Interphase fluorescence in situ hybridization (iFISH) is increasingly used for the identification of BCR/ABL gene rearrangements in chronic myeloid leukemia (CML) and acute lymphoblastic leukemia (ALL). In the present study, we have explored the incidence of both typical and atypical iFISH patterns of BCR/ABL gene rearrangements in a series of 168 consecutive BCR/ABL+ patients – 135 CML, 31 precursor B-ALL and two acute myeloblastic leukemia (AML) cases – and established their underlying genetic alterations through further molecular and chromosome analyses. Two different FISH probes (Vysis Inc., Downers Grove, IL, USA) were used: the LSI BCR/ABL dual color extra signal (ES) and the dual color dual fusion BCR/ABL probe (D-FISH). Our results show that most BCR/ABL+ patients (83%, including 88% of all CML, 61% of ALL and one of two AML) displayed typical iFISH patterns of either Major (M) BCR/ABL (87% of CML, 13% of ALL and one of the two AML) or minor (m) BCR/ABL gene rearrangements (1% of all CML and 48% of ALL cases) with the two probes. Further molecular and cytogenetic studies confirmed the presence of such typical rearrangements in all except one of these ALL cases who had coexistence of an MBCR/ABL and an mBCR/ABL gene rearrangement together with monosomy 9. In the remaining 29 cases (17%), up to five different atypical iFISH patterns were detected with the ES probe. Atypical iFISH patterns were most frequently due to additional numerical changes – most often supernumerary Philadelphia (Ph) chromosome (7%) but also gain or loss of chromosome 9 (1%) or 22 (1%). Deletion of 9q sequences proximal to the breakpoint were also frequently observed with the ES probe (8%). Application of the D-FISH probe showed that in most of these latter cases (5%) deletion of 22q sequences distal to the breakpoint also occurred. The remaining cases with atypical iFISH had cryptic insertion of BCR in 9q34 (1%). Exact interpretation of each iFISH pattern was supported by FISH on metaphases and molecular determination of the BCR breakpoint. In summary, our results indicate that despite the high incidence of typical iFISH patterns of BCR/ABL gene rearrangements, atypical patterns are also found in BCR/ABL+ acute leukemias; the precise definition of the alteration present in individual cases is dependent on metaphase studies and molecular definition of the breakpoint.
For many years, conventional karyotyping has been used as the sole diagnostic tool for t(9;22). However, it has several limitations that may lead to failure for detecting BCR/ABL gene rearrangements in around 5% of all chronic myeloid leukemia (CML) patients1,2,3,4 and a relatively high proportion of acute lymphoblastic leukemia (ALL) cases.4,5 In addition, about 5% of CML cases carry ‘masked’ translocations that can only be detected by molecular techniques such as fluorescence in situ hybridization (FISH) or RT-PCR.6
The first generation of BCR/ABL single fusion FISH probes detected the fusion gene with high specificity but with a low sensitivity (false positive rate ⩾5%).7 A new generation of FISH probes has been developed with probes extending up to 450 Kb proximately to the ABL gene on 9q34 and in some probes also with distant coverage on 22q.8 The rational was to define the t(9;22) by two FISH events: a fusion signal and an extra signal (ES) corresponding either to the remaining probe on the 9q+ or to a second fusion on the 9q+, according to the breakpoint localization in the BCR gene. With these new probes – dual color extra signal (ES) and dual color dual fusion (D-FISH) – the cutoff rate for false positives has dropped significantly to <1%.9 With the ES probes, the pattern of interphase FISH (iFISH) signals observed is distinct for the major (M) and minor (m) breakpoint in the BCR gene (MBCR or mBCR), whereas the D-FISH probe design does not distinguish between these two events. Additional variations to the typical iFISH patterns can be caused by numerical changes such as gain of the Philadelphia (Ph) chromosome, and to deletions of sequences proximal to the breakpoint on chromosome 9 or distal to the breakpoint on chromosome 22.8,9,10,11,12,13,14
The aim of this study was to determine the incidence of atypical iFISH patterns of BCR/ABL gene rearrangements and to establish the specific underlying molecular abnormalities carried by the neoplastic cells, in a large series of 168 consecutive BCR/ABL+ leukemias, which included CML, precursor B-ALL and AML cases.
Material and methods
A total of 168 consecutive patients – 135 CML, 31 precursor B-ALL and two AML – who were studied at the onset of the disease and who showed BCR/ABL gene rearrangements by iFISH, were included in the study. Diagnosis of CML and ‘de novo’ acute leukemias was performed according to the FAB15 and the WHO16 classifications, respectively; the EGIL criteria17 were used for the immunological classification of acute leukemias.
