Chromosome 22 breakpoints in variant Philadelphia translocations and Philadelphia-negative chronic myeloid leukemia

Chromosome 22 Breakpoints in Variant Philadelphia Translocations and Philadelphia-Negative Chronic Myeloid Leukemia P. J. Browett, H. M. G. Cooke, L. M. Secker-Walker, and J. D. Norton

ABSTRACT: The standard t(9;22)(q34;q11) found in Philadelphia (Ph) chromosome positive chranic

myeloid leukemia (CML) involves a highly restricted (5.8 kb) chromosome 22 breakpoint cluster region (bcr), which results in the formation of a chimeric gene comprising e x o n s from the 5' e n d afbcr and protooncogene c-abl coding sequences from chramasame 9. In a survey of 21 patients with hematologic a n d clinical features of CML we detected rearrangement of the chromosome 22 bcr by g e n e probe analysis in all cases, including 16 with a standard t(9;22), two with variant Ph translocations [t(lO:22)(q26;ql l);t(11;22)(p15;q11)], one with a complex Ph translocation [t(9;11;22)(q34;q13;q11}], one with a complex translocation a n d a masked Ph[t(9;14;22) (q34;q24;q11)], and one Ph-negative case with a t(1;9)(p32;q34). These abservations further substantiate the suggestion that, despite karyotypic heterogeneity, a c o m m o n underlying molecular lesi on, the bcr-abl gene chimera, is involved in the disease pathogenesis of CML.

INTRODUCTION Over 90% of cases of chronic myeloid leukemia (CML) are characterized by the Philadelphia (Ph) chromosome. This c o m m o n l y arises from a reciprocal translocation between the long arms of chromosomes 9 and 22 [1] and results in the juxtaposition of the c-abl protooncogene from chromosome 9 with part of the breakpoint cluster region" (bcr) (also termed Phl) gene on chromosome 22 [2, 3]. The resultant hybrid bcr-abl gene encodes for a novel mRNA of 8.5 kb and an altered c-abl phosphoprotein of 210 kd with increased tyrosine kinase activity [4-7]. The breakpoint on c h r o m o s o m e 22 occurs within a defined region of 5.8 kb termed the bcr, and a rearrangement within this area can be mapped by Southern blot hybridization techniques in most cases of Ph-positive CML [8, 9]. Recent studies have shown that similar breakpoints within bcr may occur in some cases of CML that lack the Ph

From the Academic Department of ltaematology, Royal Free. llospital School of Medicine. London, England.

Address reprint requests to: Dr. L.;',I. Seeker-Walker, Academic Department of Haemato]ogy, Royal Free Hospital School of Medicine, Pond Street, Hampstead, London NW3 2QG. England. Received June 22, 1988; accepted September 28, .1988.

169 © 1989 Elsevier Science Publishing Co., Inc. 655 Avenue of the Americas, New York, NY 10010

Cancer Genet Cytogenet 37:169-177 (1989) 0165-4608/89/S03.50

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P.I. Browett et al. chromosome, particularly those with clinical and hematologic features typical of Phpositive CML [10-16]. In a small number of these Ph-negative cases it has been possible to demonstrate either translocation of the c-abi protooncogene from chromosome 9q to c:aromosome 22q, a novel mRNA of 8.5 kb, or a p210, suggesting a c o m m o n underlying molecular mechanism in disease pathogenesis that results from the formation of a chimeric bcr-abl gene [11, 12, 14, 161. Variant Ph translocations t(v;22), where v represents any chromosome other than 9, and c o m p l e x Ph transtoca:ions riO;v;22) may occur in a p p r o x i m a t e l y 8% of Phpositive CML cases [27-19]. However, the significance of the additional chromosomal abnornmlities in these cases is unclear, although limited studies have shown bcr breakpoints with evidence of a resultant bcr-abl chimera [16, 20, 21]. In order to explore these issues further we analyzed bcr gone rearrangement as a marker of the bcr-abl fusion in cases of CML that are welt characterized ciinically and either lack the Ph c h r o m o s o m e or have a variant or complex Ph translccation. The results are compared with those obtained from a larger series of cases with the standard Ph chromosome.

