Molecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: genesis and deletion of derivative chromosome 9

Cancer Genetics and Cytogenetics 194 (2009) 30e37

Molecular cytogenetic characterization of variant Philadelphia translocations in chronic myeloid leukemia: genesis and deletion of derivative chromosome 9 Ayda Bennoura,*, Halima Sennanaa, Mohamed Adne`ne Laatirib, Moez Elloumic, Abderrahim Khelifb, Ali Saada a

Cytogenetics Division, Department of Cytogenetics, Molecular Genetics, and Biology of Reproduction, Farhat Hached Hospital, Sousse 4000, Tunisia b Department of Hematology, Farhat Hached Hospital, Sousse, Tunisia c Department of Hematology, He´di Chaker Hospital, Sfax, Tunisia Received 5 March 2009; received in revised form 2 May 2009; accepted 24 May 2009

Abstract

The mechanisms for the formation of variant Philadelphia (Ph) translocations that occur in 5e10% of patients with chronic myeloid leukemia (CML) are not fully characterized. Studies on the prognosis of these variant translocations have yielded conflicting results, especially regarding imatinib outcome and the status of deletions on the derivative chromosome 9. To shed light on these controversial subjects, we sought to analyze all variant translocation cases presented at diagnosis and identified in our institution between the years 2001 and 2008. Of 336 CML patients who presented at diagnosis and were studied by conventional cytogenetics and fluorescence in situ hybridization (FISH), 25 patients (7.44%) exhibited variant Ph-rearrangements. All chromosomes could be implicated in variant Ph rearrangements, with 32 breakpoints defined. Their distribution was located preferentially in the CG-richest regions of the genome. Deletions on der(9) were observed in 15 of the 25 cases (60%), a greater proportion in typical Ph translocations (12e15%). Both oneand two-step mechanisms were encountered in our series, as well as multiple-step mechanisms, which originate more complex rearrangements. Higher prevalence was observed for the two-step mechanism (56%). Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and 9q34 deletion status. Ó 2009 Elsevier Inc. All rights reserved.

1. Introduction Chronic myeloid leukemia (CML) is a clonal myeloproliferative disorder characterized by the presence of Philadelphia chromosome (Ph), resulting from the t(9;22) (q34;q11.2) [1]. The crucial pathogenetic consequence of this translocation is the formation of a novel and chimeric BCR/ABL gene in the breakpoint region of the derivative chromosome 22 [2]. In 5e10% of cases, variant rearrangements involving 9q34, 22q11.2, and one or more additional genomic regions generate this chimeric gene [3]. Variant Ph chromosome translocations may be caused by insertion or other mechanisms [4,5]. There is open debate about the formation of these rearrangements: some researchers [6e9] have invoked a one-step mechanism, wherein chromosome * Corresponding author. Tel. and fax: þ216-73-219488. E-mail address: [email protected] (A. Bennour). 0165-4608/09/$ e see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2009.05.010

breakage occurs on three different chromosomes simultaneously in a three-, four-, or five-way translocation, then reciprocally rejoin at the same time. Others [10,11] have suggested a two-step mechanism, in which a standard two-way t(9;22) is followed by subsequent translocation involving additional chromosomes, and some recent studies [3,9] have reported both mechanisms in the same patient. It has been suggested that the formation of variant translocation in a two-step mechanism is similar to or is in essence a clonal evolution [12]. Clonal evolution typically coincides with or precedes accelerated phase or blast crisis of CML. Therefore, an inherent implication of the two-step mechanism is that variant translocations might be associated with a poorer prognosis [4]. These translocations result in the BCR/ABL fusion on chromosome 22, on 9q34, or variants that can be perceived by fluorescence in situ hybridization (FISH) [13]. FISH analysis of BCR and ABL rearrangements using dual-color probes is an effective tool not only for the localization of

