On the genesis and prognosis of variant translocations in chronic myeloid leukemia

Cancer Genetics and Cytogenetics 173 (2007) 97e106

On the genesis and prognosis of variant translocations in chronic myeloid leukemia Madhavi Gorusua, Peter Bennb, Zihai Lia, Min Fangb,* b

a Neag Comprehensive Cancer Center, University of Connecticut Health Center, MC1614, Farmington, CT 06030 Division of Human Genetics, Department of Genetics and Developmental Biology, University of Connecticut Health Center, BB#5, 263 Farmington Avenue, Farmington, CT 06030-6140

Received 1 September 2006; received in revised form 6 October 2006; accepted 13 October 2006

Abstract

Variant translocations involving 9q, 22q, and at least one additional genomic locus occur in 5e10% of patients with chronic myeloid leukemia (CML). The mechanisms for the formation of these variant translocations are not fully characterized. Studies on the prognosis of these variant translocations revealed conflicting results. In addition, deletions in the derivative chromosome 9 are reportedly more frequent among variant translocation cases. We analyzed cytogenetic and FISH data from 22 CML patients with variant translocations tested at our laboratory. Deletions were observed in 6 of the 14 cases with FISH data available (43%), consistent with the literature and higher than in typical translocation cases (12e15%). Sequential changes of 9q deletions are possible and could be acquired as the disease progresses in addition to simultaneous formation of the Philadelphia chromosome with the deletion. Variant translocation CML patients with a deletion showed a worse cytogenetic response 1 year after therapy than those without a deletion (P ! 0.05). Variant translocations may be formed by either a one-step or a two-step mechanism. Proper assessment of the prognostic significance of variant translocations requires better categorization of these translocations based on their mechanisms of genesis and the deletion status. Ó 2007 Elsevier Inc. All rights reserved.

1. Introduction Chronic myeloid leukemia (CML) is characterized by the presence of the Philadelphia chromosome (Ph), typically derived from a reciprocal translocation between a chromosome 9 (band q34) and a chromosome 22 (band q11.2) [1]. In 5e10% of the CML cases, one or more additional chromosome loci are involved in the translocations, which are termed variant or complex translocations [2]. Occasionally, chromosomal changes are submicroscopic and appear to be Ph-negative. These are also termed cryptic translocations or masked Ph chromosomes [2,3]. Masked Ph chromosome translocations may be caused by insertion or other mechanisms [4]. Nevertheless, these translocations result in the fusion of BCR and ABL1 genes, which can be detected by fluorescence in situ hybridization (FISH) or polymerase chain reaction (PCR) [3,5]. How the variant translocations form is controversial [6,7]. Many favor the one-step mechanism, wherein chromosome breakage occurs on three different chromosomes * Corresponding author. Tel.: (860) 679-1697; fax: (860) 679-3616. E-mail address: [email protected] (M. Fang). 0165-4608/07/$ e see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.cancergencyto.2006.10.006

simultaneously in a three-way translocation, for instance, and reciprocally rejoin at the same time [7,8]. Others proposed a two-step mechanism, in which a standard two-way t(9;22) translocation is followed by subsequent translocations involving additional chromosomes [9] (Fig. 1). The two-step mechanism suggests that the formation of a variant translocation is similar to or is in essence a clonal evolution [2,5]. 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. The literature is inconsistent on whether variant translocations confer the same clinical course and outcome as standard ones. Most results were based on case reports or small series. Earlier studies concluded that a variant translocation showed no differences in the disease course of CML and had no effect on prognosis, compared with a standard translocation [8,10e12]. More recent studies, however, suggest that variant translocations are associated with an adverse outcome [13e16]. Recently, deletions of the derivative chromosome 9 sequences, der(9), have been reported [17,18]. Some studies

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Fig. 1. Mechanisms of genesis for variant translocations in chronic myeloid leukemia. Chromosome 9 is represented in gray, chromosome 22 is represented in black, and the remaining variant chromosome involved is represented in white. The upper panel illustrates the one-step mechanism; the lower panel, the two-step mechanism (see Introduction section). The segments in the open boxes exchange material as shown by the curved arrows. The letters represent the probe colors used in FISH studies: R for red, labeling the ABL1 gene sequence region; G for green, labeling the BCR gene sequence region; and F for fusion. The normal chromosome homologs are not shown.

