
8 BM
9 PB
27 9 6 4 3 1 60 2 0
1 1 1 1 1 0 14 0 1
8 9 9 9 9 0 6 4 75
9 6 3 3 3 1 39 1 0
1 7 1 1 1 0 9 0 0
6 7 6 6 6 0 82 6 5
12 15 11 11 10 3 65 7 1
3 4 4 3 3 0 91 2 11
5 0 0 0 1 0 52 0 0
89 91 5 4 2
92 37 42 18 40
1 61 3 1 8
81 85 73 15 52
53 42 47 29 15
88 74 82 80 63
74 65 74 50 66
44 2 9 0 69
65 90 45 44 31
91 96 55 10 nt
0 36 60 80 3
82 88 85 5 5
16 31 67 26 8
19 80 46 58 4
2 92 71 66 2
9 83 65 68 7
0 46 67 nt nt
41 64 68 39 2
Surface antigens:
Lymphoid CD 7 CD2 CD5 CD3 TCR ct~ TCR 3,~5 CD 4 CD 8 CD19
Myeloid/monocytoid CD 33 CD 13 CD 1lb CD 14 CD 15
Others CD 34 HLA-DR CD 56 CD 16 CD 57
BM, Bone marrow; PB, peripheral blood; nt, not tested
[2]. S u d a n b l a c k staining a n d u l t r a s t r u c t u r a l cytochemist r y as further d i a g n o s t i c tests were n o t p e r f o r m e d . Six patients o l d e r t h a n 60 years received a c o m b i n a t i o n t h e r a p y with d a u n o r u b i c i n , vincristine, a n d A R A - C . A l l o t h e r patients were t r e a t e d with A R A - C , d a u n o r u b i cin, a n d etoposide. I n three cases (nos. 1, 8, a n d 9) no h e m a t o l o g i c a l response was observed. Two o t h e r patients (nos. 2, a n d 3) h a d a s h o r t c o m p l e t e remission b u t t h e n d i e d o f relapse (no. 2) a n d f u n g a l sepsis after c o n s o l i d a tion c h e m o t h e r a p y (no. 3). A l l o t h e r p a t i e n t s showed p a r t i a l remissions with a r e d u c t i o n o f the blast cell c o u n t in the b o n e marrow. O n l y one p a t i e n t w h o s e disease developed f r o m a m y e l o d y s p l a s t i c s y n d r o m e has now survived 11 m o n t h s f r o m diagnosis; all o t h e r p a t i e n t s died within 1 - 9 m o n t h s after diagnosis.
Immunophenotype A c c o r d i n g to i m m u n o p h e n o t y p i n g , nine o f 43 A M L p a t i e n t s expressed CD56 o n m o r e t h a n 2 0 % o f Ficolls e p a r a t e d b o n e m a r r o w a n d / o r p e r i p h e r a l b l o o d cells. I n six cases, b o n e m a r r o w aspirates a n d p e r i p h e r a l b l o o d s a m p l e s were investigated simultaneously. Since the leukemic cells d i d n o t c a r r y the l y m p h o i d a s s o c i a t e d antigens CD19 a n d CD7, or o t h e r T-cell m a r kers like CD3, CD8, CD2, in eight cases the leukemias were n o n l y m p h o i d (Table 2). Remarkably, in p a t i e n t 3 m o s t C D 3 4 + leukemic cells stained for the CD56, a n d in
Table 3. Double labeling of CD34- and CD56-positive cells (% positive cells) Patient no.
