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Although the dic(9;20)(p11–13;q11) is a recurrent chromosomal abnormality in paediatric B-cell precursor acute lymphoblastic leukaemia (BCP ALL), occurring in approximately 2% of the cases, its molecular genetic consequences have not been elucidated. In the present study, high-resolution genome-wide array-based comparative genomic hybridisation (array-CGH) and fluorescence in situ hybridisation (FISH) were used to characterise the 9p and 20q breakpoints (BPs) in seven childhood BCP ALLs with dic(9;20), which was shown to be unbalanced in all of them, resulting in loss of 9p13.2-pter. Five of the cases had loss of 20q11.2-qter, whereas two displayed gain of 20cen-pter. All BPs on 9p clustered in a 1.5 Mb segment of the sub-band 9p13.2; in three of the cases, the 20q BPs mapped to three adjacent clones covering a distance of 350 kb at 20q11.2. Thus, the aberration should be designated dic(9;20)(p13.2;q11.2). One of the ALLs, shown to have a complex dic(9;20), was further investigated by FISH, revealing a rearrangement of the haemapoietic cell kinase isoform p61 (HCK) gene at 20q11. The disruption of HCK may result in a fusion gene or in loss of function. Unfortunately, lack of material precluded further analyses of HCK. Thus, it remains to be elucidated whether dic(9;20)(p13.2;q11.2) leads to a chimaeric gene or whether the functionally important outcome is loss of 9p and 20q material.
The dicentric chromosome abnormality dic(9;20)(p11–13;q11) was first reported as a non-random aberration in B-cell precursor acute lymphoblastic leukaemia (BCP ALL) one decade ago (Rieder et al, 1995; Slater et al, 1995), and to date, almost 50 cases have been published (Mitelman et al, 2006). The reason for the rather recent detection of this recurrent abnormality is undoubtedly that it is a subtle rearrangement that may be mistaken for monosomy 20 by G-banding alone. Thus, fluorescence in situ hybridisation (FISH) analyses are necessary for accurate identification of the abnormality, with dic(9;20) ‘masquerading’ as monosomy 20 and a concomitant deletion of 9p (Rieder et al, 1995; Slater et al, 1995; Heerema et al, 1996; Clark et al, 2000).
The dic(9;20) is frequently the sole cytogenetic change (Mitelman et al, 2006), and is hence most likely a primary abnormality strongly associated with the leukaemogenic process (Rieder et al, 1995; Slater et al, 1995; Clark et al, 2000), although it has been detected in single cases that also harboured the primary translocations t(8;14)(q24;q11) (Clark et al, 2000) or the t(9;22)(q34;q11) (Rieder et al, 1995). Additional changes are present in approximately half of the cases, with trisomy 21 being particularly common, followed by gains of chromosomes X, 8 and 20 (Clark et al, 2000; Mitelman et al, 2006).
The dic(9;20) is more common in paediatric ALLs (2%) than in adult cases (<1%), and seems to be more frequent in females, with female ratio of 1·9 (Mitelman et al, 2006). The median age at diagnosis is 4 years and the median leucocyte count is 20–30 × 109/l (Johansson et al, 2004). Apart from one T-lineage ALL, all reported cases had a BCP immunophenotype that was generally positive for TdT, HLA-DR, CD10, CD19 and CD24, and negative for myeloid markers (Rieder et al, 1995; Slater et al, 1995; Heerema et al, 1996; Clark et al, 2000; Raimondi et al, 2003). The prognostic impact of dic(9;20) is still unclear, but although many cases display non-standard risk clinical features, most reported patients have attained complete remission and it has been suggested that the overall survival of patients with dic(9;20) may be good (Rieder et al, 1995; Slater et al, 1995; Heerema et al, 1996; Clark et al, 2000; Raimondi et al, 2003). Considering that monosomy 20 in ALL may represent unrecognised dic(9;20), it is in this context noteworthy that −20 as the sole change has been associated with a relatively favourable outcome (Betts et al, 1990; Silengo et al, 1992).
Previous FISH analyses, using chromosome painting and centromeric probes, have revealed that the dic(9;20) contains centromeres of both chromosomes 9 and 20 that results in loss of 9p and 20q material (Rieder et al, 1995; Slater et al, 1995; Heerema et al, 1996; Clark et al, 2000). However, the dic(9;20) has, as yet, not been characterised in detail at the molecular level. It is therefore unknown whether it results in a fusion gene, akin to the PAX5/ETV6 chimaera generated by the dic(9;12)(p13;p13) in ALL (Strehl et al, 2003), or whether the pathogenetically important outcome of the dic(9;20) is loss of genetic material, e.g. deletion of tumour suppressor genes. We have carried out a detailed molecular analysis of the dic(9;20) in seven paediatric BCP ALLs, using array-based comparative genomic hybridisation (array-CGH) and locus-specific FISH.
