C o m b in e d m e th o d fo r s im u lta n e o u s m o rp h o lo g y ,
im m u n o p h e n o ty p e
a n d k a ry o ty p e (M A C ) in le u k e m ia s
Discipline of Hematology
and Hemotherapy,
Escola Paulista de Medicina-UNIFESP-
São Paulo, Brazil
In th e p re s e n t s tu d y , a c o m b in e d m e th o d (C M ) fo r a tta in in g s im u lta n e o u s id e n tific a tio n o f le u k e m ic c e ll m o rp h o lo g y , k a ry o ty p e a n d im m u n o p h e n o ty p e h a s b e e n e v a lu a te d in 2 1 p a tie n ts w ith a c u te le u k e m ia a n d 1 w ith C M L in b la s t c ris is w e re s tu d ie d fo r m o rp h o lo g y , c ito c h e m is try , im m u n o p h e n o ty p e a n d k a ry o ty p e . K a ry o ty p e w a s p e rfo rm e d in a b o n e m a rro w s a m p le b y u s in g c o n v e n tio n a l te c h -n iq u e s . I-n e a c h c a s e , d ire c t m e th o d (D M ) a -n d /o r th re e c u ltu re s w e re trie d . T h e C M c o -n s is te d i-n s e p a ra ti-n g a s m a ll p a rt o f th e m a te ria l re s u ltin g fro m a n y o f th e c u ltu re s o r D M , p re p a rin g s lid e s th ro u g h c y to s p in a n d im m u n o p h e n o ty p in g th ro u g h A P A A P m e th o d u s in g th e s a m e m o n o c lo n a l a n tib o d ie s (M o A b ) a s fo r d ia g n o s is . In 1 4 c a s e s , th e m e ta p h a s e s p ro v e d p o s itiv e to th e M o A b : in 4 , th e c e lls w ith a b n o rm a lity h a d th e ir o rig in d e fin e d ; in o th e r 4 th e k a ry o ty p e w a s n o rm a l p re v e n tin g a n y id e n tific a tio n ; 6 c a s e s h a d m in im a l a b n o r-m a litie s n o t v is ib le th ro u g h C M ; a n d in tw o c a s e s a b n o rr-m a l k a ry o ty p e s w e re d e te c te d o n ly in th e c u ltu re s w ith G M -C S F : ' T h is s tu d y s h o w e d th a t C M is fe a s ib le in c a s e s w h e re e v id e n t n u m e ric a l o r s tru c tu ra l c h ro m o s o m a l a b n o rm a ltie s a re p re s e n t.
U N IT E R M S : L e u k e m ia , c h ro m o s o m e s , im m u n o p h e n o ty p e .
IN T R O D U C T IO N
S
le u k e m ia se v e r a l o f th ea r ec y to g e n e tico f c lin ic a I im p o r ta n c e ,a b n o r m a litie s o n c ein a c u teth e y . a s s o c ia te w ith d is tin c tiv e m o d e s in w h ic h th e d is e a s e p r e s e n ts its e lf a s w e ll a s to p r o g n o s tic f a c to r s .1 .2T h e a c q u is itio n o f im m u n o p h e n o ty p in g a n d c y to g e n e tic d a ta h a s le d to a m o r p h o lo g ic ,
im m u n o p h e n o ty p ic a n d c h r o m o s o m a l ( M I C ) c la s s if ic a tio n
3 .4 .5 w h ic h r e la te s m o r p h o lo g ic a l s u b g r o u p s w ith in F A B
s u b ty p e s w ith s p e c if ic k a r y o ty p e a b n o r m a litie s .
M e th o d s th a t a llo w th e s im u lta n e o u s a n a ly s is o f m o r p h o lo g y , im m u n o p h e n o ty p e a n d k a r y o ty p e ( M A C ) h a v e b e e n d e s c r ib e d a n d d e s e r v e p a r tic u la r a tte n tio n . 6 .7 .X .9 .1 O
A d d re ss fo r co rre sp o n d e n ce :
M aria de Lourdes Lopes F errari C hauffaille R ua B otucatu, 740, 3 ºandar
S ão P aulo/S P - B rasil - C E P 04023-006
T h e id e a o f c o n c o m ita n tly s tu d y in g s u c h a s p e c ts is o f m a jo r in te r e s t e s p e c ia lly in le u k e m ia s , a s it c o u ld p o s s ib ly in d ic a te th e c e ll lin e a g e r s w h ic h p r e s e n t c h r o ln o s o ln a l a b n o r m a litie s . I t m a y a s w e ll in d ic a te th e in v o lv e ln e n t o f d if f e r e n t c e lllin e a g e s th u s c o r r o b o r a tin g th e h e te r o g e n e ity o f le u k e m ia s . lO A n d f in a lly , th e c o m b in e d I n e th o d ln a k e s
th e a n a ly s is o f a p a r tic u la r c e ll p o s s ib le .
