Cytogenetic pattern of acute lymphoblastic leukemia (ALL) in the Yogyakarta and Central Java Province, Indonesia

(1)

2@

Haryana et al. Med

J

Univ Indon

Cytogenetic

Pattern

of Acute

Lymphoblastic Leukemia

(ALL)

in

the

Yogyakarta

and

Central

Java Province, Indonesia

Sofia

M. Haryana*, E.Suryadi**,

Sultana

M. Husein***, Radjiman*

ABSTRAK

Penelitian

ini

bertujuan untuk tnelihat ganbaran sitogenetika penderita

ALL

(Acute Lynphoblttstic Leukenia). Ganrbaran sitogenetika ini penring untuk tnenentukan penatalaksanaan penderita tersebut. ùnpat belas pasien ALL yang berobat ke runah sakit

di

Yogyakarta

lan

Setnarang sejak

Juni

1992 sampai Novetnber

tg92

telah diperiksa ganbaran sitogenetikanya dengan netode G-banding. Hasil peneriksaan tnenunjukkan 4 penderita dengan kelainan struktural kronrosottt berupa t(9;22) (q34.1;q11.21) pada 2

pasien, t(4;1

l)

(q23) pada 1 pasien, det 11, pI3-14 pada I pasien; 9 penderita dengan kelainaniunlah kronoson berupu hiperploidi, potiploidi, tetraploidi, atau ltotlosolti, 3 diuntaranT,a tennasuk kelainan kronoson ganda (struktural dan nunerikal); 4 penderita tidak ilitentukan kelainan krontoson. Pada penderita dengan hanya hiperploidi > 5O kronosortt dan ganbaran sitogenetika nonnal setelah pengobaran nenunjukkan remisi/retnisi parsialis. Sedangkan 3 penderita dengan kelainan krouosottt ganda menuniukkan perialanan penS,akir lebih progresif. DisituputkaÀ bahwa kelainan struktural pada ALL nentputtyai arti penling dalant nenenluknn penggolongan

dan prognosis penyakit.

ABSTRACT

The

ain of

this investigation is to analyse the cytogenetics of ALL patients. Cltosenelic analysis is inportant in deterntining the ,nanage,ileyû of those patients. Fourleen ALL patients, hospitalised in several hospitals in Yog1,ofta71a and Setnarang since June 1992 to Novetnber 1992, v'ere studied by G- banding,nethod. The results shov,ed 4 patients u,ithstructural abrutnnalitr- î(9;22) (q34.1;q I 1.21)

(2patients),t(4;tI)(q2t;q23)(Iparienr),arddel 11,p13-pl4(Ipatient);g

ltutienrwithnunerical al.tnonnalitt'suchashyperploidl', polypktitll,, rcrraploiù1, and tnonosorttl', three of thenr shou'ed double abnornulities (slrudural and nunerical); 4 putients wilh nonnal karl,otype. Patienrs v'ich previously have hyperpktidy > 5O chrotnoxtn and normal karyotype, afier lreatnte,Tt shou'ed re,ilissioty'partial

itt ALL is inporrunt

b

deternrine the groult and prognosis

tf

the diseo^se.

Key words :

ALL,

c)'rogenerics - translocatiott - deletion -hyperploidl'- classifcatiott - prognosis.

INTRODUCTION

Cancer is the result of

accumulation

of

multiple genetic

changes.l Each alteration,

whether

an initiating

or

a progress.ion

event, may

be mediated through a

gross

chromosal

change and

therefore

has the

potential

to be cytogenetical

ly visible.2

The common

tumor schromosomal

abberrations are

classified

as struclural or numeric.

Structural

al-terations include translocations, inversions, deletions,

irsertions

and amplifications,

whereas

nunlerical

ab-normalities are

losses

or

duplication

of

whole

chromosomes.3 In hematological malignancy

such as

leukemia, analysis

of

the

chromosomes

also has been

extensively studied, which revealed that almost all

leukemia carry

a

karyotype abnormality.