MATERIALS AND METHODS Patients Cytogenetic and molecular investigations were performed on peripheral blood or belle marrow samples obtained from 21 patients with clinical and hematologic features of CML in either chronic phase or blast crisis [22].

Cytogenetic Analysis Cytogenetic analysis was carried out on unstimulated cultures of peripheral blood or bone marrow harvested directly or after 2 4 - 7 2 hours in culture [23]. In case 5, a bone marrow s a m p l e obtained prior to allogeneic bone marrow transplantation (BMT) was anaiyzed after rescue from a frozen sample and was cultured for 72 hours with phytohemagglutinin and interlcakin 2[24].

bcr Analysis High m o l e c u l a r weight DNA was prepared from whole bone marrow or peripheral blood by using standard techniques. DNA (5-6 ttg) was digested with each of the restriction enzymes EcoRI, BciI, BamHI, BglII, or HindlII, and after separation of the fragments by electrophoresis through 0 . 7 - 1 % agarose gels, transferred to nitrocellulose filters by the method of Southern [25]. Pooled normal h u m a n peripheral blood was used as a control in all experiments. Probes were labeled with 32p-dCTP to a specific activity of 3 - 5 x 10' cpm//zg by a modification of the random otigopriming method [26]. Hybridization and autoradiography were performed as described previously [24]. Two probes were employed in this study, as shown diagramatica!ly in Figure 3A; all DNA samples were initially hybridized with a 3' bcr probe (bcr-1), a 1.2-kb HindIII/BglII genomic fragment (Oncogene Science Inc). A 5' cDNA bcr probe (bcr-2) was used on those samples in which bcr rearrangement could not be demonstrated with the 3' bcr-1 probe.

171

bcr Rearrangement in Variant Ph CML

RESULTS

Clinical and Cytogenetic Findings Of 21 patients presenting with clinical and hematologic features of CML, 16 (cases 6 21) were found to have a standard t(9:22)(q34;q11) Ph translocation, four (cases 1-4) had variant or c o m p l e x Ph translocations, and one (case 5) was Ph negative. The salient clinical and laboratory features of the variant and Ph-negative cases are presented in Table 1. Cases 1 and 2 were analyzed in lymphoid blast crisis that had followed a chronic phase of 17 years and 10 months, respectively. The other three patients (one child and two adults) were in chronic phase with peripheral blood and bone marrow appearances indistinguishable from those seen in standard Ph-positive chronic-phase CML. All patients initially showed a good response to b u su l p h an or hydroxyurea, although considerable variation in the duration of chronic phase was noted. The partial karyotypes of the Ph-variant and Ph-negative cases are presented in Figures 1 and 2, and the findings are summarized in Table 2. In the two variant cases (cases 3 and 4) c h r o m o s o m e s 10 and 11, respectively, were the recipients of 22ql 1ter. In cases 1 and 2, chromosomes 11 and 14, respectively, were involved in complex translocations in addition to c h r o m o s o m e bands 9q34 and 22q11. In case 2 this c o m p l e x translocation resulted in a masked Ph chromosome. The Ph-negative case (case 5) had a t(1;9) at presentation, with evidence of clonal ev o l u t i o n prior to BMT. In a normal bone marrow sample taken 3 months after allogeneic BMT from a h u m a n leukocyte antigen (HLA) identical, mixed leukocyte culture (MLC) nonreactive sister, a normal female population with one XY,t(1;9) cell in one of 15 metaphases examined was found. This case is fully reported elsewhere [27].

bcr Analysis In all five cases with either Ph-variant or Ph-negative CML, a breakpoint within the 5.8-kb bcr of c h r o m o s o m e 22 was demonstrated with two or more restriction enzymes. The approximate regions to w h i c h the breakpoints were mapped are sh o w n in Figure 3A. The distribution of breakpoints between exons 1 and 4 was similar to that seen in the 16 cases of standard Ph-positive CML, all of w h i ch showed bcr rearrangement (data not shown). Figure 3B panel 1, lanes 1 - 5 show representative Southern blots of the variant and Ph-negative cases following digestion with the

Table 1.

