Cytogenetic Analysis of Different Cellular
Populations in Chronic
Myelomonocytic Leukemia
Antonio Cuneo, Paolo Tomasi, Luisa Ferrari, Massimo
Balboni, Nadia Piva, Franca Fagioli,
and Gianluigi Castoldi
ABSTRACT: Kuwotypes of ditllerent cellulur populati(ms made ufter s e p u r a / i o n of b o n e m t l r r o a cells
o n a g r a d i e n t of P e r c o l l w e r e evuluutod in seven p a t i e n t s a)fe(:ted by c h r o n i c myelomanocytie
leukemia diagnosed occarding to FAB crilerio. M c g a k a r 3 o c y / c s , mnnocytic cells, und gr(nm-
lacytic and erythroid precursors were prefere, utiully collected a f t e r cctllrifugalion betweee
density l a y e r s of 1045 1050 mo/ml. 1050 I060 mg/ml, ul~d 1065 1070 mg/ml, respectively.
The enriched cell fl'uctiuns were cultured separutely ulld submitted lo crtogenelic im~estigu-
l i o n a f t e r sbart-term culture. Some chramosomc u b e n ' a l i o l ~ s (5(I ,+8) were observed h~ idl
cclluh," f r a c t i o n s in three p a t i e n t s , thus providing cytogenctic e~ idence af the invnh,cmenl ot
u common progenitor s t e m cell in this myelodysplustic disorder. On the other hun& chro-
m o s o m e abnormalities such as del(3)lq21j uml del(ll)(q23) ( i p p e a r e d to be clm(illcd to the
megakuryacytic and tim monocytic fractions, rCsl)ectix'ely, in two putients. It is coneeir(ll~lc
thut lineugc-restricled uberr(ltions may develop us u c(mscqtw, ucc, o1" u multistep e l ( r e a l m o -
luIion and n l a v s h o w e cl(ise relatilmship with the hemapaietic di.lll'e, res~tiutive f)rllcesses.
INTRODUCTION
Chronic m y e l o m o n o c y t i c l e u k e m i a (CMMol) is a myeloproliferative disorder re- cently included by the FAB Cooperative Group among the m y e l o d y s p l a s t i c syn- dromes [1]. This classification, w h i l e introducing a sharper definition of the disease, has provided the basis for studies leading to a better delineation of its clinical and biologic aspects [2, 3]. The cytogenetic features of CMMoL have recently been eval- uated in studies carried out on a large series of patients, confirming the observation of previous reports [3, 4] of the occurrence of nonrandom c h r o m o s o m e abnormali- ties in about 30% of the cases. Most of these aberrations are not specific for this disease and appear to be shared by other m y e l o i d malignancies [5, 6].
Since CMMoL is considered a disorder of a pluripotent progenitor stem cell [71, the i n v o l v e m e n t of multiple h e m o p o i e t i c lineages is a c o m m o n feature in most of the patients. Thus the cytogenetic analysis of bone marrow cells separated on a density gradient [8] in order to obtain relatively pure cell populations may repre-
From the Department of Hematology, University of Ferrara, Italy.
Address requests for reprints to Dr. Antonio Cuneo, Cattedra di Emotologia. Universita' di Ferrara, C.so Giovecca 203, 44100 Ferrara, Italy.
Received May 3, 1988; accepted August 4, 1988.
