Derivative Chromosome 9 Deletions Are a Significant Feature Of

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Leukemia (2005) 19, 564–571 & 2005 Nature Publishing Group All rights reserved 0887-6924/05 $30.00 www.nature.com/leu Derivative chromosome 9 deletions are a signiﬁcant feature of childhood Philadelphia chromosome positive acute lymphoblastic leukaemia

HM Robinson1, M Martineau1, RL Harris1, KE Barber1, GR Jalali1, AV Moorman1, JC Strefford1, ZJ Broadﬁeld1, KL Cheung1 and CJ Harrison1

1Leukaemia Research Fund Cytogenetics Group, Cancer Sciences Division, University of Southampton, Southampton, UK

Deletions from the derivative chromosome 9, der(9), of the production of a p210BCR–ABL protein. In the remaining two- translocation, t(9;22)(q34;q11), at the site of the ABL/BCR thirds of ALL patients and rarely in CML, the breakpoint arises fusion gene, have been demonstrated by fluorescence in situ hybridisation (FISH), in both Philadelphia chromosome (Ph)- further upstream, between exons e2’ and e2, known as the positive chronic myeloid leukaemia (CML) and acute lympho- minor bcr (m-bcr). The translocation involving this breakpoint BCRÀABL 5 blastic leukaemia (ALL). In CML they occur in 10–15% of cases produces a p190 protein. and appear to indicate a worse prognosis, whereas in ALL, the FISH has shown that deletions involving the reciprocal ABL/ situation is unclear. This study presents the findings of dual BCR fusion gene occur at the site of the translocation breakpoint fusion FISH used to detect such deletions in a series of 27 BCR/ on the derivative chromosome 9, der(9), of the t(9;22) in both ABL-positive childhood ALL patients. Metaphase FISH was CML and ALL. In CML they were observed by using a dual essential for the accurate interpretation of interphase FISH 6 signal patterns. Three cases (11%) had a single fusion signal, colour dual fusion approach. This technique allows for the resulting from deletions of the der(9). Three other patients with detection and (where metaphases are present) location of both variant translocations and one with an insertion, also had a the BCR/ABL and ABL/BCR fusion genes.7 In cells with a single fusion, but with no evidence of deletions. Gain of a deletion, one or both of the signals corresponding to ABL/BCR fusion in approximately one-third of patients indicated a are absent from the der(9). These findings were correlated with second Ph, which appears to be a diagnostic marker of Ph- molecular studies indicating loss of the ABL/BCR fusion positive ALL. This study shows that the incidence of deletions 6 from the der(9) in childhood ALL is at least as high as that transcript in a number of patients. However, not all patients reported for CML. who lack expression of the ABL/BCR fusion gene at the Leukemia (2005) 19, 564–571. doi:10.1038/sj.leu.2403629 molecular level have a visible deletion by FISH.8 FISH studies Published online 17 February 2005 showed that 10–15% of patients with CML had a deletion Keywords: Philadelphia chromosome positive acute lymphoblastic involving ABL/BCR, and that the incidence was higher among leukaemia; deletions; fluorescence hybridisation ABL/BCR in situ those patients with variant Ph translocations.6 The most significant clinical finding was that, in CML, the deletions were associated with a worse prognosis.9–12 More recent inves- tigations have shown that these deletions also occur in adult Introduction ALL, although no association with outcome has yet been established.13,14 Fluorescence in situ hybridisation (FISH) is increasingly being In the study presented here, a series of 27 BCR/ABL positive, used to identify significant gene rearrangements in acute childhood ALL patients were investigated using dual colour dual leukaemia. A range of commercial probes are now available, fusion FISH, with a commercially available probe, to determine providing rapid and accurate detection systems. These include a the incidence of ABL/BCR deletions. Comparison of FISH signal number of probe systems, which have been developed for the patterns, at both interphase and metaphase with the G-banded detection of the BCR/ABL fusion gene. This gene, usually karyotypes, revealed unexpected findings. located on the Philadelphia chromosome (Ph), arises from a reciprocal translocation between chromosomes 9 and 22 at breakpoints q34 and q11, respectively, t(9;22)(q34;q11), and Materials and methods results in the fusion of the 30 segment of the ABL gene to the 50 segment of BCR. The rearrangement, the hallmark of chronic Patients myeloid leukaemia (CML), also occurs in 11–30% of adults1 and 3–5% of children with acute lymphoblastic leukaemia (ALL).2,3 4 Patients with a confirmed diagnosis of Ph-positive ALL entered In ALL it is associated with a poor prognosis in both age groups. to one of the National Cancer Research Institute (NCRI) ALL Expression of the fusion gene in ALL, results in two types of treatment trials, ALL97 or ALL2003, for children aged 1–18 chimaeric mRNA, dependent upon the location of the break- years inclusive, with available material, were included in this point within the breakpoint cluster region (bcr) of the BCR gene. study. They had been previously identified as Ph-positive by In a third of patients with ALL and the majority of those with conventional cytogenetics and/or interphase FISH. Demo- CML the breakpoint occurs within a 5.8 kb region spanning BCR graphic, clinical and survival data were provided by the Clinical exons 12–16 (exons b1–b5), known as the major bcr (M-bcr). Trial Service Unit (CTSU), Oxford. The translocation involving this breakpoint results in the