In all cases, heparin-anticoagulated fresh bone marrow (BM) aspirate samples were obtained at diagnosis and used for the cytogenetic studies described below.
BM aspirate samples were cultured for 24 and 48 h following standard procedures.18 Chromosomes were stained by Giemsa and the karyotypes described according to the ISCN recommendations.
FISH analysis for the t(9;22) translocation was initially performed on both interphase nuclei and metaphase chromosomes using the LSI-BCR/ABL dual color ES probe (Vysis Inc., Downers Grove, IL, USA) (Figure 1). In addition, the LSI-BCR/ABL D-FISH DNA probe (Vysis Inc.) (Figure 1) was also used in a subgroup of 49 patients – 37 CML, 11 precursor B-ALL and one AML ANL. Whenever appropriate, the CEP 9 probe (Vysis Inc.) and a biotinylated probe that simultaneously identifies the centromeric repetitive DNA sequences of chromosomes 14 and 22 (Cambio, Cambridge, UK) were also used.
FISH analyses were performed on BM cells prepared according to conventional cytogenetic techniques and placed on slides. Hybridization with fluorochrome-labelled FISH probes was performed according to the recommendation of the manufacturers with slight modifications.19 For the biotinylated probe, appropriate immunologic detection of hybridization was performed using avidin–FITC.7
Fluorescence signals were evaluated using a BX60 fluorescence microscope (Olympus, Hamburg, Germany) and a × 100 oil objective. For all slides measured, the number of unhybridized cells in the areas assessed was <1% and only those spots with a similar size, intensity and shape were counted (minimum of 200 cells/sample). The iFISH patterns obtained in normal cells – two green, two red, no fusion signals – are shown in Figure 2 for both the ES (pattern N) and the D-FISH (pattern n) probes. Based on the analysis of nuclei from 20 BCR/ABL− BM samples, a case was considered to be BCR/ABL+ once the percentage of BCR/ABL+ nuclei was ⩾0.9% (mean number plus two standard deviations of nuclei showing a typical MBCR/ABL iFISH pattern).
Molecular analysis of BCR/ABL transcripts
RNA was extracted from washed BM mononuclear cells by the guanidium thiocyanate method.20 RT-PCR analysis of the fusion gene transcripts was carried out according to the guidelines of the Biomed-1 Concerted Action.21 This procedure includes a reverse transcription of the RNA and a double round PCR of the cDNA, with the identification of the amplicons through agarose gel electropheresis and ethidium bromide staining. The procedure also includes the check of the RNA integrity through amplification of the normal ABL RNA and a control PCR (shifted PCR) for confirmation or exclusion of false-positive results.
The great majority of the patients analyzed (139/168, 83%) including most CML (119/135, 88%) and ALL (19/31, 61%) and one of two AML cases, displayed typical iFISH patterns of BCR/ABL gene rearrangements involving either the MBCR (pattern A from Figure 2; n=122) or the mBCR breakpoints (pattern B from Figure 2; n=17), with the ES probe. Pattern A consisted of one fusion-der (22)-, one green-nonrearranged 22- and two red-der (9) plus the nonrearranged chromosome 9- signals; in pattern B the red signal corresponding to der(9) is replaced by a second fusion. Most cases with a typical MBCR/ABL pattern (pattern A) corresponded to CML cases (117 out of 122 MBCR/ABL+ cases; 96%) and only a few were patients with ALL (n=4) or AML (n=1). In turn, all except two cases with a typical mBCR/ABL iFISH pattern (pattern B) corresponded to ALL patients (n=15); the other two were CML. Additional iFISH analyses with the D-FISH probe were performed in 20 and six cases with typical patterns A and B, respectively. All these cases showed an identical and typical iFISH pattern with this probe (pattern ab from Figure 2), this probe not allowing distinction between MBCR and mBCR breakpoints.
As may be seen in Table 1, further metaphase and molecular studies showed that all except one case with typical iFISH patterns had conventional t(9;22) involving either the MBCR (pattern A in Figure 2) or the mBCR breakpoints (pattern B in Figure 2); in the remaining case metaphase studies showed that pattern B was because of a double Ph associated to loss of der(9); coexistence of both p190 and p210 transcripts were found in this ALL patient.
