Heterogeneity of lineage involvement by trisomy 8 in myelodysplastic syndrome. A multiparameter analysis combining conventional cytogenetics, DNA in situ hybridization, and bone marrow culture studies

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Heterogeneity of Lineage Involvement by Trisomy 8 in

Myelodysplastic Syndrome

A Multiparameter Analysis Combining Conventional Cytogenetics,

DNA In Situ Hybridization, and Bone Marrow Culture Studies

Franca Fagioli, Antonio Cuneo, Antonella Bardi, Maria Gretel Carli,

Renato Bigoni, Rosa Balsamo, Rita Previati, Isabella Pazzi,

Grazia Roberti, Gian Matteo Rigolin, and Gianluigi Castoldi

ABSTRACT: To better understand the role of trisomy 8 in myelodysplastic syndrome (MDS), we performed

a m u l t i p a r a m e t e r analysis c o m b i n i n g conventional c h r o m o s o m e studies (CCS), fluorescence in situ hy-

bridization (FISH), and bone marrow (BM) culture studies in two patients with MDS evolving into acute myeloid l e u k e m i a (AML).

A m o s a i c i s m ofa cytogenetically normal clone and a clone with trisomy 8 was detected in both patients throughout the course of the disease, a finding confirmed by FISH on BM cells. The relative size of the

trisomic clone increased from 52% to 71% (p < 0.0001) and from 53% to 69% (p = 0.001) of all BM cells

at the t i m e of the l e u k e m i c switch in patients 1 and 2, respectively.

Combined FISH and i m m u n o p h e n o t y p i n g of BM cells showed i n v o l v e m e n t of the granulomonocytic lineage in patient 1 and i n v o l v e m e n t of erythroid cells as well as of the granulomonocytic lineage in pa- tient 2. O n l y disomic l y m p h o c y t e s were detected in both patients.

FISH on single hemopoietic colonies grown in semisolid media detected trisomic CFU-GM and disomic

BFU-E in patient 1, whereas a proportion of CFU-GM and BFU-E deriving from the trisomic clone was

detected in patient 2. However, the percent of trisomic colonies was lower than the percent of involved granulomonocyte precursors and involved erythroblasts, as detected by c o m b i n e d FISH and im-

m u n o p h e n o t y p i n g on fresh BM samples.

We have t h u s shown heterogeneity of lineage i n v o l v e m e n t by trisomy 8 in MDS undergoing transforma-

tion into AML. A l t h o u g h preferential growth of disomic clones m a y occur in vitro, the finding of an in-

creased size of the trisomic clone at the t i m e of l e u k e m i c switch suggests that these cells had proliferative

advantage in vivo over cells w i t h o u t trisomy 8.

INTRODUCTION

A number of non-random chromosome changes have been described in myelodysplastic syndrome (MDS) [1], reflecting the heterogeneity of the clinicobiologic features of this myeloid neoplasia characterized by ineffective hemopoiesis resulting in progressive cytopenia affecting one or more cell lineages. Trisomy 8 is one of the most frequently occurring numerical change in MDS, and does not appear to be restricted to any of the cytologic subsets identified by the FAB group in 1982 [2].

From the Institute of Hematology, University of Ferrara, Ferrara, Italy.

Address reprint requests to: Dr. Antonio Cunea, Institute of Hema-

tology, University of Ferrara, Via Savonarola, 9, 44100 Ferrara, Italy.

Received July 5, 1994; accepted October 25, 1994.

Cancer Genet Cytogenet 82:116-122 (1995) © Elsevier Science Inc., 1995

655 A v e n u e of the Americas, N e w York, NY 10010

Cytogenetic studies of different cell populations obtained by separation over a density gradient, as well as chromosome analysis of single hemopoietic colonies, documented that trisomy 8 may involve both the erythroid series and the granulomonocytic lineage [3, 4]. However, recent investiga- tions combining fluorescence in situ hybridization (FISH) and membrane immunophenotyping to detect numerical aberrations in interphase nuclei documented the heterogeneity of distribution of this chromosome anomaly in the different hemopoietic lineages [5, 6].