CD34+ CD56-
CD34- CD56+
1
50
6
2 3 4 5 6
1 4 48 19 0
63 8 35 20 65
CD34+ CD56+ 40 1 81 3 2 0
7
3
38
0
8 9
nt 29
nt 35
nt 14
nt, not tested
d o u b l e labelings m o s t l y also for the CD13 antigen. In addition, m o r e t h a n 50% o f these blasts were also f o u n d to co-express CD19 (Fig. 1). I n p a t i e n t 1 the C D 5 6 + blast cells co-expressed the CD33 a n d CD13 antigens as a sign o f b e l o n g i n g to the m y e l o i d lineage. I n d o u b l e stainings these blasts were likely to overlap with a p r o p o r t i o n o f the C D 3 4 + cells (50%) which p a r t l y expressed the T-cell antigen CD7 (25%), this is k n o w n to occur in 10% o f acute m y e l o i d leukemias [12]. Five p a t i e n t s (nos. 1, 3, 4, 5, a n d 9) showed an a b n o r m a l n u m b e r o f C D 3 4 + blasts in the b l o o d or b o n e m a r row. D o u b l e labeling experiments revealed t h a t variable
81
A
B
i
9 I
I-4
2
I
I
i
i
i
64
~4
-
IgGl
~
128
1~
CD56
FITC
1~
224
-
FITC
D
C
c~ H
?,
%O
f
I
$
i.cl
M
6.t
96
i
l
128
16~
IgG1
"l
i
192
-
'~
224
FITC
64
~6
T28
16e
CD56
l~e
-
;24
FITC
Fig. 1 A-D. Double labeling contour display of cells from patient 3 stained with anti-CD56 (x-axis) and anti-CD34 (y-axis) or anti-CD19 (y-axis) moabs: A and C negative controls with FITC- and PE-conjugated isotype-identical second antibodies; B > 80% cells co-express CD56 and CD34; D > 60~ cells carry the CD19 and CD56 antigen
percentages (2~ of the CD56+ blasts carried the CD34 antigen (Table 3). In four patients (nos. 2, 6, 7, and 9) CD56+ blasts were shown to express the monocytic differentiation marker CD14 in addition. In all cases excepting patients 1 and 3 a portion of the CD56+ blasts also stained for the differentiation antigen CD15 of mature myeloid cells. In patient 6 double labelings revealed the co-expression of CD14 and CD15 on the CD56+ cells. In contrast, CD15 was not found on CD34+ cells by double stainings. Seven of the nine patients expressed CD4 on most CD56+ blasts. In all patients the CD56+ blasts also carried HLA-DR, but only to a variable extent HLA-DQ or -DP antigens. CD57, present on about 60% of NK cells, was not found on the CD56+ blasts of the cases analyzed. CD16 as a late myeloid and NK-cell differentiation antigen was expressed on more than 20% of cells in all cases, excepting patients 1 and 3.
Discussion
According to the previous report of Griffin et al. [41, nine of 59 patients (16O7o)with A N L L and three of 15 patients (20~ with chronic myeloid leukemia expressed the CD56 antigen on their leukemic blast cells. In this study, the CD56 + leukemias were observed with a similar frequency of 21% in an equally large group of patients with ANLL. Although the expression of CD56 on leukemic cells thus appears to be a fairly common event, there is not much known about the precise phenotype and clinical course of these patients. In a case report by Tauchi et al. [13], a CD56+ leukemia was classified as M5a-AML with an immunophenotype o f the monocytic cell lineage, based on the expression of CD4 and CD36 and the lack of T-cell receptor rearrangement. Among our nine CD56+ patients, one (no. 8) suffering from an M5aAML showed a CD14-, CD4 + immunophenotype which
