Perilobar nephrogenic rests are nonobligate molecular genetic precursor lesions of insulin-like growth factor-II-associated Wilms tumors

Abstract

Purpose: Perilobar nephrogenic rests (PLNRs) are abnormally persistent foci of embryonal immature blastema that have been associated with dysregulation at the 11p15 locus by genetic/epigenetic means and are thought to be precursor lesions of Wilms tumor. The precise genomic events are, however, largely unknown.

Experimental design: We used array comparative genomic hybridization to analyze a series of 50 PLNRs and 25 corresponding Wilms tumors characterized for 11p15 genetic/epigenetic alterations and insulin-like growth factor-II expression.

Results: The genomic profiles of PLNRs could be subdivided into three categories: those with no copy number changes (22 of 50, 44%); those with single, whole chromosome alterations (8 of 50, 16%); and those with multiple gains/losses (20 of 50, 40%). The most frequent aberrations included 1p- (7 of 50, 14%) +18 (6 of 50, 12%), +13 (5 of 50, 10%), and +12 (3 of 50, 6%). For the majority (19 of 25, 76%) of cases, the rest harbored a subset of the copy number changes in the associated Wilms tumor. We identified a temporal order of genomic changes, which occur during the insulin-like growth factor-II/PLNR pathway of Wilms tumorigenesis, with large-scale chromosomal alterations such as 1p-, +12, +13, and +18 regarded as "early" events. In some of the cases (24%), the PLNRs harbored large-scale copy number changes not observed in the concurrent Wilms tumor, including +10p, +14q, and +18.

Conclusions: These data suggest that although the evidence for PLNRs as precursors is compelling, not all lesions must necessarily undergo malignant transformation.

Figure 1. Genetic and epigenetic means of IGF2 overexpression in PLNRs

(A) Loss of heterozygosity at 11p15 in a hyperplastic PLNR19 (H&E, ×100), as seen by H19 methylation and allelic imbalance. In addition there is increased IGF2 mRNA (RT-PCR) and protein (IHC, ×400). (B) Loss of imprinting at 11p15 in a sclerosing / regressive / adenomatous PLNR24 (H&E, ×100), as seen by high levels of H19 methylation and the absence of allelic balance. There is also increased IGF2 mRNA (RT-PCR) and protein (IHC, ×400). Hypermethylation (blue boxes) at CpG islands (shaded) at the H19 DMR was assessed by pyrosequencing of the reverse strand. Polymorphic probe D11S4465 showing one or two peaks on an ABI7700 Genetic Analyser. NK = normal kidney from same patient as PLNR. * = statistically significant (p<0.05, t test).

Figure 2. Copy number changes in PLNRs

Genome plots from aCGH with log₂ ratios for each clone (x axis) plotted according to chromosomal location (y axis). The centromere is represented by a horizontal line. Points are coloured green and red to represent gains and losses, respectively. (A) Sclerosing PLNR47 (H&E, ×100), demonstrating no large-scale changes in DNA copy number; (B) Sclerosing PLNR16 (H&E, ×100), showing a single alteration, loss of 1p36; (C) Sclerosing/adenomatous PLNR08 (H&E, ×100), harbouring multiple changes including loss of 1p36, gain of 12 and loss of 19.

Figure 3. Summary of copy number changes by aCGH in PLNRs

The proportion of tumours in which each clone is gained (green bars) or lost (red bars) is plotted (y axis) for each BAC clone according to genomic location (x axis). Vertical dotted lines represent chromosome centromeres.

Figure 4. Comparison of genomic profiles of PLNRs and Wilms tumours

(A) Genome plots for the hyperplastic PLNR19 and associated mixed Wilms tumour WT19 from the same patient, with identical copy number profiles - +6, +7, −18, +22q. (B) Genome plots demonstrating molecular evolution during Wilms tumorigenesis in a single patient, with additional copy number changes (+1q, −16q) in the blastemal/mixed Wilms tumour WT15 compared with the sclerosing PLNR15 (+12, +17, +18). Log₂ ratios for each clone (x axis) plotted according to chromosomal location (y axis). The centromere is represented by a horizontal line; (C) Categorical analysis of copy number changes between PLNRs and Wilms tumours, calculated by Fishers exact tests on the segmented values for each clone. Those with a permutation corrected p value of less than 0.05 are plotted (odds ratio, y axis) according to genomic location (x axis). (D) Paired SAM analysis of copy number changes between matched PLNRs and Wilms tumours. Those with a false discovery rate of less than 0.1 are plotted (SAM score, y axis) according to genomic location (x axis).

Figure 5. Similarity scores for pairs of PLNRs and Wilms tumours

Horizontal lines represent the distribution of similarity scores for each individual PLNR and all profiled Wilms tumours based on the aCGH data (orange circles). The appropriately matched tumour is plotted as a closed circle. Grey circles represent cases where the PLNR harboured no copy number changes detected by aCGH, blue circles highlight rests with genomic alterations.