2 9
:c, 1989 Elsevier Science Publishing Co., IIK:. Cancer Gelmt (]ytogenel 37:2!) 37 { 1!1891 655 Avenue of the Americas. New York. NY 101)111 [1165 460g,'gl,$(13.511
¢.,o Table 1 Laboratory features in seven patients with CMMoL BM aspiraton Patient Hb WBC %Mono-I Platelets Sermn Unfraetionated BM N{}. {}f sex/age {gdl} ( × 10"/1,) [t}B) { x 10TI,] lvs{}zvme M/E %m{}n{}-I Cytoh}gi{: features kary{}lypes (24-48 1]) cells 1. F/7,q vr 9.5 25 22 200 30 mg/I, 8/1 25 Megaloblastc}id {:hange NN 1 l of erythroid 45,XX. 16 3 precursors 5% 46,XX.del{11)(q23) 1 atypical monocvtes 2. M/59 vr 9.7 21.2 111 650 29 mg:L 6:1 28 Micromegakaryocytes, NN 12 numerous 46.XY,del(3}(q21 ) 2 sideroblasts 47.XY, + 21 1 3. M/64 vr 9.9 b.8 41 180 16 mg/I~ 7 I 16 Nuclear ervthroid NN 8 aberrati{}ns-- 46,XY,del{5)(q31} 4 {}{:{:asi{mal rinpe{l 47,XY, ~ 21},{l{A(5){q311 2 sider{}bIasts 44,XY. l,q, 22,del{5){q31} 1 4. M/68 vr 12.1 5.1 32} 290 nd 5/1 13 ltyp{}granular nd neutr{}phil leukoc~ tos 5. F/61 yr !1.9 12.5 15 158 /I mg/L 6/1 18 Hypogranular and NN 9
hyposegmented neutrophil leu
ko{:vtes 6. F/54 vr 11}.4 8,4 20 85 19 mg/I~ 8/1 22 Ityp{}lobular 46,XX, ~ 8. 18 4 neutrophils-- 47,XX. ~ 8 3 nu{:h}ar NN 5 dyseryti]ropoiesis 7. F/77 yr 11.1 14.5 11} 163 9 mg/L 5/1 9 Multinuclear NN 11 erythroblasts Abbre~i.lMns: Mono i, c:elb, ~>J tim {I]{ll]{){]\r[]{: lim~age: PI~, perilAmral Mood, BXI, b<me IIIHII{}~,\: ~ft[[Jl]l hs{}zv[]lt!, [1v ~) ~} mg 1,: M E, m\eh}-t~r',throid ratio: rid. not done: X*, nornlal karyotvpu.
Cytogenetics of Enriched Cell Fractions in CMMoL
31
sent an interesting tool for the assessment of the d i s t r i b u t i o n of the chromosome abnormalities a n d the detection of lineage-restricted aberrations. The results of a cytogenetic study performed on bone marrow cells after separation on a Percoll d e n s i t y gradient are presented a n d discussed in this report.
PATIENTS AND METHODS
The patients were a d m i t t e d to our i n s t i t u t i o n b e t w e e n January 1984 a n d December 1986, a n d diagnosis was made in accordance with the FAB criteria [1]. Bone mar- row aspiration smears were stained both with the May G r u n w a l d - G i e m s a m e t h o d a n d with cytochemical reactions [Prussian Blue, S u d a n Black B (SBB), n a p h t h o l AS-D chloroacetate esterase (NASDCAE), a n d a l p h a - n a p h t h y l acetate esterase (ANAE) with a n d w i t h o u t fluoride inhibition].
Separation of Bone Marrow Cells
M o n o n u c l e a r cells (30 x 106) were layered on top of a Percoll gradient in sterile p o l y p r o p y l e n e tubes a n d centrifuged at 600 g for 30 m i n u t e s . The densities used were 1070 mg/ml, 1065 mg/ml, 1060 mg/ml, 1055 mg/ml, 1050 mg/ml, a n d 1045 rag/ Figure 1 Cellular populations at different Percoll densities are shown, a. 106(}-1f]50 mg/ml fraction. The cytospin preparation was stained with combined reactions for ANAE and NASDCAE. A scattered graimlar posilivity for the nonspecific esterase is present in most ol the elements. A granulocytic precursor positively stained for NASDCAE is arrowed, b. Cells collected at 1050 1[]45 mg/ml density stained with anti-factor Vlll-related antigen monoclonal antibody coupled with the immunoalkaline phosphatase technique are shown.