Consequently, the biologic significance of trisomy 8 in MDS remains poorly understood. We therefore designed a study combining conventional chromosome analysis, FISH, and immunophenotyping, as well as bone marrow culture studies, in two patients with MDS evolving into acute myeloid leukemia (AML) with trisomy 8, to analyze the distribution of this numerical aberration in different hemopoietic

0165-4608/95/$09.50 SSDI 0165-4608(94)00228-Y

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T r i s o m y 8 in MDS 117 lineages a n d to better u n d e r s t a n d its p o s s i b l e role in the nat-

ural history of MDS u n d e r g o i n g t r a n s f o r m a t i o n into AML.

PATIENTS A N D METHODS

Two patients w i t h MDS evolving into A M L carrying t r i s o m y 8 were s t u d i e d . Diagnoses were m a d e a c c o r d i n g to the FAB criteria [2, 7]. Cytogenetic, FISH studies, a n d in vitro assays of p r o g e n i t o r cell growth were p e r f o r m e d as s u m m a r i z e d below.

Patient 1

This 65-year-old patient was referred to our institute in Janu- ary 1992 for a n e m i a and s p l e n o m e g a l y (4 cm below left costal margin). The b l o o d c o u n t revealed h e m o g l o b i n (Hb) 8 g/dL, platelet (Plt) 40 x 109/L, white b l o o d cell (WBC) 2 x 109/L, w i t h 3% blasts. The BM was hypercellular, w i t h h y p e r p l a - sia of the g r a n u l o m o n o c y t i c lineage and relative r e d u c t i o n of the e r y t h r o i d series (see Table 1). N u c l e a r as well cyto- p l a s m i c d y s e r y t h r o p o i e s i s was a p p a r e n t in 20% erythroblasts. The majority of n e u t r o p h i l s were hypogranular. Megakaryocytes were r e d u c e d a n d a moderate i n c r e a s e of l y m p h o c y t e s a n d p l a s m a cells was observed. Based on the p r e s e n c e of 27% blasts in the BM, a d i a g n o s i s of refractory a n e m i a w i t h excess of blasts in t r a n s f o r m a t i o n (RAEB-t) was made.

T h e p a t i e n t r e q u i r e d intensive s u p p o r t i v e t h e r a p y t h r o u g h o u t the course of the disease. Progressive r e d u c t i o n of mature forms of the gmnulocytic lineage concomitant w i t h a gradual increase of blast cells was r e c o r d e d over a 2-month period, resulting in leukemic switch w i t h 45% BM blast cells w i t h m y e l o m o n o c y t i c features.

Therapy w i t h cytosine a r a b i n o s i d e (Ara-C) 10 mg/m 2, by intravenous infusion, t w i c e a day for 10-14 clays was given. Six courses were a d m i n i s t e r e d b e t w e e n May 1992 a n d A p r i l 1993, o b t a i n i n g o n l y p a r t i a l short-lasting r e d u c t i o n in the n u m b e r of blast cells. Progressive s p l e e n e n l a r g e m e n t de- v e l o p e d a n d the p a t i e n t d i e d 18 m o n t h s after d i a g n o s i s w i t h d i s e a s e p r o g r e s s i o n .

Patient 2

This 67-year-old patient was admitted to our institute in Febru- ary 1992 for p e t e c h i a e of the p e l v i c limbs. The b l o o d count

revealed p a n c y t o p e n i a : Hb 8.5 g/dL, Plt 50 x 109/L, WBC 3 x 109/L, w i t h 18% granulocytes, 76% l y m p h o c y t e s , 4% monocytes, a n d 2 % blasts. The b o n e marrow sample, hypercellular w i t h trilineage myelodysplasia, revealed 24% blasts. These findings were c o m p a t i b l e w i t h a diagnosis of RAEB-t. Progressive leftward shift of g r a n u l o c y t o p o i e s i s w i t h increase of BM blast cells to 38% was observed d u r i n g a 3-month p e r i o d . Blast cells were o c c a s i o n a l l y g r a n u l a r a n d s h o w e d S u d a n black-B positivity. These findings were consistent w i t h a transformation of MDS into AML without matu- ration. T r e a t m e n t w i t h Ara-C, 10 m g / m 2, by intravenous infusion, t w i c e a day for 7-10 days was a d m i n i s t e r e d . Eleven courses of low-dose Am-C were given b e t w e e n M a r c h 1992 a n d October 1993, o b t a i n i n g a r e d u c t i o n in the n u m b e r of BM blast cells. The p a t i e n t is p r e s e n t l y alive a n d well, w i t h a cytologic p i c t u r e consistent w i t h RAEB.