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the BCR/ABL fusion gene, but also for the detection of ABL deletion on the derivative chromosome 9 [14,15]. Recently, these deletions have been described by FISH with frequencies of 15% [14], and this incidence has been shown to vary for different cytogenetic subgroups of CML, with a significantly higher incidence of deletion in patients with variant Ph translocations [4,14]. A controversial hypothesis has been proposed concerning the onset of deletions on der(9) [9]. Most authors have considered that deletions on der(9) are associated with a shorter duration of chronic phase and shorter survival in patients treated with interferon therapy [16e19], although it has been suggested that an effective treatment with a selective tyrosine kinase inhibitor such as imatinib mesylate against the activity of BCR/ABL in CML [20] might counter adverse prognostic factors such as der(9) deletions [21]. To date, it remains controversial whether patients with a der(9) deletion have a different outcome if treated with imatinib mesylate. To shed light on these controversial subjects, we sought to analyze all the variant translocation cases of CML identified by our cytogenetics laboratory between the years of 2001 and 2008.

2. Patients and methods 2.1. Patients From January 2001 to August 2008, we examined bone marrow samples from 336 CML patients with chronic myeloid leukemia; of these, 25 patients, with variant chromosomal rearrangements, were in chronic phase at diagnosis (Table 1). 2.2. Conventional cytogenetics Conventional cytogenetic analysis was performed on bone marrow specimens after 24e48 hours of unstimulated culture. The cells were cultured and processed by R-banding. At least 20 metaphases were captured and analyzed. Karyotypes were described according to ISCN 1995 [22]. 2.3. Fluorescence in situ hybridization FISH analyses were performed according to the manufacturer’s recommendations. The Vysis locus-specific extra-signal LSI BCR/ABL ES dual-color translocation probe (Abbott Molecular, Des Plaines, IL), labeled with SpectrumGreen and SpectrumOrange, was used to locate the BCR/ABL fusion gene. At least 20 metaphases per patient were analyzed. Interphase nuclei were also analyzed in some cases presenting uncommon signal patterns or in case of lack of metaphase chromosomes. To rule out false positive results, a cutoff level was established at 3%, based on analysis of bone marrow samples from two healthy patients (200 interphase nuclei counted). A SpectrumAqua

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probe covering the ASS1 gene to the 50 ABL sequence on chromosome 9q34 was also included with the BCR/ABL ES probes, to assist in the detection of 9q deletions. To elucidate the genesis of variant translocations, wholechromosome paints (WCP) were complementarily used. All probes were provided by Vysis and the hybridization and detection were performed according to the manufacturer’s protocols. Images were captured and processed with Axioscope 2 and Cytovision 3.93 (Applied Imaging, Santa Clara, CA).

3. Results 3.1. Cytogenetics profile Out of 336 CML patients, a total of 25 CML cases with variant translocations were identified. Patient characteristics are summarized in Table 1. Of the 25 cases with variant Ph rearrangements, 11 had a simple variant translocation, 13 had a complex variant translocation, and one (case 6) had a masked variant Ph translocation. Simple variant translocations appeared to involve 22q11.2 and another chromosome other than chromosome 9. Complex variant translocations appeared to involve three or more chromosomes: 9, 22, and one or more others. A majority of variant translocations (22 of the 25 cases) were involved in three-way translocation (i.e., chromosomes 9, 22, and one other), two variants involved four-way translocation (cases 11 and 21), and only one case involved fiveway translocation (case 15). Breakpoints involved in the variants are listed in Table 1 and are illustrated in the ideogram in Figure 1. We did not detect any rearrangement occurring repeatedly in our cohort of 25 patients. We did, however, identify several chromosomes, chromosomal regions, and breakpoints that were involved in variant Ph rearrangements more than once (Fig. 1). Besides chromosomes 9 and 22, chromosome 1 was the most frequently involved in variant Ph rearrangements, at six times. Chromosomes 4, 12, and 13 were involved four times. Chromosome 3 was involved three times. The other chromosomes were involved only once or twice. Chromosomes 5, 8, 14, 15, 16, 18, 20, and Y were never involved. We defined 32 breakpoints in all, in a nonrandom pattern of distribution (Table 1; Fig. 1). 3.2. FISH The LSI ES-BCR/ABL probe allowed the detection of the BCR/ABL fusion gene on the Ph chromosome in all cases but one. In case 6, the BCR/ABL fusion gene was located on the short arm of chromosome 3. Distinct WCP FISH signal patterns suggested either a one-step or a two-step mechanism for the formation of variant translocation. A one-step mechanism could be generated after three or more chromosomes break at the

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Table 1 Cytogenetic and FISH results for 25 cases of chronic myeloid leukemia, with mechanism of variant translocation and locus designation of the additional chromosome involved in the Ph translocation Case

Karyotype at diagnosis

Locus

WCP signal

Mechanism

Deletion on der(9)?

