Although trisomy 8 as the sole chromosome aberration is the most common numerical abnormality in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS), little is known about its pathogenetic effects. Considering that +8 is a frequent secondary change in AML/MDS, cryptic – possibly primary – genetic aberrations may occur in cases with trisomy 8 as the apparently single anomaly. However, no such hidden anomalies have been reported. We performed a high-resolution genome-wide array-based comparative genome hybridization (array CGH) analysis of 10 AML/MDS cases with isolated +8, utilizing a 32K bacterial artificial chromosome array set, providing >98% coverage of the genome with a resolution of 100 kb. Array CGH revealed intrachromosomal imbalances, not corresponding to known genomic copy number polymorphisms, in 4/10 cases, comprising nine duplications and hemizygous deletions ranging in size from 0.5 to 2.2 Mb. A 1.8 Mb deletion at 7p14.1, which had occurred prior to the +8, was identified in MDS transforming to AML. Furthermore, a deletion including ETV6 was present in one case. The remaining seven imbalances involved more than 40 genes. The present results show that cryptic genetic abnormalities are frequent in trisomy 8-positive AML/MDS cases and that +8 as the sole cytogenetic aberration is not always the primary genetic event.
Acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS) are characterized by acquired clonal chromosome aberrations closely associated with the leukemogenic process.1 One of the most common of these is trisomy 8, which is present as the sole change in 5–7.5% and together with other aberrations in 10–15% of cytogenetically abnormal cases.1, 2 As a single anomaly, +8 is associated with de novo AML/MDS, possibly also with previous exposure to organic solvents, and displays a higher frequency in elderly AML patients, occurring in all AML and MDS subgroups.2, 3 There is some controversy as to the prognostic impact of isolated trisomy 8 in AML, with different studies reporting an intermediate or poor outcome.4, 5 In MDS, such cases are assigned to the intermediate cytogenetic subgroup.6
Next to nothing is known about the pathogenetic effects and the functional outcome of +8. In fact, trisomy 8 may not be sufficient to initiate leukemia. For example, although individuals with constitutional mosaicism for +8 are at an increased risk for hematologic malignancies, these disorders arise several years after birth.7 Also, Nilsson et al.8 have shown that +8 is a late event in MDS with this trisomy as the sole change, that is, hematopoietic stem cells without +8 are part of the MDS clone. Furthermore, it is known that the presence of +8 leads to a general overexpression of genes residing on this chromosome, suggesting gene dosage effects, but these chromosome 8 genes do not dominate the specific gene expression signature of trisomy 8-positive cases, which may indicate that additional changes are present.9 Taken together with the fact that trisomy 8 is a common secondary change in AML and MDS,1 it seems likely that other cryptic genetic changes – not detectable by standard cytogenetic methods such as G-banding – may be present in cases with an extra chromosome 8 as the seemingly sole aberration.10 Possible hidden abnormalities could be point mutations and cytogenetically unidentifiable chromosome aberrations resulting in fusion genes, small deletions, or amplifications. Somatic mutations in, for example, CEBPA and RUNX1 have been reported in AMLs/MDSs with +8.11, 12 However, these are not specific for, or even more common in, cases with isolated trisomy 8. As regards cryptic chromosome aberrations, several studies have addressed this issue by using various fluorescence in situ hybridization (FISH)-based techniques, but hidden changes have not been identified in cases with trisomy 8 as the sole aberration.10, 13
Array-based comparative genome hybridization (array CGH) is a relatively novel method for detecting copy number changes.14 This technique has proved to be an important supplement to G-banding to screen for chromosome aberrations, and several investigators have utilized array CGH to characterize amplicons and homozygous deletions in various neoplasms and to identify constitutional translocation breakpoints by detecting concomitant deletions.15, 16 However, only a few studies have used genome-wide array CGH to screen for neoplasia-associated abnormalities that are cryptic by G-banding analysis,17, 18 and none of these have included AML or MDS cases. The recent development of an array CGH platform with partly overlapping bacterial artificial chromosome (BAC) clones, covering at least 98% of the human genome, enables detection of genomic imbalances involving as little as 100 kb.19, 20 In the present study, this high-resolution genome-wide array CGH was applied in 10 AMLs and MDSs with trisomy 8 as the seemingly sole genetic change to search for cryptic chromosome aberrations.