~ r
3 2 A. Cuneo et al.
ml from the bottom to the top, a n d the v o l u m e of each layer was 1.2 ml. Fraction- ated cellular p o p u l a t i o n s were collected at the interfaces of the different densities, w a s h e d twice in TC M e d i u m 199 (GIBCO), a n d cultured in RPMI-1640 m e d i u m (GIBCO) s u p p l e m e n t e d with 15% fetal calf serum a n d antibiotics at 37°C i n 5% CO2 h u m i d i f i e d atmosphere. Cytospin preparations obtained from each culture were stained by means of cytochemical a n d c y t o i m m u n o l o g i c procedures [periodic acid- Schiff, NASDCAE, ANAE (+ NaF), anti-factor VIII related antigen m o n o c l o n a l anti- body c o u p l e d with i m m u n o a l k a l i n e phosphatase technique] for a cytologic charac- terization.
Cytogenetic Investigation
Each cell fraction was processed for chromosome analysis according to c o n v e n - tional procedures after 6-72 h o u r s ' culture i n c u b a t i o n . Air-dried slides were sub- mitted to ASG a n d t r y p s i n b a n d i n g m e t h o d s [9, 10]. As m a n y metaphases as pos- sible of each preparation were analyzed a n d karyotyped according to the ISCN n o m e n c l a t u r e [11].
RESULTS
The data c o n c e r n i n g the clinical a n d hematologic appearance at p r e s e n t a t i o n con- firmed the existence of distinct characteristics in this myelodysplastic s y n d r o m e [2]. Bone m a r r o w was h y p e r c e l l u l a r in all the patients, with a marked increase of g r a n u l o m o n o c y t i c precursors. Five patients presented i m p o r t a n t dyserythropoietic features; dysgranulopoiesis (three cases) a n d marked dysmegakaryocytopoiesis (one case) were also observed. Detailed hematologic data a n d unfractionated b o n e mar- row karyotypes are given in Table 1.
The separation of bone marrow cells was successful in all patients, a n d the en-
Table 2
Distribution patterns of various ceil p o p u l a t i o n s in seven patients with CMMoL assessed by cytochemical and c y t o i m m u n o l o g i c techniquesPrevailing cytologic type at Perco[l densities Patient 1070 1065 mg/ml "1060 1050 rag/m[ -"1050 mg/ml 1. Ebls: 21% Mono-l: 48% PM-M: 72% Mybls: 33% 2. Ebls: 27% Mono-l: 43% PM-M: 68% Mybls: 41% 3. gbls: 48% Mono-l: 18% PM-M: 45% Mybls: 71% 4. Ebls: 44% Mono-l: 56% PM-M: 50% Mybls: 40% 5. Ebls: 28% Mono-l: 71% PM-M: 61% Mybls: 26% 6. Ebls: 44% Mono-l: 22% PM-M: 54% Mybls: 76% 7. gbls: 31% Mono-l: 40% PM-M: 63% Mybls: 47% Mono-[ + Mvbls: 70'14 Mk-l: 22% Mono-I + Mvbls: 54% Mk-I: 38% Mono-l+Mvbls: 81% Mk-I: 11% Mono-I + Mvbls: 86% Mk-l: 8% Mono-I + Mvbls: 80% Mono-I + Mvbls: 78% Mk-l: 10% Mono-I + Mvbls: 73% Mk-l: "l.q%
Difteronlial on cytospin preparation slides (200 cells observed).
Abbreviations: Ebls. Erylhroblasts: PM-M. pronlvelocvtes and myelocytes: Morn>l, ~x;lls ol the monocytic lineage: Mybls. myeloblasts: Mk-l. <ells ot tim megakarvocylic lineage.
Cytogenetics of Enriched Celt Fractious in CMMoL
33
r i c h e d c e l l u l a r fractions c o l l e c t e d b e t w e e n the 1970-1065, 1 0 6 0 - 1 0 5 5 - 1 0 5 0 , and 1050-1045 m g / m l densities w e r e suitable for cytogenetic investigation. As r e v e a l e d by the staining p r o c e d u r e s , these fractions w e r e c o n s t i t u t e d by erythroblasts and g r a n u l o c y t i c precursors, by myeloblasts and m o n o c y t i c cells, by megakaryocytes, and by both m y e l o i d and m o n o c y t i c i m m a t u r e cells, respectively, (Fig. 1). The dis- tribution pattern of various cell p o p u l a t i o n s is given in Table 2.