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The salient results of the present array-CGH and FISH analyses of seven paediatric dic(9;20)-positive BCP ALLs were the high frequency of cryptic deletions, the consistent losses of 9p and 20q material, the clustering of breakpoints both at 9p and at 20q, and the possible rearrangement of the HCK gene at 20q.
All but one of the ALLs harboured chromosomal losses not identified by conventional G-banding analyses (Tables I and II). The only recurrent change was the deletion involving the IGH@ locus at 14q32.33, found in four of the cases. This, most probably, reflects a somatic immunoglobulin rearrangement clonotypic for the leukaemic blasts. Hence, we deem it unlikely that these 14q32 deletions were causally associated with the leukaemia development. The other cryptic deletions involved 4p11-12, 10q11, 12q23-24 and 15q26 (Table II). The molecular genetic consequences and the leukaemogenic impact of these remain to be elucidated, but it may be noteworthy that deletions involving these chromosome bands have been reported in a handful of BCP ALLs previously, although not in the context of dic(9;20) (Mitelman et al, 2006). The CDKN2A gene at 9p21 was deleted in all cases; this was expected considering that the BPs at 9p were proximal to this locus. However, it was homozygously lost in case 7, which hence harboured a cryptic del(9)(p21p21). Previously, a similarly cryptic homozygous CDKN2A deletion has been reported in one dic(9;20)-positive ALL (Andreasson et al, 2000). This ALL subgroup may, to some extent, be associated with homozygous CDKN2A loss. Obviously, this needs to be investigated in a larger series, in which cryptic deletions of this gene should be actively searched for by FISH or molecular genetic means.
The present investigation clearly demonstrates that the dic(9;20) is unbalanced, leading to loss of 9p13.2-pter in all cases and to deletion of 20q11.2-qter in five of them. The two exceptions were cases 1 and 6, which harboured two normal chromosomes 20 (Table I); gain of the entire 20p was seen in these (Fig 1 and Table II). Thus, the pathogenically important outcome of dic(9;20), without additional copies of chromosome 20, may be the simultaneous loss of tumour suppressors or haploinsufficiency of genes located distal to 9p13.2 and 20q11.2, although the possibility of a fusion gene cannot be excluded. In fact, the observed clustering of breakpoints at 9p13.2 and at 20q11.2 not only means that the cytogenetic description of this abnormality should be dic(9;20)(p13.2;q11.2) but also, and more importantly, that it may rearrange genes located at the BPs. However, it should be stressed that although the BPs clustered, they were non-identical (Table II and Figs 2 and 3), being distributed in a 1·5 Mb segment in 9p13.2 and a 350 kb segment in 20q11.2. The identification of several separate breakpoints within the 9p13 region could indicate that it is particularly prone to chromosomal breakage and recombination, something that would explain why 9p is the most common chromosome arm involved in dicentric chromosomes in ALL (Raimondi et al, 2003; Mitelman et al, 2006).
To date, we know of only two dicentric aberrations that have been shown to result in fusion genes, namely the dic(8;11)(p12;q14) [ODZ4/NRG1] in breast cancer (Liu et al, 1999) and the dic(9;12)(p13;p13) [PAX5/ETV6] in BCP ALL (Strehl et al, 2003). Regarding the dic(9;20), a possible target gene in 9p13 could be the PAX5 gene, previously shown to be involved not only in the dic(9;12) but also in various B-cell lymphomas with t(9;14)(p13;q32) (Poppe et al, 2005) and to be of great importance in B-cell differentiation (Busslinger, 2004). However, the 9p BPs in the dic(9;20) cases mapped hundreds of kb distal to PAX5, making it a less likely candidate, although one cannot exclude a position effect leading to aberrant expression of this gene in ALLs with dic(9;20). With regard to possible target genes in 20q11.2, four characterised genes are known to be located in the 350 kb segment to which the BPs clustered in three of the seven cases, namely DNMT3B, CMMD7, BAK1 and MAPRE1 (Fig 2). However, FISH analysis of case 5 (Fig 4) disclosed a possible rearrangement of another gene – the HCK gene, which maps 600 kb proximal of these genes. Unfortunately, lack of material precluded any further studies of this gene, and it remains to be elucidated whether it is involved in a fusion gene or if loss of function is the functionally important outcome. It should be stressed, however, that none of the other dic(9;20)-positive cases harboured BPs close to HCK, as ascertained by array-CGH. This, together with the fact that the BPs were non-identical, although clustered, in the seven investigated cases, may suggest that loss of 9p and 20q material is the pathogenetically important consequence of the dic(9;20)(p13.2;q11.2).