In the remaining 29 cases (17%) – 16 CML, 12 ALL and one AML – up to five different atypical iFISH patterns were detected with the ES probe (patterns C to G in Figure 2). In order to identify the exact molecular/genetic abnormalities involving the BCR and ABL genes in each of these atypical iFISH patterns, further PCR studies, conventional cytogenetics and/or FISH analyses of both metaphase and interphase chromosomes 9 and 22, were performed.
As illustrated in Figure 2 and shown in Table 1, pattern C (two fusion, one green and two red signals) was found in five cases (3%) (three CML and two ALL) all of whom had p210 transcripts compatible with the occurrence of an MBCR breakpoint; metaphase studies performed in these cases confirmed the presence of an extra fusion signal, as compared to a typical iFISH MBCR/ABL pattern (pattern A), because of the presence of a supernumerary Ph (Table 1). In turn, all cases showing nuclei with three fusion signals associated with one red and either one (pattern D; n=8, 5%) or two (pattern E; n=1, 1%) green signals were ALL and had p190 transcripts/mBCR breakpoint; analysis of metaphase chromosomes confirmed in all except one of these ALL cases that the additional third fusion signal, as compared to typical mBCR/ABL iFISH pattern B, was because of a supernumerary Ph; in the other case, who had atypical pattern D, instead of corresponding to an additional Ph, the third fusion was because of an additional der(9) In the ALL case who displayed pattern E with a double Ph, the second green signal (BCR probe) observed was because of an additional gain of a nonrearranged chromosome 22.
Among all atypical iFISH patterns, pattern F (one fusion, one red and one green signals) was the most frequently observed: 14 cases (8%), corresponding to 13 CML and one AML. Despite having an identical iFISH pattern with the ES probe, this group was shown to be genetically rather heterogeneous. Accordingly, metaphase studies showed that in some of these CML cases with p210 transcripts (11/13 cases) extensive deletions of 9q sequences proximal to the breakpoint occurred explaining the loss of one red signal – as compared to typical MBCR/ABL iFISH pattern A; in contrast, in the other two CML patients the presence of a single red signal was because of the existence of cryptic insertion of BCR in 9q34. In the remaining patient with atypical iFISH pattern F corresponding to an AML, p190 transcripts were detected and loss of a fusion signal, as compared to typical mBCR/ABL iFISH pattern B, was explained by the existence of an extensive deletion of der(9q). The application of the D-FISH probe in these cases further showed the existence of deletions on 22q sequences distal to the breakpoint – pattern f2 from Figure 2 – in nine of the 11 CML cases who had del(9q) but not in the other two cases.
Pattern G (two fusions, one green and no red signal with the ES probe) was found in one ALL case who had p190 transcripts. Metaphase studies confirmed the absence of an ABL probe-derived red signal because of del(9q) of the nonrearranged chromosome 9.
In the present study, we show that the use of a new generation of BCR/ABL FISH probes in interphase nuclei of a large series of BCR/ABL+ leukemias is associated with the observation of a variable number of different iFISH patterns. The most frequently detected patterns with the ES probe corresponded to typical BCR/ABL gene rearrangements involving the MBCR (one fusion, one green (BCR probe) and two red (ABL probe) signals) and the mBCR (two fusion, one green and one red signals) breakpoints; as expected, distinction between these two breakpoint regions could not be achieved with the D-FISH probe. Although both patterns A and B were found both in CML and ALL cases, the former was more frequently observed in CML while the latter was usually associated with ALL. Interestingly, in all ALL showing a typical MBCR/ABL pattern, iFISH studies showed the presence of MBCR/ABL gene rearrangements not only in blast cells but also in residual neutrophils (data not shown). These results could suggest that these patients might actually correspond to blast crisis of CML arising from a multipotent stem cell, who present as an apparently ‘de novo’ ALL.
Since additional karyotypic changes (eg supernumerary Ph, gain or loss of chromosomes 9 and 22, as well as deletions of 9q and 22q) can occur in BCR/ABL+ CML, ALL and AML cases,8,9,10,11,12,13,14 other atypical iFISH patterns could be expected as observed in around one-sixth of all cases studied; conversely, cases with additional cytogenetic changes could present as a typical pattern and potentially be misdiagnosed with iFISH. Exact interpretation of the atypical iFISH patterns obtained in individual cases was dependent on the analysis of metaphases complemented with molecular definition of the breakpoint. Accordingly, metaphase analyses associated to molecular studies showed that in cases with atypical iFISH patterns presence of an additional Ph and deletions of 9q sequences proximal to the breakpoint, were the two most frequent underlying genetic abnormalities. However, none of these two abnormalities was associated with one single atypical iFISH pattern.