The cytogenetic investigations w e r e p e r f o r m e d after a 6-hour i n c u b a t i o n from the erythroblast e n r i c h e d fraction, after 2 4 - 4 8 hours from the m o n o c y t i c fraction, and after 2 4 - 7 2 hours from the m e g a k a r y o c y t e - e n r i c h e d cellular p o p u l a t i o n . Results were reported in Table 3. All the cultured cellular fractions e x h i b i t e d a trisomy of c h r o m o s o m e 8 in two patients (cases 4 and 6) and a t e r m i n a l d e l e t i o n of c h r o m o - some 5 w i t h breakpoint at band q31 in one patient (case 3). On the other hand, s o m e c h r o m o s o m e aberrations, such as del(3)(q21) (patient 2, Fig. 2) and del(11)(q23) (patient 1, Fig. 3) p r e s e n t e d a more selective distribution i n v o l v i n g the megakaryocyte- and m o n o c y t e - e n r i c h e d cell fractions, respectively.
D I S C U S S I O N
Standard cytogenetic t e c h n i q u e s do not p e r m i t r e c o g n i t i o n of the cytologic type of the d i v i d i n g cells, thus causing a major p r o b l e m in the analysis of m y e l o p r o l i f e r a - rive disorders w h e r e the i n v o l v e m e n t of a c o m m o n progenitor stem cell gives rise to a m u l t i l i n e a r h e m p o i e t i c i m p a i r m e n t . Recently, the i n t r o d u c t i o n of a m e t h o d for the s i m u l t a n e o u s study of cell m o r p h o l o g y and karyotype offered the c h a n c e to assign n u m e r i c a l c h r o m o s o m e aberrations to i m m u n o l o g i c a l l y identified cell lines [12]. Alternatively, a c h r o m o s o m e analysis of e n r i c h e d cellular fractions after sep- aration on a density gradient might represent an effective c o n t r i b u t i o n to a better Table 3 Karyotypic patterns of e n r i c h e d cell fractions in seven patients with CMMoL
Percoll densities (rag/hill--culture times (hr)
No. No. No.
of of of
Patient cells 1070 1065 (6 hr) cells 1060-1050 (24 48 hr) (:ells 1 0 5 0 -1045 (24 72 hr)
1. 9 NN NN 1 46.XX,del(5)(q311 46,XX. 10,+21.deI{5}(q31) 45,XX. 16 2, 10 NN NN 46,XY,del(3){q21 47,XY, + 21 3. NN 46,XY.del(5){q31 44,XY, 19, 22,del(5}(q31) 47,XY, - 14 4. 47,XY.+8 50,XY,+8,+ 12, + 13,+ 2O NN 5. 6. 4 NN 3 46,XY,del(5)(q31) 2 47,XY. + 20,del(5)(q31) 2 45,XY. 15 1 47,XY, + 8 5 46,XY,+ 8 , - 22 2 46,X, Y,+8,del(20)(p121 3 45.XY,- 13 10 NN 4 46,XX, + 8 . - 18 2 47,XX,+ 21 3 NN 9 NN 6 NN 6 4 46.XX.del(11](q23) 2 2 46,XX,- 6. + 20 3 9 NN 11 2 47,XY,+ 21 4 1 8 NN 7 5 46.XY,del(5)(q31) 1 1 46.XY,+20, 22,del(5)(q31) 2 2 46,XY,t(l:2)(q32:q36) 2 2 47,XY, + 8 5 5 45,X, Y , + 8 , - 2 2 2 3 NN 3 8 NN - - Inadequate sample 4 45,XX,+8, 18, 20 2 47,XX,+8 2 46,XX,+8, 18 1 NN 3 NN lO NN 7 NN
34
A. Cuneo et al.I.
t
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X YF i g u r e 2 The karyotype obtained from 1050-1045 mg/ml megakaryocyte-enriched cell frac- tion (patient 2) is shown. The terminal deletion of chromosome 3 is arrowed.
