The karyotype of Philadelphia chromosome-negative, bcr rearrangement-positive chronic myeloid leukemia

The Karyotype of Philadelphia ChromosomeNegative, bcr Rearrangement-Positive Chronic Myeloid Leukemia M. E. Weinstein, A. Grossman, M. A. Perle, P. L. Wilmot, R. S. Verma, R. T. Silver, Z. Arlin, S. L. Allen, E. Amorosi, S. E. Waintraub, L. R. Shapiro, and P. A. Benn

ABSTRACT: Philadelphia (Ph) chromosome negative chronic myeloid leukemia (CML) can be distinguished f r o m clinically similar disorders on the basis of the presence of rearrangement of the breakpoint cluster region (bcr) of chromosome 22. We have identified six patients with Phnegative CML, each with bcr rearrangement. Apparently normal karyotypes were observed in two cases, and a third contained a rearrangement that did not appear to involve chromosomes 9 or 22. The other three c a s e s had translocations involving chromosome band 9q34 but no case contained the common derivative chromosome 9pter-~9q34: :22qll--~22qter. One case appeared to contain either a deletion of an unrearranged bcr locus in approximately 50% of cells or duplication of rearranged bcr, both 5' and 3' of the chromosome 22 breakpoint. Considerable complexity exists in the types of genetic changes that can juxtapose bcr and the c-abl oncogene in CML. Based on the molecular and cytogenetic analyses of these and other cases described in the literature, we conclude that most cases of true Ph-negative CML arise from submicroscopic genetic exchanges rather than m a s k i n g of simple t(9;22)(q34;q11) translocations by secondary rearrangements.

INTRODUCTION T h e l e u k e m i c c e l l s of m o s t 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 l e u k e m i a ( C M L ) c a r r y a t ( 9 ; 2 2 ) ( q 3 4 ; q 1 1 ) [1]. T h e d e r i v a t i v e c h r o m o s o m e 22 [the P h i l a d e l p h i a c h r o m o s o m e (Ph)] c o n t a i n s t h e c-abl o n c o g e n e t r a n s l o c a t e d f r o m c h r o m o s o m e 9 to t h e b r e a k p o i n t c l u s t e r r e g i o n (bcr) of c h r o m o s o m e 22 [2]. T h e c h i m e r i c bcr/abl g e n e e n c o d e s for a bcr/abl p r o t e i n w i t h t y r o s i n e k i n a s e a c t i v i t y [3]. T h i s bcr/abl e x c h a n g e is s t r o n g l y i m p l i c a t e d i n t h e p a t h o g e n e s i s of C M L [4].

From the Lifecodes Corporation, Valhalla (M. E. W., A. G.. P. A. B.). Westchester County Medical Center, and New York Medical College, Valhalla (P. L. W., Z. A., L. R. S./, NY, New York University Medical Center. New York (M. A. P., E. A.), Long Island College Hospital--SUNY tIealth Science Center Brooklyn (R. S. V.), New York Hospital Cornell Medical Center. New York (R. T. S.), North Shore University Hospital--Cornell University Medical College, New York (S. L. A.), NY, and Holy Name Hospital. Teaneck, NJ (s. E. W.).

Address requests for reprints to M. E. Weinstein, Ph.D., Lifecodes Corporation, Saw Mill River Rd., Valhalla, N Y 10595. Received April 29, 1988; accepted June 30, 1988.

223 ,~c;1988 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas. New York, NY 10010