Correspondence: Dr CJ Harrison, Leukaemia Research Fund Cytoge- Cytogenetics netics Group, Cancer Sciences Division, University of Southampton, MP 822 Duthie Building, Southampton General Hospital, South- Conventional cytogenetic analysis was carried out on diagnostic ampton SO16 6YD, UK; Fax: þ 44 23 8079 6432; E-mail: [email protected] bone marrow and/or peripheral blood samples by the UK Received 19 August 2004; accepted 12 November 2004; Published regional cytogenetics centres. Members of these laboratories online 17 February 2005 form the UK Cancer Cytogenetics Group. G-banded slides were Chromosome 9 in Ph-positive ALL HM Robinson et al 565 reviewed by the Leukaemia Research Fund UKCCG Karyotype enumerate individual chromosomes.19 A total of 100 interphase Database in Acute Leukaemia (Database)15 as part of their nuclei were scored for each centromeric probe. routine review of all cases entered to ALL97. Slides from case Whole chromosome paints (wcp) (STARnFISH, Cambio, UK) 7092, an ALL2003 patient, were not available for review, thus and Multiplex-FISH (M-FISH), using the SpectraVysion 24 an incomplete karyotype (inc), based on metaphase FISH colour chromosome painting kit (Vysis, UK), were applied to analysis only, was given. Karyotypes were described according metaphases from patient 5643, in order to clarify the G-banded to the International System of Human Cytogenetic Nomencla- karyotype. Subtelomeric probes, specific for the short and long ture (ISCN, 1995).16 arms of chromosome 9 (9p and 9q) (QBiogene, UK), were sequentially hybridised onto the same metaphases. Detailed investigation of the BCR/ABL rearrangement in this patient was carried out by FISH with specific overlapping PAC probes 0 0 FISH covering the ABL gene. PAC probe dJ1132H12 maps 5 to 3 and includes ABL intron 1 while dJ835J22 maps 50 to 30 and includes ABL exon 11.20 The probes were directly labelled with Spectrum FISH experiments were performed on the same fixed cell Red and Spectrum Green, respectively, by Nick translation suspensions as used for cytogenetic analysis. Commercial (Vysis, UK) and hybridised to slides using a modified method for probes were hybridised according to the manufacturers’ noncommercial probes, as previously described.21 instructions. Where possible 200 interphase nuclei were scored per test and the signal patterns for all cells were recorded. Initial FISH analysis, for identification of BCR/ABL fusion, was Results performed using the LSIs BCR/ABL Dual Colour, Single Fusion s Translocation probe (SF) (Vysis, UK), or the LSI BCR/ABL ES Among 2096 patients entered to ALL97 and 179 with complete Dual Colour Translocation probe (ES) (Vysis, UK) or both. s data, entered to ALL2003 by 30 June 2004, 49 patients were Ph- Further analysis was carried out with the LSI BCR/ABL Dual positive. In total, 27 of these children (18 males and nine Colour, Dual Fusion Translocation probe (DF) (Vysis, UK). In all females), with fixed cell suspensions available for further three probes, ABL sequences were labelled with Spectrum investigation, were included in this study. The median age Orange, providing a red signal, and BCR sequences were was 6 years (range 1–15 years), the mean white blood cell count labelled with Spectrum Green, providing a green signal. was 128 Â 109/l, and the immunophenotype was pre-B or Although the size and extent of the sequences included in the common ALL. probes varied, as indicated in Figure 1, all normal cells showed two red and two green signals (2R2G) in interphase. The designs of the SF and the ES probes allow detection of breakpoints Cytogenetics involving the m-bcr of the BCR gene as well as the BCR/ABL fusion.17,18 The interphase FISH patterns expected with each Cytogenetic data and FISH results from at least two probes were probe are shown in Table 1. For those cases with standard and available for the majority of patients. The karyotypes and FISH nonstandard signal patterns observed in interphase, the number signal patterns from the different probes are shown in Table 2. and relative size of the signals were confirmed, where possible, Patients are ordered according to their interphase FISH signal on representative metaphase cells from the same samples. This pattern with the DF probe. The translocation t(9;22)(q34;q11) allowed the location of the individual signals to be determined appeared as the sole chromosomal abnormality in four patients in relation to relevant chromosomal abnormalities. (3192, 3537, 4331, 4622). Complex-variant Ph translocations In two patients (4582, 4239), the Chromoprobe Multiprobe-It were observed in three patients (4018, 4239, 4844). There was system (Multiprobe-I) (Cytocell, UK), which comprises centro- no cytogenetic evidence of a Ph translocation in one patient meric probes specific for each chromosome pair, was used to (5643) with a pseudodiploid karyotype. Additional chromosomal