Hematological

ma

lignancy

particularly

leukemia

is

still

a

problem in Indonesia.

Cases

recorded

at

Dr'

Sardjito

Hospital

in

the Yogyakarta province from

1987

to

1990 included Hodgkin lymphoma (6.29%),

(2)

VoL 3, No 4, October-Decenùer 1994

non

Hodgkin

lymphoma (46.85%),

mulriple

myeloma

(3.14%), Iymphoid

leukemia

(2t.86%),

myeloid

leukemia (19.8670) and

other types

of

leukemia

(1.987o).' The frequency

of

nralignancy

cases

from

year

to

year was increasing.

In

1987

there

were

884 cases,

in

1988 I 164 cases,

in

1989

ll90

and

in 1990

1282 cases.

The

total

number

of

malignancy

cases at

Dr.

Sardjito

Hospital

Yogyakarta

*.ré

+SZO

"ur"r.4'5

In

Semarang,

Sunjoyo reported that there were

84

cases

of

hematological

malignancies

between

1984-1986, included

non Hodgkin lymphoma (42.9%),

myeloid leukemia

(32.47o),

lymphoblastic

leukemia

(17.27o),

multiple

myeloma (3.7%) and Hodgkin

lymphoma (2.3%)."

The diagnosis

of

leukemia

in yogyakarta or

In-donesia

in

general is based on the morphological

find-ing

of

blood

lineage

cells,

in blood or

bone marrow

smear stained

with routine staining

such as Giemsa,

Wright, May Grunwald,

and examined_under

regular

light

microscope

(FAB

Classification).5

Therapy using standard

regimen therapy showed

no satisfactory

results;'

this may be due to the

timing

of

patients

arrival at the hospital. Usually

_patients came to the hospital at the terminal state

of

the disease

or might

be due

to

innaccuracy

of

diagnosis.

In

lggg

a

group

of

hematologist

proposed a

new classification

of

leukemia

based

on morphology,

immunophenoty-ping

and citogenetics (MIC).7 Classification

which

merely

based

on_morphology could not classify

_the

type

of

leukemia.T

Using cyiàgenetic analysis

will

in-crease the accuracy

ofth

_erapy

can be-determined

and

edict-ed.8'e'lo

In

Indonesia,

th

using

cytogenetic analysis

has

The aim

of this

study is

to analyse the

cytogenetic

pattern

of ALL

patients

in

yogyakarta and

Central

Java,

Indonesia,

in

the hope that therapy could

be

correctly given

to the patient and prognosis

of

the disease can be

predicted.

I\{ATERIALS

AND

N,TETHODS

Samples

One

ml of

heparinized peripheral blood

_{and 0.5}

ml

of

bone marrow aspira{e

fronr patients diagnosed

as

leukemia

at Dr.

Sardjito Hospital

(yogyakarta),

Kariadi

Hospital

(Semarang), and Telogorejo Hospital (Semarang).

Methods

Arresting

: Colcemid was used

in this investisation

as

arresting agent to stop

mitosis

at

metaphas".S

Col".-ALL C1'togenetic

Pattenr

201

mid

is a

mitotic

spindle

inhibitor,

which

can

block

the

formation

of

spindle

fibres. Normally, spindle fiber

was attached

to

the centromere

of

each

chromosome

before

mitotic division

and

pull

the chromatids

apart

by contraction

at anaphase,r

Hypotonic Shock

: The

hypotonic solution

used was

0.075M

KCL

since it has been

known,

that this solution

is the least damaging to chromosomes

substructure.

A

hypotonic

solution

is

a

solution with

a lower salt

con-centration

than

that

of

the cytoplasm

of

cells,

so there

will

be a movement

of

HzO

from

the hypotonic

solu-tion into

the cell.3

S

rxatron :Theïxahve

so)utlon ,rru,

ïr"ù)y

,nuà"

ol3

parts

of

absolute methanol

to I

part of glacial

acetic acid. The

volatile

alcohol

plays a

role in flattening

and spreading the chromosonres

during air

drying.