Patients, materials and methods
The study comprised four MDSs and six AMLs with trisomy 8 as the single abnormality, including four males and six females, with a median age of 70 years (range 28–94) (Table 1). All but one of the cases were de novo AML or MDS; patient 3 had received radio- and chemotherapy because of polycythemia vera. Apart from +8, cases 3 and 7 had subclones containing trisomy 21 and monosomy 14, respectively (Table 1). Cases 1–3, 6 and 8 had previously been analyzed with multicolor FISH as well as with metaphase FISH with partial chromosome painting and subtelomeric probes for 8p and 8q and with gene-specific probes for FGFR1, MYST3 (MOZ), RUNX1T1 (ETO) and MYC, without revealing any additional abnormalities.10
Array-based comparative genome hybridization
DNA was extracted from bone marrow (BM) or peripheral blood cells, obtained at the time of diagnosis and stored at −80°C. In case 5, a sample obtained after transformation of chronic myelomonocytic leukemia (CMML) to AML was utilized. Male reference genomic DNA was used in all hybridizations (Promega, Madison, WI, USA). Labeling, slide preparation and hybridization were performed as described by Jönsson et al. (submitted). In brief, 1.0 μg leukemic and 1.2 μg reference DNA was labeled by random priming with Cy3 and Cy5, respectively, using the BioPrime Array CGH Genomic Labeling Module (Invitrogen, Carlsbad, CA, USA). Unincorporated nucleotides were removed with the CyScribe GFX Purification Kit (Amersham, Little Chalfont, UK). Labeled test and reference DNA was combined with 100 μg Cot-1 DNA, dried by speed vacuum centrifugation, and redissolved in 55 μl of hybridization solution (50% formamide, 10% dextran sulfate, 2 × SSC, 2% sodium dodecyl sulfate (SDS), and 10 μg/μl yeast tRNA). Slides containing the 32K array set, consisting of 32 433 BAC clones (BACPAC Resources, Oakland, CA, USA) and covering at least 98% of the human genome,19, 20 were produced at the SWEGENE DNA microarray resource center at Lund University, Sweden. The slides were crosslinked and treated with the Pronto Microarray Reagent System (Corning, Acton, MA, USA). Hybridization took place in a humidified chamber at 37°C for 48–72 h. The slides were washed in 2 × SSC, 0.1% SDS for 15 min, followed by 2 × SSC, 50% formamide (pH 7.0) for 15 min at 45°C, 2 × SSC, 0.1% SDS for 30 min at 45°C, and in 0.2 × SSC for 15 min at room temperature.
The analyses of microarray images were performed with the GenePix Pro 4.0 software (Axon Instruments, Foster City, CA, USA). For each spot, the median pixel intensity minus the median local background for both dyes was used to obtain a test over reference gene copy number ratio. Data normalization was performed per array subgrid using lowess curve fitting with a smoothing factor of 0.33.21 To identify imbalances, the MATLAB toolbox CGH plotter was applied, using moving mean average over three clones and limits of log2 >0.2.22 Classification as gain or loss was based on (1) identification as such by the CGH plotter and (2) visual inspection of the log2 ratios. In general, log2 ratios >0.5 in at least four adjacent clones were considered to be deviating. Ratios of 0.5–1.0 were classified as duplications/hemizygous deletions; ratios >1.0 were classified as amplifications/homozygous deletions. All normalizations and analyses were performed in the BioArray Software Environment database.23
Fluorescence in situ hybridization
To confirm the array CGH findings, metaphase or interphase FISH was performed on BM cells that had been stored in fixative. Signals were detected using an Axioplan 2 fluorescence microscope (Carl Zeiss, Jena, Germany) and analyzed with the CytoVision Ultra system (Applied Imaging, Newcastle Upon Tyne, UK). Bacterial artificial chromosomes and P1 artificial chromosomes (PACs) (Table 2; BACPAC Resources) were applied. In addition, probes for the ETV6/RUNX1 genes, the chromosome 16 centromere, and the subtelomeric regions of 4p and 13q (Vysis, Downers Grove, IL, USA) were used. Trisomic metaphases were identified with a centromeric probe for chromosome 8 (Vysis).
The array CGH analyses confirmed most of the aberrations identified by G-banding, that is, trisomy 8 was detected in all cases and gain of chromosome 21 in case 3. However, −14, which by G-banding was present in 3/24 metaphases in case 7, was not revealed by array CGH (Table 1).