Colony A s s a y

In vitro growth of e r y t h r o i d c o l o n i e s (BFU-E) a n d of g r a n u l o m o n o c y t i c c o l o n i e s (CFU-GM) was assayed accord- ing to the m e t h o d s d e s c r i b e d by Iscove et al. [8, 9]. In the BFU-E growth assay e r y t h r o p o i e t i n (Boehringer M a n n h e i m ) was a d d e d to give a final c o n c e n t r a t i o n of 2 U/ml. Recom- b i n a n t h u m a n g r a n u l o c y t e - m a c r o p h a g e c o l o n y - s t i m u l a t i n g factor (rhGM-CSF, 10 ng/mL, Boehringer M a n n h e i m , Ger- many) was u s e d to stimulate growth of CFU-GM.

C h r o m o s o m e Studies on B o n e Marrow Cells

Bone marrow cells were c u l t u r e d for 24 a n d 48 h o u r s without mitogens. Synchronization w i t h methotrexate and thymi- d i n e was p e r f o r m e d . M e t a p h a s e s were G - b a n d e d w i t h Wright's stain. C h r o m o s o m e aberrations were d e s c r i b e d ac- c o r d i n g to the International System for H u m a n Cytogenetic N o m e n c l a t u r e [10].

Cytogenetic Studies of I n d i v i d u a l CFU-GM and BFU-E

To analyze the karyotype of the progeny of single hemopoietic c o l o n i e s g r o w n in s e m i s o l i d m e d i a a n d to assess the n u m - ber of BFU-E a n d CFU-GM w i t h a n d w i t h o u t t r i s o m y 8, conventional cytogenetic as well as FISH studies of h e m o p o i e t i c c o l o n i e s were p e r f o r m e d . C h r o m o s o m e analysis from in- d i v i d u a l c o l o n i e s were p e r f o r m e d on days 8 to 10 for CFU- GM a n d on days 12 to 13 for BFU-E. C o l c e m i d (100 ~tL of a

Table 1 BM, c y t o g e n e t i c , a n d i n t e r p h a s e c y t o g e n e t i c f i n d i n g s in two p a t i e n t s w i t h MDS evolving into AML

Patient, % Bls/% GM cells/ No. abnormal/total no. sex/age (yr) % Ebls (BM) a Diagnosis BM karyotype metaphases

No. of fluorescent spots in interphase cells b 1 2 3 1, M/65 27/53/11 RAEB-t 47,XY, + 8 25/43 6 42 52 45/43/10 AML-M4 47,XY, + 8 33/39 8 21 71 2, M/67 24/54/26 RAEB-t 47,Y,der(X)t(X;1)(q27;q25), + 8 16/23 6 41 53 38/47/23 AML-M1 47,Y,der(X)t(X;1)(q27;q25), + 8 19/21 4 27 69

Abbreviations: Bls, blast cells; GM cells, cells of the granulomonocytic lineage; Ebls, erythroblasts. ° Differential on 300 BM cells; other cells were megakaryocytes, lymphocytes, and plasma cells.

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0.1 p~g/mL solution) was a d d e d to c u l t u r e s 6 hours p r i o r to the e n d of the i n c u b a t i o n p e r i o d . S i n g l e c o l o n i e s were indi- v i d u a l l y a s p i r a t e d a n d transferred into an E p p e n d o r f tube c o n t a i n i n g 80 I~L of 0.075 KCL. After i n c u b a t i o n (30 minutes, 37°C), the entire content of the E p p e n d o r f t u b e was transferred onto a microscope slide pre-treated w i t h poly-L-lysine. T h e s l i d e s were i n c u b a t e d (30 minutes) in a moist chamber. Methanol/acetic a c i d (3:1) fixative was d r o p p e d onto the area of the s l i d e s w h e r e the c o l o n y was p l a c e d . The m e t a p h a s e s

suitable for analysis were G - b a n d e d w i t h Wright stain a n d k a r y o t y p e d .

