A cytogenetic and molecular analysis of five variant Philadelphia translocations in chronic myeloid leukemia

A Cytogenetic and Molecular Analysis of Five Variant Philadelphia Translocations in Chronic Myeloid Leukemia Christine M. Morris, Ingrid Rosman, Susan A. Archer, Jill M. Cochrane, and Peter H. Fitzgerald

ABSTRACT: Three patients had complex translocations involing 9q34, 22q11, and a third chromosome (Xq11, 7q11.2, or 15q11.2). Two patients had apparently simple variant Philadelphia (Ph) translocations, t(19;22) and t(11;22), with no obvious involvement of chromosome 9, and the Ph was masked in the t(11;22). In situ hybridization studies showed transposition of the abl gene from chromosome 9q34 to the breakpoint cluster region (bcr) of chronmsome 22 in all five patients; this was confirmed by rearrangements of the bcr gene in leukemic DNA. In situ hybridization also showed that the bcr-3' and c-sis probes consistently translocated to recipient chromosomes X, 1, 7, 11, and 15, whereas IgCh remained on chromosome 22q. These results confirm that association of abl and bcr is a consistent feature of chronic myeloid leukemia irrespective of the cytogenetic presentation and support the conclusion of Hagemeijer that all simple variant Ph translocations are, in fact, complex and involve at least three chromosomes.

INTRODUCTION The P h i l a d e l p h i a c h r o m o s o m e (Ph) is f o u n d in m o r e t h a n 90% of p a t i e n t s w i t h c h r o n i c m y e l o i d Leukemia (CML) [3]. In most cases the Ph is one p r o d u c t of a reciprocal t(9;22)(q34;q11), k n o w n as the s t a n d a r d Ph t r a n s l o c a t i o n [4, 5]. Variant Ph t r a n s l o c a t i o n s h a v e b e e n d e s c r i b e d in a s m a l l p r o p o r t i o n ( 3 % - 8 % ) of patients w i t h CML [5-12]. At the c y t o g e n e t i c level, t h e s e variants u s u a l l y i n v o l v e c h r o m o s o m e 22 a n d s o m e c h r o m o s o m e o t h e r t h a n 9 (simple variant Ph translocations), or one or m o r e c h r o m o s o m e s in a d d i t i o n to c h r o m o s o m e s 9 and 22 ( c o m p l e x variant translocations). O n rare occasions, the Ph m a y be m a s k e d by the a t t a c h m e n t of material from a n o t h e r c h r o m o s o m e . In all variant Ph r e a r r a n g e m e n t s , the b r e a k p o i n t on c h r o m o s o m e 22 is w i t h i n b a n d q11, the s a m e as in the s t a n d a r d t(9;22) [8, 13]. In r e c e n t years, the m o l e c u l a r e v e n t s that o c c u r as a result of the s t a n d a r d Ph t r a n s l o c a t i o n h a v e b e e n d e f i n e d in c o n s i d e r a b l e detail. Consistently, the p r o t o o n c o g e n e c-abl is t r a n s l o c a t e d from its usual p o s i t i o n at c h r o m o s o m e 9q34 to the d e l e t e d 22q, a d j a c e n t to a n d 3' of a t r u n c a t e d b r e a k p o i n t cluster region) (bcr) gene

From the Cancer Society of New Zealand Cytogenetic and Molecular Oncology Unit, Christchurch Hospital, Christchurch, NZ. Address requests for reprints to Dr. C. M. Morris, Cancer Society of New Zealand Cytogenetics Unit, Christchurch Hospital, Christchurch, New Zealand. Received February 29, 1988; accepted June 8, 1988.

179 © 1988 Elsevier Science Publishing Co.. Inc. 655 Avenue of the Americas, New York. NY 10010

Cancer Genet Cytogenet 35:179-197 (i988) 0165-4608/88/S03.50

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[14-16]. This translocation results in the expression of an abnormally large 8.5-kb hybrid bcr-abl mRNA and a new bcr-abl hybrid protein [17-25]. The bcr-3' sequences are relocated onto chromosome 9, along with the r e m a i n d e r of the 22q arm. Translocation of c-abl to within bcr has also been demonstrated in CML patients who have variant or masked Ph translocations [1, 2, 26-32] and in patients with Ph-negative CML who are karyotypically normal [33, 34]. These studies suggest that chromosome 9 is involved in all variant translocations, both simple and complex, but further work is needed to substantiate this. We describe four patients with CML and one with essential t h r o m b o c y t h e m i a (ET] who d i s p l a y e d variant Ph translocations in their leukemic cells, including results of chromosome in situ and DNA molecular h y b r i d i z a t i o n studies using probes for the abl, sis, bcr, and IgCX genes.