demonstrated that CML patients with such deletions had much shorter survival than those without the deletion [17e19]. Those initial studies reported that der(9) deletions were more frequent in patients with variant Ph translocations [17,18], and many other groups have since supported these findings [15,19e24]. We might therefore hypothesize that variant translocations are associated with a poor prognosis due to increased frequency of der(9) deletions in these cases. Earlier studies, however, were based on patients treated with hydroxyurea (HU), interferon a, or both; it has been suggested that an effective treatment such as imatinib mesylate (Gleevec; Novartis, East Hanover, NJ), which is a potent tyrosine kinase inhibitor selectively against the activity of BCR/ABL1 in CML [25], might be able to overcome adverse prognostic factors such as der(9) deletions [26]. To date, it remains controversial whether patients with a der(9) deletion have a different outcome if treated with imatinib mesylate. To help shed light on these controversial subjects, we sought to analyze all the variant translocation cases of CML identified by a Connecticut cytogenetics laboratory between the years of 1991 and 2005. Our findings suggest that (i) 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 mechanisms is also possible) and (ii) variant translocations in CML are associated with an adverse outcome. In addition, our data challenge the notion that all der(9) deletions occur at the formation of Ph chromosome; rather, we found that these chromosomal deletions may occur as a sequential process.

2. Materials and methods Cytogenetic analysis was performed according to standard procedures with the G-banding method. For FISH studies, either the BCR/ABL dual-color, dual-fusion (DF) translocation probe or the BCR/ABL dual-color extra signal (ES) probe was used (both from Abbott Molecular/ Vysis, Des Plaines, IL). These probe sets allow the identification of the BCR/ABL1 fusion gene based on the juxtaposition of fluorescently labeled BCR and ABL1 signals. Furthermore, a SpectrumAqua probe (Aq) covering the argininosuccinate synthase gene (ASS ) 50 to the ABL1 sequence on chromosome 9q34 was also included with the BCR/ABL DF probes to assist in the detection of 9q deletions and the identification of the Ph chromosome in the interphase nuclei [27]. Hybridization and washing procedures were performed according to the manufacturer’s recommendations (Abbott-Vysis). In general, 200 nuclei (both interphase and available metaphase nuclei) were scored for FISH signal patterns. The false-positive cutoff was established for each probe by taking the scores from 20 normal control specimens to calculate the mean plus three standard deviations for positive signals. Positive cells within the cutoff range that might represent hybridization artifacts and random colocalization were excluded from the results. Therapeutic response criteria using cytogenetic data were according to the literature. In brief, complete response was defined as no Ph-positive cells present; major response was defined as 1e34% Ph-positive cells present; minor response was defined as 35e90% Ph-positive cells present; and no response if 90e100% Ph-positive cells remain.

M. Gorusu et al. / Cancer Genetics and Cytogenetics 173 (2007) 97e106

Weighted average frequency of deletions in variant translocation cases and standard translocation cases of CML was calculated for each category by dividing the number of cases with a deletion by the total number of cases in the category. Two-sided Fisher exact tests were performed, with a P-value of !0.05 considered statistically significant. Analysis of 1-year response rates was based on two groups, response versus no response. 3. Results 3.1. Cytogenetics profiles A total of 22 CML cases with variant translocations were identified (Table 1). Chromosomes 1, 11, and 17 were each involved in three cases. Chromosomes 9 (breakpoint other than q34) and 12 were involved twice. Chromosomes X, 2, 4, 7, 8, 13, 14, 19, 21, and 22 were each seen in a single case. Fourteen cases were processed as part of the initial diagnosis. Eight were follow-up CML cases, including three patients already in the accelerated phase or blast crisis. Among the 14 initial diagnosis cases, 1 (7%) showed clonal evolution at the first cytogenetic study, 2 (14%) later developed clonal evolution, and another 2 (14%) acquired independent clonal aberrations after clearance of the Phchromosome upon treatment (Table 2). Overall, among all cases first assessed at our laboratory, 3 out of 22 (14%) Table 1 Karyotype summary of the 22 variant translocations in chronic myeloid leukemia Case