Glacial

acetic acid

will

rupture

the membrane

of

the cell.

Slide

Preparation

and

Staining

: Slides were washed

in

distilled

water,

dried

at

room temperature

and

stained

with Giemsa.ll

Giemsa

Staining and Banding

Giemsa staining is a method to stain the chromosomes.

Giemsa stain

is a

Romanowsky-type

dye

mixture.

Azure B

and eosin in the Giemsa bound to

DNA.ll

The

solutions

needed are :

l.

Giemsa

stain

concentrate, made

by

aclding 1.0 g

Giemsa

powder

to

66

ml

methanol and 66 ml

glycerin, stirred

for 2

days

at room

temperature,

prepared

at

least

2

weeks

befôre

use. Stored

in

a

dark container

in

a refrigerator.

2.

Phosphate

buffer (pH

6.8), which

consists

of

0.025M

KHzPO+

(3.4 gll),and

rhe

pH

was adjusted

using 50% NaOH.

Giemsa

staining solution

was prepared by

nrixing

5

ml

of

Gienrsa

stain

concentrate and

45 ml of

phosphate

buffer (pH

6.8).

Giemsa banding

(G

banding)

is

a

nrethod to

ob_

tain

bands

in

chromosomes

by treating

slides

with

a protease

such as

trypsin or

incubate the slides

in

hot

salinecitrate. The

chromosome

banding pattern

ob-tained reflects both

structural

and

functional

composi-tion of

the chromosonl.r. lo'l I

Solutions needed are

:

l.

Giemsa

stain concentrate

2.

Phosphate

buffer (pH

6.8)

(3)

-202

HarT,ana et al.

Working staining solution

is prepared

by mixing 26 ml

buffer (pH

6.8), 7 ml methanol, 0.6-l.0 ml lOX trypsin

EDTA

and

0.8 ml

Giemsa

stain

concentrate,

Chromosome Analysis

Chromosomes are prepared by

direct

bone

marrow

and

peripheral

blood culture developed in our laboratory.

Marrow cells were cultured

in RPMI-1640

medium

supplemented

with

75To fetal calf serum. Only + g. ₁

ml of

sedimented cells were processed

from

each

centrifuge tube.

The cells were

exposed

to colcemid

(0.06 mg/ml)

for 25 minutes and then

to

hypotonic

solution

i.e.

KCI

(0.075

mol/l) for a total of 32 minutes

(including mixing,

standing and centrifuge time).

Slides were

fixed in 3:

I

methanol

acetic

acid (v/v)

and

dried on a hot plate. Slides were subjected to

Giemsa

staining

and

banding.

For each

case, 10-20 metaphases

(mean

l5)

were

studied by direct microscopy

to determine the modal

number and to

identify the

malignant

stem

line.

Karyo-type

were prepared

from

photographic prints.

Chromosomal abnormalities

were

classified according

to the

International Conventions.l0'

I l

' 12 The

definition

of an abnormal

stem

line

was

defined according

to that

proposed

by the

Second

International workshop

on

chromosome

in leukemia.

A

case was

considered

ab-normal

if

it

had

an

abnornral clone,

regardless

of

the

proportion of normal

metaphase. l3

RESULTS

The patients were

diagnosed when

they

were

hospitalised. There

were

14 patients

analysed

cytogenetically. Out of those 14 patients 8

were males

and 6 were females

(Table

l).

The range

of the age

was between 2-2O yearc

old.

There were 4 patients under 5,

7

patients

between 5

and 12,

and 3 patients more than

12

years

old

(Table 2). The diagnosis was

based on

FAB classification (morphology)

by'Wright

or Giemsa

staining.