82 slightly differed from that described by Tauchi et al. [13] due to surface staining for CD33, thus indicating a myelomonocytic cell type. Although all other CD56+ leukemias carried myelomonocytic cell surface markers, they did not constitute an immunophenotypically unique subgroup of A N L L . At least two major subtypes of CD56+ blasts were distinguished, according to the expression of the early hematopoietic surface antigen CD34. In the C D 3 4 + as well as the C D 3 4 - group the CD56+ leukemias further appeared to differ according to the expression of the CD14 and/or CD15 antigens on variable percentages of CD56 + blasts, indicating maturation in the myelomonocytic lineages. CD15 was not expressed on C D 3 4 + cells (patient 9), but it was repeatedly found on the C D 5 6 + cell population. Double labelings revealed that subpopulations of C D 5 6 + blasts may carry either CD14 or CD15 (patient 2), or that CD14 and CD15 might be expressed simultaneously on CD56+ blasts (patient 6). In seven of nine cases the C D 5 6 + cells carried the surface marker CD4, suggesting a differentiation toward the monocytic lineage. Co-expression of the T-cell differentiation antigens CD7 and CD4 was observed in patient 1 with an undifferentiated form of acute myeloid leukemia [2], where the co-expression o f CD7 may even be higher than 10070 [1,121. Another case (no. 3) showed an unusual reproducible staining with different moabs against the CD19 antigen. In both patients (nos. 1 and 3) the C D 5 6 + blast cells may be related to a very immature differentiation stage of hematopoietic precursors. Interestingly, CD16 was not expressed in these two cases. The different immunophenotypes of the CD56 + leukemias did not correlate with a specific FAB-classified leukemic subgroup, since they were associated with M0, M2, M4, or M5. In contrast to a previous short communication [14], CD56+ leukemias further did not appear to reveal a higher complete response rate to chemotherapy in comparison with other A M L patients. On the contrary, one might even suspect an even worse prognosis for C D 5 6 + acute leukemias, although the small number of patients analyzed in this study and the bias toward older patients do not allow us to make definite statements on prognosis. So far, the C D 5 6 + leukemias might represent rare precursors of NK cells which, although present in the normal bone marrow, are difficult to detect by routine analysis. Since N C A M appears to be involved in the intercellular contact mechanism, expression of CD56 could also be related to specific activation stages of myelomonocytic cells during differentiation. Further experimental analysis has to establish functional significance of this molecule on a subgroup of A N L L .
Acknowledgement. We thank P. Neumann for her excellent technical assistance.
References 1. Bennett JM, Catovsky D, Daniel M-T, Flandrin G, Galton DAG, Gralnick HR, Sultan C (1991) Proposal for the recognition of minimally differentiated acute myeloid leukaemia (AML-MO). Br J Haematol 78:325-329 2. Catovsky D, Matutes E, Buccheri V, Shetty V, Hanslip J, Yoshida N, MoriUa R (1991) A classification of acute leukaemia for the 1990s. Ann Hematol 62:16-21 3. Gadd S (1989) Cluster report: CD14. In: Knapp W (ed) Leukocyte typing IV. Oxford University Press, Oxford, pp 787798 4. Griffin JD, Hercend T, Beveridge R, Schlossman SF (1983) Characterization of an antigen expressed by human natural killer cells. J Immunol 130:2947-2951 5. Hercend T, Griffin JD, Bensussan A, Schmidt RE, Edson MA, Brennan A, Murray C, Daley JF, Schlossman SF, Ritz J (1985) Generation of monoclonal antibodies to a human natural killer clone. J Clin Invest 75:932-943 6. Knapp W (1989) International workshop and conference on human leukocyte differentiation antigens. Oxford University Press, Oxford 7. Lanier LL, Le AM, Civin CI, Loken MR, Phillips JH (1986) The relationship of CD16 (Leu-11) and Leu-19 (NKH-1) antigen expression on human peripheral blood NK cells and cytotoxic T-lymphocytes. J Immunol 136:4480-4486 8. Lanier LL, Le AM, Ding A, Evans EL, Krensky