Indication

1

Accelerated phase New dx Post BMT

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

Karyotype

46,XY,t(4;9;22)(q21;q36;q11),del(17)(p12)[14]/45,sl, 18[3]/45,sdl1,add(17)(p12),del(17)(p12) 46,XX,t(1;9;22)(q34;q11)[20] 46,XX,t(2;9;22)(q11;q34;q11.2),del(6)(p21)[9]/ 46,XX[1] New dx 46,X,t(X;9;22)(q24;q34;q11.2)[20] New dx 46,XY,t(9;16;9;22)(p22;p13.1;q34;q11.2)[5] Blast crisis 46,XY,t(1;9;22)(p36.3;q34;q11.2)[15] MPD 46,XX,t(7;9;22)(q11.2;q34;q11.2)[20] CML 46,XY,t(8;9;22)(p23;q34;q11.2)[18]/46,XY[3] CML 46,XX,t(9;21;22)(q34;q22;q11.2) CML 46,XY,t(9;22;17)(q34;q11;p13)[21] New dx 46,XY,t(9;22;14)(q34;q11;q24)[20] New dx 46,XX,t(9;22;17)(q34;q11;p13)[20] New dx 46,XY,t(9;22;13)(q34;q11.2;q14) New dx 46,XY,t(9;22;11)(q34;?q11.2;q13)[20] New dx 46,XY,t(9;22;12)(q34;q11.2;p13)[20] New dx 46,XY,t(9;22;19)(q34;q11.2;p13.3)[20] New dx 46,XX,t(1;9;22)(p34.3;q34;q11.2)[18]/ 46,sl,del(X)(p11.4),add(17)(q23)[2] CML 46,XY,t(9;22;11)(q34;q11.2;q13)[3]/46,XY[17] New dx 46,XY,t(9;22;12)(q34;q11.2;q24.3)[20] CML 46,XY,t(9;22;9)(q34;q11.2;p13)[19]/46,XY[1] New dx 46,XY,t(9;22;11)(q34;?q11.2;q13)[20] New dx 46,XY,t(9;22;17)(q34;q11.2;q23)[19]/46,XY[1]

Abbreviations: BMT, bone marrow transplantation; CML, chronic myeloid leukemia; dx, diagnosis; MPD, myeloproliferative disease.

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demonstrated clonal evolution, namely, cases 1, 3, and 17 (Table 1). 3.2. FISH studies demonstrate the mechanism of genesis of variant translocations Out of the 22 variant translocation cases, 14 had FISH studies performed. Distinct FISH signal patterns (summarized in Table 3) suggest either a one-step or a two-step mechanism for the formation of variant translocations [6,7,28,29]. As illustrated in Figure 1, if three (or more) chromosomes break at the same time and reciprocally rejoin (i.e., with one-step breakage and mismatch reunion), the signal pattern would be one fusion, two green, and two red (1F2G2R) with the DF probe. Indeed, 50% of the cases showed this signal pattern (Fig. 2a). Sometimes, however, we see a signal pattern of 2F1G1R, consistent with a classical t(9;22) translocation (Fig. 2b). On metaphase chromosomes of such a variant translocation, surprisingly, the second fusion signal does not reside on the derivative chromosome 9. It is instead on the third chromosome (Figs. 2b and 2c). This can be explained by a two-step mechanism: first, the standard t(9;22) is formed, leading to the BCR/ABL1 fusion on der(22) and the ABL1/BCR reciprocal fusion on der(9); second, the segment of der(9) including the ABL1/BCR fusion exchanges material with the third chromosome, resulting in a final der(9) with no ABL1/ BCR fusion signals (Fig. 1). From our data set, the one-step mechanism appears to be more common than the two-step mechanism (50% vs. 21.4%). Three cases (9, 17, and 20) showed FISH signal patterns suggestive of both mechanisms in the genesis of variant translocations (Table 3). In case 20, 75% of cells showed abnormal signal patterns consistent with BCR/ ABL1 fusion at initial diagnosis, in the proportion 45% demonstrating the 1F2G2R signal pattern (consistent with the one-step mechanism) and 30% demonstrating the 2F1G1R signal pattern (consistent with the two-step mechanism). After imatinib mesylate therapy for 1 year, positive cells were reduced to 5%, as assessed with FISH; half of these were 1F2G2R cells and half were 2F1G1R cells. Case 14 is unusual, in that both chromosome 22 homologs appeared normal with G-banding chromosome analysis, despite the discernible abnormalities in chromosomes 9 and 11 (Fig. 3a). FISH analysis established the presence of the BCR/ABL1 fusion; however, the fusion appeared on the derivative chromosome 11, der(11), and, on one of the apparently normal chromosome 22 homologs, no BCR or ABL1 signal was observed (Fig. 3b). Therefore, this is a masked variant translocation with a probable multiplestep genesis of the translocation, as depicted in Figure 3c. 3.3. Deletions of the BCR or ABL1 locus are more frequent in variant translocation CMLs Because each of the studies reporting the prevalence of 9q deletions in variant translocations included only a small