MATERIALS AND METHODS Patients Table 1 summarizes the hematologic findings at diagnosis for the five patients discussed in this report. Patients 2-5 were diagnosed as CML; cases 2 and 4 have since died in accelerated phase and acute blast crisis, respectively. Patient 1 has shown clinical t h r o m b o c y t h e m i a without evidence of leukemia for 9 years, a feature that is described elsewhere [35].

Metaphase Preparations Leukemic cells from peripheral blood or bone marrow were cultured for 24 or 48 hours in Chang's m e d i u m with 10% AB serum a d d e d [36], or cultured and synchronized according to the method of Yunis [37] and Webber and Garson [38], with m i n o r modifications. Cells were prepared for cytogenetic analysis by standard hypotonic and fixation procedures, and were trypsin G b a n d e d [39].

In Situ Hybridization The probes p A B l - s u b 9 [40], bcr-3' and c-sis (Oncogene Science Inc, USA), and IgCX p l A 5 [41] were labeled by nick translation using 3H-dATP and 3H-dCTP nucleotides (Amersham) to a specific activity of 1-2 x 107dpm/~g [42] and h y b r i d i z e d in situ to chromosomes as p r e v i o u s l y described [32]. Chromosomes were G b a n d e d after autoradiography by the m e t h o d of Cannizzaro and Emanuel [43].

Southern Blot Hybridization High molecular weight DNA was extracted from leukocytes derived from bone marrow or peripheral blood buffy coat samples from the leukemic patients, digested with selected restriction endonucleases (BamHI, BglIII, HindIII, or EcorRI) and electrophoresed on 0.8% agarose gels. The separated DNA fragments were transferred to Gene Screen Plus m e m b r a n e (New England Nuclear) and h y b r i d i z e d with the 32p-labeled bcr-3' probe. Filters were w a s h e d as r e c o m m e n d e d in the manufacturers protocol and exposed to x-ray film for 1-4 days before developing.

RESULTS The cytogenetic findings for the five patients are presented in Table 2; results from chromosome in situ and Southern blot hybridization experiments are presented in Table 3.

ET CML CML CML CML

1 2 3 4 5 Normal range:

F/27 F/32 M/62 M/55 M/25

Sex/Age (yr) 134 145 115 142 102 135-175

Hemoglobin (g/L) 12.2 19.4 172.0 175.0 10.7 4.0 11.0

WBC × 109/L 7.7 13.6 111.8 80.5 8.2 2.0-8.0

N e u t r o p h i l s × 109/L 0.244 1.164 5.16 3.5 -<0.01-0.1

Basophils x 10~/L

2030 607 333 550 351 150-450

Platelets × 109/L

Abbreviations: CML, chronic myeloid leukemia; ET, essential thrombocythemia; NA, nut available; NAP, neutrophil alkaline phosphatase; WBC, White blood cell count.

Diagnosis

H e m a t o l o g i c f i n d i n g s in d i a g n o s t i c b l o o d of five p a t i e n t s w i t h v a r i a n t P h t r a n s l o c a t i o n s

Patient n u m b e r

Table 1

7 14 6 NA 5 15-100

NAP

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c . M . Morris et al.

11

12

6

7

8

t0

t3

t4

15

t6

17

18

19

20

22

¥

X

t .'

21

Figure 1 G-banded karyotype of a leukemic cell from patient 1: t(X;9;22)(q11;q34;q11). Arrows indicate the location of breakpoints on rearranged chromosomes.