The s a m e p r o c e d u r e was a d o p t e d to p r e p a r e s l i d e s from single h e m o p o i e t i c c o l o n i e s for the p u r p o s e of FISH s t u d y

(see below).

Fluorescence In Situ Hybridization

For the d e t e c t i o n of t r i s o m y 8 in interphase cells from BM

samples a n d from single h e m o p o i e t i c colonies, the chromosome 8-specific centromeric probe (Oncor Inc., Gaithersburg, MD) was used. The p e r c e n t a g e of i n t e r p h a s e cells w i t h t r i s o m y 8 was assessed by FISH on BM cells at d i a g n o s i s a n d at l e u k e m i c s w i t c h in b o t h patients. S l i d e s p r e p a r e d from BM s a m p l e s for c o n v e n t i o n a l cytogenetic analysis, aged at r o o m t e m p e r a t u r e for 5-15 days, were treated w i t h RNAse (RNAse A, 100 I~g/mL in 2 x SSC), d e h y d r a t e d in alcohol, a n d d e n a t u r e d in 70% f o r m a m i d e at 70°C for 2 minutes. The b i o t i n y l a t e d probe (100 ng/slide) was d i s s o l v e d in 45 ~tL hy- b r i d i z a t i o n m i x t u r e (total volume) consisting of 60 % forma- m i d e in 2 x SSC, 10%wt/vol dextran sulphate, a n d 30 ~g s a l m o n s p e r m DNA.

Following d e n a t u r a t i o n at 75°C for 5 minutes, 45 ~tL of h y b r i d i z a t i o n m i x t u r e were layered onto each slide. The slides were covered w i t h a 24 x 50 m m coverslip a n d sealed w i t h rubber c e m e n t (San.ford, Bellwood). H y b r i d i z a t i o n was allowed to p r o c e e d overnight at 37°C in a moist chamber. The s l i d e s were t h e n w a s h e d in four c h a n g e s of 50% f o r m a m i d e at 45°C for 3 m i n u t e s a n d in four c h a n g e s of 2 x SSC at 45°C for 3 minutes. Following a 20-minute b l o c k i n g step w i t h 5 % non-fat d r y m i l k p o w d e r in 2 x SSC, the s l i d e s were incubated w i t h FITC-avidin (Vector, Burlingame, CA) (5 ~tg/mL in 4 x SSC/5% non-fat d r y milk) for 20 minutes.

S i g n a l a m p l i f i c a t i o n was obtained w i t h a layer of bio- tinylated a n t i - a v i d i n m o n o c l o n a l a n t i b o d y (Vector, Burlin- game; 5 ~tg/mL), followed by i n c u b a t i o n w i t h FITC-avidin.

P r o p i d i u m i o d i d e (0.5 ~g/mL) was u s e d as a counterstain. Slides were e x a m i n e d u n d e r a fluorescence microscope (Di- a p l a n Leitz); 200 BM i n t e r p h a s e cells w i t h w e l l - d e l i n e a t e d f l u o r e s c e n t spots were c o u n t e d in each patient for signal screening. At least 50 i n t e r p h a s e cells from each single h e m o p o i e t i c colony were observed.

Combined Immunophenotyping and FISH

To d e t e r m i n e the percentage of erythroblasts, granulocyte precursors, a n d l y m p h o c y t e s carrying t r i s o m y 8, c o m b i n e d i m m u n o p h e n o t y p e / F I S H s t u d y was p e r f o r m e d on BM cells at diagnosis.

F o l l o w i n g s e p a r a t i o n over a F i c o l l g r a d i e n t , t h e m o n o n u c l e a r cells were incubated w i t h the following mono- c l o n a l antibodies: anti-CD13 (My7, Coulter Inc.) for granulo- cytic lineage, anti-CD14 (My4, Coulter Inc., Hialeah, FL) for m a t u r e m o n o c y t i c cells, a n t i - g l y c o p h o r i n (OKRBC, Ortho Diagnostic System Inc., Raritan, NJ) for e r y t h r o i d lineage, anti-CD2 (Tll, Dakopatts, C o p e n h a g e n , Denmark) labeling T l y m p h o c y t e s , and anti-CD19 (Leu12, Becton-Dickinson, M o u n t a i n View, CA) labeling B l y m p h o c y t e s . To m i n i m i z e non-specific Fc binding, cell s u s p e n s i o n s were preincubated w i t h 5% n o r m a l rabbit s e r u m (Dakopatts).