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25

46,XX,t(9;7;22)(q34;p21;q11.2)[16] 46,XX,t(9;21;22)(q34;q22;q11.2)[21] 46,XY,t(11;9;22)(q12;q34;q11.2)[19] 46,XX,t(6;9;22)(q22;q34;q11.2)[16] 46,XY,t(X;9;22)(p22;q34;q11.2)[20] 46,XX,t(3;9;22)(p14;q34;q11.2),del(2)(p24)[16] 46,XY,t(3;9;22)(q26;q34;q11.2)[15] 46,XX,t(1;9;22)(p36;q34;q11.2)[20] 46,XY,t(9;13;22)(q34;q13;q11.2)[20] 46,XY,t(4;9;22)(q27;q34;q11.2)[17] 46,XY,t(9;12;12;22)(q34;q21;p12;q11.2)[18] 46,XX,t(10;9;22)(q25;q34;q11.2)[18] 46,XX,t(9;17;22)(q34;q23;q11.2)[20] 46,XX,t(3;9;22)(q26;q34;q11.2)[18] 46,XX,t(1;1;2;9;12;13;22)(q24;q31;p21;q34;q11.2)[17] 46,XY,t(4;9;22)(q13;q34;q11.2)[15]/47,idem,þ8[4] 46,XX,t(9;17;22)(q34;q22;q11.2)[17] 46,XX,t(6;9;22)(q21;q34;q11.2)[16] 46,XX,t(9;12;22)(q34;p13;q11.2)[15] 46,XX,t(9;12;22)(q34;q13;q11.2)[15] 46,XY,t(1;1;9;22)(p34;q42;q34;q11.2)[16] 46,XY,t(4;9;22)(q34;q34;q11.2)[20] 46,XY,t(1;9;22)(p35;q34;q11.2)[10]/45,idem,Y[5] 46,XX,t(9;19;22)(q34;q13;q11.2),17[13]/46,XX[3] 46,XY,t(9;13;22)(q34;q31;q11.2)[18]

7p21 21q22 11q12 6q22 Xp22 3p14, 2p24 3q26 1p36 13q13 4q27 12q21, 12p12 10q25 17q23 3q26 2p21, 1q24, 1q31, 12p12, 13p12 4q13 17q22 6q21 12p13 12q13 1p34, 1q42 4q34 1p35 19q13 13q31

ins(7;22) t(21;22) ins(9;11) ins(9;6) ins(X;22) ins(3;9) ins(3;22) ins(1;22) ins(9;13) ins(4;22) t(9;12), t(12;22) ins(9;10) t(9;17) ins(9;3) ins(1;9), t(12;13), t(1;2) ins(4;22) ins(22;17) ins(6;22) ins(9;12) t(9;12) ins(1;22), t(1;9) ins(9;4) ins(1;22) ins(9;19) ins(9;13)

1 step 1 step 2 step 2 step 1 step 2 step 1 step 1 step 2 step 1 step multiple step 2 step 2 step 2 step 2 step 1 step 2 step 1 step 2 step 2 step multiple-step 2 step 1 step 2 step 2 step

yes yes yes yes no yes yes yes yes no yes no no no no yes no yes no no no yes yes yes yes

All cases were in chronic phase at diagnosis. Abbreviations: FISH, fluorescence in situ hybridization; WCP, whole-chromosome paint.

