Here we present the first genome-wide profiling of copy number changes in PLNRs and associated Wilms tumours. The proposed precursor status of both PLNRs and ILNRs has been largely defined by epidemiological (21
) morphological (3
), but only recently by molecular (6
) evidence. Here we demonstrate clear clonal progression on the basis of copy number profiles of PLNRs and Wilms tumours.
The route by which Wilms tumours arise from ILNRs is believed to be accompanied by mutations and deletions of WT1
at 11p13 at an early developmental stage (5
), followed by mutation and nuclear localisation of β-catenin during the progression to Wilms tumour (25
), with Wnt pathway activation and downstream transcription factor activation. This sequence of events is strongly associated with a stromal histology in the subsequent Wilms tumour (11
By contrast, PLNRs and the Wilms tumours accompanying or arising from them have been associated with dysregulation at the 11p15 locus by genetic and epigenetic means, leading to an overexpression of IGF2 (12
). The molecular events accompanying this IGF2-driven pathway have not been elucidated, although it has previously been suggested that Wilms tumours with wild-type WT1
(presumably largely developed via the PLNR pathway) harboured more genetic alterations than those with WT1
mutations (ILNR pathway) (26
We have determined the temporal order of changes in DNA copy number which occur during the IGF2/PLNR pathway of Wilms tumorigenesis. Large scale chromosomal alterations such as gain of 12, 13 and 18, and loss of 1p, were frequently observed in PLNRs and can be regarded as ‘early’ events. This is in contrast to other common Wilms-associated alterations - gain of 1q, 6 and loss of 10p and 16q, which were not observed in our PLNRs, and are therefore ‘later’ events in the proposed multistep model of Wilms tumour development.
It is of interest to note the disparity in timing between the copy number changes most convincingly associated with treatment failure and tumour relapse. Although loss of 1p was frequently observed in PLNRs, gain of 1q and loss of 16q were restricted to Wilms tumours. These three events have shown concordance in previous Wilms tumour profiling experiments, suggesting a common mechanism (14
). The data provided here suggest a sequence in which the copy number loss of 1p occurs prior to a coordinated +1q/−16q, at least via the IGF2/PLNR pathway. The implication of this is that we would therefore expect the formation of an isochromosome 1q to also be associated with the WT1/ILNR pathway, although this remains to be determined.
Genetic loss at 16q has been previously associated with LOI at 11p15 (20
), with the gene encoding CTCF, a regulator of imprinting at the H19/IGF2
locus, found on 16q22. Our data add weight to the evidence (6
) that despite this, 16q abnormalities are later events, and are preceded by genetic and epigenetic up-regulation of IGF2
. In the single PLNR in which we observed not a copy number change, but allelic loss, of 16q, the associated Wilms tumour was of anaplastic histology, a subtype strongly associated with mutations in TP53
). We were not able to determine the timing of the event in this case. Other correlates of IGF2
LOI in Wilms tumours have been reported as 11q loss and trisomy 12 (20
). While we observed copy number gain of chromosome 12, and both LOH and loss at 11q in our rests, suggesting these are early events with the IGF2/PLNR pathway, neither correlated directly with LOI.
In around half of the PLNRs we profiled, no large scale changes in DNA copy number were observed. This is around twice the proportion of Wilms tumours that gave similarly copy neutral aCGH profiles (14
) or normal karyotypes (30
). Almost all of these cases, however, exhibited high levels of IGF2 expression, either due to LOI or LOH (and assumed UPD). Although this suggests that in some cases the genetic/epigenetic targeting of IGF2
is sufficient in its own right to drive the tumorigenic process through to blastemal cell persistence, which additional abnormalities may be required for tumour progression are yet to be identified. Alterations at the genetic, epigenetic, transcript, small RNA and/or post-translational modification levels may all play a role. In any case, the observation that those rests which were actively proliferating harboured more genomic alterations, and more closely resembled their associated Wilms tumours, suggests that in a proportion of cases at least, there are additional alterations driving clonal expansion and selection for the malignant phenotype (31
In a small number of cases, the genomic profiles of PLNRs did not match with either the associated Wilms tumour, or with a topographically distinct rest. Given the often multifocal nature of PLNRs, this is perhaps not surprising, although it has not been demonstrated before. This evidence seems to suggest that PLNRs are non-obligate precursors, in that not all lesions necessarily develop into Wilms tumours, a fact clear from the seminal early studies by Beckwith, who reported that approximately 1 in 100 kidneys at birth harbour nephrogenic rests, while the incidence of Wilms tumour is only 1 in 10,000 (3
). We provide a molecular underpinning of this observation in patients who did develop Wilms tumour, demonstrating that certain lesions, often containing +10p and/or +14, may be genomic dead-ends, as their associated tumour, and Wilms tumours in general, did not harbour these alterations. It is possible that genomic gains at these loci may even actively promote regression or involution of the rest, an intriguing thought given that they are otherwise commonly lost in Wilms tumours themselves. By contrast, the frequent occurrence of +18 in these and other PLNRs and Wilms tumours suggest it to be in some instances a non-critical mutation in sporadic IGF2/PLNR Wilms tumorigenesis.
Although Wilms tumours have been classified as “PLNR-like” and “ILNR-like” on the basis of their associated precursor lesion, and appear to evolve by different genetic and epigenetic pathways, the two routes may incorporate significant cross-talk. The novel X-chromosome tumour suppressor gene, WTX
, acts as a negative regulator of β-catenin in the cytoplasm, and WTX
mutations may also drive nuclear localisation of β-catenin and subsequent up-regulation of its transcriptional targets (32
). Unfortunately we have not been able to address whether WTX
deletion occurs early in development, and is thus present in the PLNRs, as our array platform does not contain a suitable BAC clone at the Xq11 locus. Additionally, WT1 itself may act as a transcriptional repressor of both IGF2
) and its receptor, IGF1R
), which may in turn mediate the nuclear translocation of β-catenin (35
). The IGF2/IGF1R axis has recently been identified as playing a direct and central role in the self-renewal of embryonic stem cells (36
). Immature blastemal cells, stem cells of the developing kidney, are more likely to predominate in the IGF2/PLNR Wilms tumours (12
) and IGF1R may also be up-regulated in Wilms tumour blastema by genomic means (13
). Deconvoluting the critical initiating steps and functional endpoints of these two developmental pathways is a key challenge for Wilms tumour biology.