Patient 1

Cytogenetics. All fourteen G-banded metaphases scored from a diagnostic bone marrow specimen taken in November 1978 were Ph-positive. The 2 2 q - chromosome was one product of a complex translocation involving chromosomes 9, 22, and X: t(X;9;22)(Xpter~-~Xq11::22q11--~22qter;9pter--,9q34::Xq11-~Xqter;22pter22q11::9q34-9qter) (Fig. 1), as reported elsewhere [44]. Band lq34 was not positively identified on the Ph ( 2 2 q - ) chromosome, but was presumed to be present on the assumption that the translocation was reciprocal. This translocation was present in all metaphases (Table 2). Blood lymphocytes cultured for 48 hours with phytohemagglutinin (PHA) had a normal female karyotype (Table 2). Molecular studies. Specific in situ hybridization of the abl gene probe was detected on chromosome 9q34 and on the Ph chromosome (Table 3; Fig. 2). There was no significant labeling on the derivative 9q + chromosome. This result showed that the c-abl gene was translocated from 9q34 to the Ph chromosome and proved that the rearrangement was reciprocal. Both bcr-3' and sis probes hybridized strongly to the normal chromosome 22 and to the 22q material translocated to the long arm of one X chromosome (Table 3; Fig. 2). Neither probe hybridized significantly to the Ph chromosome. These results were consistent with the breakpoint on the Ph being in the bcr of band 2 2 q l l and confirmed that the deleted part of chromosome 22 was relocated to the derivative X chromosome. The IgC)t probe hybridized to the normal chromosome 22 and the Ph chromosome in region 2 2 q l l (Table 3; Fig. 2). Southern blots showed bcr rearrangement with three restriction enzymes (Table 3; Fig. 3a), and confirmed that the Ph breakpoint was in the bcr of chromosome 22.

183

Variant Ph T r a n s l o c a t i o n s in CML

Table 2

C y t o g e n e t i c findings for five p a t i e n t s w i t h CML or ET and a v a r i a n t Ph t r a n s l o c a t i o n

Patient number

Clinical stage

1

ET-diag

2

CML-diag

CML-AP 3

CML-diag

4

CML-diag

CML-MBC 5

CML-diag

Specimen

Date

Karyotype

Number metaphases

BM PB(+PHA) PB BM

11/21/78 12/12/78 1/30/84 8/14/84

46,X,t(X;9;22)(q11;q34;q11) 46,XX 46,X,t(X;9;22) 46,XX,t(9;22;15)(q34;q11;q11.2) 46,XX 46,XX,t(9;22;15) 46,XX 46,XX,t(9;22;15) 46,XX,t(9;22;15) 46,XX,t(9;22;15) 46,XX,t(9;22;15) 46,XY,t(7;9;22)(q11.2;q34;q11) 46,XY,t(7;9;22)(q11.2;q34;q11) 46,XY,t(11;22)(q13;q11) 46,XY 46,XY,t(11;22) 46,XY,t(11;22) 46,XY,t(11;22) 46,XY,t(11;22) 47,XY, + 8,t(19;22)(q13;q11) 46,XY,t(19;22) 47,XY, + 8,t(19;22) 46,XY,t(19;22) 47,XY, + 8,t(19;22) 46,XY,t(19;22) 47,XY, + 8,t(19;22) 46,XY,t(19;22) 47,XY, + 8,t(19;22)

14 15 1 26 4 3 1 9 1 25 20 10 30 24 1 3 29 19 28 20 10 2O 3 29 1 27 2 3

PB

8/14/84

PB BM PB BM PB BM

11/29/84 1/31/85 5/29/86 7/31/86 8/24/87 8/24/87 9/9/83

PB PB BM PB PB

9/9/83 5/21/84 4/16/86 6/3/86 5/30/85

BM

6/17/85

PB

2/25/86

PB

3/25/86

PB

11/27/86

Abbreviations: diag. first diagnosed; CML-MBC, myeloid blast crisis; CML-AP, accelerated phase; BM, bone marrow; PB( + PHA), peripheral blood stimulated with phytohemagglutinin; PB, peripheral blood, unstimulated.