T h e m a in d if f e r e n c e s o f th e c O ln b in e d ln e th o d w h e n c o m p a r e d to th e c o n v e n tio n a l te c h n iq u e a r e : h y p o to n ic tr e a tm e n t ( u s in g d is tille q w a te r in s te a d o f K C l) , w h ic h le a v e s c y to p la s ln a n d c e ll m e m b r a n e in ta c t, th r o u g h th e u tiliz a tio n o f c y to s p in in th e p la c e ' o f a ir d r y in g S ln e a r s a n d th e a b a n d o n o f a c id f ix a tiv e s . lO
F e w s tu d ie s o n th ~ a p p lic a tio n o f th is m e th o d to le u k e m ic s a m p le s h a v e b e e n p u b lis h e d u p to th e p r e s e n t m o m e n t, m o s tly a n a ly s in g c a s e s w h e r e n U ln e r ic a l c h r o m o s o m a l a b n o r m a litie s o c c u r r e d w ith r e s u lts o f ln u c h c o n s e q u e n c e .9•IO B y m e a n s o f th is te c h n iq u e th e
in v o lv e m e n t o f m u ltip le lin e a g e s ( i.e . e r y th r o c y tic , m y e lo m o n o c y tic a r m e g a k a r y o c y tic ) h a s b e e n
elelTIOnstrateel in some cases of acute myeloiel Ieukemia
(AML) with lTIOnOsomy 7. However,
not alI the cases of
AML with trisomy 8 elemonstrateel such involvement.
7We believe that the regular application of this lTIethoel,
though
tilTIe consuming,
woulel proviele
invaluable
information
on leukelTIic cell origino Thus, we inteneleel to .
elevelop anel evaluate the feasibility ofthis CM as a regular
proceelure,
using
a alkaline
phosphatase
anti-alkaline
phosphatase
(APAAP)
kit.
M A T E R I A L A N D M E T H O D S
Twenty-one patients with acute leukemia (18 AML, 2
acute IYlTIphoielleukelTIia - ALL- anel 1 lTIixeelleukelTIia)
anel 1 with chronic myeloiel leukelTIia (CML) in blast crisis
who
were
aellTIitteel to Hospital
S. Paulo
have
been
investigateel, or those whose samples (cases 16 anel 20) had
been sent to us for analysis, from June to December
1992.
The average age of these subjects was 32 years (ranging
frOlTI2 lTIonths to 65 years), being 9 male anel 13 felTIale.
The acute leukemia
was diagnoseel in the presence
ofblast cells in the patients'
peripheral blood or more than
30% of thelTI in bone
marrow.
11.12 Bone
lTIarrOW and
peripheral blood Sl11earSwere stained for Leishman, SuelalTI
Black B (SBB), Peroxielase
(POX), periodic
aciel Schiff
(PAS),
naftol-ASD
esterase
(ASD),
alfa naftil acetate
esterase
(aES),
NaF anel acid phosphatase
(Fác)13 for
lTIorphological
and cytochemical
diagnosis
of the acute
leukemia
and classified
according
to FAB.
1 1 . I 4 . 1 5 . lóBone lnarrow were collected with heparin, separated
by Ficoll Hypaque
(Phannacia
AB/ Wintrobe)
gradient,
and
then
divided
for
3 different
purposes:
for
imlnunophenotyping,
cytogenetic
and CM.