Specific b i n d i n g of the p r i m a r y a n t i b o d y was v i s u a l i z e d w i t h rabbit a n t i - m o u s e i m m u n o g l o b u l i n conjugated w i t h tetramethyl r h o d a m i n e isothiocyanate (TRITC) (Dakopatts). Cytospin preparations were m a d e from each i m m u n o l a b e l e d cell s u s p e n s i o n a n d fixed by i m m e r s i o n for 30 m i n u t e s in m e t h a n o l / a c e t i c acid at a c o n c e n t r a t i o n 19:1 a n d by a sec- o n d i m m e r s i o n for 15 m i n u t e s in m e t h a n o l / a c e t i c a c i d at a c o n c e n t r a t i o n 3:1.

The same h y b r i d i z a t i o n protocol as d e s c r i b e d above was e m p l o y e d for the detection of trisomy 8 in i m m u n o l a b e l e d cells. No counterstaining was used. At least 100 i m m u n o l a - b e l e d cells w i t h w e l l - d e l i n e a t e d signals were observed for each m o n o c l o n a l a n t i b o d y e m p l o y e d .

RESULTS

Cytogenetic and i n t e r p h a s e cytogenetic f i n d i n g s are de- s c r i b e d in Table 1. Results of c o m b i n e d i n n u n o p h e n o t y p i n g a n d DNA in situ h y b r i d i z a t i o n a n d FISH on single colonies are p r e s e n t e d in Tables 2 a n d 3.

C h r o m o s o m e studies on single colonies y i e l d e d 2-7 (me-

Table 2 P r o g e n i t o r assays and c y t o g e n e t i c studies on i n d i v i d u a l colonies in two patients w i t h RAEB-t

Patient Progenitor assays a Karyotype of individual colonies b diagnosis CFU-GM BFU-E CFU-GM BFU-E

1 RAEB-t 90 42 47,XY, + 8 (4) 46,XY (20) 46,XY (16)

2 RAEB-t 105 20 47,Y,der(X), + 8 (3) 47,XY,der(X), + 8 (1) 46,XY (15) 46,XY (19) Each value shows the mean of quadruplicate cultures. Normal value for CFU-GM: 120-200/105 BM mononuclear cells. Normal value for BFU-E, 70--120/105 BM cells.

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T r i s o m y 8 in MDS 119 d i a n 4) a n a l y z a b l e m e t a p h a s e s from e a c h colony. The s a m e

karyotype was present in all metaphases obtained from a single colony. Salient results in each p a t i e n t are s u m m a r i z e d below.

Patient 1

Trisomy 8 was p r e s e n t in 25/43 of a n a l y z e d m e t a p h a s e s at diagnosis, a figure c o n f i r m e d by FISH studies revealing 52 % interphase cells w i t h t h r e e fluorescent signals (Table 1). The relative size of the BM t r i s o m i c c l o n e was f o u n d to be in- c r e a s e d by FISH to 71% of all i n t e r p h a s e cells at l e u k e m i c s w i t c h (p < 0.0001), w h e n 33/39 m e t a p h a s e s w i t h + 8 were o b t a i n e d (see Table 1).

C o m b i n e d i m m u n o l o g i c a n d FISH analyses showed three f l u o r e s c e n t signals in 49% CD13 + cells a n d in 39% CD14 + cells. Conversely, no positive cell w i t h markers for the l y m - p h o c y t e a n d e r y t h r o i d l i n e a g e d i s p l a y e d t h r e e f l u o r e s c e n t signals. Cytogenetic analysis o n s i n g l e c o l o n i e s s h o w e d the p r e s e n c e of 4/20 (20%) CFU-GM w i t h t r i s o m y 8, w h i l e this a b e r r a t i o n was not p r e s e n t in any of the BFU-E cells exam- ined. S i m i l a r f i n d i n g s were o b t a i n e d by FISH o n the single colonies, as s h o w n in detail in Table 3.