a 9q34

Cen 5’ 3’ Tel ASS 1b ABL 1a 2 11 DF Probe (~650 kb) ES Probe (~650 kb) SF Probe (~300 kb) b 22q111 m-BCR m-BCR

Cen 5’ 3’’ Tel IgLV BCR

DF Probe (~600 kb) DF Probe (~600 kb) ES Probe (~300 kb) SFF Probe (~300 kb)

Figure 1 Schematic illustration of commercial FISH probe systems used for the detection of BCR/ABL rearrangements. (a) Maps of ABL (chromosome 9) and (b) BCR (chromosome 22), showing relative positions of BCR/ABL probes. Commercial probes used: LSIs BCR/ABL Dual Colour, Single Fusion Translocation probe (SF), LSIs BCR/ABL ES Dual Colour Translocation probe (ES), LSIs BCR/ABL Dual Colour, Dual Fusion Translocation probe (DF). Breakpoints in the BCR gene breakpoint cluster region arrowed.

Leukemia Chromosome 9 in Ph-positive ALL HM Robinson et al 566 Table 1 Signal patterns from FISH probe systems detecting BCR/ABL rearrangements

Cytogenetic interpretation Single fusion Extrasignal dual Dual fusion dual dual colour probe (SF) colour probe (ES) colour probe (DF)

Normal 2R2G 2R2G 2R2G

Ph positive (standard)a 1R1G1F(M-bcr) 2R1G1F(M-bcr) 1R1G2F 1R2G1F(m-bcr) 1R1G2F(m-bcr)

Additional copy of Ph chromosome 1R1G2F(M-bcr) 2R1G2F(M-bcr) 1R1G3F 1R2G2F(m-bcr) 1R1G3F(m-bcr)

Ph-positive with deletion of der(9) 1R1G1F 1R1G1Fb 1R1G1Fb 2R1G1Fc 2R1G1Fc 1R2G1Fd 1R2G1Fd

Ph-positive 3-way translocation 1R1G1F(M-bcr) 2R1G1F(M-bcr) 2R2G1F 1R2G1F(m-bcr 2R2G1F(m-bcr) R ¼ red (ABL signal); G ¼ green (BCR signal); F ¼ fusion signal. aReciprocal Ph translocation with no deletion detectable by FISH. bDeletion of ABL/BCR fusion. cDeletion of 30BCR from ABL/BCR. dDeletion of 50ABL from the ABL/BCR.