same time and reciprocally rejoin; a two-step mechanism could be generated by two successive rearrangements. First, the standard t(9;22) is formed, leading to the BCR/ABL fusion gene on der(22) and the ABL/BCR reciprocal fusion on der(9). Second, the segment of der(9) or der(22) exchanges material with the variant chromosome. From the WCP FISH study, we observed two main rearrangements. The first was insertion of chromosomal material from chromosome 22 into the variant chromosome. This was observed in 9 of the 25 patients. In this pattern, the BCR/ABL fusion gene is located on der(22). The WCP analysis did not reveal rearrangements involving the variant chromosome and chromosome 9 for the 9 cases in which a 22q chromosomal material was located on the variant chromosome. This suggests that three breaks have occurred on chromosomes 9, 22, and the variant chromosome and that 9q34~qter rejoined 22q11 at the same time as 22q11~qter inserted into the variant chromosome. This pattern is indicative of a one-step mechanism formation (Fig. 2). Conversely, insertion of chromosomal material from variant chromosome into der(9), observed in our series in 9 of the 25 patients, seems to be a genetic event secondary to the t(9;22)(q34;q11.2), thereby implying a second breakpoint in 9q34. The location of BCR/ABL fusion gene on the der(22) implies that t(9;22) happens first, with

subsequent translocation between chromosome 9 and the variant chromosome removing parts of the variant chromosome on der(9) chromosome. Two cases (11 and 21) showed both rearrangements coexisting: an insertion of parts of chromosome 22 on the variant chromosome and insertion of chromosomal material of the variant chromosome on der(9), suggesting that other, more complex multiple-step mechanisms can be involved in the formation of variant translocation. In case 11, variant translocation involved both chromosomes 12 (in addition to chromosomes 9 and 22). At first, patient 11 seemed to have quite a complicated variant translocation, but then examination of both chromosomes 12 and of chromosomes 9 and 22 with WCP FISH for 9, 12, and 22 revealed reciprocal translocation between chromosomes 9 and 12dand, in the same metaphases, another translocation between chromosomes 12 and 22. Thus, variant rearrangement in case 11 resulted from three reciprocal translocations, respectively between chromosomes 9 and 22, chromosomes 9 and 12, and chromosomes 12 and 22. This kind of rearrangement requires two breaks on each chromosome 9 and 22; thus, a BCR/ ABL fusion gene was located on der(22) and chromosomal material was exchanged reciprocally between chromosomes; the t(9;22) could not happen simultaneously with the other translocations, and therefore this variant rearrangement was a result of multiple-step mechanism formation.

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Fig. 1. Composite karyogram indicating breakpoints (dots) involved in variant Philadelphia rearrangements in a Tunisian cohort of chronic myeloid leukemia patients.

Case 21 had variant translocation between both chromosomes 1 with chromosomes 9 and 22. Extra-signal FISH analysis revealed BCR/ABL fusion gene on der(22), and WCP for chromosomes 1, 9, and 22 revealed insertion of parts of chromosome 22 on one chromosome 1, which could be generated at the same time as t(9;22) (i.e., insertion of segment 9q34 on chromosome 22 and translocation of segment 22q11 on chromosome 1) while a reciprocal translocation took place between chromosome 9 and the other chromosome 1. This latest translocation was an event that should occur independently of t(9;22), because it requires a second breakpoint on 9q34. Thus, rearrangements in this case are the result both of mechanisms with one step, for the formation of t(9;22) and simultaneously insertion of 22 segment on chromosome 1, and of two steps, with t(1;9) as a second event to the first rearrangement. Two other cases (15 and 17) presented unusual WCP FISH signal patterns. Case 15 had translocation of part of chromosome 9 on chromosome 1, suggesting that two breaks took place on chromosome 9. Thus, this translocation occurred in a second step after the standard t(9;22).

The fusion BCR/ABL signal was on der(22), and chromosome 22 seems not rearranged with chromosome 1. Case 17 presented another unusual rearrangement. WCP for chromosomes 9, 17, and 22 revealed, in addition to t(9;22), translocation of part of chromosome 17 to chromosome 22, suggesting that two breaks took place on chromosome 22. The LSI ES-BCR/ABL extra-signal probe revealed the fusion signal (BCR/ABL) on der(22). In both cases 15 and 17, these observations point to a two-step rearrangement process. Cases 13 and 20 both disclosed common reciprocal translocation involving chromosome 9 with, respectively, chromosomes 17 and 12, which is considered a rare WCP signal pattern in our group. This signal pattern requires two breaks on chromosome 9. Chromosome 22 seems rearranged only with chromosome 9, and there were no chromosomal material from the variant chromosome on 22. This observation is in favor of a common two-step mechanism (Fig. 3). In one 6, the karyotype was modified after applying the WCP FISH analysis. A longer chromosome 9 with both chromosomes 22 of normal size and a shorter chromosome