Immunophenotyping
was performed
by indirect
ilnlnunofluorescence,
using
the following
MoAb:
CD
33(My 9), CD 13 (My 7), CD 14 (Leu 9), CD 19 (B4), CD
41 (plt I), CD 10 (calla), SIg (Coulter) and CD 7 (Dako,
Beckton&Dickson)(Table
I). Positivity
was considered
when more than 20% of cells were stained. Bone lnarrow
cells separated by Ficoll-Hypaque
gradient were washed 3
tilnes in phosphate
buffered
solution (PBS), resuspended
in RPMI
1640 medium (final concentration=
2x 1Oócells/
lnl) and divided into different tubes with mouse antibody
and incubated for 30 lTIinutes at 4°C. Then fIuorescent
anti-1110use antibody was added for 30 min in a cold CalTIera.
The ceIls were washed in PBS for 2 tilTIeS,the slides prepared
and 100 ceIls were counted in fluorescent
microscope.
17For cytogenetic
and CM studies, bone lnarrow cells
were cultivated
in a 25 ClTI3tissue culture flask with Sml
RPMI 1640 (Sigma) medium, pH
7.0,200/0
fetal calf serUln
(Cultilab),
L-glutamine
2mM
(Sigma)
and
penicillin-streptomicin
(1 OOU; 1OOug/mI). Depeneling on the amount
of lTIaterial obtained
(visual observation),
2 or 3 cultures
were set up: a short term culture (S), a synchronised
culture
(MTX),
a culture
with GM-CSF
(GM)
and/or
a elirect
methoel (DM). The cultures were prepareel as follows:
S
culture: was cultivated for 24 hours at 37°C; MTX culture:
ametopterine
(MethotrexateR-Leelerle)
was aelded 5 hours
after set up (final concentration=10-
7M) for 17 hours at
37°C
anel
thymidine
(Sigma)
was
added
(final
concentration=
10-
5M) for the last 5 hours; GM-culture:
GM-CSF (Amgen)(1 Oul/ml) was aeleleelfor 24 hours; DM:
colchicine
(Colcemiel-Gibco)
was ilTIlTIediatly added.
'x
Colchicine
(0,4ul/ml)
was added for the last two hours to
alI cultures anel then the material was spun at 1200r/nlin
for 8 min anel the sobrenaelant elischarged. For the CM 5,
elrops (Pasteur pipette) were taken out froln the lTIaterial
left from each culture or DM and put in 0.51TIIhypotonic
solution (elistilleel water). The cells were counteel in Cehn
CC 510
eletronic
counter
and
adjusted
to a final
concentration
of 2 to 20xl03 cellshTIlTI.3 50 uI of this
solution was put into cytospin CalTIeraS and spun at 400r/
lTIin for 4 mino Meanwhile,
cells
were being
counted
(around 5 to S minutes) hypotonization
was taking place.
The slides were fixeel in cold fonTIalin-acetone
solution
for 1 lTIin at the moment of use and then washed
in Tris
buffered
solution
(TBS) pH 7.6 with 2.5% hUlTIan AB
serum (4:1) for 40 mino Then 20 uI OflTIOUSetnonoclonal
antibody
(diluteel 1: 10) was dropped
over the cells and
incubated
for 60 lnin. After TBS wash rabbit anti-tnouse
Ig antibody (Dako Z 259) diluted 1: 10 in TBS with hunlan
AB SerUlTI(4: 1) for 40 min was added. Another TBS wash
and the APAAP complex
(Dako D 651) was put over the
celIs for 40 lTIin diluteel I :50 in TBS. After wash alkalin
phophatase
substrate
was added
diluteel
in TBS
with
levamisole
I mM for 20 mino After final wash, the slides
were dried and stained for Harris Helnatoxilin
for 5 lnin.
Blast celIs positivity
was observed
at the lnicroscope
as
welI as positive and negative lnetaphases.
Metaphases
that
had chromosomal
abnormalities
were identifieel. Positive
control was done with lnarrow slides frotn the satne patient.
The specimens
left over frotn the cultures
or DM were
handled
for conventional
cytogenetic
analysis,
that is:
hypotonization
with KCI 0.075M
(I Otnl) for 20 tnin,
fixation with methanol-acetic
acid solution (I :3)( 1OtnI), 3
washes
in fresh made fixative,
anel in the next elay the
slides
were
made,
aged
and
bandeei
for trypsin
G
banding.19.