Large-scale abnormalities detected by the array CGH, not corresponding to subclones seen with G-banding, included higher ratios, possibly representing gains, of the whole chromosome 16 and 19q in cases 8–10 and of 21q in case 10. The possibility of small subclones with +16 and +21q was investigated with interphase FISH in case 10, the only case for which material was available for analysis. No hidden subclones were detected.
Abnormalities not expected to be detectable by G-banding were found in nine of the 10 cases using array CGH. Cases 3 and 7 displayed one change, cases 1, 4 and 6 two changes, case 5 three changes, cases 8 and 10 six changes and 10 changes in case 9. In total, the array CGH analyses identified 23 different intrachromosomal imbalances; 11 gains and 12 losses (Table 2). All gained clones displayed ratios indicative of low-copy number changes, that is, duplications, ranging in size from 0.4 to 1.9 Mb (median 1.0 Mb); all deleted clones were apparent hemizygous deletions, ranging in size from 0.2 to 4.9 Mb (median 1.5 Mb). Fifteen of the aberrations were found in single cases; the remaining ones in 2–4 cases. All but two involved regions containing known genes. Eleven imbalances overlapped partly or completely with previously described genomic copy number polymorphisms (CNPs; Table 2).24
Metaphase FISH was utilized to investigate five of the 11 gains detected with array CGH, including all four gains not corresponding to previously described CNPs. These were a 1.7 Mb gain at 1p35.3, a 1.3 Mb gain at 5q35.1, an 0.4 Mb gain at 8q21.2 in case 10, a 1.0 Mb gain at 15q21.3 and an 0.5 Mb gain at 19p13.1 (Table 2). All except the gain at 8q21.2 displayed a normal hybridization pattern with FISH. The latter, which was present in cases 3 and 10, displayed a very high log2 ratio with array CGH, visible even within the elevated baseline of the trisomic chromosome 8. Fluorescence in situ hybridization showed increased signal intensity of the probe in one homologue in metaphases with disomy 8 and in two homologues in metaphases with trisomy 8. Furthermore, because of the possibility of cryptic, unbalanced translocations involving distal 4p and 13q in case 10, which displayed slight increases in the ratios close to these telomeres, subtelomeric probes were applied. No hidden rearrangements were found, and no difference in signal intensity was noted between the homologues.
Eight of the 12 detected copy number losses were investigated with metaphase or interphase FISH, including seven of the eight losses not corresponding to previously known CNPs (the remaining loss could not be investigated due to lack of material). These were 1.4 and 1.6 Mb losses encompassing the same 2q33.2 region in cases 8 and 9, respectively, a 2.1 Mb loss at 4q13.1, a 1.9 Mb loss at 6q24.1 in case 9, a 1.8 Mb loss at 7p14.1 in case 5 (Figure 1), an 0.3 Mb loss at 7q11.21 in case 8, a 1.1 Mb loss at 9p21.3 in case 9, a 1.5 Mb loss at 12p13.2 in case 10 and an 0.8 Mb loss at 17q24.3 in case 1 (Table 2). The FISH analyses showed no losses of the 2q33.2 and the 4q13.1 regions in case 8 or of the 6q24.1 and the 9p21.3 regions in case 9. The remaining changes were confirmed to be hemizygous deletions. A similar 2q33.2 deletion was detected in a subclone in addition to the trisomy 8 in case 9. For the chromosome 7 losses, deletions were detected in all disomic as well as in all trisomic metaphases, suggesting that these abnormalities were either CNPs or leukemia-associated aberrations preceding +8. Notably, the latter was confirmed for the del(7)(p14p14) in case 5; FISH on a sample taken before CMML transformation displayed no hemizygous deletion (Table 1 and Figure 1b and c). To investigate the possibility of this change being generally associated with transformation of CMML to AML, seven cases of CMML that had transformed were screened with metaphase FISH. No additional deletion was found (data not shown). The breakpoints of the 7p14 deletion in case 5 were confirmed with FISH to be in, or proximal to, the PAC RP4-570D2, and in, or distal to, the BAC RP11-762A4. For the 12p13.2 loss, only metaphases with trisomy 8 could be investigated. Hence, it remains unknown whether this deletion occurred prior to the +8. The 17q24.3 deletion was not seen in metaphases from case 1, but was detected in approximately 35% of the interphase cells.