Table 1. Patients with

ALL

Sex

Med

J Univ lndon

The

cytogenetic

analysis on

peripheral blood

and

bone marrow

samples

were carried

out on at least

10

metaphases

from

one

slide. The findings

were as

fol-lows (Table 2)

:

-

1

patient

of

46

XY, t(4;ll)

(q21;q23)

from

peripheral blood

and 53

XXY

from

bone

marrow.

-

I

patient

of

46 XY t(9;22) (q34.1;qI1.2l)

(Phila-delphia chromosome

positive)

from peripheral

blood

and 56

XY

from

bone

marrow.

-

I patient of t(9;22) (q34.1;q11.21)

fromperipheral

blood,

Ph+

from

bone

marrow.

-

7 patients

of

abnormality in chromosome number

from

bone

marrow

i.e. 52

XY

mozaic,

91 XX

polyploidy

mozaic, hyperploidy

>

50

chromo-somes

mozaic, 87 XY polyploidy

mozaic (257o),92

XXYY

tetraploidy mozaic, and monosomies

45

XX-7

and 45

)tY-17

.

-

4 patients showed no structural and numerical

chro-mosome

abnormality.

Tablc 2. Agc, Scx, Cytogcnctic l'inding ol ALL

No.of Agc

Scx

paticnt (ycas)

Cytogcnctic

linding

Sourco

20 6

ll

4 9 6.5 M M M F F F M M

46 XY, nrozaic, -52

XY

BM

46 XX, nrozaic,

9l XX

plyploidy

BM 46 XX

46 XY i(4;l | )(q2 I .q23)/s3

XXY

P/BM

(4+ l;5+ l;6+ I ;l I + l;l 6+ I ;2 l+ I _;X+t)

45

XX-7

BM

46 XY t(9;22)(q34.l.ql l.2l)(Ph+)/ P/BM

56 XY hypcrploidy (5+l)(6+l)(8-l)

( l3+ l)( l4+ l)( l6+ l)(17-l)(18+2)

(2 t+3)(22+2)

46 XY, hypr:rploidy > 50 Chromosomc BM 46 XY

46 XY, 87 XY polyploi<Iy

25%

BM a6 xY r(9;22)(q34. l.q I 1.2)iPh+

46 XX 46 XX

46 XX,92 XXYY tclraploidy

4-5 XY- l7 dcl I I ,p I 3-p 14

P/BM

BM =

sourcc of karyotyping analysis is l'rom tronc ntarrow

P/BM

=

sourcc of karyotyping analysis is liont krnc nrarrow and pcriphcral bltnd

=

nralc

=

fcnrale

t4M

24F

35F

46M

55F

63M

% 20 t9 7. 8. 9. 10.

I l.

t2. t3. 14. Male Female 57.1 42.9 BM BM Total M F n = number of patienl

(4)

Vol 3, No 4, October-Decenber 1994

Table 3. The Impact of cytogenetic finding and outcome

No. of

patient

Cytogenetic finding Outcome

ALL Cytogenetic

Pattern

203

abN =

abnornral

ND = no lollow

up

str

= slruclural

N=normal

D=dicd

num=nunrcrical

No follow up paticnts (21.a%); complctc renrission (35.7%) and dicd (42.86%)

DISCUSSION

The data obtained

from

the

Third

International

workshop on

chromosome

in Leukemia,

comprising

330 children and adults

with

ALL

showed

that

the

specific

karyotype

patterns

provided significant

inde-pendent

prognostic

information,

in regard to

remission

duration

and

survival.

l0' I I'13'14'

Our

investigation consisted

only

of l4

ALL

patients,

which

was

very small to

asses

the impact

of

the karyotype on

the progressiveness

of

the

disease,

But

we hope

it

might give

a general idea

of

the pattern

of

cytogenetics

in

Central

Java

and

Yogyakarta

province.

Our findings

showed several translocations.