Accordingly, gain of a Ph was usually characterized by the presence of an additional fusion signal in both MBCR/ABL+ and mBCR/ABL+ cases, with the two probes. However, among cases in the latter group, gain of an extra fusion signal was not specific of supernumerary Ph, since it was also observed in association with gain of der(9). In turn, not all ALL cases showing a supernumerary Ph had this atypical iFISH pattern: in one case, supernumerary Ph was associated with an additional gain of the nonrearranged chromosome 22 that translated into a different iFISH pattern; in another patient, gain of a Ph was associated with del(9q) and; in a third ALL, a double Ph coexisting with both p210 and p190 mRNA transcripts in association with monosomy 9 was observed leading to a typical iFISH pattern. The presence of a double Ph in CML as well as in ALL has been previously reported as a secondary genetic change.22 In line with this, in almost all cases with a supernumerary Ph, nuclei carrying a double Ph chromosome coexisted with a variable proportion of nuclei displaying typical iFISH patterns. In line with the previous observations, the presence of a double Ph with mBCR breakpoints was exclusively found among ALL patients while gain of a second Ph in MBCR/ABL+ cases was observed both among CML and ALL cases. Interestingly, our results suggest that among MBCR/ABL+ cases studied at diagnosis, the presence of a double Ph would be more frequently observed in ALL than in CML.
Coexistence of p190 and p210 transcripts have been reported in ALL23 and could be due either to the presence of a double Ph chromosome, each carrying a different breakpoint, or, more commonly, to the presence of a typical MBCR/ABL translocation in which the p190 is generated by alternative splicing of MBCR/ABL mRNA.24
Der(9q) deletions were found only among MBCR/ABL+ CML and in one AML; with the ES probe they constantly translated into loss of one red signal from der(9q). A similar incidence of del(9q) to that found in our study has been previously described in CML.8,13,14 Such deletions typically expand up to 5.5 Mb14 on chromosome 9q sequences proximal to the breakpoint site of the translocation, therefore, hampering visualization of the residual ABL probe on der(9). Interestingly, all MBCR/ABL+ nuclei from all these CML cases showed del(9q) confirming that both abnormalities – t(9;22) and del(9q) – might represent a single one-step event.14 Most CML cases with del(9q) also had deletions of 22q sequences distal to the breakpoint. As expected, this latter abnormality could only be detected with the D-FISH probe and it also involved all BCR/ABL+ nuclei. Although both mBCR/ABL and MBCR/ABL gene rearrangements have been reported in AML,25 to the best of our knowledge this is the first report in which coexistence of an extensive del(9q) and mBCR/ABL gene rearrangements are described in AML.
Despite the association found between loss of a red signal in MBCR/ABL+ nuclei and extensive del(9q), this atypical iFISH pattern was not specific of this cytogenetic abnormality. Accordingly, an identical pattern was found in two CML cases who were Ph-negative and had no del(9q). Informative metaphase FISH analyses showed in both cases a cryptic insertion of BCR in 9q34. The exact mechanisms responsible for the aberrant localization of the BCR/ABL fused gene on chromosome 9 instead of chromosome 22 are not entirely clear. Previous reports on Ph− CML cases in which the BCR/ABL fusion gene is located on chromosome 9q34 suggest that either an interstitial insertion of the BCR gene from chromosome 22 into the ABL gene on chromosome 9 or two successive translocations including a standard t(9;22) followed by a translocation between der (9q) and der(22q) could occur.26 In the two cases reported here, insertion of 5′ BCR sequences could not be confirmed through chromosome painting (data not shown) probably because of the small size of the fragment inserted in the ABL gene. It should be noted that no interphase nuclei with a typical MBCR/ABL iFISH pattern were observed in any of these cases, which could help to explain the underlying mechanism for this genetic abnormality.
In summary, our results indicate that although iFISH can correctly diagnose many BCR/ABL+ patients, exact interpretation of individual cases requires the application of the probes on metaphases complemented with molecular definition of the breakpoint.
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MD Tabernero is supported by a grant from the MCYT, programa Ramón y Cajal. JM Sayagues is supported by a grant (02/9103) from Ministerio de Sanidad y Consumo, Madrid, Spain.
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Primo, D., Tabernero, M., Rasillo, A. et al. Patterns of BCR/ABL gene rearrangements by interphase fluorescence in situ hybridization (FISH) in BCR/ABL+ leukemias: incidence and underlying genetic abnormalities. Leukemia 17, 1124–1129 (2003) doi:10.1038/sj.leu.2402963
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