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100 Table 2 Cases with clonal evolution Case

Indication

Karyotype

1 3 5a 5b 6 7a 11a 15 17

Accelerated phase Post BMT Follow-up Blast crisis Blast crisis Follow-up Follow-up Follow-up New diagnosis

46,XY,t(4;9;22)(q21;q36;q11),del(17)(p12)[14]/45,sl,18[3]/45,sdl1,add(17)(p12),del(17)(p12) 46,XX,t(2;9;22)(q11;q34;q11.2),del(6)(p21)[9]/46,XX[1] 46,XY,t(9;16;9;22)(p22;p13.1;q34;q11.2)[3]/46,sl,add(19)(p13.3)[8]/49,sdl1,þ8,þadd(19)(p13.3),þ21[2]/46,XY[7] 49,XY,þ8,t(9;16;9;22)(p22;p13.1;q34;q11.2),þ19,add(19)(p13.3)2,þ21[20]þ46,XY[1] 46,XY,t(1;9;22)(p36.3;q34;q11.2),der(9)t(1;9;22),þ2mar[cp6] 46,XX,del(16)(q22)[5]/46,XX[15] 46,XY,del(11)(q23)[3]/46,XY[17] 46,XY,inv(3)(q21q26.2),t(9;22;12)(q34;q11.2;p13)[20] 46,XX,t(1;9;22)(p34.3;q34;q11.2)[18]/46,sl,del(X)(p11.4),add(17)(q23)[2]

a

These cases developed independent clonal aberrations without the Ph chromosome.

number of cases [15,17,19e22], we summarized data from 13 articles and calculated the weighted average frequency of der(9) deletion in both variant and standard Ph translocations (Table 4). As shown, deletion 9q occurs in ~15% of CML cases in general. The frequency for deletion observed in standard Ph translocation was 12.7%, whereas the weighted average frequency for deletion observed in variant translocation was 40.4% (P ! 0.001). Thus, the literature is clear on the increased prevalence of deletions among variant translocation CML. Among the 14 cases with FISH data from our laboratory, deletions were observed in 6 cases (43%). This is consistent with the rate reported in the literature and is significantly higher than that reported for standard translocations (P ! 0.01). The combination of the BCR/ABL DF probes and ASSAq probe allowed us to distinguish whether the ABL1 locus or the BCR locus was deleted in a Ph-positive cell (Fig. 4). It appears that deletions can occur at either locus with approximately equal frequency (Table 3).

3.4. Variant translocation CML patients with a deletion showed worse response to treatment To determine the outcome associated with variant translocations, we assessed the cytogenetic response at 1 year after therapy in our CML patients with variant translocations. Because clinical and survival data were limited in our data set and the cytogenetic response at 1 year after therapy has been shown to correlate well with survival [30,31], we analyzed the cytogenetic response and further compared the two groups with and without deletions (Table 3). Among the six cases demonstrating a deletion, four cases have no available data and the remaining two cases were found to be essentially unresponsive to therapy. One of the cases (case 15) had no cytogenetic response at all and subsequently acquired clonal evolution within a year; the patient eventually died at 2 years post diagnosis, despite interferon a and imatinib therapy. The other (case 18) had initial response and was Ph-negative for a short period of time. Relapse quickly set in before the end of the first year, however, and the patient received bone marrow transplant.