Cytogenetic
abnonnalities
were elescribeel
following
the International
Systetn
for Chrolllosollle
Nomenclature
recotntnendations.20
C H A U F F A I L L E , M . L . L . F . ; C O U T I N H O , v . ;Y A M A M O T O , M . & K E R B A U Y , J . - C o m b i n e d m e t h o d f o r s i m u l t a n e o u s m o r p h o l o g y , i m m u n o p h e n o t y p e a n d k a r y o t y p e . ( M A C ) , i n l e u k e m i a s
RESULTS:
T ab Ie 1 sh o w s th e cIassificatio n o f leu k em ias, cy to ch em ical reactio n s an d im m u n o p h en o ty p e. C ase 1 w as a M 2 A M L w ith ch aracteristic m o rp h o Io g y sh o w in g A u er ro d s an d p seu d o -C h ed iak i-H ig ash i an o m aly . T h e
k ary o ty p e sh o w ed Io ss o fY ch ro m o so ln e an d tran sIo catio n t(8 ;2 1 ), an an o m aIy seen in aro u n d
120/0
o fM 2 leu k em ias. C ase 2 w as an A L L in reIap se w ith a h y p erd ip lo id k ary o ty p e an d G b an d in g w as n o t o b tain ed d u e to fu zzy ch ro m o so m es. C ase 3 w as a ty p icaI M 3 w ith t( 1 5 ;I 7 ). . C ases 4 an d 5 w ere M 4 an d M 2 Ieu k em ias resp ectiv ely ,w ith a v ery p o o r b an d in g m ak in g it im p o ssib le to fin d
Table 1
C ytochem ical reactions, FAB classification and im m unophenotype of the leukem ias
CASE DIAG NO STIC PO X SBB PAS aES NaF ASD AcF CD33 CD13 CD14 CD10 CD19 CD7 CD41 Slg
1 AM L M 2 99°10 100°10 10°10 95°10 95°10 72°10 52°10 25°10 26°10 13°10
o
o
o
o
2°1o2 ALL L 1 r - NEG 10°10 - - - - O O O 70°10 78°10 <1 °10 <1 °10 O
3 AM L M 3 100°10 100°10 DIF NEG NEG - - 92°10 86°10 1°10 100010d 3°1o <1 °10 <1 °10 O
4 AM L M 4 40°10 44°10 NEG 58°10 inhib - - 94°10 77°10 64°10 1°10 8°1o 10°10 3°1o 11°10
5 AM L M 2r 60°10 70°10 DIF TEC TEC - - 87°10 76°10 O O O O O O
6 AM L M 1 NEG 96°10 NEG NEG NEG 10°10 NEG 70°10 37°10 O O O O O O
7 AM L M 2Eo 100°10 100°10 NEG - - - + + O O O O O O
8 AM L M 1 40°10 90°10 NEG 30°10 30°10 - NEG 45°10 44°10 5°1o O O O <1 % O
9 AM L M 3v 98% 99°10 DIF DIF DIF - NEG O 40°10 O O O O O O
10 AM L M 4L+ 71 °10 79°10 10°10 69°10 inhib 5% - 61 °10 15°10 60°10 O 23°10 O O O
11 ALLL1B NEG NEG NEG - - - NEG O O O 63°10 25°10 15°10 O O
12 AM L M 3 100°10 100°10 DIF 85°10 85% - - 89°10 18°10 O O O O O O
13 AM L M 4 5°1o - - 51 °10 inhib 9°1o - 15°10 48°10 28°10 <1 °10 <5°1o 4% 1°10 O
14 AM L M 1 90°10 100°10 NEG NEG NEG NEG 79°10 41°10 <1 °10 O <1 °10 45°10 <2°10 O
15 AM L M 1 98°10 99% 2°1o NEG NEG 1°10 - 77°10 <5°1o <5°1o O O 40% O <5%
16 M ixed L NEG NEG 39% 55°10 inipar NEG - 24% 22°10 <5°1o 37°10 35°10 12°10 <2°10 11°10
17 AM L M 2 84°10 96°10 DIF NEG NEG 50% NEG 21 °10 22°10 3°1o O 8°1o 1°10 1% O
18 AM L M 4 5°1o 20°10 DIF 56°10 inhib - - 66°10 4% 16°10 O O O O O
19 AM L M 1 94°10 100°10 DIF DIF DIF 23°10 - O O O O O O O O
20 AM L M 4Eo 63°10 92°10 NEG 70% inhib 21 °10 - 26% 27°10 2°1o O O 11°10 O O
21 CM L M BC - 79°10 NEG - - - - 67°10 31 % 6% O O 11°10 O O