Patient 2

Cytogenetics. T w e n t y - n i n e of 34 m e t a p h a s e s a n a l y z e d from b o n e m a r r o w a n d leuk e m i c b l o o d c u l t u r e s w e r e P h - p o s i t i v e w h e n l e u k e m i a was d i a g n o s e d (Table 2). T h e Ph c h r o m o s o m e was d e r i v e d from a c o m p l e x t r a n s l o c a t i o n i n v o l v i n g c h r o m o s o m e s 9, 15, and 22 (Fig. 4). M o s t of the long arm of one c h r o m o s o m e 15 was t r a n s l o c a t e d o n t o the e n d of one c h r o m o s o m e 9. T h e d e l e t e d part from c h r o m o s o m e 22 a p p e a r e d to substitute the d e l e t i o n f r o m c h r o m o s o m e 15 and, a l t h o u g h not c y t o g e n e t i c a l l y visible, a small part of r e g i o n 9q34 was p r e s u m e d to be t r a n s l o c a t e d to the e n d of the q arm on the Ph c h r o m o s o m e . The t r a n s l o c a t i o n was d e s c r i b e d thus: t(9;22;15)(9pter~9q34::15q11.2-~l 5qter;22pter~22q11::9q34-~9qter;15pter--~ 15q11.2::22q11-~22qter). S u b s e q u e n t c h r o m o s o m e studies s h o w e d the s a m e karyot y p e in all m e t a p h a s e s a n a l y z e d (Table 2). The difficulty of d i s t i n g u i s h i n g the n o r m a l c h r o m o s o m e 22 from the d e r i v a t i v e c h r o m o s o m e 15 p o s e d a p r o b l e m for c h r o m o s o m e in situ studies using o n c o g e n e probes (Fig. 4). A l t h o u g h s y n c h r o n i z e d c u l t u r e s w e r e a n a l y z e d , good high-resolution G - b a n d e d m e t a p h a s e s w e r e not obtained. M e t h y l g r e e n / 4 ' . 6 - d i a m i d i n o - 2 p h e n y l - i n d o l e (MG/DAPI) specifically stains the c e n t r o m e r i c h e t e r o c h r o m a t i n of

t(X;9;22) t(9;22;15) t(7;9;22) t(11;22) b t(19;22)':

1 2 3 4 5

9q+ ,22q-,Xq 9q+,22q-,15q9 q + ,22q , 7 q 22q+,11q2 2 q - , 1 9 q + , l p + 'j

D er iva t ive chromosomes ET CML CML CML CML

Clinical stage 9,22q 9,22q9,22q9,22q+ 9,22q-

abl

were not distinguishable (see text results, this patient).

'~ lp ~ visible on retrospective analysis of HR-G banded metaphases.

': Full interpretation from molecular results: t(l:19;9;22)(p36;q13;q34;q11).

Full interpretation from molecular results: t[9;11:22)(q34;q13:ql 1).

" Chromosomes 22 and 15q

22,Xq 22,15q - " 22,7q22.22q ,11q 22,1p+

bcr-3' 22,Xq -22,7q 22,22q + 22Ap+

sis 22,22q 22,22q - ~ 22,22q 22,22q + 22,22q-

C~.

In situ h y b r i d i z a t i o n : c h r o m o s o m a l l o c a l i z a t i o n of

R -R R R

Ba m H I

R R R R --

BglII

R R R -R

EcoRI

N -N -R

HindIII

S o u t h e r n blot h y b r i d i z a t i o n - b c r - 3 ' restriction enzymes used

studies on leukemic cells from five patients with CML and variant Ph translocations

Abbreviations: N. germ line fragment only; R. abnormal fragment size detected.

Variant Ph translocation

R e s u l t s of m o l e c u l a r h y b r i d i z a t i o n

P atie nt numbers

Table 3

185

Variant Ph Translocations in CML

abi

j, X

X/22

9

9//X

22

22q-

bcr-$

[IIi~FI~II[]~1~ X

X

II~ll-llll] l

CIIII]~I

X/22

X,/22

fTll~-Al X/22

9

9

~JliiltZl[!.lim][]