Table 3 Summary of available FISH results and treatment responses Response at 1 year Case

FISH result

4 7 8 9 13 14 15

1F2G2R 1F2G2R 2F1G1R 1F1G2R 1F2G2R 1F1G1R 1F2G2R

16 17 18 19 20 21 22

1F2G2R with DF probe 88% 1F2G2R, 8.5% 2F1G1R, and 2% 1F1G2R with DF probe 1F2G2R with DF probe, later 26% 1F2G1R 2F1G1R with DF probe, F on der(12) 45% 1F2G2R and 30% 2F1G1R with DF probe 80% 1F1G2R and some 2F1G1R with DF probe 1F2G2R with DF probe

with with with with with with with

Mechanism DF probe DF probe DF probe, F on der(8); 7.5% 1F2G1R1Aq ES probe; 1F2G2R and 2F1G1R with DF probe DF probe ES probe, F on der(11) DF probe; 36% 1F1G2R at 21 months

1 step 1 step 2 step Mixed 1 step ?multistep 1 step

1 step Mixed 1 step 2 step Mixed 2 step 1 step

Deletion

ABL/ASS

ABL/ASS BCR

BCR ABL

BCR

Therapy

Cytogenetic

Molecular

HUeIFN Imatinib n/a ImatinibeHSP n/a n/a Imatinib

Minor Complete n/a Complete n/a n/a No response; clonal evolution Complete n/a Relapsed n/a Complete n/a Complete

n/a Complete n/a Still þ n/a n/a þ

Imatinib n/a ImatinibeHSPeBMT n/a ImatinibeHSP n/a Imatinib

Still þ n/a þ n/a n/a n/a 2-log reduction

Abbreviations: Aq, aqua; BMT, bone marrow transplantation; DF, dual color, dual fusion; ES, extra signal; F, fusion; FISH, fluorescence in situ hybridization; G, green; HSP, heat-shock protein; HU, hydroxyurea; IFN, interferon a; n/a, not available; R, red; þ, positive for BCR/ABL1 fusion gene expression.

Fig. 2. Distinct FISH signal patterns suggest either a one-step or a two-step mechanism for the formation of variant translocations in chronic myeloid leukemia. (a) Hybridization with the LSI BCR/ABL dual-color, dual-fusion translocation (DF) probe revealed that a 1F2G2R signal pattern would suggest a onestep formation of a variant translocation. (b) The 2F1G1R signal pattern suggests a two-step genesis of a variant translocation. The BCR/ABL1 fusion signal was located on the Ph chromosome. The ABL1/BCR reciprocal fusion signal was translocated to the short arm of a chromosome 8 [der(8)]. The remaining ABL1 (R) and BCR (G) signals were on the normal chromosomes 9 and 22, respectively. (c) G-banding karyotype of the variant translocation, as shown in (b).

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Among the eight cases without a deletion, two had no data and the remaining six cases all had cytogenetic responses. Five patients treated with imatinib (some of whom were also study participants in a clinical trial of HSP70 immunotherapy [30]) showed complete cytogenetic response, whereas only one patient treated with interferon a showed minor cytogenetic response. It is not surprising that imatinib treatment led to a superior response; this has been well documented [32e34]. Overall, our data are consistent with the hypothesis that CML patients with 9q deletion respond less favorably to therapy than those without the deletion (P ! 0.04). Our data further demonstrated that deletions can be a sequential process. Case 15 showed no deletions at initial diagnosis; at 21 months, however, interphase FISH analysis revealed 36% of the abnormal cells with a signal pattern consistent with a deletion (Table 3). Case 18 was similar, with no deletions at initial diagnosis but showing a deletion pattern in 26% of the abnormal cells at 3 months after diagnosis. Although patients analyzed in different stages of the disease exhibited no significant difference in frequencies of deletionsd5 of 13 (38%) at chronic phase versus 2 of 2 (100%) at progression to accelerated phase or blast crisisda trend is noticeable (P 5 0.20).

4. Discussion

Fig. 3. An unusual case of cryptic variant translocation in chronic myeloid leukemia. (a) Both chromosome 22 homologs appeared normal under G-banding analysis, despite discernible abnormalities in chromosomes 9 and 11. (b) FISH analysis with the LSI BCR/ABL extra signal (ES) probe confirmed the BCR/ABL1 fusion, which was localized on der(11), the derivative chromosome 11. One of the apparently normal chromosome 22 homologs showed neither BCR nor ABL1 signal. (c) Schematic of a possible mechanism of how this variant translocation was formed. A submicroscopic deletion and insertion of the ASS/ABL1 sequence from a chromosome 9 to a chromosome 22 took place, resulting in BCR/ABL1 fusion. Subsequently, a one-step breakageereunion involving three chromosomes led to the translocation of the entire BCR/ABL1 fusion onto a chromosome 11 and the formation of the final derivative chromosome 9 with 11q sequences but no ASS/ABL1 sequences. The final derivative 22, although morphologically normal, contained no BCR sequences.