AM, Clayberger C, Phillips JH (1987) Expression of Leu-19 (NKH-1) antigen on IL 2-dependent cytotoxic and non-cytotoxic T cell lines. J Immunol 138:2019-2023 9. Lanier LL, Testi R, Bindl J, Phillips JH (1989) Identitiy of Leu-19 (CD56) leukocyte differentiation antigen and neural cell adhesion molecule. J Exp Med 169:2233-2238 10. Mt~ller C, Ziegler A, Muller C, Hadam M, Waller HD, Wernet P, Mt~ller G (1985) Divergent expression of HLA-DC/MB, -DR, and -SB region products on normal and pathological tissues as detected by monoclonal antibodies. Immunobiology 169:228-249 11. Mfiller GA, Miiller C, Risler T (1984) Charakterisierung glomerul/~rer Halbmonde bei rapid progressiver Glomerulonephritis (RPGN) mit Hilfe monoklonaler Antik6rper. Verh Dtsch Ges Inn Med 90:1013-1016 12. Norton JD, Campana D, Hoffbrand AV, Janossy G, CoustanSmith E, Jani H, Yaxley JC, Prentice HG (1988) Rearrangement of immunoglobulin and T-cell antigen receptor genes in acute myeloid leukemia with lymphoid-associated markers. Leukemia 1:757-761 13. Tauchi T, Ohyashiki K, Ohyashiki JH, Kawanishi 5(, Kimura Y, Saito M, Nakazawa S, Kawai Y, Toyama K (1990) CD4+ and CD56+ acute monoblastic leukemia. Am J Hematol 34: 228 14. Willman CL, Stewart CC, Elias L, Potter J, Kopecky K, StockNovak D, Head D, Weick JK, Grever MR (1988) Identification of new biological subtypes of acute leukemia co-expressing NK (LEU19) and myeloid cell surface antigens: a Southwest Oncology Group (SWOG) study. Blood 77 [Suppl 1]: 236 (abstract) 15. Ziegler A, Heinig J, Mtiller C, Gitz H, Thinnes FP, UchanskaZiegler B, Wernet P (1986) Analysis by sequential immunoprecipitations of the monoelonal antibodies Tt~22, 34, 36, 37, 39, 43, 58 and YD 1/63. HLK directed against human HLA class-II-antigens. Immunobiology 171:77-92
Annals of
Hematology 9 Springer-Verlag 1992
Original article Phenotypic and clinical heterogeneity of CD56-positive acute nonlymphoblastic leukemia M.A. Reuss-Borst, B. Steinke, H.D. Waller, H.J. Biihring, and (2. A. Miiiler Medical University Clinic of Ttibingen, Second Department of Internal Medicine, Tt~bingen, Federal Republic of Germany Received August 19, 1991/Accepted December 18, 1991
Summary. The precise phenotype and clinical course are described of a subgroup of acute nonlymphoblastic leukemias (ANLL) expressing the NK-cell differentiation antigen CD56. As previously reported, C D 5 6 + leukemias occurred in a frequency of about 20% of A N L L cases showing clinical and immunophenotypical heterogeneity. Carrying various myelomonocytic markers, all cases were diagnosed to be of nonlymphoid origin. Positive or negative expression of CD34 allowed us to distinguish two major subtypes of C D 5 6 + leukemias representing immature and more differentiated cells carrying further differentiation antigens (CD14 and/or CD15) of the myelomonocytic lineages. These phenotypes correlated with the M0, M2, M4, and M5 leukemias of the FAB classification.
NK cells [5, 7]. CD56 has also been detected on small lung cell carcinomas as well as on the immature leukemia cell line KGla [ 7 - 9 ] . In 1983, Griffin et al. [4] reported that about 15% of acute nonlymphoblastic leukemias (ANLL) express CD56. However, aside from one case report on a C D 5 6 + acute monoblastic leukemia with phenotypical characteristics of immature monocytes [13], the lineage and differentiation specifity of these acute leukemias has so far not been analyzed in detail. In this study nine patients with CD56+ acute myeloid leukemia were investigated for their precise immunophenotype and individual hematological course to evaluate the clinical and lineage entity of this group of leukemias.