Array CGH is a powerful technique for detection of unbalanced chromosome abnormalities. In the present study, we investigated the possibility of cryptic genetic changes in 10 AML/MDS cases with trisomy 8 as the apparently sole aberration, utilizing a recently developed platform which covers at least 98% of the human genome and enables detection of genomic imbalances involving as little as 100 kb. The analysis identified an extra chromosome 8 in all 10 AML/MDS cases as well as trisomy 21 in case 3, confirming the karyotypes. Loss of chromosome 14 was not identified in case 7, in spite of the cytogenetic finding of a subclone with monosomy 14. However, −14 was only found in 3/24 metaphases with G-banding (Table 1); such a low frequency is probably below the detection level of array CGH.
The analyses suggested gains of the whole chromosome 16 and of 19q in cases 8–10 and gain of 21q in case 10, none of which had been detected by G-banding. To investigate the possibility of subclones containing these imbalances, interphase FISH was performed in case 10. No such subclones were detected; these ‘imbalances’ were probably artifacts. This underscores the importance of verifying unexpected array CGH findings with alternative techniques, such as FISH. The reason for this discrepancy is presently unclear, but likely reflects poor array CGH hybridization. In fact, systematic locus-specific ratio fluctuations affecting chromosomes 16 and 19 have previously been reported in conventional CGH analyses.25
In addition to the large-scale chromosome aberrations, array CGH revealed cryptic intrachromosomal imbalances, ranging in size from 0.2–4.9 Mb, in 9 of the 10 AML/MDS cases. In total, 23 different duplications (n=11) and hemizygous deletions (n=12) were found (Table 2). Metaphase FISH analysis was performed to investigate five of the 11 gains detected by array CGH, namely those involving 1p35.3, 5q35.1, 8q21.2, 15q21.3 and 19p13.1 (Table 2). This analysis disclosed signals only at the expected loci, indicating the presence of segmental duplications. For the 8q21.2 imbalance increased signal intensities on two of the chromosome 8 homologues in metaphases with trisomy 8 revealed duplication of the homologue with the gain. This would suggest that the duplication occurred prior to the trisomy 8. However, this is a well-known CNP.24 Eight of the 12 losses detected by array CGH were investigated with metaphase or interphase FISH, four of which were confirmed to be hemizygous deletions (Table 2). The 2q33.2 and 4q13.1 losses in case 8 and the 6q24.1 and 9p21.3 deletions in case 9 detected by array CGH could not be confirmed by FISH. However, a similar 2q33.2 deletion was seen in a subclone in case 9, and the 17q24.3 deletion in case 1 was only found in interphase cells, suggesting that some of the discrepancies between array CGH and FISH may be explained by subclones containing deletions not being detected by FISH. Excluding the discrepant results, a total of 20 intrachromosomal imbalances were present among the 10 cases.
Eleven of the above-mentioned 20 changes overlapped partly or completely with previously described CNPs in the human genome (Table 2).24 Thus, these aberrations were most likely constitutional. Despite the fact that several recent investigations have shown that CNPs are quite common,26 the phenotypic effects – if any – of these variants are as yet unknown. Because genes are frequently included in the duplicated or deleted regions, dosage effects resulting in, for example, increased susceptibility to cancer is an intriguing possibility.26 The identified CNPs representing duplications in our cases lead to gain of several potentially oncogenic genes (Table 2). However, a larger number of hematologic malignancies as well as control samples must be investigated before any conclusions can be drawn regarding the possible etiologic or pathogenetic impact of gene dosage effects resulting from these, or other, CNPs.
Nine aberrations, occurring in altogether four cases (nos. 1, 5, 9 and 10), did not correspond to known CNPs (Table 2), and may thus have been neoplasia-associated. The number of intrachromosomal imbalances varied from 1 to 5 changes per case. Of these cases, only one was an MDS; the remaining were AMLs. Thus, it is possible that cryptic genomic imbalances are more common in trisomy 8-harboring AMLs than in MDSs. Some of these imbalances may represent previously undetected CNPs, but this is not likely the case for all the detected changes, because two of them were found in more than one of the AMLs. CNPs of such a high frequency would probably already have been reported, unless certain genomic variants are associated with a higher risk to develop AML/MDS.26 Furthermore, one of the deletions detected with array CGH – the one involving the ETV6 gene – is a well-known abnormality in hematologic malignancies, and the loss at 7p14.1 was shown with FISH (Figure 1) to be confined to the leukemic metaphases (see below). Thus, we conclude that array CGH analysis reveals cytogenetically cryptic leukemia-associated aberrations in a high proportion (40%) of myeloid malignances with trisomy 8 as the sole cytogenetic anomaly.