Translocation

of

t(4;11)

(q2l;q23)

was found

in

1

patient. This type

of

translocation was common in

congenital

ALL

and

ALL

in

young

infants.ls

Translo-cation

of t(a;l1)

has

als_o

been reported related

to exposure to carcinogens. to

T*o

patients showed

trans-location of t(9;22) (q34.I;ql1.2l) called

ph + ph

+

ALL

besides

CML

(chronic

and about 6% occurs

in

ion.of

(4;l l)

and

t(9;22)

indi-.ir.

14'16

Those patients

after

an

inductive remission showed a partial remission

but then they were

hospitalized

with

CNS (central

nervous

system) leukemia and

died

of

cerebral bleeding.

Hy-perploidy

with

chromosome number

of

470- 50 or > 50

is conrmonly

p.ssociated

with B

lineage

and rare

in

T-lineage ALL.|4

Some

of

the patients-also showed

hy-perploidy

more than

50

chromosome,

but we

are not

sure whether

these

patients were

of B

lineage,

since

immunophenotyping

has

not

been

performed.

Hyperploidy

indicated

good

prognosis especially

in

mozaic

with normal

karyotype.E'e

In

this study we

found five

patients

with

normal karyotype

or in

mozaic

with hyperploidy,

achieved complete remission;

they

were

3

patients

with normal karyotype,

I

patient

with

normal karyotype and hyperploidy (mozaic) and

1

patient

with polyploidy.

One

patient

with

monosomi-7

achieved

short

remission and then

died. While

3

patients

which

consists

of

I

patient

with

hyperploidy

(mozaic),

I

patient

with

polyploidy 25%,1

patient

with

tetraploidy (mozaic) could not be followed up.

This

finding

indicates that cytogenetic analysis

has an

im-portant role

in predicting the prognosis

and

also

in

determining

the

high risk

group

from

general

popula-tion.

Patients

with

chromosome

abnormalities

espe-cially with

translocation

or

deletion

of

whole

chromo-some

(monosomy) should

be

categorized

as

high

risk

patient eventhough

Hb, WBC

and tota.l

platelets

were

in

norn.ral range,

and regimen therapy

chosen

should

be

given intensively.

l.

2. 3. 4. 5. 6. 7. I 9. 10. 11. 12. 13.

t4.

abN (num) abN (num) N

abN (str+nunr) abN (num) abN (str+num) abN (num) N abN (num) abN (str) N N abN (nurn) abN (str+nurn)

ND Remission Rernission D Remission

-

D D Remission D ND p.Remission

-

D Remission Remission ND D

-

N

- rcmission -

abN

_{- rcnrission}

-N

-dicd

-

abN

- died

3 paticnts : 21.4%

3 paticnts:21.4% I paticnt

:

7.14%

5 paticnts : 35.'/ l%

Table 4. Age and karyotype abnorrnality

No.of patient Age Karyotype N/abN

l.

2. J. 4. 5. 6. '7. 8. 9. 10. 11. t2. 13.

t4.

4 4 5 6 5 3 20 6 11 4 9 6.5 20

l9

abN (nurnerical,gain) abN (numerical,gain) N abN (structural+numerical,gain) abN (nurnerical,loss)

abN (structural +numerical,gain) abN (nurnerical,gain)

N abN (nurnerical,gain) abN (structural) N N abN (nurnerical,gain) abN (strtrctural+numerical,loss)

Abnornral karyotypc : Age undcr-5 ycas : 4 paticnts (40%)

-5 - l2 ycani : 3 palicnts (30%)

> 12 ycan : 3 paticnts (30%)

Norrnal karyotypc : Agc untlcr -5 ycars : - (0%)

5 - l2 ycars:4 paticnts (100%)

> 12 ycars: - (o%)

(5)

Haryana et al. Med

J

Univ Indon

È.ia CM I "'- '

-Ten$^l : l'*::*:

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*24''.-Figure 1. Karyory^pe of an ALL-LI patient showing a) 46 XY,

t(4;lI)(q2l;q2j)

and

b) 53 XXYfrot,t bone nnrrow culture

Karyotyping was done _frontG-banded chro,,toso,ile

t

:

translocation q

:

long ann of the chronosonte G

:

Giensa

ALL-LI

:

Acute lynrphoblasric leukenia type L-1

,nnêe?./,

, 'ç21 ---22... y. !