9/X

~IIII~P, l ~ I I ] ~ 9

~1~

~lJ~

22

22q-

9/X

9/X

22

0,' 22

22q-

0,J 22q-

Figure 2 Distribution of labeled sites on chromosomes X, 9, 22, and derivatives from patient 1 after hybridization with probes for abl (partial representation of a total 298 grains scored from 148 leukemic metaphase cells), bcr-3' (101 metaphases, 157 grains), c-sis (62 metaphases, 112 grains), and IgCk (43 metaphases, 65 grains). chromosome 15 [45], but i n d i v i d u a l variations occur in the intensity of staining [46]. Unfortunately, centromeric staining of the derivative chromosomes 15 was indistinguishable from other acrocentric chromosomes in metaphase cells from patient 2 (data not shown). Results of the c h r o m o s o m e in situ studies are presented without distinguishing chromosomes 22 and 1 5 q - . Molecular studies. Chromosome in situ h y b r i d i z a t i o n studies showed that v-abl

h y b r i d i z e d to the normal c h r o m o s o m e 9 band q34 and to the Ph c h r o m o s o m e ( 2 2 q - ) (Table 3; Fig. 5). The bcr-3' probe h y b r i d i z e d to c h r o m o s o m e 22 and/or the 1 5 q - chromosome, and the IgCk probe h y b r i d i z e d to chromosome 22 and/or 1 5 q and the Ph c h r o m o s o m e (Table 3; Fig. 5). The bcr gene was rearranged in leukemic DNA (Table 3; Fig. 3b).

186

c.M.

b

d

0

/ :ii~!il ii!?I

M o r r i s et al.

: ii:~~ii:~i~i ~,!:i~,!ii~I,!~,~!

e M

B

H

23~

q.5~

6.6;

Rt

4

2/1~,:

F i g u r e 3 Southern blot analysis of BamH1 (B), BglII (Bg), EcoRl (E), and HindIlI (H) digested leukemic DNA of patients 1-5 (a-e) hybridized with a 1.2-kb bcr-3' probe. Germ line (N) and rearranged (R) alleles are marked for each digest. M, HindlII digest of X phage DNA.

F i g u r e 4 Partial G-banded karyotype of the variant Ph t(9;22;15)(q34;q11;q11.2) observed in the leukemic ceils of patient 2. Arrowheads indicate breakpoints on rearranged chromosomes 9 and 15. Note the difficulty of distinguishing the 1 5 q - derivative and normal chromosome 22 (asterisk).

9

m

m

•

•

15

22

18 7

Variant Ph Translocations in CML

9/15

15

15/22

22

I

J

22q-

bcr- 3'

•

i I II •

9

I i i

9/15

15

15/22

22

L

I

22q-

c~

[~lMi

Illi•

I i']

9/15

15

15/'22

I

22

22q-

J

Figure 5 Regional assignment of labeled sites on chromosomes 9, 15, 22, and derivatives of patient 2 after hybridization with probes for abl (partial representation of a total 204 grains scored from 106 leukemic metaphase cells), bcr-3' (60 metaphases, 98 grains), and IgCX (60 metaphases, 132 grains). The cytogenetic in situ and DNA hybridization studies together indicated that c-

abl had shifted from chromosome 9 to the Ph chromosome with consequent rearrangement of the bcr and were consistent with relocation of bcr-3' to the derivative chromosome 15, along with the translocated distal part of chromosome 22q. Patient 3

Cytogenetics. All metaphase cells derived from the diagnostic bone marrow and peripheral blood were Ph-positive. The Ph was one product of a complex translocation involving chromosomes 7, 9, and 22, with the form t(7:9;22)(7pter--~

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c . M . Morris et al.

7q11.2::22q11--~22qter~9pter--~9q34::7q11.2--~ 7qter;22pter-~ 22q11::9q34--*9qter) (Table 2; Fig. 6). Molecular studies. The abl probe hybridized specifically to c h r o m o s o m e 9q34 and to the Ph chromosome (Table 3; Fig. 7). The bcr-3' and c-sis probes showed strong hybridization to the normal c h r o m o s o m e 22 and to the 22q material on the derivative 7 q - chromosome. The IgCX probe hybridized to chromosome 2 2 q l l and to the Ph chromosome (Table 3; Fig. 7). The bcr gene was rearranged (Table 3; Fig. 3c). Patient 4