changes were seen in 15 patients in all or in a proportion of interphase populations that patients 7092 and 4412 had cells, with an extra copy of the Ph, der(22)t(9;22)(q34;q11), in common (2R1G1F, 2R1G2F). The loss of the 30BCR signal being the most common secondary abnormality in eight from the der(9) in patient 7092, was confirmed on metaphases patients, including four with high hyperdiploidy (3715, 4102, (Figure 2c and d). In this patient the red signal on the der(9) 4684, 4914). Cytogenetic analysis failed in two patients (4582, chromosome was consistently smaller and was not clearly 4786), while a normal karyotype was seen in two others (3005, visible in all interphase cells, indicating that the deleted region 3864). might include some sequences from the 50 part of ABL in addition to 30BCR. The SF probe confirmed in patient 4412 that there was an intact fusion on the Ph, indicating the presence of Breakpoint in m-bcr: detection by FISH 50BCR sequences, in spite of there being no suitable metaphases for analysis with the DF probe. The additional fusion signal, seen A total of 25 patients were tested with the SF and/or ES probes, in interphase in some cells of both patients, proved on of which 15 showed a FISH signal pattern consistent with a metaphases to be a second Ph. Without metaphases in these breakpoint within m-bcr of 1R2G1F or 1R2G2F with the SF two cases to confirm that the common 2R1G2F interphase signal probe (13 patients), or 1R1G2F with the ES probe (two patients) pattern indicated the loss of 30BCR, one might have been misled (Table 2). Since the signal from the 30 BCR fragment with both into underestimating the incidence of deletions in ALL, or probes is always smaller than the normal BCR signal, non- misinterpreting that the deletion arose as a secondary event to detection of this small signal may sometimes be due to limited the translocation. resolution and should not be interpreted as representative of an A single green and a single fusion signal (2R1G1F) were M-bcr breakpoint without supporting evidence from additional observed in the interphase cells from patient 5643 (Figure 2e). molecular analyses. Thus 10 patients without a signal pattern There was no evidence of a Ph in the abnormal metaphases of indicative of an m-bcr breakpoint and two others not tested with this patient by conventional cytogenetics. Sequential metaphase either probe were reported as not known (NK). FISH with the DF probe and M-FISH, showed the fusion signal to be located on an apparently normal chromosome 22 and the ABL signals to the 9q34 bands of two copies of chromosome 9, DF FISH one of normal appearance, the other with an interstitial deletion in the short arm (Figure 2e). This indicated that BCR/ABL fusion The presence of the BCR/ABL fusion gene was confirmed by had resulted from a submicroscopic insertion of chromosome 9 FISH using the DF probe in all 27 patients. A variety of material into chromosome 22. Metaphase FISH, with individual interphase FISH signal patterns were observed as shown in PAC probes covering the ABL gene, indicated that there was no Table 2. A single fusion signal, rather than the standard two, was deletion from the der(9) since both 50 and 30 sequences of the detected in nine patients. Loss of a fusion signal (1R1G1F) and gene were present on both copies of chromosome 9, with 30 its component parts were seen in two patients, but only at sequences also on the der(22) (Figure 2f). The insertion event interphase (Figure 2a). One had a normal karyotype by G- was confirmed by further sequential hybridisation of 9p and 9q banding (3005) and the other a failed cytogenetic result (4786). subtelomeric probes, which showed that the telomeric regions In three patients (4412, 4483, 7092), the single fusion signal of both copies of chromosome 9 were intact (Figure 2g). In this resulted from deletion of a component of the ABL/BCR fusion. case, although both the SF and DF probes had identical non- Loss of a red signal at interphase in patient 4483 was confirmed standard interphase patterns, only metaphase FISH was able to in metaphase (Figure 2b) to correspond to deletion of the identify the location of the fusion. chromosomal region including the 50ABL sequences from the The single fusion signal in the 2R2G1F pattern observed at der(9). Loss of a green signal was seen from each of the two interphase in three patients (4018, 4239, 4844) was due to

Leukemia Chromosome 9 in Ph-positive ALL HM Robinson et al 567 Table 2 Karyotypes and related FISH signal patterns for different BCR/ABL detection systems