22 AM L M 2 55°10 40°10 NEG NEG NEG - - 41°10 24°10 21 °10 O O 10°10 O O
aES= Alfa naftil acetate esterase
SBB= Sudam Black B
- = reaction not done
CD=cluster designation
DIF= difuse positivity
AcF= acid fosfatase
inhib= reaction inhibited
M 4L+= M 4 with Iym phoid com ponent
ASD= naftol ASD acetate esterase
TEC= technical problem
0= negative result
M BC= m yeloid blast crisis
Eo= eosinophilia
inipar= partial inhibition
L+= Iym phoid com ponent
r= relapsed leukem ia
Slg= surphace im unoglobuline
PAS= Periodic Acid Schiff
M 3v= M 3 hypogranular variant
NaF= inhibition by NaF
PO X= peroxidase
N EG = negati.ve reaction
São Paulo M edicai Journal/RPM 115(1): 1336-1342, 1997 CHAUFFAILLE, M .L.L.F.; CO UTINHO , v.;YAM AM O TO , M . & KERBAUY, J. -Com bined
A m o n g th e 2 2 p a tie n ts stu d ie d , 1 8 w e re A M L , b e in g M l, M 2 a n d M 4 e q u a lly fre q u e n to F e w c a se s o f A L L in
th is se rie s is e x p la in e d b y th e p re p o n d e ra n c e o f a d u lt
p a tie n ts a d m itte d to o u r in firm a ry .
T h e re w a s 6 3 % o f k a ry o ty p e a b n o n n a litie s. In T h e
F o u rth In te rn a tio n a l W o rk sh o p in L e u k e ln ia s,2 4 5 0 % o f
A M L p re se n te d k a ry o ty p e a b n o rm a litie s. In la te r stu e lie s,
a p p ro x im a te ly 7 0 to 8 0 % o f c a se s o f A M L a n d 5 0 % o f
A L L h a v e c lo n a l a c q u ire d c h ro m o so ln a l a b e rra tio n o f
le u k e m ic c e llS . 1 .2 4 .2 5 .2 6 .2 7
M A C m e th o d id e n tifie s th e In a lig n a n t c e ll a n e l
d e te rm in e s its lin e a g e sim u lta n e o u sly in th e in ta c t c e lls.
T h is h a s b e e n w e ll d e m o n stra d e d b y o th e r a u th o rs7 .~ .I()
U sin g M A C m e th o d w ith a n A P A A P k it, w e w a n te e l
to c h e c k its fe a sib ility a s a ro tin a ry p ro c e d u re . W e c o u le l
d e fin e th e lin e a g e a s m y e lo ie l in k a ry o ty p ic a lly a b n o n n a l
c e lls o n ly in fe w c a se s a m o n g 2 2 c o n se c u te v e ly stu e lie e l.
It b e c a rn e e v id e n t th a t c a se s w ith n o rm a l k a ry o ty p e a re
n o t su ita b le fo r th is p ro c e d u re sin c e th e re re m a in s a s to
d o u b t w h e th e r th e c y to g e n e tic a lly n o rm a l c e ll b e lo n g s to
th e m a lig n a n t c lo n e o r if it re p re se n ts th e n o n n a l re sid u a l c lo n e . In c a se s 1 5 a n d 1 8 , w ic h d id n o t p re se n t k a ry o ty p e a b n o rm a litie s, w e o b se rv e d th a t a ll m e ta p h a se s in th e slie le s
w ith C D 1 3 a n d C D 3 3 w e re p o sitiv e to th e se In y e lo id
m a rk e rs. S o w e c o u ld im a g in e th a t in th e se c a se s n o n n a l k a ry o ty p e w a s re p re se n tin g m a lig n a n t c lo n e . A lth o u g h o n e
c a n n o t b e c e rta in th a t c y to g e n e tic a lly n O rIn a l c e lls iso la te d
stru c tu ra l a b n o rm a litie s. C a se 6w a s a M l
