Significance of TP53 Mutation in Wilms Tumors with Diffuse Anaplasia: A Report from the Children's Oncology Group

Abstract

Purpose: To investigate the role and significance of TP53 mutation in diffusely anaplastic Wilms tumors (DAWTs).

Experimental design: All DAWTs registered on National Wilms Tumor Study-5 (n = 118) with available samples were analyzed for TP53 mutations and copy loss. Integrative genomic analysis was performed on 39 selected DAWTs.

Results: Following analysis of a single random sample, 57 DAWTs (48%) demonstrated TP53 mutations, 13 (11%) copy loss without mutation, and 48 (41%) lacked both [defined as TP53-wild-type (wt)]. Patients with stage III/IV TP53-wt DAWTs (but not those with stage I/II disease) had significantly lower relapse and death rates than those with TP53 abnormalities. In-depth analysis of a subset of 39 DAWTs showed seven (18%) to be TP53-wt: These demonstrated gene expression evidence of an active p53 pathway. Retrospective pathology review of TP53-wt DAWT revealed no or very low volume of anaplasia in six of seven tumors. When samples from TP53-wt tumors known to contain anaplasia histologically were available, abnormal p53 protein accumulation was observed by immunohistochemistry.

Conclusions: These data support the key role of TP53 loss in the development of anaplasia in WT, and support its significant clinical impact in patients with residual anaplastic tumor following surgery. These data also suggest that most DAWTs will show evidence of TP53 mutation when samples selected for the presence of anaplasia are analyzed. This suggests that modifications of the current criteria to also consider volume of anaplasia and documentation of TP53 aberrations may better reflect the risk of relapse and death and enable optimization of therapeutic stratification. Clin Cancer Res; 22(22); 5582-91. ©2016 AACR.

Figure 1. TP53 Mutations in DAWTs

Illustrated is the location of the TP53 mutations (n=61) within the known p53 protein domains. The number of variants detected for each is provided in the parenthesis. TAD = Transcription-Activation Domain; DNABD = DNA binding domain; TM = Tetramerization Domain.

Figure 2. Association between stage, TP53 status and Disease Free Survival

Kaplan-Meier curve of Disease Free Survival (DFS) (in years), comparing DAWTs with TP53 mutation and/or copy loss and DAWTs without either. Numbers at risk are listed at the bottom of the graphs. All patients who relapsed or progressed also died.

Figure 3. Comparison of Copy number variations and gene expression in DAWTs with and without TP53 abnormalities

A. Copy number variations (CNVs; defined as segments with ≥ 8 markers and log2 ratio ≥ +0.5 or ≤ −0.5) identified in DAWTs with TP53 abnormalities (top panel) or without TP53 abnormalities (bottom panel). Each row represents one DAWT. Segments in red represent CN loss and those in blue CN gain. The seven TP53-wt samples showed fewer copy number alterations in their genome when compared to the DAWTs carrying TP53 mutations and/or copy loss, and are similar to those previously reported in FHWTs. The tumors with TP53 abnormalities had recurrent loss of chromosomes 4q, 14q, 16q, 17p, and 22. B. Hierarchical analysis of genes differentially expressed in TP53 mutant DAWT: Statistical Analysis for Microarray (SAM) comparing the seven TP53-wt tumors with the remaining 31 tumors identified 35 genes differentially expressed (SAM q<0.10 and Student’s t-test BH corrected p value < 0.05). Hierarchical analysis of these 35 genes shows all to be upregulated in the tumors lacking TP53 mutation or 17p13 copy number loss (red is upregulation and blue indicates downregulation). These include several genes located on 17p, and other genes associated within apoptotic pathways known to be regulated by p53 (such as BAX and CDKN1A).

Figure 4. Abnormal protein accumulation by p53 immunohistochemistry

Archival tissue blocks containing histologic evidence of anaplasia were available for three of the seven DAWTs determined to be TP53-wt by the absence of TP53 mutation and copy number loss in a single randomly selected frozen tumor sample (the remaining four cases did not have archival tissue within the biopathology center containing clear histologic evidence of anaplasia). (Original magnification 40X for all.) A) PAJMLZ: The block containing anaplasia shows diffuse abnormal p53 protein accumulation. B) PALEZT: The block contains anaplastic cells that were stromal that surrounded non-anaplastic blastemal cells. Immunohistochemistry shows nodules of blastemal cells that were largely negative for p53 protein, with anaplastic stromal cells containing marked accumulation of p53 protein. C) PAJNRH: The block had a microscopic focus of anaplasia, which shows intense abnormal nuclear p53 staining.

Figure 5. Tumor heterogeneity and sample selection in detecting TP53 abnormalities

The predominant underlying cause of failure to detect TP53 abnormalities is heterogeneity in tumors with a low volume of anaplasia. The diagnosis of DAWT is established by a pathologist after analyzing multiple representative slides taken from all regions of the tumor. In contrast, single randomly selected tissue samples are taken for banking or biologic studies without knowledge of the histology. In some cases anaplasia may be diffuse, but may still be due to a very small percentage of cells, making it difficult to detect TP53 abnormalities. If there are scattered nodules of anaplasia, area(s) of anaplasia may not be selected; or there might be only a very small focus of anaplasia within the sample selected, insufficient for the detection of TP53 abnormalities.