Patient BCR bp SF BCR/ABL probe Karyotype number by FISH ES DF

3005 NK 2R1G1F 1R1G1F 46,XX[32] 4786 NK 1R1G1F 1R1G1F Fail 4483 m-bcr 1R2G1F 1R2G1F 46,XY,t(9;22)(q34;q11)[4]/45,idem,-Y[10] 7092 NK 2R1G1F/ 2R1G1F/a 45-47,XY,t(9;22)(q34;q11),+der(22)t(9;22),inc[6] 2R1G2F 2R1G2Fa 4412 NK 1R1G1F/ 2R1G1F/ 47,XX,del(9)(p11),t(9;22)(q34;q11),+der(22)t(9;22)(q34;q11)[7] 1R1G2F 2R1G2F 5643 NK 2R1G1F 2R1G1F 46,XX,del(9)(p1?p1?),dic r(14)(q?),ins(22;9)(q11;q3?4q34)[11] 4018 NK 2R1G1F 2R2G1F 46,XX,der(9,11,12,22)t(9;11)(q22;q2?1)t(9;22;12)(q34;q11;p13)[4] 4239 m-bcr 1R2G1F 2R2G1F/ 46,XY,t(9;22;17)(q34;q11;q21)[2]b 1R2G2F 2R2G2F 4844 NK 2R2G1F 46,XY,t(9;22;10)(q34;q11;q22)[5]/46,idem,-13,der(19)t(13;19)(q12;p13),del(20)(q1?), -der(22)t(9;22;10)(q34;q11;q22),+mar1,+mar2[5]/45,idem,der(20)t(20;21)(q1?;q1?),- 21[2] 5673 m-bcr 2R2G1F 2R1G2F/ 46,XX,t(9;22)(q34;q11),i(9)(q10)[2] 1R2G1F 1R1G2F 2896 m-bcr 1R2G2F 1R1G3F 47,XX,t(9;22)(q34;q11),+der(22)t(9;22)[11] 3715 m-bcr 1R2G2F 1R1G3F 55,XY,+X,+2,+4,+4,+8,t(9;22)(q34;q11),+13,+14,+21,+der(22)t(9;22)[6] 4102 m-bcr 1R2G2F 1R1G3F 54-55,XX,+2,+del(3?)(q2),+4,+6,del(9)(p?1),t(9;22)(q34;q11),-10,+13,+14,+21, +der(22)t(9;22),+mar,+mar [cp7] 4289 NK 1R1G1F/ 1R1G2F/ 45-47,X,-X,t(9;22)(q34;q11)/47,idem,+der(22)t(9;22)[cp7] 1R1G2F 1R1G3F 4582 m-bcr 1R2G1F/ 1R1G2F/ Failc 1R2G2F 1R1G3F 4684 NK 1R1G3F 59-61,XY,+X,+Y,+2,+4,+5,+6,+8,t(9;22)(q34;q11),+10,+10,+17,+18,+21, +der(22)t(9;22)[cp4]/46,XY[4] 4914 m-bcr 1R2G2F 1R1G3F 1R1G3F 53,XY,+2,+4,t(9;22)(q34:q11),+10,+17,+18,+der(22)t(9;22)(q34;q11)[4] 2685 m-bcr 1R2G1F 1R1G2F 46,X,+X,der(Y)t(Y;17)(q12;q1?1),t(9;22)(q34;q11),-17,add(18)(q23),der(19)t(1;19) (q?;p13)[3]/47,idem,+mar[3] 2792 m-bcr 1R1G2F 1R1G2F 45,X,-Y,t(9;22)(q34;q11),add(9)(q10),der(19)t(Y;19)(q11;q13.?4)[10] 3192 NK 1R1G1F 1R1G2F 46,XY,t(9;22)(q34;q11)[4] 3537 m-bcr 1R2G1F 1R1G2F 46,XY,t(9;22)(q34;q11)[2] 3795 m-bcr 1R2G1F 1R1G2F 46-48,XY,-7,t(9;22)(q34;q11),i(21)(q10),+mar,+mar[cp10] 3864 m-bcr 1R1G2F 1R1G2F 46,XY[27] 4331 NK 1R1G1F 1R1G2F 46,XY,t(9;22)(q34;q11)[10] 4463 NK 1R1G1F 1R1G2F 46,XY,der(9)t(9;22)(q34;q11)del(9)(p11p22)inv(9)(p22q31),der(22)t(9;22),add(16)(q2?)[7] 4622 m-bcr 1R2G1F 1R1G2F 46,XY,t(9;22)(q34;q11)[3] 4709 m-bcr 1R2G1F 1R1G2F 46,XY,t(9;22)(q34;q11),add(13)(p1?)[3]/46,XY,idem,der(11)(1;11)(q1?;p15)[2]/ 46,XY,der(9)t(9;22)(q34;q11),der(22)t(1;22)(q1?;p1?)t(9;22)(q34;q11)[3] bp ¼ breakpoint; SF ¼ single fusion dual colour; ES ¼ extra signal; DF ¼ dual colour dual fusion; R ¼ red (ABL signal); G ¼ green (BCR signal); F ¼ fusion signal; NK ¼ not known; Bold ¼ cases with a single fusion with the DF probe. aOne red signal smaller. bMultiprobe-I conﬁrmed +X,+11,+18,+21,+21,+22. cMultiprobe-I conﬁrmed +X,+Y,+2,+4,+6,+8,+10,+14,+17,+18,+20,+21,+21,+22. Normal (2R2G) FISH signal patterns and normal populations by cytogenetics have not been included.