Several issues concerning variant translocations of CML remain unresolved, especially regarding the mechanisms of genesis and the prognostic significance. We analyzed 22 cases of variant translocation CML identified in our laboratory. Although all chromosomes have been described as participating in variant translocations, the breakpoint distribution does exhibit a nonrandom pattern, and has been reported to locate preferentially in the CG-richest regions of the genome, typically represented by the light bands in G-banded chromosomes [35]. Examination of the frequency of chromosomes and breakpoints involved among our cases provides support to these proposals (Table 1). With the BCR/ABL DF translocation probe, we were able to implicate the potential mechanisms for the formation of variant translocations in each of our CML patients (Fig. 1 and Table 3). Both one-step and two-step mechanisms occur, with the one-step more common, perhaps because fewer breakpoints are required. In three cases, we obtained FISH results consistent with both mechanisms, suggesting the possibility of a mixed mechanism. This finding is, to our knowledge, unique. An alternative explanation is that the 2F1G1R signal pattern came from cells with a standard t(9;22) instead of a variant three-way translocation. Supporting evidence includes the coexistence of metaphase t(9;22) and t(9;22;V) (where V represents the variable chromosome involved in the translocation), demonstrated by G-banding in a few reported cases [6,15]. We did not, however, see any t(9;22)

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Table 4 Frequency of der(9) deletion in chronic myeloid leukemia (CML) cases der(9) deletion, no. of cases (n) References

Standard CML

Variant CML

Total

Dewald et al., 1999 [44] Herens et al., 2000 [45] Sinclair et al., 2000 [17] Huntly et al., 2001 [18] Kolomietz et al., 2001 [19] Storlazzi et al., 2002 [38] Cohen et al., 2001 [40] Primo et al., 2003 [46] Lee et al., 2003 [41] Reid et al., 2003 [15] Aoun et al., 2004 [47] El-Zimaity et al., 2004 [10] Costa et al., 2006 [20] Weighted average

d d 6 25 19 7 d 11 16 d 17 d d 101

d d 10 16 4 3 d d 0 20 0 6 2 61

28 5 16 39 23 10 14 11 16 20 17 6 2 207

(34) (212) (140) (66) (135) (59) (152)

(798), 12.7%

metaphase with G-banding in the three cases at initial diagnosis or in follow-up studies. In addition, the proportion of cells with the two distinct FISH patterns did not change after imatinib treatment in case 20 (suggesting that imatinib mesylate targets the BCR/ABL1 positive cells regardless of the mechanism of genesis). It might seem paradoxical that the two different FISH patterns were associated with only one abnormal karyotype by G-banding. Therefore, there might be greater complexity than has previously been recognized. In some instances, the genesis of the variant translocation is clearly more complex; it may involve insertion and deletion, and results in a cryptic or masked translocation (Fig. 3). An array of

Fig. 4. FISH signal patterns distinguish ABL1 deletion from BCR deletion chronic myeloid leukemia. (a) Signal pattern 1F1G2R with the BCR/ABL DF probe suggests the BCR deletion. (b) FISH with the ASS-Spectrum Aqua probe (Aq) shows two Aq signals. (c) Signal pattern 1F2G1R with the BCR/ABL DF probe suggests the ABL1 deletion, which is confirmed by the ASS FISH (d).

(22) (41) (9) (5)

(0) (54) (0) (13) (7) (151), 40.4%

(147) (51) (56) (253) (250) (71) (94) (135) (59) (54) (152) (13) (7) (1,342), 15.4%