Cytogenetics. Twenty-six of 27 metaphases scored from bone marrow cells taken when leukemia was diagnosed showed the t(11;22)(q13;q11) (Fig. 8). The breakpoint on chromosome 22 at q l l was the same as that seen in the standard Ph translocation, but material from c h r o m o s o m e 11q was attached at the c h r o m o s o m e 22 breakpoint site and gave a masked Ph chromosome. There was no obvious involvement of c h r o m o s o m e 9 in the translocation (Fig. 8). No further karyotype changes appeared during the blastic phase (Table 2). Molecular studies. The abl probe h y b r i d i z e d specifically to chromosome 9q34 and to the breakpoint junction of the derivative c h r o m o s o m e 22q+ (masked Ph chromosome) in metaphase cells prepared from leukemic peripheral blood (Table 3; Fig. 9). Thus, although c h r o m o s o m e 9 was not visibly involved in this rearrangement, the abl gene from chromosome 9 was relocated to the 22q+ chromosome. The bcr3' probe hybridized to the normal c h r o m o s o m e 22, to the derivative chromosome

Figure 6 Partial G-banded karyotype of the variant Ph translocation of patient 3: t(7;9;22)(qll.2;q34;q11). Arrowheads indicate breakpoints on rearranged chromosomes.

l 7

9

189

Variant Ph Translocations in CML

L •

-:---

7q

9

9,1.

7q~

9

9q*

22

Ph

22

Ph

r"

7

I 7

7q-

9

9q*

22

Ph

7

7q-

9

9q÷

22

Ph

F i g u r e 7 Regional assignment of grains on chromosomes 7, 9, 22, and derivatives of patient 3 after hybridization with probes for abl (partial representation of a total 148 grains scored from 50 leukemic metaphase cells/, bcr-3' (70 metaphases, 151 grains), IgCk (50 metaphases, 94 grains), and c-sis (91 metaphases, 164 grains). Arrowheads indicate breakpoints on rearranged chromosomes 7q and 9q +.

190

c . M . Morris et al.

1

6

2

7

3

S

4

|

g

13

22

19

9

11

12

17

18

,

¢"

Y

X

9

Figure 8 {Top) G-banded karyotype of leukemic metaphase cell from patient 4: t(11;22)(q13;q11). Arrowheads indicate breakpoints on rearranged chromosomes. (Bottom) Apparently normal chromosomes 9 from two other leukemic metaphase cells. 22q+ in the vicinity of the breakpoint, and to the derivative chromosome 1 1 q (Table 3; Fig. 9). Hybridization to the two rearranged sites indicated that either duplication of bcr-3' sequences had occurred during the rearrangement, or the breakpoint on chromosome 22 had occurred within the region of the bcr-3' probe. The c-sis probe hybridized to the normal chromosome 22 at bands q12-13 and to the part of chromosome 22 relocated to the derivative chromosome 1 1 q - (Fig. 9). The IgCX probe hybridized specifically to the normal chromosome 2 2 q l l and to the masked Ph chromosome, just proximal to the breakpoint (22q11) Table 3; Fig. 9). DNA extracted from leukemic blood cells and hybridized with the bcr-3' probe showed a germ line band and two rearranged bands in BamHI and BglII digests (Table 3; Fig. 3d), indicating a rearrangement within the region of the bcr-3' probe, as suggested by chromosome in situ hybridization.

191

Variant Ph Translocations in CML

abl

9

11

11/22

22

22/11

9

11

11/22

22

22/11

bcr- 3

sis

9

11

9

11/22

11

11/22

22

22/11

22

22/11

Figure 9 Regional assignment of labeled sites on chromosomes 9, 11, 12, and derivatives of patient 4 after hybridization with probes for abl (partial representation of a total 274 grains scored from 135 leukemic metaphase cells), bcr-3' (94 metaphases, 146 grains), c-sis (79 metaphases, 146 grains), and IgCX (30 metaphases, 52 grains). Arrowheads indicate breakpoints on derivative chromosomes 11 and 22. Patient 5 A detailed description of the cytogenetic and in situ hybridization studies for this patient has been published elsewhere [32]. In brief, the patient presented with what appeared to be a simple variant Ph translocation, t(19;22)(q13;q11) (Table 2). In situ hybridization studies revealed m o v e m e n t of the c-abl oncogene from a cytogenetically normal chromosome 9 to the Ph, and bcr-3' and c-sis probes hybridized to the distal end of chromosome l p (Table 3). Retrospective high-resolution G-banding studies supported the conclusion that this was, in fact, a complex translocation