Cam:er Genet Cytogenet 35:223 229 (1988) 0165-4608/88/S03.50

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Rare patients w i t h CML a p p a r e n t l y lack a Ph c h r o m o s o m e , but h a v e a disease that is i n d i s t i n g u i s h a b l e f r o m P h - p o s i t i v e CML [5]. B e c a u s e certain types of m y e l o p r o l i f e r a t i v e s y n d r o m e s and s o m e m y e l o d y s p l a s t i c s y n d r o m e s m a y be difficult to d i s t i n g u i s h from CML, a p r o b l e m has existed in p r e c i s e l y i d e n t i f y i n g " t r u e " cases of P h - n e g a t i v e CML [6, 7]. Recently, it has b e e n s h o w n that D N A analysis for bcr r e a r r a n g e m e n t a l l o w s d e f i n i t i v e d i s c r i m i n a t i o n b e t w e e n CML and c l i n i c a l l y similar d i s o r d e r s [8]. A n essential genetic alteration in CML m a y therefore be the juxtaposition of bcr and c-abl and this m a y be p r e s e n t e v e n in the a b s e n c e of c y t o g e n e t i c e v i d e n c e of the t r a n s l o c a t i o n [9]. P h - n e g a t i v e CML m a y arise as a result of m a s k i n g of the Ph c h r o m o s o m e following a d d i t i o n a l t r a n s l o c a t i o n . M i n u t e r e a r r a n g e m e n t s not detectable by c y t o g e n e t i c analysis c o u l d also a c c o u n t for s o m e cases of P h - n e g a t i v e CML. We d e s c r i b e h e r e i n six cases of P h - n e g a t i v e CML and r e v i e w the k a r y o t y p e s of 29 other cases d e s c r i b e d t h u s far in the literature. The r e a r r a n g e m e n t s that can p r o d u c e P h - n e g a t i v e CML m a y s o m e t i m e s be h i g h l y c o m p l e x , i n v o l v i n g translocations, insertions, d e l e t i o n s , and d u p l i c a t i o n s .

MATERIALS A N D METHODS The six patients w i t h P h - n e g a t i v e CML w e r e identified t h r o u g h r o u t i n e referral to four c y t o g e n e t i c s laboratories. Clinical and h e m a t o l o g i c data for t h e s e six patients are s u m m a r i z e d in Table 1. C y t o g e n e t i c analysis was p e r f o r m e d on b o n e m a r r o w or p e r i p h e r a l b l o o d samples c u l t u r e d for 0 - 4 8 h o u r s in the absence of m i t o g e n . Essentially s t a n d a r d techn i q u e s w e r e u s e d for c h r o m o s o m e p r e p a r a t i o n s and a n a l y s e s [10]. R e a r r a n g e m e n t of the bcr of c h r o m o s o m e 22 was d e t e c t e d by S o u t h e r n blot analysis u s i n g m e t h o d s d e s c r i b e d e l s e w h e r e [11]. D N A was d i g e s t e d w i t h restriction e n z y m e s BgllI, BamHI, HindIII, and XbaI. For e a c h case, t w o bcr probes w e r e used; a 1.2-kb HindIII/BgllI f r a g m e n t f r o m the 3' end of bcr, and either a 5' probe consisting of a 1.9-kb BglII/HindIII f r a g m e n t or a 5' 2.5-kb BgllI/BamHI fragment. In each case, r e a r r a n g e m e n t was detectable w i t h m o r e t h a n one restriction e n z y m e and all testing was carried out in d u p l i c a t e .

Table 1

H e m a t o l o g i c data for six patients w i t h P h - n e g a t i v e CML Differential distribution (%)b

Blood count × 109/L Patient

Sex/Age

Spleen ~

Platelets

WBC

B/P

M/M

N

E

B

LAP

Survival b

1 2 3 4 5 6

M/47 F/58 F/70 M/43 M/72 F/43

Enlarged 0 0 0 0 0

396 322 399 512 421 507

68.6 21.9 26.0 17.5 38.0 44.2

0 0 11 0 0 0

8 O 20 0 16 11

78 73 53 58 64 72

3 1 0 0 2 3

0 0 6 5 2 3

NK NK 0 28 7 13

43 40 43 8 7 0

Abbreviations: B/P, blasts plus promyelocytes; M/M, myelocytes plus metamyelocytes; N~ neutrophils; E, eosinophils; B, basophils; NK, not known. °Spleen size, centimeters below the costal margin. bSurvival from diagnosis to study date in months. NK, not known.

Ph-Negative CML

Table 2

225

Cytogenetic and m o l e c u l a r genetic results bcr Rearrangement 5' probe

Patient 1 2 3 4 5 6

Karyotype 46,xY 46,XX,t(9;12) (q34;q13) 46,XX,t(9;11) (q34;q11) 46,XY,t(8;9) (q22;q34) 46,XY,t(20;21} (q11;q22) 46,XX

bcr Rearrangement 3' probe

BgllI

BamHI

HindIII

XbaI

BglII

BamHI

HindlIl

XbaI

+ +

NT

-

+ +

+ +

+ +

+

+ +

+

+

+

÷

+

--

_

+

+

-

+

+

+

--

_

+

+

NT

NT

+

÷

+

--

+

+

-

+

NT

NT

+, rearrangement detected; - , no rearrangement detected; NT. not tested.