Key words: I m m u n o p h e n o t y p e - CD56 - Acute nonlymphoblastic leukemia
Material and methods Patients
Introduction During the Fourth International Conference on H u m a n Leukocyte Differentiation Antigens [6] the antigen CD56 was distinguished by six monoclonal antibodies (Leul9, N901, FP2-11.14, T-199, NKH1A, L185) and established to be identical with one (mol. wt. 140 kD) of the three isoforms of the neuronal cellular adhesion molecule (NCAM). This molecule, primarily expressed on normal and malignant neuroectodermal cells, is also referred to as a pan-natural-killer (NK) cell marker, since it is found on all cells mediating n o n - M H C - restricted cytotoxicity. In particular, in normal peripheral blood CD56 is expressed on a small subset of lymphocytes representing
Address for correspondence: C.A. MUller, Section for Transplan-
tation Immunology and Immunohematology, Second Department of Internal Medicine, Medical University Clinic, Otfried-MtillerStrasse 10, W-7400 Ttibingen, Federal Republic of Germany
Among 43 patients with acute nonlymphoblastic leukemia presenting at the University Medical Clinic, Dept. II, in Tt~bingen (Germany) between January 1989 and May 1991, nine patients (21%) were found to express the CD56 antigen on more than 20~ of their mononuclear cells in peripheral blood and bone marrow. Four patients were male and five were female, their ages ranging from 39 to 72 years (Table 1). Monoclonal antibodies (moabs)
For immunophenotyping the following monoclonal antibodies were used routinely: WT31 (a[3-T-cell-receptor), Leu19 (CD56), Leul2 (CD19), Leul5 (CDllb), LeuM9 (CD33), My10 (CD34) were purchased from Becton Dickinson (Sunnyvale, CA, USA); My7 (CD13) was derived from Coulter Immunology (Hialeah, FL, USA); OKTll (CD2), OKT3 (CD3) were purchased from Ortho Diagnostics (Raritan, N J, USA); and TA1061 (761-T-cell-receptor) was bought from T Cell Sciences (Cambridge, MA, USA). CD16 was derived from Dianova (Hamburg, FRG) and HNK-1 (CD57) was provided by the Fourth Workshop on Human Leukocyte Differentiation Antigens. TT1 (CD4), TT2 (CD8) [11], Tt~71 (CD5), Tt193 (CD7), 11C2 (CDllb), 46All (CD13), 6F3 (CD15), and TM1 (CD14) [3], as well as the HLA class-II antigen-specific antibody Tt~36 (anti-DR common) [15], were derived from our laboratory.
79 Table l. Clinical and hematological characteristics of the patients Patients 1
2
3
4
5
6
7
8
9
Clinical data Sex (m/f) Age (years) Skin infiltrates MDS 1 Remission2 Survival (months)
f 72 2
f 72 + _ + 6
m 39 _ + 6
m 67 + _+ 9
m 63
f 67
f 54
m 54
m 75
--
+
+
--
+
5
6
114
1
1
Peripheral blood WBC ( x 109/1) Blasts (%) Hb (g/100 ml) Platelets ( x 109/1)
28 72 10.2 129
39.4 49 9.2 39
19.4 60 7.9 29
129 88 10.6 21
34 20 9 189
26.7 72 10.1 195
4.6 7 6 105
10 78 10 90
10.8 22 4.8 98
Bone marrow Blasts (%) FAB subtype 3 Auer rods
75 M0 -
60 M4 -
72 M2 +
70 M2 -
58 M2
85 M4
50 M4
95 M5a
65 M4
] Myelodysplastic syndrome 2 Remission: -, none; _+, partial; +, complete 3 French-American-British classification 4 Still alive
Indirect immunofluorescence analysis Mononuclear cells obtained by Fieoll-hypaque density gradient centrifugation from heparinized peripheral blood and bone marrow aspirates were labeled by the indirect immunofluorescence technique with the use of moabs as first, and F(ab)2 fragments of fluoresceinisothiocyanate (FITC)-conjugated goat-anti-mouse immunoglobulins (Dianova, Hamburg, FRG) as second antibody layer. Double staining experiments were performed with a mixture of the specific moabs as first, and a mixture of FITC and phycoerythrin (PE)-conjugated goat-anti-mouse immunoglobulin isotype specific antibodies (Southern Biotechnology, Birmingham, USA) as second antibody layer. Fluorescence was evaluated on a FACS IV cell sorter (Becton Dickinson, San Jose, CA, USA), as previously described [10]. Dual scatter gates (forward x 90 ~ scatter) were set to electronically select subpopulations, of which the fluorescence distribution was analyzed. The green fluorescence of the FITC-labeled cells was measured through a 530-nm band pass filter and the yellow fluorescence of PE-stained compounds through a 570-nm band pass filter. After proper compensation with control beads, the fluorescence intensities (log scale, 4 decades) and the scatter signals (linear scale) were analyzed by a data lister and evaluated on an IBM-AT using inhouse programs.