Figure 1 Combined FISH and immunophenotyping on BM cells

from patient 2: a CD13 + cell with three fluorescent signals using a chromosome 8-specific probe is shown.

P a t i e n t 2

A 47,Y, der(X)t(X;1)(q27;q25), + 8, karyotype was detected in 16 of the 23 m e t a p h a s e s a n a l y z e d from BM s a m p l e s at diagnosis. FISH analysis revealed the p r e s e n c e of t h r e e f l u o r e s c e n t signals in 53% of the b o n e m a r r o w cells. Trisomic cells increased to 69% in the leukemic phase (p = 0.001), w h e n 19/21 m e t a p h a s e s s h o w e d the s a m e c l o n a l a b n o r m a l i t i e s detected at d i a g n o s i s (see Table 1). C o m b i n e d i m m u n o l o g i c a n d FISH analysis (Fig. 1) d e m o n s t r a t e d the p r e s e n c e of t r i s o m y 8 in 62% CD13 + cells, in 27% CD14 + cells, and in 21% OKRBC + cells. T and B lymphocytes d i d not have evidence of trisomy 8.

Cytogenetic s t u d i e s on single c o l o n i e s revealed the pres- e n c e of the 47,Y, der(X)t(X;1)(q27;q25), + 8, karyotype in 3/18 (16.6 %) of the CFU-GM, a f i n d i n g c o n f i r m e d by FISH analysis s h o w i n g 4/20 t r i s o m i c CFU-GM. E v i d e n c e of t r i s o m y 8 in 1/10 a n d in 1/20 i n d i v i d u a l BFU-E was p r o v i d e d by FISH a n d by c o n v e n t i o n a l c h r o m o s o m e analysis, respectively. Detailed results are s u m m a r i z e d in Tables 2 a n d 3.

DISCUSSION

A l t h o u g h t r i s o m y 8 is one of the most frequently o c c u r r i n g c h r o m o s o m e c h a n g e in MDS, its role in the n a t u r a l history of this primitive h e m o p o i e t i c stem cell disorder r e m a i n s elu- sive. I n d e e d , w h i l e debate still s u r r o u n d s its p o s s i b l e prog- nostic s i g n i f i c a n c e [11, 12], reports s h o w i n g m u l t i l i n e a g e i n v o l v e m e n t by t r i s o m y 8 in MDS w i t h progressive a c c u m u - l a t i o n of the t r i s o m i c c l o n e in the BM have b e e n p u b l i s h e d [5, 6, 13, 14], along w i t h studies d o c u m e n t i n g that fluctua- tions of t h e size of the t r i s o m i c c l o n e may o c c u r rather fre- q u e n t l y w i t h o u t c o r r e l a t i o n w i t h d i s e a s e evolution [15, 16]. By c i r c u m v e n t i n g the n e e d for metaphases, FISH enables cytogenetic s t u d y of adequate n u m b e r s of i n t e r p h a s e cells, t h u s p e r m i t t i n g the e s t i m a t i o n of the c l o n e size as well as the assessment of the distribution of chromosome aberrations in i m m u n o l a b e l e d cells [17, 18]. In a d d i t i o n , the s t u d y of h e m o p o i e t i c c o l o n i e s grown in s e m i s o l i d m e d i a can be eas- T a b l e 3 C o m p a r i s o n of c o m b i n e d FISH a n d i m m u n o p h e n o t y p i n g w i t h FISH on s i n g l e

h e m o p o i e t i c c o l o n i e s to assess l i n e a g e i n v o l v e m e n t in two p a t i e n t s w i t h RAEB-t

Monoclonal antibody tested Signal screening in immunolabelled cells a 1 2 3 FISH o n i n d i v i d u a l c o l o n i e s b No. of CFU-GM

with three spots/ total no. assessed

No. of BFU-E with three spots/ total no. assessed Patient 1 Anti CD13 10 41 49 Anti CD14 8 53 39 Anti-glycop horine 9 91 0 Patient 2 Anti CD13 9 29 62 Anti CD14 6 67 27 Anti-glycophorine 10 69 21 5/20 0/12 4•20 1110

a No CD2-positive and CD19-positive cells with three signals were detected (100 cells observed).

b More than 80% cells with three signals were found in each colony classified as trisomic; more than 80% cells with two signals were found in each colony classified as disomic.