separation of the two components of the ABL/BCR fusion. The the presence of an isochromosome 9, i(9)(q10), seen by pattern was the result of variant three-way Ph translocations. cytogenetics in which the duplication of the long arm of Metaphase FISH proved that the red ABL signal of the second chromosome 9 accounted for the extra ABL signal. A second fusion was retained on the der(9), while the green BCR one was population observed in this patient with the standard 1R1G2F relocated to the third chromosome involved in the translocation pattern, indicating a single copy of ABL, may represent a non- (chromosome 10, patient 4844, chromosome 12, patient 4018). dividing population without i(9)(q10), suggesting that this No metaphases were available for FISH studies in patient 4239. abnormality may be a secondary chromosomal change. Interphase FISH did however reveal a second population with Metaphase FISH was needed in order to distinguish this case two fusions (2R2G2F) in this patient, while Multiprobe-1 from the two deletion cases with the same 2R1G2F pattern, showed a hidden hyperdiploid population, including a third which had arisen by a different mechanism, namely deletion of copy of chromosome 22 centromere. The second interphase the green part of the der(9) fusion with acquisition of a second fusion was thus consistent with being an extra copy of the Ph. copy of the Ph, as described above. Although conventional cytogenetics identiﬁed the variant Seven patients showed a 1R1G3F pattern, associated with the translocations, only metaphase FISH demonstrated that the gain of a fusion signal in all (4684, 2896, 3715, 4102, 4914) or ABL and BCR components of the der(9) fusion had remained in some of the cells (4289, 4582). This correlated with an intact. additional fusion signal seen with either the SF or ES probes in Patient 5673 showed a 2R1G2F pattern indicating an all cases except 4684. Metaphase FISH and G-banded analysis additional copy of the ABL signal. This was consistent with in six of these patients conﬁrmed the presence of an additional

Leukemia Chromosome 9 in Ph-positive ALL HM Robinson et al 568

Figure 2 Representative cells showing results of hybridisations with BCR/ABL probes. In all cells: normal chromosome 9 has a red signal; normal chromosome 22 has a green signal. (a) BCR/ABL-positive interphase cell hybridised with DF probe, showing loss of a fusion signal (patient 3005). (b–d) BCR/ABL-positive metaphases hybridised with DF probe (b) loss of red 50ABL signal from der(9) (arrowed) (patient 4483). (c) and (d) loss of green 30BCR signal from der(9) chromosome (arrowed) (patient 7092), in (c) there is an additional copy of the Ph. (e) BCR/ABL fusion (arrowed) on an apparently normal chromosome 22, together with two copies of ABL (red) and a single copy of BCR (green) on chromosomes 9 and 22 followed by sequential M-FISH (patient 5643). (f) PAC probes, dJ1132H12 (red) and dJ835J22 (green), hybridised to metaphases showing the presence of both PACs on the two copies of chromosome 9 and dJ835J22 on the ins(22) (arrowed) (patient 5643) (g) partial karyotype of the two copies of chromosome 9, hybridised with p (green) and q (red ) subtelomeric probes (patient 5643).