Results
Clinical presentation and hematological course T h r e e (nos. 2, 3, a n d 5) o f nine p a t i e n t s evaluated presented at the h e m a t o l o g y d e p a r t m e n t with unspecific s y m p t o m s o f fever, night sweat, a n d weakness. Patients 2 a n d 5 showed leukemic infiltrates o f the skin at the t i m e o f first clinical a d m i s s i o n (Table 1). F o u r o t h e r p a t i e n t s (nos. 4, 6, 7, a n d 9) were p r i m a r i l y d i a g n o s e d as suffering
f r o m a m y e l o d y s p l a s t i c s y n d r o m e ( M D S ) a n d developed acute l e u k e m i a a few m o n t h s later. O n e p a t i e n t (no. 1) was f o u n d to have a leukocytosis with myeloblasts in the differential c o u n t o f p e r i p h e r a l b l o o d cells on a r o u t i n e check u p b u t h a d no clinical s y m p t o m s . I n a n o t h e r p a tient (no. 8) acute m y e l o i d l e u k e m i a was f o u n d to cause a c h l o r o m a with s u b s e q u e n t p a r a p l e g i a . Petechiae a n d h e m a t o m a s due to t h r o m b o c y t o p e n i a were n o t i c e d in three p a t i e n t s (nos. 2, 3, a n d 4). In all except one p a t i e n t (no. 7) p e r i p h e r a l leukocytosis with blast cell counts between 7~ a n d 88O7o was observed. I n four cases (nos. 2, 6, 7, a n d 9) b o n e m a r r o w smears stained with May-Grt~nwald-Giemsa revealed p o l y m o r p h i c m o n o c y t o i d blasts beside t y p i c a l myeloblasts. Since on c y t o c h e m i c a l stains m y e l o p e r o x i d a s e ( M P O ) a n d ~ - n a p h thyl-acetate-esterase ( a - N A E ) were positive, these cases were d i a g n o s e d as M 4 leukemias, a c c o r d i n g to the FAB classification. I n three p a t i e n t s (nos. 3, 4, a n d 5) b o n e m a r r o w aspirates revealed p o l y m o r p h i c m y e l o i d blast p o p u l a t i o n s with granules, A u e r rods (no. 3), a n d an increased n u m b e r o f promyelocytes. A c c o r d i n g to their m o r p h o l o g y a n d c y t o c h e m i c a l staining, these cases were classified as M 2 leukemias. Case 8, d i a g n o s e d as suffering f r o m M 5 a leukemia, showed large M P O - n e g a t i v e , b u t a - N A E strongly positive blasts with slightly basophilic, wide c y t o p l a s m as the d o m i n a n t cell p o p u l a t i o n . I n p a t i e n t 1, 75~ o f the cells on the b o n e m a r r o w s m e a r represented large u n d i f f e r e n t i a t e d blast cells. Cytoc h e m i c a l stainings p r o v i d e d n o f u r t h e r characteristics o f the l e u k e m i a s w h i c h was therefore d e f i n e d as A M L - M O
80 Table 2. Immunophenotype of peripheral blood or bone marrow cells (% positive cells) Patients 1 BM
2 BM
3 PB
4 BM
5 BM
6 PB
7 BM
8 BM
9 PB
27 9 6 4 3 1 60 2 0
1 1 1 1 1 0 14 0 1
8 9 9 9 9 0 6 4 75
9 6 3 3 3 1 39 1 0
1 7 1 1 1 0 9 0 0
6 7 6 6 6 0 82 6 5
12 15 11 11 10 3 65 7 1
3 4 4 3 3 0 91 2 11
5 0 0 0 1 0 52 0 0
89 91 5 4 2
92 37 42 18 40
1 61 3 1 8
81 85 73 15 52
53 42 47 29 15
88 74 82 80 63
74 65 74 50 66
44 2 9 0 69
65 90 45 44 31
91 96 55 10 nt
0 36 60 80 3
82 88 85 5 5
16 31 67 26 8
19 80 46 58 4
2 92 71 66 2
9 83 65 68 7
0 46 67 nt nt
41 64 68 39 2
Surface antigens:
Lymphoid CD 7 CD2 CD5 CD3 TCR ct~ TCR 3,~5 CD 4 CD 8 CD19
Myeloid/monocytoid CD 33 CD 13 CD 1lb CD 14 CD 15
Others CD 34 HLA-DR CD 56 CD 16 CD 57
BM, Bone marrow; PB, peripheral blood; nt, not tested
[2]. S u d a n b l a c k staining a n d u l t r a s t r u c t u r a l cytochemist r y as further d i a g n o s t i c tests were n o t p e r f o r m e d . Six patients o l d e r t h a n 60 years received a c o m b i n a t i o n t h e r a p y with d a u n o r u b i c i n , vincristine, a n d A R A - C . A l l o t h e r patients were t r e a t e d with A R A - C , d a u n o r u b i cin, a n d etoposide. I n three cases (nos. 1, 8, a n d 9) no h e m a t o l o g i c a l response was observed. Two o t h e r patients (nos. 2, a n d 3) h a d a s h o r t c o m p l e t e remission b u t t h e n d i e d o f relapse (no. 2) a n d f u n g a l sepsis after c o n s o l i d a tion c h e m o t h e r a p y (no. 3). A l l o t h e r p a t i e n t s showed p a r t i a l remissions with a r e d u c t i o n o f the blast cell c o u n t in the b o n e marrow. O n l y one p a t i e n t w h o s e disease developed f r o m a m y e l o d y s p l a s t i c s y n d r o m e has now survived 11 m o n t h s f r o m diagnosis; all o t h e r p a t i e n t s died within 1 - 9 m o n t h s after diagnosis.