le u k e m ia w ith a n o rm a l k a ry o ty p e . C a se 7 w a s a M 2 A M L w ith 8 % o f m a rro w
e o sin o p h ils a n d sh o w e d a n o e n tire ly
n o rm a l k a ry o ty p e . C a se 8 w a s a M l
su b ty p e w ith lo ss o f p a rt o f th e lo n g a rm
o f c h ro m o so ln e 5 . C a se 9 w a s a v a ria n t
M 3 b u t t(1 5 ; 1 7 ) w a s n o t se e n . C a se 1 0
w a s a n in fa n t w ith le u k e m ia p re se n tin g
t( 4 ; 1 1 ) a n d c la ssifie d a s m o n o c y tic
su b ty p e w ith ly m p h o id c o m p o n e n t
fo llo w in g H U R W T Z & M IR R O 2 1
c rite ria . C a se 1 1 w a s a B c e ll A L L su b ty p e
L I w ith lo ss o f a c h ro m o so m e o f g ro u p
C a n d tra n slo c a tio n in v o lv in g lo n g a rm
o f c h ro ln o so m e 1 . C a se 1 2 w a s a M 3 w ith
d e le tio n o f p a rt o f lo n g a rm o f
c h rO ln o so m e 6 a n d a d d itio n a ly , in so m e
m e ta p h a se s m o n o so m y 5 . C a se 1 3 w a s a
Figure 1 - E x a m p le o f c a s e 2 1 s h o w in g a m e ta p h a s e p o s itiv e to C O 3 3 . T h e a r r o w
M 4 le u k e m ia w ith m o n o so m y 7 in a in d ic a te s th e P h ila d e lp h ia c h r o m o s o m e . P o s itiv e a n d n e g a tiv e in te r p h a s e s a r e a ls o
p a tie n t w ith p re v io u s e x p o sitio n to to x ic s e e n .
a g e n ts. C a se 1 4 w a s a M l w ith m a te ria l
a d d e d to th e sh o rt a rm o fc h ro m o so m e 1 6 . C a se s 1 5 a n d
_D_IS~~_U_S~S_I~~N_:~~~~~~~~~~~~~
1 8 w e re M l a n d M 4 re sp e c tiv e ly , w ith n o rm a l k a ry o ty p e s.
C a se 1 6 h a s b e e n p u b lish e d a n d w a s a n A L L d ia g n o se d
a n d tre a te d w ith e p ip o d o p h y llo to x in so m e y e a rs e a rly a n d
n o w p re se n tin g re la p se o r a se c o n d a ry m ix e d le u k e m ia
w ith t(9 ;2 2 ) a n d a d d itio n a ly , in so m e ln e ta p h a se s,
ln o n o so ln y 1 3 2 2 .C a se 1 7 w a s M 2 w ith h y p o d ip lo id ia . C a se
1 9 , M l w ith n o rm a l k a ry o ty p e , th o u g h n e g a tiv e sh o w e d a
im ln u n o p h e n o ty p e p re se n te d o v e rt p o sitiv e p e ro x id a se
(9 4 % ). C a se 2 0 w a s a ty p ic a l M 4 w ith e o sin o p h ilia a n d
c e n tra l n e rv o u s sy ste m in filtra tio n a n d in v e rsio n o f
c h ro m o so m e 1 6 . C a se 2 1 w a s a C M L in m y e lo id b la st
c risis w ith o n ly th e p e rsiste n c e o f P h ila d e lp h ia
c h ro m o so m e . C a se 2 2 w a s a M 2 in a 5 3 -y e a r-o ld p a tie n t
w ith a c o m p le x k a ry o ty p e w ith m o n o so m ie s 2 a n d 7 ,
tra n slo c a tio n s in v o lv in g 1q a n d th e p re se n c e o f a m a rk e r
c h ro ln o so m e . S o m e o f th e se p a tie n ts (c a se s:
1 ,3 ,4 ,6 ,1 2 ,1 4 ,1 5 ,1 7 ,1 8 a n d 1 9 ) h a v e b e e n stu d ie d
c o rre la tin g su rv iv a l tÍln e a n d k a ry o ty p e a b n o rm a litie s. 2 3
T a b le 2 sh o w s th e k a ry o ty p e a n d th e re su lts o f C M .