Ph with the third fusion signal located on it. In the seventh chromosome 22. These ﬁndings demonstrated the presence of a patient (4582) with a failed cytogenetic result, conﬁrmatory hidden hyperdiploid clone with chromosomal gains similar to evidence came from complementary interphase FISH with the other four hyperdiploid cases in the series. Since these four Multiprobe-I, which showed gain of centromeric signals cases also had three fusion signals in association with an extra indicative of trisomies for a number of chromosomes including Ph, the interphase FISH results for patient 4582 including an

Leukemia Chromosome 9 in Ph-positive ALL HM Robinson et al 569 extra chromosome 22 centromere, implied the presence of a for adult ALL by Specchia et al13 and Kolomietz et al.11 A lower second Ph. incidence of 1.5% was reported by Reid et al14 in their The remaining 10 patients showed the standard pattern of predominantly adult series, while no cases were detected in the 1R1G2F, indicating the presence of both the BCR/ABL and ABL/ series investigated by Lee et al.31 This variation may be a BCR fusions. These results were consistent with the cytogenetic consequence of the small numbers of patients studied, or of a findings of a balanced t(9;22)(q34;q11), apart from one case bias in selection. Alternatively, as suggested from our study, (3864) with a normal karyotype, in which no abnormal there is a possibility that some deletions may be masked by a metaphases were seen either by G-banded analysis or FISH, predominant secondary population with two fusion signals. It indicating that the abnormal clone was restricted to the non- has been proposed that der(9) deletions are more commonly dividing population. associated with M-bcr breakpoints.13 Therefore, the lower incidence reported by Reid et al,14 may reflect the smaller number of patients in their series with M-bcr rearrangements. Discussion Our study is incapable of providing support, since the assign- ment of breakpoints within BCR was made by FISH and deletion This study records the detailed interphase FISH patterns and of ABL in association with an m-bcr breakpoint was only chromosomal locations of probes to the BCR and ABL genes, in confirmed in a single case. a series of children with Ph-positive ALL. We identified a An additional fusion signal at interphase correlated with a significant number of cases with deletions from the der(9), as second copy of the Ph. There was evidence of a second copy of well as variant translocations without apparent deletions from the Ph in a third of the patients in this study, which compares either chromosome 9 or 22. We recorded the first insertion with the results of a previous series of similar size.18 In contrast, event in a child with ALL using FISH, while the extra copy of the the presence of two Ph at diagnosis of CML was much rarer. This Ph seen in several cases, may prove to be a feature of diagnosis implies that the presence of an additional Ph may be a in this disease. diagnostic marker of Ph-positive ALL. A third of the patients showed a single fusion with the DF In CML, der(9) deletions have been associated with a probe rather than the expected two, in all, or in a majority of worse prognosis.9–12,23 The poor outcome has been linked to cells. In three patients, the fusion on the der(22) together with patients with a visible deletion by FISH. Cases of Ph-positive loss of either the red or the green signal from the der(9), CML have been reported, which lack expression of the ABL/BCR indicated a deletion of sequences including 50ABL or 30BCR transcript but have no visible deletions.8,27 Such submicro- from the breakpoint region of the der(9). The variable extent of scopic deletions, below the level of detection by FISH, the deletions exhibited in this study and other small ser- have been identified in a small series of patients whose ies9,11,13,14 indicate that they are likely to be as molecularly prognosis was poor.9 A candidate tumour suppressor gene, heterogeneous as those reported in CML.6,13,20,22 In agreement PRDM12, was identified in these cases32 and subsequently with Huntly et al9 for CML, we found the deletions to be present shown to be deleted in a case of Ph-positive CML with a variant in all cells of our ALL patients. Thus they must have occurred at translocation.33 the same time as the original translocation event, and not as the No association with survival has yet been established for result of clonal evolution. deletions of der(9) in ALL. The identification of tumour Three other patients with a single fusion signal had variant suppressor genes in CML and the determination of their translocations in which the fusion signal from the der(9) was potential role in prognosis may also have implications for ALL. split between this chromosome and the third partner, with no Ph-positive ALL has an extremely poor outcome, which may be evidence of accompanying deletions. These findings, albeit in attributable to the loss of unidentified tumour suppressor genes. only a small number of patients, contrast with the situation in Although we can draw no conclusions about prognosis owing to CML, in which a higher incidence of deletions was demon- the short follow-up time, it is of interest that four of our patients strated in those with variant Ph translocations.23 with deletions did not show a good response, based on the One patient among the series with a single fusion signal percentage of blasts at day eight of treatment (unpublished showed, from a combination of FISH techniques, that the BCR/ observations). ABL fusion had arisen from insertion of part of the ABL gene into Parallel cytogenetic, interphase and metaphase FISH studies chromosome 22, with no accompanying deletion of the with probes specific for the BCR and ABL genes on a series of corresponding sequences from the der(9). A number of cryptic children with Ph-positive ALL provided detailed results, which insertions of either BCR into ABL at 9q34, as reviewed by Primo could not have been achieved from any of the techniques et al,24 or rarely ABL into BCR at 22q11,25 have been reported in independently. This study demonstrates the importance of CML, with the insertions confirmed by dual fusion FISH.7,18,25– supplementing conventional cytogenetics with FISH for the 28 An insertion is sometimes seen as the end point of a series of correct identification of BCR/ABL positivity in children with events. Thus, in our case, either the der(9) chromosome which ALL. It confirms that conventional cytogenetics provides useful provided ABL sequences to chromosome 22 could have been supporting evidence in cases with a visible chromosomal lost, with subsequent duplication of the normal copy of abnormality and, as emphasised by other workers,9,12,18,24,28,34 chromosome 9, or else there could have been a partial shows that metaphase FISH should always be employed in duplication of ABL sequences on the der(9), which preceded interpreting interphase FISH results. Thus it could not be their insertion into chromosome 22. The deletion of the short assumed that, in our two cases with no available metaphases arm of one of the copies of chromosome 9 in this patient is a or additional, supporting interphase FISH, the complete loss of known, secondary feature of childhood Ph-positive ALL both components of the second fusion was due to deletions from associated with a worse prognosis.29 the der(9). Although a strong association has been established The frequency of deletions from the der(9) in CML has been between loss of ABL/BCR signals and deletions from the der(9), estimated to be 10–15% from a number of studies.6,9–12,30 The we have clearly demonstrated that alternative explanations incidence reported for ALL is more variable. In our study, the exist. With the introduction of new treatments for Ph-positive figure of 11% for confirmed deletions is similar to that reported patients, this approach will have particular relevance, should