techniques, including FISH with whole chromosome painting or bacterial artificial chromosome (BAC) and yeast artificial chromosome (PAC) clones, multipoint interphase FISH, M-FISH, and PCR, has been used to delineate the breakpoints and chromosomes involved in such cases [15,36]. Evaluation with interphase FISH alone may not be fully informative when a double population coexists. Why is der(9) deletion more common in variant translocations than in standard t(9;22)? It has been hypothesized that each recombination event has a probability of erroneous repair leading to genomic loss next to the breakpoint, potentially involving tumor suppressor genes [10,24]. Therefore, with more recombination events involved, a variant translocation has a greater chance of genomic loss. Genomic instability has also been proposed [15]. New findings of large deletions on other chromosomes involved in variant translocations in addition to der(9q) deletion are consistent with these hypotheses [23,37]. Deletions generally occur at the sequence 50 to ABL1 or the sequence 30 to the BCR gene, although exceptions have been reported [24]. The size of the deletion is variable, and the minimal deleted region has been reported to contain the ASS gene on chromosome 9 and the immunoglobulin lambda-like polypeptide 1 isoform (IGLL1) gene on chromosome 22[38]. Although the probes we used do not precisely identify the location of the deletion or deletions, they allowed us to distinguish whether the ABL1 locus or the BCR locus is deleted (Fig. 4). It appears that deletions can occur at either locus with approximately equal frequency (Table 3). Because of the two-step mechanism whereby the ABL1/BCR fusion sequence is translocated to the third chromosome, deletions of either ABL1 or BCR sequences, along with the adjacent loci, may happen on the third chromosome rather than on der(9). For example, in case 8, we observed that 7.5% of the abnormal cells were missing the red signal from the fusion on der(8), resulting in the 1F2G1R signal pattern (consistent with a deletion). This finding

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was corroborated by the FISH result using the ASS-Aq probe, which showed a single hybridization signal instead of two seen in normal control cells (Table 3). In case 21, among the cells showing the 1F1G2R signal pattern, we noticed on the metaphase FISH that the missing green signal was from the fusion on der(11), consistent with a deletion of the BCR sequence (Table 3). Genomic deletions on chromosomes other than der(9) have been reported by another group [23,37]. We acknowledge that our study is limited by its retrospective nature with ascertainment of cases over an extended time span, small sample size, and incomplete clinical information. Nevertheless, the data were revealing and contribute to the clarification of certain controversial points in our understanding of CML. We confirmed that deletions of the ABL1 or BCR locus are more prevalent in variant translocation CML cases (Table 4). Our data also indicated statistically significant worse therapeutic responses (P ! 0.04) among these patients, compared with those without a deletiondeven when treated with a relatively superior regimen such as imatinib mesylate (Table 3), which has been proposed to be capable of overcoming adverse prognostic features [26]. These results need to be confirmed in a larger cohort of patients without selection bias and under uniform treatment protocols. One of our future goals is to correlate the deletion status with real-time quantitative PCR data (Q-PCR) over the disease course [39]. Case 22 at initial diagnosis showed one cell with 1F2G1R1Aq, which is consistent with a deletion but may also reflect the colocalization of two ASS/ABL1 signals. This patient showed complete cytogenetic response at 1 year after the initial diagnosis and imatinib therapy. At that same time, however, Q-PCR yielded a less than 2-log reduction. It has been reported that a 3-log reduction predicts good long-term disease-free survival, whereas a less than 2-log reduction might suggest early relapse and poor outcome associated with Abl kinase domain mutation and imatinib resistance [33]. Therefore, this single cell might represent a true deletion cell. Repeat FISH analysis is necessary to see if more such cells would be observed. In addition, our data challenge the notion that all deletions happen at the same time as formation of the Ph chromosome [18,19,38,40]. We have shown in the same sample (cases 15 and 18) that some cells showed a deletion pattern and others did not. A few recent reports described similar findings even with metaphase FISH analysis [41e43]. Therefore, it is worth performing FISH studies at different post-therapy stages and to correlate with Q-PCR results. We speculate that the presence or emergence of deletion 9q will predict a less steep drop of Q-PCR value over the treatment course and therefore will result in a suboptimal therapeutic response. A poor therapeutic response in variant translocation CML is likely caused by a greater frequency of der(9) deletions associated with variant Ph translocations. Reid et al. [15] declared that der(9) deletions are the key prognostic factor in variant CML.

In summary, we have analyzed 22 variant translocation CML cases regarding the genesis and prognosis of variant translocation CML. Our data are consistent with the model that variant translocations convey a less favorable therapeutic response and, subsequently, a worse outcome, because the increased activity of genomic rearrangement may lead to a higher risk of deletions of important genes at the loci adjacent to the breakpoints. Proper assessment of the prognostic significance of variant translocations thus requires better categorization of these translocations based on their mechanisms of genesis and their deletion status.

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