definition of the cytogenetic aspects of the different h e m o p o i e t i c lineages in this group of h e m a t o l o g i c malignancies. As far as the cytogenetic results are concerned, our data parallel those given in previous reports [5, 13, 14]: nevertheless, some c o m m e n t s s h o u l d be m a d e regarding the m e t h o d o l o g i c approach. One patient (case 2) p r e s e n t e d relevant d y s m e g a k a r y o c y t o p o i e s i s with high platelet counts associated with a 3q21 aberration detected in m e t a p h a s e s p r e f e r e n t i a l l y arising in the 1050 1045 mg/ml fraction. Since n o r m a l k a r y o t y p e s were e x h i b i t e d by e r y t h r o i d and im- mature m y e l o i d cells, this a b n o r m a l i t y seems to be confined to the megakaryocyte- e n r i c h e d cell fraction. This c l i n i c a l - c y t o g e n e t i c a s s o c i a t i o n has been d e s c r i b e d in different m y e l o p r o l i f e r a t i v e d i s o r d e r s [13, 15-18], suggesting the existence in this region of a regulatory locus for t h r o m b o p o i e s i s .
A b n o r m a l i t i e s of c h r o n m s o m e 11 have been reported only o c c a s i o n a l l y in CMMoL and m y e l o d y s p l a s i a [19,201, w h e r e a s 11q23 rearrangements are c o n s i d e r e d t y p i c a l of l e u k e m i c proliferations with m o n o b l a s t i c differentiation [21-231. One pa- tient (case 1) w i t h h y p e r p l a s t i c features of the m o n o c y t i c lineage and an increase of i m m a t u r e m o n o c y t e s e x h i b i t e d a clone carrying an 1 1 q - a b n o r m a l i t y among the m e t a p h a s e s arising in the m o n o c y t i c c e l l u l a r fraction. This cytogenetic finding is not s u r p r i s i n g for a patient with a d y s p l a s t i c m o n o c y t i c lineage since its relation- ship with the differentiation of m o n o c y t e s has a l r e a d y been p o i n t e d out [24, 251. Interestingly, this a b n o r m a l i t y was o n l y p r e s e n t in the p r e p a r a t i o n h a r v e s t e d after 48 h o u r s ' i n c u b a t i o n and was not detected after 72 h o u r s ' c u l t u r e in the 1045-1050
Cytogenetics of Enriched Cell Fractions in CMMoL
35
-,11
t
11
i t F
1 2 3 4 56
7
8
9
10
11
12
13
14
15
16
17
18
D ' ~
~ ' ~
~ "
~ ' i ' ~
20 21 22 X YThe karyotype obtained from 1060-1050 mg/ml monocyte-enriched cell fraction (patient 1) showing a terminal deletion of chromosome 11 is presented.
19
Figure 3
mg/ml fraction where 20% monocytic cells were present. This finding seems to em- phasize the role played by culture time in cytogenetic analysis and gives evidence of the different mitotic activity of bone marrow cells during short-term culture [26]. It is thus likely that the kinetic features and low proliferative capacity that seem to characterize this clone [24] impede its detection in cytogenetic preparations ob- tained from whole bone marrow samples, possibly accounting for its absence in previous cytogenetic reports.
Some of the chromosome aberrations detected in our patients ( + 8 , 5 q - ) are typ- ical of leukemic myeloid proliferations and have alrady been described in this my- eloproliferative disorder. Their distribution appeared not to be correlated with the cytologic composition of the cellular fractions, thus providing cytogenetic evidence for the involvement of a common progenitor stem cell in this myelodysplastic syn- drome [27]. It is conceivable that some steps in the progression of the disease might be represented by the occurrence, in either a multipotent or a committed hemo- poietic progenitor, of genetic aberrations causing growth advantage or maturation impairment of single marrow cell lineages.
36
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