192

c . M . Morris et al. q~

C i , , , = = .. ~

9

,,== =~

lp+

I

19

19q* ~

22

2?.q-

Figure 10 Regional assignment of grains on chromosomes ~, 9, 19, 22, and derivatives of patient 5 after hybridization with an IgCX probe (partial representation of a total 189 grains scored from 50 leukemic metaphase cells). involving four chromosomes, t(1;19;9;22)(p36;q13;q34;q11). Further to the earlier report, m o l e c u l a r h y b r i d i z a t i o n studies have confirmed the in situ IgCk site on c h r o m o s o m e 22 and the Ph c h r o m o s o m e (Table 3; Fig. 10), and that the bcr gene was rearranged in Southern blot studies (Table 3; Fig. 3e).

DISCUSSION Three of our patients had c o m p l e x translocations involving three chromosomes and gave derivative 9 q + and typical Ph chromosomes. The other two patients apparently had simple variant Ph translocations with no obvious involvement of chromosome 9; the Ph chromosome was masked in one of them. In situ h y b r i d i z a t i o n studies showed m o v e m e n t of the abl gene from chromosome 9 to the bcr of chromosome 22 in leukemic cells of all five patients, and this was confirmed by rearrangements of the bcr gene. Bartram et al. [26] first demonstrated translocation of c-abl from c h r o m o s o m e 9 to 2 2 q - in two CML patients with complex variants clearly involving c h r o m o s o m e 9 (Table 4). It was not surprising to find the abl gene from c h r o m o s o m e 9 on the Ph chromosome in these complex variants if it is assumed that they are reciprocal rearrangements. Indeed, prior to the a p p l i c a t i o n of molecular techniques, the existence of complex variants was considered good evidence that a p p o s i t i o n of 9q34 and 2 2 q l l was critical for the d e v e l o p m e n t of CML [13, 47]. Simple variant Ph translocations are characterized by their apparent lack of involvement of c h r o m o s o m e 9 in the Ph translocation, and they have posed a problem in determining a uniform model for the Ph chromosome. However, the in situ finding of c-abl on c h r o m o s o m e 22q in our two patients and in five others (Table 4) clearly showed that the distal part of c h r o m o s o m e 9 participated in these Ph translocations. These results a m p l y justify the conclusion of Hagemeijer et al. [1] that all simple variant Ph translocations are, in fact, complex and involve at least three chromosomes. The diagnosis of CML in the Ph-negative patient 4 suggested that the t(11;22)(q13;q11) present in his leukemic cells involved a masked Ph chromosome. Although chromosome 9 was not visibly involved by the translocation, c-abl was relocated on the 22q+ derivative and m a p p e d at an interstitial site adjacent to the 2 2 q l l breakpoint. A break of the bcr gene within the region of the probe used, as shown by in situ h y b r i d i z a t i o n and Southern blot studies, closely localized the translocation site and showed that the c-abl gene had inserted next to 5'-bcr in this patient. The karyotype of his leukemic cells might thus be described as 46,XY,t(9;11;22)(q34;q13;q11), with the Ph chromosome masked by translocated material from c h r o m o s o m e 11q. Hagemeijer et al. [2] have also reported movement

9,22q 9,22q9,22q 9,22q 9,22q9,22q9,22q 9,22q + 9,22q +

9q + ,22q - ,lp 9q+ ,22q-,11q-

9q - ,22q - ,4p + 9q - ,22q - ,7p + 9q ,22q , 1 2 p + 2 2 q - ,21q+ 2 2 q - ,22q+

22q + ,6p 9q + ,22q+ ,lp - ,3p + ,5p -

coabl

° subtle deletion of chromosome 9 detected by high-resolution banding techniques.

Abbreviations: ISH. in situ hybridization; SCH, segregation analysis in somatic cell hybrids.

Complex t(1;9;22)(p32;q34;q11) t(9;11;22)(q34;q12;q11) Apparently simple t(4;9;22)(p16;q34;q11) ~' t(7;9;22)(p22;q34;q11) a t(9;12;22)(q34;p13;q11] ° t(21;22)(q22;q11) t(22;22)(q13;q11) Masked t(6;22)(p21;q11) t(1;3;5;9;22)(p32;pl 3; q12;q34;q11)

Derivative chromosomes

22,6p -

22,9

bcro3'

22,1p 22,11q -

c-sis

[2] [2]

[1] I29] [1) [27] [30]

ISH ISH ISH ISH SCH ISH ISH

[26] [26]

[Ref.]