In 1 4 c a se s th e re w a s so m e p o sitiv ity to o n e o r m o re M o A b a p p lie d o n th e In e ta p h a se . B u t o n ly in 4 c a se s (1 3 ,1 7 ,2 1 a n d 2 2 ) (F ig . 1 ) c o u ld w e p re c ise ly d e fin e to w h ic h lin e a g e
b e lo n g e d th e c e ll w ith c y to g e n e tic a b n o rm a lity . A m o n g
th e re stin g 1 0 c a se s, in 4 th e k a ry o ty p e w a s n o rm a l (c a se s
7 , 9 ,1 5 a n d 1 8 ) a v o id in g id e n tific a tio n s a n d in 6 , sm a ll
a b n o n n a litie s w e re im p o ssib le to b e se e n th ro u g h C M
(c a se s 1 ,8 ,1 2 ,1 4 ,1 6 a n d 2 0 ). In c a se s 8 a n d 1 2 , k a ry o ty p e
a b n o rm a lity w a s se e n o n ly in G M c u ltu re s.
C H A U F F A IL L E , M .L .L .F .; C O U T IN H O , v .;Y A M A M O T O , M . & K E R B A U Y , J . -C o m b in e d
m e th o d fo r s im u lta n e o u s m o r p h o lo g y , im m u n o p h e n o ty p e a n d k a r y o ty p e ( M A C ) in le u k e m ia s
Table 2
Karyotype and combined method results
C A S E A R Y O T Y P E C U L T U R E C O M B IN E D M E T H O D
C D
13 14 33 41 7 10 19
1 4 5 ,X ,-Y ,t(8 ;2 1 )(q 2 2 ;q 2 2 ) M T X + +
S O +
-D M O O O O O
~ 5 5 ,X X M T X
D M
~ 4 6 ,X X ,t(1 5 ;1 7 )(q 2 2 ;q 2 1 ) S
-
OD M O O
4 4 6 ,X Y S O
D M
~ 4 6 ,X X S
D M
~ 4 6 ,X X M T X
G M
t7 4 6 ,X X M T X + - +
G M O O
D M O O O
a
4 6 ,X Y ,d e l(5 )( q 2 3 ) M T X + - +G M +
-D M O
~ 4 6 ,X Y M T X +
-G M +
-D M
1 0 4 6 ,X X ,t(4 ;1 1 )(q 2 1 ;q 2 3 ) M T X
-
-S
1 1 4 5 ,X Y ,t(1 ;? ),-C S O O
1 2 4 6 ,X X ,d e l(6 )(q 1 5 )/ M T X O
4 6 ,X X ,-5 ,d e l(6 )(q 1 5 )
G M +
-1 3 4 5 ,X X ,-7 M T X + +
GM +
-1 4 4 6 ,X X ,1 6 q + S + +
-GM + - +
-1 5 4 6 ,X X GM
o
o
s
+1 6 4 6 ,X Y ,t(9 ;1 1 )(p 2 1 ;q 2 3 )/ G M O
4 5 ,X Y ,t(9 ;1 1 )(p 2 1 ;q 2 3 ),-1 3
S + + O
1 7 3 8 ,X Y /4 1 ,X V S + - +
G M + +
1 8 4 6 ,X Y G M + +
1 9 4 6 ,X X G M O O O
D M O O
o
~O 4 6 ,X X ,in v (1 6 )(p 1 3 q 2 2 ) S + - O O
G M +
D M
o
o
o
~ 1 4 6 ,X Y ,t(9 ;2 2 )(q 3 4 ;q 1 1 ) S + - + - +
-~ 2 4 5 ,X X ,t(1 ;? )(q 1 2 ;? ),-2 ,-7 ,+ m a r S + -
o
o
L e g e n d : M T X = c u ltu re w ith a m e to p te rin e ; S = s h o rt te rm c u ltu re ; G M = c u ltu re w ith G M -C S F ; D M = d ire c t m e th o d ; + p o s itiv e s ta in ; - n e g a tiv e s ta in ; + - = c o n c o m ita n t p re s e n c e o f p o s itiv e a n d n e g a tiv e m ito s is ;
o
a b s e n c e o f m ito s is .S ã o P a u lo M e d ic a i J o u rn a llR P M 1 1 5 (1 ): 1 3 3 6 -1 3 4 2 , 1 9 9 7 C H A U F F A IL L E , M .L .L .F .; C O U T IN H O , V ; Y A M A M O T O , M . & K E R B A U Y , J . - C o m b in e d
fro ln tu m o r sam p les reaIly reflectth e k ary o ty p e p ro file o f
th e tu m o r.