Hematologic and clinical features of Ph-variant and Ph-negative cases of CML

Case

Age/sex

Hb (g/dl)

1 2 3 4 5

40/F ~ 54/M 8/M 72/F 26/M

7.9 10.8 8.0 10.8 10.0

Platelets ( × 10~/L)

WBC ( × 109/L)

Spleen

(cm}

Stage

Survival (months from diagnosis)

36 23 984 848 427

188 240 240 51 150

+7 cm +10 + 15 0 +15

Lymphoid blast crisis Lymphoid blast crisis Chronic Chronic Chronic

194" 19 b 9t 48 c 17

° Analyzed in lymphoid blast crisis followingchronic phase of 17 years. b Presented in lymphoid blast crisis followingchronic phase of 10 months. c Died of colon carcinoma while in stable chronic phase.

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Figure I Partial karyotypes and diagrammatic representations of variant (cases 3 and 4), complex (case 1), and masked (case 2} Ph translocations. The juxtaposition of bands 9q34 and 22qll proposed in cases 3 and 4 cannot be visualized cytogenetically.

enzyme ]3glII and h y b r i d i z a t i o n with a 3' bcr-1 probe. In panels 2 and 3 (Figure 3B) bcr analysis of DNA following digestion with HindItI, prepared from case 5 i m m e d i ately before and 3 months after allogeneic BMT is presented. Prior to BMT there was a novel rearranged bcr fragment of equivalent intensity to germ line, indicating an essentially 100% clonal population. Engraftment was d o c u m e n t e d at 3 weeks; how-

173

Figure 2

Table 2

Partial karyotype of Ph-negative CML case 5.

C h r o m o s o m e findings in P h - v a r i a n t and P h - n e g a t i v e CML

Case

Stage

Tissue

Cells (no.)

Karyotype

Lymphoid blast crisis

BM

Lymphoid blast crisis

BM

26 1 12 8

Chronic Chronic Chronic

PB BM PB

Pre-BMT

BM

Post-BMT

BM

46,XX,t(9;11;22)(q34;q13;q11) 46,XX 46,XY,t(9;14;22)(q34;q24;q11) 47,XY,der(1),der(2),der(7),der(8), der(9), t(9;14;22),der(10), +der(22) 46,XY,t(!0;22)(q26;q11) 46,XX,t(il;22)(p15;q11) 46,XY,t(1;9)(p32;q34) 46,XY 46,XY,t(i;9)(p32;q34] 46,XY,t(1;9) with clonal evolution 46,XY XY,t(1;9) 46,XX

22 12 4 14 5 17 5 1 14

174 A bcr

Bg Bg exorls

Bg BgBg Bg

:

breakpoints

f ii:'/

;H 5

BI

8

1

~=~

cases 1 , 3 , 4 cases

B

bcr-1

B

BC

1" ~ : "1 2

"

4

B

~C .... ....

Bg

.

1 2 3

B probes

BgBg

H

;

Ikb

2,5

probe

~,oX" O~'~

Cases

pre - BMT

p o s t - BMT

kb

4-2-

Bgl II

H i n d III

F i g u r e 3 (A) Restriction map of the bcr locus of chromosome 22q. The exons are represented as solid vertical bars and the restriction endonucleases are as follows: E, EcoRI; Bg, BglII; B, BamHI; H, HindfII; Bc, BclI. The structural relationship of the 3' (probe 1) and 5' (probe 2) bcr probes is indicated. The solid boxes Below the map represent the approximate positions of the breakpoints of the variant Ph and Ph-negative CML cases. (B) bcr analysis by Southern blot hybridization. Panel 1 shows representative autoradiographs of the variant and Ph-negative cases of CML (cases 1-5). Control (C) DNA was prepared from pooled normal human peripheral blood; next to the control is a case of CML with a standard t(9;22). Panel 2 shows results of analysis of DNA prepared from case 5 pre- and post-BMT. The additional bands in the postBMT analysis were present in all lanes in this experiment including the control, as shown, and were due to a plasmid contaminant. The size of the 8ermline fragments is indicated in kilobase (kb) pairs.