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ily performed by FISH, allowing for the comparison of the percent of trisomic cells in the in vitro culture system usually adopted for colony assays and in uncultured BM samples. Because few patients with MDS and trisomy 8 have been studied, we designed this multiparameter analysis of the distribution of trisomy 8 in erythroid, granulomonocytic, and lymphoid lineage in two patients with MDS evolving into AML. Discussion will focus on the issue of lineage involvement by trisomy 8 in these patients apparently running the same clinical course, and on the comparative analysis of the percentage of trisomic cells in BM cells identified immuno- logically, as well as in single CFU-GM and BFU-E.

Both disomic and trisomic cells were detected in BM cells in our patients by conventional chromosome analysis and by FISH, showing a fairly good correspondence between the percentage of metaphase cells with trisomy 8 and the percentage of interphase nuclei with three fluorescent signals. This finding confirms that FISH allows for the reliable detection of trisomy 8 in interphase cells in MDS [19]. It is worth noting, however, that caution should be employed when in- terpreting these data since errors in assessing the abnormal- to-normal metaphase ratio may occur frequently [12]. In ad- dition, the FISH artefact rate has been shown recently to be relatively high, suggesting the need for an automated signal evaluation [20].

In view of the mosaicism of the disomic and trisomic clone in BM samples, it was of interest to assess which cell popu- lation carried trisomy 8 in these patients. B and T lymphocytes in our patient did not carry trisomy 8, as shown by combined FISH and immunophenotyping. This finding is in keeping with previous reports consistently showing that chromosome aberrations in MDS are usually restricted to the nonlymphoid lineage [21-23]. However, molecular genetic studies of the nature of lymphopoiesis in MDS have shown that lymphocytes may be part of the clonal proliferation in a fraction of MDS [24, 25], raising the possibility that clonal hemopoiesis may precede the development of chromosome aberrations in some patients [26].

Data obtained by combined immunophenotyping and FISH in this study provide evidence for the involvement of the granulomonocytic lineage by trisomy 8 in our patients. By contrast, erythroid cells carrying trisomy 8 were only seen in patient 2, trisomic erythroblasts not having been detected by double FISH and immunophenotyping in patient I in the present study. Different hypothetical scenarios may be en- visaged to account for the heterogeneity of lineage involvement by trisomy 8 in our two patients.

Trisomy 8 may represent a clonal abnormality affecting a stem cell capable of differentiating along the granulomonocytic pathway only, acquired during the evolutive phases of MDS. In patient I, it is reasonable to assume that the clonal proliferation leading to refractory anemia was initially cytogenetically normal and that trisomy 8 was acquired when progressive accumulation of blast cells, later leading to overt leukemia, started to occur. This interpretation would explain why the erythroid lineage did not carry trisomy 8 and is in keeping with the evolution pattern of MDS previously described by Tricot et al. [27].

Alternatively, trisomy 8 may be acquired in the early stages of the natural history of MDS and may involve a multipotent

stem cell giving rise to a progeny of erythroblasts and granulocytes. Data obtained from patient 2 suggest that the trisomic clone may coexist with cytogenetically normal progenitors and that disease progression may occur, possibly due to a proliferative advantage of this chromosomally abnormal clone. Indeed, a statistically significant increase in the percentage of trisomic cells in BM samples was detected by FISH in our two patients when overt leukemia developed. The above considerations, along with the analysis of the clinical course in our two patients, which proved to be rather aggressive in patient 1 and indolent in patient 2, raise the issue of clinicobiologic heterogeneity of RAEB-t, a subset of MDS possibly including cases of %arly AML;' as well as gen- uine cases of preleukemia [28].

Consistent involvement of granulocyte precursors and erythroblasts by trisomy 8 in MDS was reported in previous studies either using chromosome analysis of different cell populations obtained by separation on a density gradient [4], cytogenetic studies on progenitor cells [3], or chromosomal in situ suppression hybridization [13].