Case 10 displayed a hemizygous deletion of 1.5 Mb at 12p13.2, including ETV6 (Table 2). Cytogenetically visible hemizygous deletions involving this region are common in leukemias,1 but deletions have also been described in a handful of AMLs with cytogenetically normal chromosomes 12, including one case with +8 in addition to a del(11)(q13).27, 28 The pathogenetic importance of these deletions in AML is unknown, as is the target gene. As previously reported deletions have not all encompassed ETV6, it has been suggested that loss of CDKN1B may instead be the biologically important outcome.27 However, this gene was not included in the deleted segment in the present case. Thus, a gene located between ETV6 and CDKN1B may be the true target of the deletion in AML.
In case 5, a 1.8 Mb loss at 7p14.1 was detected (Table 2 and Figure 1). Fluorescence in situ hybridization confirmed that this was a hemizygous deletion and also showed that it was present in cells with disomy as well as trisomy for chromosome 8 in the AML sample, but not in a sample taken during the CMML phase (Table 1 and Figure 1). Thus, this deletion was not a constitutional CNP; it arose before the trisomy 8, possibly in association with the CMML transformation. To investigate whether this loss is a general phenomenon in transforming CMML, we analyzed seven additional such cases with FISH, but none of these displayed the del(7)(p14p14) (data not shown). The presently described imbalance was not visible by G-banding and has not been previously described; however, deletions involving larger 7p regions, including 7p14, have been reported in AML and MDS.1 It included the genes CDC2L5, C7ORF11, C7ORF10 and INHBA (Table 2). The telomeric breakpoint occurred close to RALA, and the centromeric breakpoint occurred in the vicinity of, or in the 3′ part of, GLI3. Although small deletions may result in fusion genes,29 RALA and GLI3 are oriented in opposite directions and are hence not able to form a fusion by this mechanism. Thus, loss of genes in this region may be the functionally important outcome of the deletion. Considering the known biological functions of the deleted genes, CDC2L5, which encodes a cyclin-dependent kinase, may be the most likely target, because this gene family is involved in cell cycle and gene expression regulation.30 Alternatively, the deletion may cause deregulation of a gene in the vicinity of the breakpoints, for example, the RALA gene. It is noteworthy that it has been reported that activation of RALA is necessary for RAS-induced tumorigenesis.31
The remaining seven copy number changes involved more than 40 genes (Table 2). Of these, three – the duplicated LAPTM5 and HSH2D (ALX) and the hemizygously deleted CD28 – have been shown to be involved in hematopoiesis and/or hematologic malignancies.32, 33
In conclusion, the present array CGH analysis revealed cryptic chromosome changes in nine of 10 AML/MDS cases with trisomy 8 as the sole cytogenetic abnormality. Excluding imbalances overlapping with previously described CNPs, novel – and possibly leukemia-associated – aberrations were found in four (40%) of the cases. Notably, at least one of these changes occurred prior to the trisomy 8. These results strongly suggest that genetic abnormalities, such as duplications and hemizygous deletions, occur in cytogenetically normal chromosomes in AML and MDS. Furthermore, our findings support the hypothesis that trisomy 8 by itself is not sufficient for leukemogenesis and that +8 may not be the primary event even when it is the sole abnormality detected by G-banding. This may explain the morphologic and clinical heterogeneity seen in AML and MDS cases harboring this chromosome aberration.
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This work was supported by grants from the Swedish Cancer Society, the Ingabritt and Arne Lundberg Foundation, and the Knut and Alice Wallenberg Foundation via the SWEGENE program.
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Cite this article
Paulsson, K., Heidenblad, M., Strömbeck, B. et al. High-resolution genome-wide array-based comparative genome hybridization reveals cryptic chromosome changes in AML and MDS cases with trisomy 8 as the sole cytogenetic aberration. Leukemia 20, 840–846 (2006). https://doi.org/10.1038/sj.leu.2404145
- acute myeloid leukemia
- myelodysplastic syndromes
- trisomy 8
- chromosome aberration
- array CGH
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