T h e sim u ltan eo u s p resen ce o f p o sitiv e an d n eg ativ e
m etap h ases in M A C m eth o d , in 1 0 cases (7 ,8 ,9 ,1 2 ,
1 3 , 1 4 , 1 7 ,2 0 ,2 1 an d 2 2 ) co u ld in d icate th e p ro liferatio n
o f ceIls o f o th er lin eag es.
It b ecam e clear th at u n ev id en t ab erratio n s ar d ip lo id
k ary o ty p e are v ery d ifficu lt to b e seen th ro u g h M A C
m eth o d , an d in th ese cases p ro b es fo r flu o rescen t
in situ
h y b rid izatio n (F IS H ) w o u ld h elp to p o in t o u t th e k aty o ty p e
ab n o rm ality (M A C F IS H tech n iq u e).
S O ln e cases, p articu larly th e ly m p h o id o n es,
p resen ted v ery few m etap h ases to an aly se th ro u g h
co n v en tio n al In eth o d as w eIl as th ro u g h M A C . P e~ h ap s
th is w as d u e to d ifferen t reaso n s su ch as lo w m ito tic in d ex ,
sln aIl am o u n t o f ceIls asp irated fro m th e p atien t, sm aIl
n llln b er o f m etap h ases p u t in to cy to sp in , am o n g o th er
tech n ical p ro b lem s.
A n o th er in terestin g p o in t to d iscu ss w as th e
o b serv atio n th at M o A b p o sitiv ity in M A C m eth o d u sin g
A P A A P w as, lo w er w h en co m p ared to in d irect
im m u n o flu o rescen ce (IF ) d an e at d iag n o stic p ro b ab ly d u e
to a d ifferen t p attern o f reactio n . 2 X IF w as d an e im m ed iatly
after asp iratio n ar co Ilectio n o f th e sam p les an d g en eraIly
p resen ted p o sitiv ity , w h ile A P A A P to o k a lo n g er tim e to
b e d an e an d co u ld p resen t d isap p earen ce o f cy to p lasln ic
ar su rp h ace an tig en reco g n ized b y C D 3 3 (M y 9 ) fo r in stan ce.2 X B esid es th at, th e ceIls stain ed fo r A P A A P h ael
b een cu ltiv ated ar su b m itted to D M , ar in o th er w o rels,
h ad g an e th ro u g h h an d lin g th at co u lel ex p lain th e altereel b in d in g s to M o A b . Y et th ese cells h ad g an e th ro u g h
h y p o to n ia an d sw eIli n g an el th u s h ad s u ffereel
m o d ificatio n s th at co u ld alter su rp h ace an tig en s.
In 2 cases (8 an d 1 2 ), k ary o ty p e ab n o n n alities w ere fo u n d o n ly in G M cu ltu re. It h as alread y b een sh o w n th at
G M -C S F m ay in d u ce p ro liferatio n o f ab n o rm al clo n o g en ic
cells in h u m an m y elo id d iseases su ch as A M L an el
m y elo d y sp lastic sy n d ro m es. 2 ó L eu k em ic cells fro ln b an e
m arro w h av e an in creased resp o n se to G M -C S F in a p attern caIled "leu k em ic sp ecific", w h ere th ere are stim u latio n o f cy to g en eticaIly ab n o rm al ceIls, p ro lif~ rativ e ad v an tag e to
m alig n an t ceIls in a m ix ed p o p u latio n (n o n n al-ab n o n n al) an d a p o ssib le in flu en ce in th e k ary o ty p e. 2 7 .2 lJ.3 0 T h is cO lllel
m ean th at G M in d u ced th e p ro liferatio n o f In alig n an t clo n al
ceIls, an d th is asp ect sh o u ld b e b etter stu d ieel in flltu re
w o rk s.
T h is stu d y sh o w ed th at th e cO ln b in ed In eth o el is feasib le in cases w ith n u m erical ar ev id en t. stru ctu ral
ch ro m o so m al ab n o rm alities', an d n o t as a ro u tin e
p ro ced u re. A sso ciated w ith p ro b es fo r F IS H , it w ill b rin g m u ch m o re in terestin g d etails.
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