bcr Rearrangement in Variant Ph CML

175

ever, analysis of DNA prepared from a bone marrow sample taken at 3 months postBMT demonstrated the persistence of a faint [relative to germ line) rearranged bcr fragment of a similar size to that seen in the pretransplant specimen. These results w o u l d be consistent with the persistence of a small clonal population with bcr rearrangement. This patient subsequently relapsed with an epidural chloroma and died in m y e l o i d blast crisis 5 months following BMT. DISCUSSION

The recent availability of molecular probes for the bcr of chromosome 22 has provided a unique marker for the standard Ph translocation, with breakpoints detectable in virtually all cases of CML with the t(9;22) [8, 9, 22]. Variant or complex translocations occur in up to 8% of cases of Ph-positive CML, and in this study we have identified similar c h r o m o s o m e 22 breakpoints in two variant Ph translocations in w h i c h the reciprocating chromosome regions were 10q26 and 11p15, respectively, and two c o m p l e x translocations (one resulting in a masked Ph chromosome), each involving breakpoints 9q34 with 11q13 and 14q24, respectively. Furthermore, the t[1;9) Ph-negative CML case proved interesting in that a chromosome 22 breakpoint was d o c u m e n t e d at the molecular level despite the presence of two cytogenetically normal c h r o m o s o m e s 22. Following allogeneic BMT, an identical bcr rearrangement was detected on analysis of DNA prepared from bone marrow in w h i c h a single XY,t(1;9) (7%) cell was found among normal female donor cells. Peripheral blood and bone marrow appearances were normal at this time; however, myeloid blast crises was d o c u m e n t e d 10 weeks later. In all the cases analyzed, hematologic and laboratory features were typical of Phpositive CML, including two cases that transformed to l y m p h o i d blast crises. However, considerable variation in the duration of chronic phase and overall survival was noted. Nonetheless, our data w o u l d be compatible with similar u n d e r l y i n g molecular events to those d o c u m e n t e d in standard Ph-positive CML, with formation of a chimeric bcr-abl gene. This is further supported by recent studies of Ph-negative CML where bcr rearrangement, and in some cases a novel mRNA of 8.5 kb a n d / o r p210, has been demonstrated in those cases with features indistinguishable from Phpositive CML [10-16]. In addition, the novel p210 has recently been reported in association with a t(21;22) [16], and Hagemeijer postulated that the c h r o m o s o m e region 9q34 is always involved in variant Ph translocations after demonstrating c-abl on 22q by in situ hybridization in two patients with variant Ph translocations [28]. However, a cautionary note is provided by the recent report of W i e d e m a n n et al. in w h i c h bcr rearrangement could not be demonstrated in a case of CML with a t(11;22)(pl 5;q'l 1)[16]. In conclusion, this study has shown similar chromosome 22 breakpoints in cases of Ph-negative and variant Ph CML to those seen in CML with a standard Ph translocation. In these cases, clinical and hematologic features were indistinguishable from those of Ph-positive CML. Although the significance of the additional c h r o m o s o m e breakpoints in these cases is not clear, the results suggest a c o m m o n u n d e r l y i n g m o l e c u l a r lesion in disease pathogenesis in cases of Ph-positive, variant, and complex Ph CML, and in some cases of Ph-negative CML. This work was supported by the Kay Kendall Leukaemia Fund, the Peter Samuel Royal Free Fund, and the Leukaemia Research Fund. P. J. B. is the holder of a Wellcome-New Zealand MRC Overseas Research Fellowship. The authors thank the following clinicians for providing samples for analysis: Dr. J. D. Matthews and Dr. P. A. Ockelford, Auckland Hospital, New Zealand; Dr. A. G. Prentice, Plymouth General Hospital; and Professor A. V. Hoffbrand and Dr. H. G. Prentice, Royal Free Hospi-

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tal, London. We also acknowledge the help of Dr. M. Parslow, Cytogenetics Laboratory, Princess Mary Hospital, Auckiand.

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