Nart

lJ niur Àiâù01

No cM : :l:'7t'i'jt1) iiq. Ftâp :

[image:5.595.83.548.61.639.2]

(6)

Vol 3, No 4, October-December 1994 ALL Cytogenetic

Pattern

2O5

ll{ilrl ùocM I 15/ft./92

inorial I 9-:.1-199A

rril

ff *,

r{

** *t

tâ.

r*

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r

f

l,

.-,

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*l

r

Ê

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;t

r{

t*

al tô 1É

rr

'.

la

,;?1 - 22 <+

l\i êrê :

1.)n1$ . 3 tsà6

Àlêr?rl

t4â *ftt n n ,l 4 Ç

-- * t? -- ;q - '- -- É 21 '*- 22

--.-î\Ttaft? :

r,r

ln. èM l{o. Êr(}r 7 at'ûal

2ç'fttl91

)*t

-9?

Figure 2. Karyoq.pe of an ALL-Ll patient shov,ing

a) 46 XY,

(9;22)(q34;qIt.2I)

(Ph+) and b) 56 XY (poliploidl,)

Karyoq'pittg was done _frontG-banded chronosone

I

:

translocation q

:

long ann of the chronosone (Ph+1

:

Philadelphia chromosone positive

G

:

Gientsa [image:6.595.114.521.67.664.2]

(7)

206

Haryana et aI. Med

J

Univ Indon

tldiljù : 11o DM : l:;/'ii/'j'i

'Ja1$ : J'it'.Hrii: Nc ?r':P :

Llatttt : \arçgal '".'-:l:eti|

Figure 3. Karyotlytittg of two ALL-LI patient showirtg a) nonosony 46 XX-7 and

b) nonosony 45

XY-I7

karyo4, pittg was done.froilt G-banded chroiltoso,,rc G

:

Giensa

ALL-LL

:

Acure lyntphobkLstic leukenia _\'pe

L-l

41 , _. un I

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.: r; i)

*& *t #*

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*{

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à

F19 2B _{ç* -." ZZ} _r.. _y

al.çtiJTi? :

ljt !:i4 :

[image:7.595.64.517.59.704.2]

(8)

Vol 3, No 4, October-Decetttber 1994

CONCLUSION.

Our results

suggested

that

multiple

abnormalities

and

monosomies

indicate poorer prognosis

compare to

hy-perploidy, poliploidy

and

normal

karyotype.

Patients

with

translocation

or deletion

cf

whole

chromosome

should be

categorized as

high

risk

patients regarldless

the

Hb,

V/BC

and

platelets

count.

Acknowledgement

We

thanked

Prof

T.Tokuhisa, Kobe

University

Medi-cal

School

for his

valuable correction

and

critics.

We

also

thank

the assistance

of Wiwied,

Suwarsih, Maya

and

Ani for

the

technical

works;

Sudaryono

for typing

the manuscript; the Director

of

Telogorejo

Hospital

and the Director

of

the

Live

Science

Laboratory

of

Gadjah

Mada University

for

providing the facilities

needed

for

this

work.

To

Prof.

Dr. A.G.

Sumantri,

Faculty

of Medicine

Diponegoro

University,

who

has

been

very supportive

in

managing

the

collaboration

between

GMU

and

Diponegoro

University,

we

wish to

express

our

sincere thanks.

We

also express

our

gratitude

to

DP4M for

rhe granr

No. 33g/p4M/DppM/

L-331llBBIl1992;

and

grant

from UGMl34l5t

M/09/01,

June

1993.

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