SCH ISH

Technique used

of v a r i a n t P h t r a n s l o c a t i o n s i n CML: P u b l i s h e d f i n d i n g s f r o m

C h r o m o s o m a l localization of:

L o c a l i z a t i o n of c e l l u l a r o n c o g e n e s o n t h e d e r i v a t i v e c h r o m o s o m e s nine cases

Variant Ph translocation

Table 4

¢.,o

194

c . M . Morris et al. of c-abl in two patients with masked Ph translocations and showed relocation of bcr-3' sequences in one of them (Table 4). The essential genomic change associated with the occurrence of CML is the juxtaposition of bcr-5' and abl gene sequences. This is consistently found in patients showing the standard t(9;22}, in patients showing variant Ph and masked Ph translocations, and even in patients who are Ph negative and show a normal karyotype. The standard t(9;22) is clearly the most cytogenetically efficient way of achieving juxtaposition of bcr-5' and abl. The complex translocations probably represent more involved cytogenetic changes that achieve the same genomic result. They may happen because breaks have occurred, simultaneously in several chromosomes at the same nuclear location. Alternatively, the breaks may be sequential in time; with the eventual juxaposition of bcr-5' and abl causing leukemic clonal proliferation. The relatively low incidence of complex translocations (although not precisely documented) is consistent with this hypothesis. The mechanism of abl movement in patients with Ph-negative CML who show a normal karyotype has not yet been explained [33]. The same mechanism of abl movement may also operate in the complex Ph translocations. It would greatly simplify the cytogenetic manipulations necessary to explain oncogene movements in some of these, as we have already noted [32]. Chromosome interchanges in human cells may be random as to chromosome site and, consistent with this, all chromosomes except Y have been described in variant Ph translocations. However, the breakpoints on chromosomes other than 9q34 and 2 2 q l l are nonrandom [12, 48, 49]. Some of these preferentially involved bands corresponds to known fragile sites, some occur in the vicinity of localized oncogenes, and others map to sites frequently involved in structural rearrangements found in different types of leukemia/lymphoma [12, 48-50]. The significance of the nonrandom involvement of some chromosome bands in variant Ph translocations is not known. There may be sequence homologies within 9q34 or 2 2 q l l that favor homologous recombination with other chromosomes, but equally some genetic rearrangements will provide a proliferative advantage to the malignant cell and preferentially bring them to attention. Two of the patients described herein had breakpoints in preferentially involved sites: band 11q13 of patient 4 and band 19q13 of patient 5, the first being one of the most fequently involved breakpoint sites in variant Ph translocations [12, 48, 49, 51-54]. Clinical or laboratory features do not distinguish these patients from other CML patients, but numbers are few, and further detailed comparative studies may disclose otherwise. The oncogenes int-1 and bcl-1 have been mapped to 11q13, but there is no evidence that these genes are involved in the variant translocations of CML [55-57]. A constitutive fragile site, fra(11)(q13) [50, 58], was carried by three patients with rearrangements of 11q13 in malignant cells [53, 58, 59]. One of these patients had CML and a variant Ph t{9;11;22) [53]. Interestingly, constitutive fragile sites are also present at band 19q13 and 7 q l l of our patients 5 and 3, respectively, although band 7 q l l is rarely involved in variant rearrangements. Consistent with other studies, the hematologic course of the CML patients presented here has been typical for the disease. Most patients with variant Ph translocations show the same clinical, hematologic, and prognostic features and types of blast crisis as those with the standard t(9;22) [8, 11, 47, 60-62]. This suggests that involvement of a third or other chromosomes is not clinically important. Our studies show that the IgC)~ region is proximal to the breakpoint site and is not involved in the variant Ph translocations, as it is not in the standard t(9;22).

Variant Ph Translocations in CML

195

Supported by the Cancer Society of New Zealand and its Canterbury and Westland Division. The authors thank Drs. M. E. J. Beard, D. C. Heaton, K. Romeril, and C. Svehla for their clinical and hematologic information, and Drs. P. E. Crossen and A. E. Reeve for the IgC~ and p A B l s u b 9 probes.

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