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Fig. 2. (A) Schematic representation of a one-step mechanism rearrangement, as occurred in case 8: three breaks occurred at the same time on chromosomes 1, 9, and 22. Reciprocal rejoining led to the variant translocation. (B) Whole-chromosome paint reveals insertion of chromosomal material from chromosome 22 on chromosome 1.

3p, in case 6, turned out to be a cryptic variant translocation among chromosomes 3, 9, and 22 (Table 1), which results from a two-step mechanism. 3.3. Deletions on der(9) and correlation with mechanism of translocation Loss of the ASS1 gene adjacent to the ABL gene was detected in 15 of our 25 cases (60%), which is a greater proportion than reported in the literature. Seven patients with deletion 9q34 exhibited a one step-mechanism of translocation formation by FISH, 7 other patients exhibited a two-step mechanism, and one patient exhibited a multiple-step mechanism. A recent study of 41 CML patients with variant Ph translocation identified 8 cases with 9q34 deletion, 7 of which were generated after a one-step mechanism [9]. The authors found no association between the variant translocation mechanism and the status of 9q34 deletion. Because each of the studies reporting the prevalence of 9q deletions in variant translocations includes a small number of cases, we considered results of several data of recent studies summarized by Gorusu et al. [4]. They demonstrated that deletion der(9q) occurs in 15% of CML in general, in 12.7% of standard Philadelphia translocations, and in 40.4% in variant Philadelphia

Fig. 3. (A) Schematic representation of a two-step mechanism rearrangement, as occurred in case 13. This mechanism may result in a succession of two different rearrangements: the first one could be the classical t(9;22)(q34;q11), followed by another reciprocal translocation between chromosomes 9 and 17. (B) Whole-chromosome paint reveals reciprocal translocation between chromosomes 9 and 17.

translocations. Thus, the literature is clear on the increased prevalence of deletions among variant translocation CML [7,23].

4. Discussion Variant Ph translocations occurring in patients with CML have been recognized for more than 20 years [24e29] and have been reported in 5e10% of CML patients. Most descriptions have been based on case reports and small series [7,30e33]. Thus, the clinical course and influence of these variants on long-term outcome is not well known, and conclusions are conflicting. Some studies have suggested that patients with variant Ph translocations may have an adverse prognosis [4,26,28,34,35], but others have suggested that these translocations have no prognostic effect [29,9]. We analyzed 25 cases of variant translocation CML identified in our laboratory. Any of the chromosomes can be involved in variant Ph rearrangements, because, although chromosomes were not described in they present