RESULTS Six patients with a clinical diagnosis of CML were identified in w h i c h the molecular genetic analysis indicated rearrangements of the bcr, but where cytogenetic analysis failed to detect a Ph chromosome. Results of cytogenetic and molecular genetic analysis are s u m m a r i z e d in Table 2. Of the six cases, two had apparently normal karyotypes (cases 1 and 6), and a third had a translocation that seemed to involve only chromosomes 20 and 21 (case 5). Three cases (2, 3, and 4) apparently had balanced translocations between chrom o s o m e band 9q34 and other chromosomes (Fig. 1). No case had the c o m m o n derivative c h r o m o s o m e 9, 9pter-~9q34: : 22q11-~22qter, or any other cytogenetic evidence to suggest that masking of the Ph c h r o m o s o m e had occurred. In each case the presence of rearrangement within the bcr was confirmed for DNA digested with more than one restriction enzyme. In one patient (case 6), rearrangement was not detectable with the 3' probe, but was demonstrated with the 5' probe. In this case it was likely that there was a deletion of part of the rearranged bcr homologous to the probe, a c o m m o n finding in bcr/abl exchanges [11, 12]. Molecular genetic results were particularly interesting for one patient (case 5). For this patient, the results from the various restriction enzyme digests placed the translocation site w i t h i n the bcr between the 5' and 3' probe regions. Surprisingly, the intensity of the autoradiograph band corresponding to bcr rearrangement was approximately twice the intensity of the band corresponding to the unrearranged bcr (Fig. 2). This difference in intensity was confirmed by densitometric analysis and was true for autoradiographs prepared using both probes. For the combinations of probes and enzymes used, the autoradiograph bands detected corresponded to sequences 5' and 3' of the breakpoint. The results, therefore, suggested that after bcr rearrangement duplication occurred both 5' and 3' of the breakpoint. Alternatively, approximately half of the cells in the sample may have had a deletion of an unrearranged bcr locus. In this patient the course of the disease was unusually rapid, resulting in blast crisis and death within 12 months of initial diagnosis.

DISCUSSION Rearrangement of the bcr appears to be highly specific for CML. To date, we have observed bcr rearrangement in all cases of Ph-positive CML, but have not observed the rearrangement in any controls or normal individuals without CML [10, 12; re-

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CASE

2

9

9

~

12

22

11

22

CASE 4

9

8

22

Figure 1 Translocations involving chromosome band 9q34 in cases 2, 3, and 4: Case 2, t(9;12)(q34;13); case 3, t(9;ll)(q34;q11); and case 4 t(8; 9)(q22;34). For each case, chromosome 22 is shown to illustrate its apparently normal banding pattern.

cent u n p u b l i s h e d observations]. W i e d e m a n n et al. [8] recently s h o w e d that Ph-negative CML can be distinguished from other hematologic disorders with similar (but not identical) clinical presentation on the basis of the presence of bcr rearrangement. It is therefore possible to reliably confirm the diagnosis of CML on the basis of the specific genetic alteration of the bcr. Using rearrangement of the bcr for confirming the diagnosis of CML, we have identified six cases of Ph-negative CML with a clinical course typically like that of the Ph-positive CML. A n additional 29 cases of bcr rearrangement-positive, Ph-negative CML have been described in the literature (Table 3). These latter cases have been diagnosed using varying criteria and were subjected to differing laboratory studies. Although the number of cases is small, several points can be made. First, a c o m m o n karyotype for Ph-negative CML is an a p p a r e n t l y normal karyotype (for example, cases 1 and 6 in Table 2). Overall, 54% of cases described so far have shown no obvious c h r o m o s o m e abnormality. Second, masked Ph chromosomes in w h i c h the simple t(9;22)(q34;q11) is followed by additional rearrangements obscuring the Ph chromosome seem to be rare. None of the six cases in this study contained the c o m m o n derivative c h r o m o s o m e 9, 9pter--~9q34::22q11--~22qter or marker chromosomes that might be interpreted as further rearrangement of a Ph chromosome. Only five of the 29 cited cases from the literature could be considered as examples of masked Ph chromosomes. Thus, Ph-negative CML seems to arise primarily from submicroscopic genetic exchanges rather than masking of simple t(9;22)(q34;q11) translocations by secondary rearrangements. Third, exchanges of minute amounts of genetic material that include the bcr and that are not detectable by cytogenetic analysis are often found in combination with microscopically ob-