Immunophenotype A c c o r d i n g to i m m u n o p h e n o t y p i n g , nine o f 43 A M L p a t i e n t s expressed CD56 o n m o r e t h a n 2 0 % o f Ficolls e p a r a t e d b o n e m a r r o w a n d / o r p e r i p h e r a l b l o o d cells. I n six cases, b o n e m a r r o w aspirates a n d p e r i p h e r a l b l o o d s a m p l e s were investigated simultaneously. Since the leukemic cells d i d n o t c a r r y the l y m p h o i d a s s o c i a t e d antigens CD19 a n d CD7, or o t h e r T-cell m a r kers like CD3, CD8, CD2, in eight cases the leukemias were n o n l y m p h o i d (Table 2). Remarkably, in p a t i e n t 3 m o s t C D 3 4 + leukemic cells stained for the CD56, a n d in
Table 3. Double labeling of CD34- and CD56-positive cells (% positive cells) Patient no.
CD34+ CD56-
CD34- CD56+
1
50
6
2 3 4 5 6
1 4 48 19 0
63 8 35 20 65
CD34+ CD56+ 40 1 81 3 2 0
7
3
38
0
8 9
nt 29
nt 35
nt 14
nt, not tested
d o u b l e labelings m o s t l y also for the CD13 antigen. In addition, m o r e t h a n 50% o f these blasts were also f o u n d to co-express CD19 (Fig. 1). I n p a t i e n t 1 the C D 5 6 + blast cells co-expressed the CD33 a n d CD13 antigens as a sign o f b e l o n g i n g to the m y e l o i d lineage. I n d o u b l e stainings these blasts were likely to overlap with a p r o p o r t i o n o f the C D 3 4 + cells (50%) which p a r t l y expressed the T-cell antigen CD7 (25%), this is k n o w n to occur in 10% o f acute m y e l o i d leukemias [12]. Five p a t i e n t s (nos. 1, 3, 4, 5, a n d 9) showed an a b n o r m a l n u m b e r o f C D 3 4 + blasts in the b l o o d or b o n e m a r row. D o u b l e labeling experiments revealed t h a t variable
81
A
B
i
9 I
I-4
2
I
I
i
i
i
64
~4
-
IgGl
~
128
1~
CD56
FITC
1~
224
-
FITC
D
C
c~ H
?,
%O
f
I
$
i.cl
M
6.t
96
i
l
128
16~
IgG1
"l
i
192
-
'~
224
FITC
64
~6
T28
16e
CD56
l~e
-
;24
FITC
Fig. 1 A-D. Double labeling contour display of cells from patient 3 stained with anti-CD56 (x-axis) and anti-CD34 (y-axis) or anti-CD19 (y-axis) moabs: A and C negative controls with FITC- and PE-conjugated isotype-identical second antibodies; B > 80% cells co-express CD56 and CD34; D > 60~ cells carry the CD19 and CD56 antigen
percentages (2~ of the CD56+ blasts carried the CD34 antigen (Table 3). In four patients (nos. 2, 6, 7, and 9) CD56+ blasts were shown to express the monocytic differentiation marker CD14 in addition. In all cases excepting patients 1 and 3 a portion of the CD56+ blasts also stained for the differentiation antigen CD15 of mature myeloid cells. In patient 6 double labelings revealed the co-expression of CD14 and CD15 on the CD56+ cells. In contrast, CD15 was not found on CD34+ cells by double stainings. Seven of the nine patients expressed CD4 on most CD56+ blasts. In all patients the CD56+ blasts also carried HLA-DR, but only to a variable extent HLA-DQ or -DP antigens. CD57, present on about 60% of NK cells, was not found on the CD56+ blasts of the cases analyzed. CD16 as a late myeloid and NK-cell differentiation antigen was expressed on more than 20% of cells in all cases, excepting patients 1 and 3.