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series (chromosomes 5, 8, 14, 15, 16, 18, 20, and Y), they have been reported in other recent studies [3,4,9]. Breaks tend to fall within the CG-richest parts of the genome. There is a fairly precise colocalization of breaks within the CG-richest areas (i.e., light bands) of the genome (Fig. 1). This correlation is not absolute, but would be consistent with the limited number of variant breakpoints in the present study and with the imprecise nature of cytogenetic breakpoint assignment. It is recognized that CG richness varies with many other features of the genome [36]; for example, it reflects increases in the density of CpG islands, genes, repetitive elements such as Alu [37,38], and recombination events [39]. CG content also varies with openness of chromatin structure [40] and transcription activity [41]. Jeffs et al. [42] studied variant BCR/ABL rearrangements in CML and found that the 30 portion of the translocated BCR gene recombined preferentially with Alu elements. More recently, Fisher et al. [36] provided a model to elucidate incidence of breaks in variant translocations in leukemia, he proposed that ‘‘open’’ chromatin, which is transcriptionally active, is relatively likely to undergo breakage and repair, with a consequent tendency to illegitimate recombination and translocation. They suggested that breaks might show a high density of Alu elements which are condensed in CG-richest chromosome regions. These theories are consistent with the present findings. With the LSI BCR/ABL ES probe and WCP FISH probes, we elucidated the mechanism for the formation of variant translocation in each of our 25 CML patients. Variant translocations may be caused by different mechanisms: several variant translocations are formed by multiple simultaneous breaks, and some arise as a result of two or more consecutive genetic events. The occurrence of just one break on each of the chromosomes 9 and 22 indicates a one-step mechanism; for example, in case 8, the WCP signals for 1, 9, and 22 reveal insertion of 22 chromosome material into chromosome 1 at the same time as the t(9;22) occurs. This rearrangement resulted from simultaneous breaks on 9q34, 22q11.2, and the variant chromosome (in this case, chromosome 1), followed by mismatch joining of the broken ends (Fig. 2). Many studies [4,7,9] favor this mechanism for the genesis of variant Ph translocations, perhaps because fewer breakpoints are required. Two or more breaks on chromosome 9 or 22 or both indicates a two-step mechanism, with the classical t(9;22) as a first event, followed by insertion between der(9) and the variant chromosome, reciprocal translocation between chromosome 22 and the variant chromosome, or both. This rearrangement suggests more than one break on each of der(9) and chromosome 22. Such a two-step mechanism has been postulated repeatedly in our study (in 14 of the 25 cases). Our data demonstrate that one-step, two-step and also multiple-step occur, but the two-step mechanism appears to be more common than the one-step mechanism (56% vs. 36%).

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In 2 of the 25 cases (8%), we obtained WCP FISH results consistent with both mechanisms in the same patient, resulting in a multiple-step mechanism. This rearrangement engages two variant chromosomes with chromosomes 9 and 22. Mechanisms of rearrangement in our cases occurred independently; they were not mixed, as described by Gorusu et al. [4]. The location of the BCR/ABL fusion gene on chromosome bands other than 22q11 represents a rare variant Ph that can be observed only by FISH. Case 6 demonstrates a masked BCR/ABL fusion gene on chromosome 3. It results from a two-step mechanism, as revealed by WCP and BAC probes [5]. Variant Ph rearrangements involve additional chromosomes and, possibly, additional genes implicated with the BCR/ABL rearrangement. This may have prognostic implications when it occurs in the setting of clonal evolution and could adversely affect outcome, particularly when using a therapy such as imatinib [43]. It is not surprising that deletions occur more frequently in variant translocation CML cases (60%), higher than in typical translocation cases (15%). These deletions have been reported to occur more frequently in patients with variant Ph translocations (40e50%) [16e18], with multiple breakage occurring on chromosomes at the same time, especially when insertions into chromosome 9 took place. In the present series, seven cases with deletions of 9q demonstrated insertion of a variant chromosome into chromosome 9 (Table 1). It has been suggested that deletions of derivative chromosome 9 account for the inferior survival of patients with variant Ph translocations [16]. Huntly et al. [17] reported a major cytogenetic response rate of 48% in patients with 9q34 deletion treated with imatinib in chronic phase, compared with 72% for patients without deletion. Findings from other recent studies [9] do not support the hypothesis that CML patients with 9q deletion respond less favorably to imatinib therapy. These studies demonstrated that the response rates, response durations and survivals were similar to those of patients with classic Ph. Thus, they suggested that imatinib should be considered in the same way as it is used for all patients with Ph-positive CML.

5. Conclusion We emphasize that whole chromosome painting (WCP) and LSI BCR/ABL dual-color, single fusion probes were valuable in determining the details of complex variant translocation observed in these cases. Our findings support that (i) any of the chromosomes can be involved in variant Ph rearrangements, and breakpoints are frequently localized in CG-richest regions of the genome; (ii) the genesis of variant translocations is via either the one-step or the two-step mechanisms (but a mixed mechanism including both one-step and two-step

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mechanisms is also possible); and (iii) 9q deletions in variant translocations are more frequent than in standard Ph translocations and they may be associated with an adverse outcome. In conclusion, we have shown how, by combining FISH studies with routine cytogenetics, a great deal of information can be gained when these tests detect and elucidate the BCR/ABL fusion gene, deletions, cryptic translocations, and complex variant translocations.

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