Microdissecting the genetic events in nephrogenic rests and Wilms' tumor development

Abstract

Nephrogenic rests are precursor lesions associated with about 40% of Wilms' tumors. This study identifies genetic steps occurring in the development of Wilms' tumor. Thirty-four Wilms' tumors with nephrogenic rests and/or areas of anaplasia were microdissected from paraffin sections to determine whether and at what stage loss of heterozygosity (LOH) occurred, using polymerase chain reaction-based polymorphic markers at 11p13, 11p15, and 16q. LOH at these loci have been identified in Wilms' tumors and are associated with identified or putative tumor suppressor genes. Three cystic nephromas/cystic partially differentiated nephroblastomas were also examined. LOH was detected in six cases at 11p13 and in six cases at 11p15, and two of these cases had LOH at both loci. All intralobar rests showing LOH also showed LOH in the tumor. A case with a small perilobar rest showed LOH of 11p13 only in the tumor. Five cases showing LOH at 16q were identified (this was identified only in the tumor, and not in the associated rest), and three of these had recurrence of the tumor. Two cases had a WT1 mutation (one germline and the other somatic), as well as LOH in both the intralobar rest and the tumor. A cystic partially differentiated nephroblastoma showed loss at 11p13 and 11p15, as well as at 16q. This study suggests that LOH at 11p13 and 11p15 and WT1 mutations are early events but that LOH at 16q occurs late in the pathogenesis of Wilms' tumor. Intralobar and perilobar nephrogenic rests are known to have different biological behaviors, and this study suggests that they are genetically different. A multistep model of Wilms' tumor pathogenesis is supported by these findings.

Figure 1.

A (case B36): Low power view of renal cortex, with a rind of perilobar nephroblastomatosis involving the upper half of the tissue. There are several hyperplastic nodules, an adenomatous nodule, and a small Wilms’ tumor (8 mm in diameter) with serpiginous blastema and pale stroma on the left. B (case B9): Low power view of intralobar rest in the middle of the field. The rest has a diffuse infiltrative pattern and is sited in the renal medulla. Renal cortex appears in the far right, and Wilms’ tumor in the upper left.

Figure 2.

a (case B14): 11p15 tetranucleotide repeat. Shown is LOH in the intralobar rest (R), as well as in the tumor (T), compared with that in the kidney (K). The heteroduplexes are clearly seen. A1, larger allele; A2, smaller allele; H, heteroduplexes. b (case B14): Acrylamide gel with products of WT1 exon 7 PCR. The tumor (T) and the rest (R) produced only a single product smaller than the product from the normal kidney (K). This demonstrates loss of both normal alleles, and that there is a mutation present in both the rest and the tumor producing the smaller product. N, normal allele; M, mutant allele. c (case B19): Acrylamide gel showing the 8-bp deletion in the normal tissue (K), rest (R), and both the left and right (bilateral) tumors (LT and RT, respectively). There is clearly loss of the normal allele in the rest and the tumors. N, normal allele; M, mutant allele. d (case B38): 16q dinucleotide repeat; agarose gel. Two alleles are clearly seen in the kidney (K), both perilobar rests (R1 and R2), and one tumor (T1). The other tumor (T2) showed LOH. M, 100 bp marker. e (case B32): 16q dinucleotide repeat. Agarose gel showing two alleles (A1 and A2) in the kidney (K) and in the dormant (D) and hyperplastic (H) rests, and only one (A2) allele in one area of the tumor (Ta). M, 100 bp marker. f (case B32): 16q dinucleotide repeat. Repeat of the PCR reaction as shown in e. Other areas of the tumor (Tb, Tc, and Td) show loss of the smaller product, as does the brain metastasis (B).

Figure 3.

Possible model of Wilms’ tumor development via intralobar or perilobar rests. Note that 16q loss and anaplasia are probably late events and independent of each other. p53 abnormalities and anaplasia appear related. It is possible that there may be no rest stage in the development of some tumors.