Discussion
According to the previous report of Griffin et al. [41, nine of 59 patients (16O7o)with A N L L and three of 15 patients (20~ with chronic myeloid leukemia expressed the CD56 antigen on their leukemic blast cells. In this study, the CD56 + leukemias were observed with a similar frequency of 21% in an equally large group of patients with ANLL. Although the expression of CD56 on leukemic cells thus appears to be a fairly common event, there is not much known about the precise phenotype and clinical course of these patients. In a case report by Tauchi et al. [13], a CD56+ leukemia was classified as M5a-AML with an immunophenotype o f the monocytic cell lineage, based on the expression of CD4 and CD36 and the lack of T-cell receptor rearrangement. Among our nine CD56+ patients, one (no. 8) suffering from an M5aAML showed a CD14-, CD4 + immunophenotype which
82 slightly differed from that described by Tauchi et al. [13] due to surface staining for CD33, thus indicating a myelomonocytic cell type. Although all other CD56+ leukemias carried myelomonocytic cell surface markers, they did not constitute an immunophenotypically unique subgroup of A N L L . At least two major subtypes of CD56+ blasts were distinguished, according to the expression of the early hematopoietic surface antigen CD34. In the C D 3 4 + as well as the C D 3 4 - group the CD56+ leukemias further appeared to differ according to the expression of the CD14 and/or CD15 antigens on variable percentages of CD56 + blasts, indicating maturation in the myelomonocytic lineages. CD15 was not expressed on C D 3 4 + cells (patient 9), but it was repeatedly found on the C D 5 6 + cell population. Double labelings revealed that subpopulations of C D 5 6 + blasts may carry either CD14 or CD15 (patient 2), or that CD14 and CD15 might be expressed simultaneously on CD56+ blasts (patient 6). In seven of nine cases the C D 5 6 + cells carried the surface marker CD4, suggesting a differentiation toward the monocytic lineage. Co-expression of the T-cell differentiation antigens CD7 and CD4 was observed in patient 1 with an undifferentiated form of acute myeloid leukemia [2], where the co-expression o f CD7 may even be higher than 10070 [1,121. Another case (no. 3) showed an unusual reproducible staining with different moabs against the CD19 antigen. In both patients (nos. 1 and 3) the C D 5 6 + blast cells may be related to a very immature differentiation stage of hematopoietic precursors. Interestingly, CD16 was not expressed in these two cases. The different immunophenotypes of the CD56 + leukemias did not correlate with a specific FAB-classified leukemic subgroup, since they were associated with M0, M2, M4, or M5. In contrast to a previous short communication [14], CD56+ leukemias further did not appear to reveal a higher complete response rate to chemotherapy in comparison with other A M L patients. On the contrary, one might even suspect an even worse prognosis for C D 5 6 + acute leukemias, although the small number of patients analyzed in this study and the bias toward older patients do not allow us to make definite statements on prognosis. So far, the C D 5 6 + leukemias might represent rare precursors of NK cells which, although present in the normal bone marrow, are difficult to detect by routine analysis. Since N C A M appears to be involved in the intercellular contact mechanism, expression of CD56 could also be related to specific activation stages of myelomonocytic cells during differentiation. Further experimental analysis has to establish functional significance of this molecule on a subgroup of A N L L .
Acknowledgement. We thank P. Neumann for her excellent technical assistance.
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