Leukemia Chromosome 9 in Ph-positive ALL HM Robinson et al 570 specific FISH patterns, particularly deletions, be found to have 10 Huntly BJ, Reid AG, Bench AJ, Campbell LJ, Telford N, Shepherd P prognostic implications. et al. Deletions of the derivative chromosome 9 occur at the time of the Philadelphia translocation and provide a powerful and independent prognostic indicator in chronic myeloid leukemia. Acknowledgements Blood 2001; 98: 1732–1738. 11 Kolomietz E, Al Maghrabi J, Brennan S, Karaskova J, Minkin S, Lipton J et al. Primary chromosomal rearrangements of leukemia The authors would like to thank the Leukaemia Research Fund for are frequently accompanied by extensive submicroscopic financial support; Dr Mariano Rocchi, Italy, for PAC probes deletions and may lead to altered prognosis. Blood 2001; 97: dJ1132H12 and dJ835J22; Dr John Crolla and his team, Wessex 3581–3588. Regional Genetics Laboratory, Salisbury, for growing and pre- 12 Cohen N, Rozenfeld-Granot G, Hardan I, Brok-Simoni F, paration of the PAC probes; the Clinical Trial Service Unit (CTSU), Amariglio N, Rechavi G et al. Subgroup of patients with Oxford, for clinical and survival data. The authors are also grateful Philadelphia-positive chronic myelogenous leukemia character- to the following cytogenetics laboratories for providing cytoge- ized by a deletion of 9q proximal to ABL gene: expression profiling, resistance to interferon therapy, and poor prognosis. netic and FISH data as well as fixed cell suspensions on this Cancer Genet Cytogenet 2001; 128: 114–119. patient series: Academic Haematology and Cytogenetics, Royal 13 Specchia G, Albano F, Anelli L, Storlazzi CT, Zagaria A, Mancini Marsden NHS Trust, Surry; West Midlands Regional Genetics M et al. 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