Two additional cases with trisomy 8 in cells of the granulomonocytic and erythroid lineage were recently found by combined immunophenotyping and DNA in situ hybridization [5, 6]. However, one patient with trisomy 8 that was confined to the erythroid lineage was also described [5], further increasing heterogeneity of lineage involvement by this chromosome aberration in MDS.

In vitro growth of hemopoietic precursors is usually im- paired in MDS, reflecting disordered in vivo proliferation. Several mechanisms are likely to be involved in the genesis of this abnormal growth and the effect of clonal chromosome changes is unclear. Therefore, the presence of a mosaicism of a disomic and trisomic clone in our two cases prompted us to assess whether colonies growing in vitro originated from chromosomally normal cells or from trisomic progenitor cells.

Conventional cytogenetic studies on single BFU-E and CFU-GM revealed that a proportion of erythroid and myeloid colonies originated from the cytogenetically abnormal stem cell in patient 2, in whom the involvement of erythroblasts and myelomonocytic cells was previously demonstrated by combined FISH and immunophenotyping.

In line with data deriving from combined immunophenotyping and FISH, some trisomic CFU-GM and only cytogenetically normal BFU-E were detected in patient I. In both patients, no additional clones with chromosome changes other than those found in direct cytogenetic harvest of BM samples were detected in progenitor cells grown in semisolid medium. This culture system, therefore, does not appear to favor the preferential in vitro growth of latent subclones with unrelated karyotypes, which have been shown recently to be detectable in MDS patients by a long-term BM culture system [29]. This finding is in keeping with a previous report [30].

For a better estimate of the number of trisomic progenitor cells growing in vitro in the MDS phase, we performed FISH on single colonies. In patient 2, the percentage of BFU-E and CFU-GM with trisomy 8 was lower than the percentage of erythroblasts and granulocyte precursors showing three fluorescent signals at combined immunologic/FISH analysis of

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T r i s o m y 8 i n MDS 121

BM cells. Likewise, preferential growth of cytogenetically n o r m a l CFU-GM was demonstrated i n p a t i e n t 1.

Therefore, this c u l t u r e system may have facilitated the growth a n d differentiation of c h r o m o s o m a l l y n o r m a l progenitor cells over trisomic cells. Potentially, modifications of the r o u t i n e c u l t u r e system adopted i n this study may be capable of further e n h a n c i n g proliferation of chromosomally n o r m a l progenitors. S w o l i n et al. [30] f o u n d i n two MDS patients w i t h a 5 q - c h r o m o s o m e a n d w i t h m o n o s o m y 7 a sig- n i f i c a n t l y lower p r o p o r t i o n of c h r o m o s o m a l l y a b n o r m a l CFU-GM w i t h respect to the p r o p o r t i o n of a b n o r m a l metaphases i n a direct BM preparation. Unlike o u r cases, a clone w i t h trisomy 8 i n a p a t i e n t w i t h MDS a p p e a r e d to have growth advantage i n vitro i n c o m p a r i s o n w i t h n o r m a l clones. I n this study, however, n o i n f o r m a t i o n is provided c o n c e r n i n g w h i c h cell lineage was involved by trisomy 8, m a k i n g c o m p a r i s o n w i t h o u r data difficult.

In c o n c l u s i o n , we have s h o w n heterogeneity of lineage i n v o l v e m e n t by trisomy 8 i n two patients w i t h MDS employ- ing a c o m b i n e d i m m u n o p h e n o t y p i n g a n d DNA i n situ hy- b r i d i z a t i o n t e c h n i q u e a n d cytogenetic study of progenitor cells grown i n semisolid media. A l t h o u g h preferential growth of c h r o m o s o m a l l y n o r m a l progenitor cells may oc- c u r i n vitro, the relative size of the trisomic c l o n e increased at the time of leukemic switch, suggesting that a positive selective pressure allowed for the e s t a b l i s h m e n t a n d expansion of trisomic cells i n vivo.

This work was supported by M.U.R.S.T., fondi 40% and fondi 60%, and by Regione ER. This work is part of the Concerted Action: "Mo- lecular Cytogenetic Diagnosis in Hematological Malignancies" within the BMH1 program of the E.V.; CT 94-1703.

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