Ph-Negative CML

1

227

2

3

4

Q

t

,O Figure 2 Autoradiograph of Southern blot for bcr analysis using the 3' bcr probe and DNA digested with BamHI. Lane 1, size standards; Lane 2, control DNA from a normal individual without CML and showing a single DNA band corresponding to approximately 3.3 kb; Lane 3, DNA from individual without CML and showing the single DNA band as observed for normal individuals; Lane 4, DNA from patient 5 with an additional DNA band approximately 7.5 kb in size. Note the intensity of the 7.5-kb band is approximately twice that of the 3.3-kb band.

vious translocation. Cases 2, 3, and 4 (Table 2) all involved chromosome band 9q34 in translocation with different chromosome regions. A n additional two published cases also involve band 9q34 (Table 3). Each of these examples presumably contains complex exchanges involving c-abl, bcr, and a third chromosomal locus and would seem to require at least four breakpoints to produce stable chromosome products. Studies using in situ hybridization techniques with probes for chromosome 9 (cabl) and chromosome 22 ( i m m u n o g l o b u l i n lambda, bcr, c-s/s) have shown that bcr/ abl chimeric DNA can be produced by reciprocal translocation between chromosomes 9 and 22 [14], by insertion of c-abl into bcr [15], or by complex translocations that combine bcr/abl at a third chromosomal site [16]. Translocations that appear to be balanced cytogenetically often contain deletions [11, 12] and, as case 5 in our study suggests, may also sometimes involve duplication. Clearly, considerable complexity exists in bcr/abl exchanges and this may be particularly true for cases of Phnegative CML. Ph-negative CML has previously been regarded as a subgroup of CML patients with a poorer prognosis [17]. This conclusion was based on studies carried out prior to the application of molecular genetics and may well be due to the i n c l u s i o n of cases that were not, in fact, CML. It remains to be established whether bcr rearrangement-positive, Ph-negative, CML is associated with a poorer prognosis by vir-

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P u b l i s h e d cases of P h - n e g a t i v e bcr r e a r r a n g e m e n t - p o s i t i v e CML

Table 3 Karyotype

Number of cases

46,XY,t(9;12)(q34;q21) 46,XY Normal 46,XY,t(9;11)(q34;q13) Normal 46,XY/46,XY del(22)(q11) 46,XY, del(16)(q22) 46,XY,t(6;22)(p21;q11) 46,XX,t(1;3;5;9;22) 46,XY,t(5;22;9)(q31;q11;q34) 46,XY,t(9;22)(q22;q11), + other abnormalities 46,XY,t(2;9) 46,XY/46,XY,t(5;6) Normal 46,XY,t(9;22)(q34;q11/, + t(5;22q - )(q11;p11) 46,XX,t(8;22)(p12;q11)

1 1 2 1 8 1 1 1 1 1 1 1 1 6 1 1

IRef.] [16] [18] [15] [19] [20, 21) [20, 21] [20.21] [22] [22] [23, 24] [24] [8] [8] [8] [25] [26]

tue of the i n c l u s i o n of cases w i t h c o m p l e x genetic exchanges. P r e l i m i n a r y e v i d e n c e suggests that these cases r e s p o n d to c h e m o t h e r a p y and b e h a v e c l i n i c a l l y as t y p i c a l P h - p o s i t i v e CML. Supported by Lifecodes Corporation, the United Leukemia Fund and the Cancer Research and Treatment Fund. The authors thank L. Soper for densitometry of autoradiographs for case 5; M. Silverstein and J. Forde for technical assistance, and S. Fortier for help in manuscript preparation.

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