Consort diagram showing the 40 cases of diffuse anaplastic Wilms tumour used for the survival analysis by <i>TP53</i> mutation and/or 17p loss.

<p>Consort diagram showing the 40 cases of diffuse anaplastic Wilms tumour used for the survival analysis by <i>TP53</i> mutation and/or 17p loss.</p

Variants with unknown function in cases with 17p loss.

<p>MAF: minor allele frequency (source: dbSNP).</p><p>Variants with unknown function in cases with 17p loss.</p

Cases of diffuse anaplastic WT with <i>TP53</i> mutations.

<p>SNV: Single Nucleotide Variation.</p>†<p>: Case that had two mutations together with <i>TP53</i> loss.</p><p>*Cases identified by deep-sequencing.</p>¥<p>Mutations found in Li Fraumeni patients (IARC TP53 Database, R17).</p><p>Cases of diffuse anaplastic WT with <i>TP53</i> mutations.</p

<i>TP53</i> Mutational Status Is a Potential Marker for Risk Stratification in Wilms Tumour with Diffuse Anaplasia

<div><p>Purpose</p><p>The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with <i>TP53</i> mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether <i>TP53</i> mutational status confers additional prognostic information.</p><p>Patients and Methods</p><p>We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for <i>TP53</i> mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. <i>TP53</i> mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling.</p><p>Results</p><p>From the 40 cases, 22 (55%) had <i>TP53</i> mutations (2 detected only after deep-sequencing), 20 of which also had 17p loss (91%); 18 (45%) cases had no detectable mutation but three had 17p loss. Tumours with <i>TP53</i> mutations and/or 17p loss (n = 25) had an increased risk of recurrence as a first event (p = 0.03, hazard ratio (HR), 3.89; 95% confidence interval (CI), 1.26–16.0) and death (p = 0.04, HR, 4.95; 95% CI, 1.36–31.7) compared to tumours lacking <i>TP53</i> abnormalities. DAWT carrying <i>TP53</i> mutations showed increased copy number alterations compared to those with wild-type, suggesting a more unstable genome (p = 0.03). These tumours showed deregulation of genes associated with cell cycle and DNA repair biological processes.</p><p>Conclusion</p><p>This study provides evidence that <i>TP53</i> mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.</p></div

Patients with diffuse anaplastic WT.

<p>(A) Event-free survival (p = 0.02) and (B) overall survival curves (p = 0.02) for patients with wt<i>TP53</i> (dashed line) versus mut<i>TP53</i> (solid line). Numbers at risk are plot every year. HR: hazard ratio, CI: confidence interval.</p

Figure 1

<p><b>A.</b> Mutant read frequency in different samples. The patient's blood samples were all taken around the time of neuroblastoma diagnosis. Numbers of sequencing reads per sample are displayed in the top level. * p = 0.01, comparing mutant read count frequencies between samples PD9058b3 and PD9058b with Fisher's exact test. Differences between PD9058b2 and sample b/b3 are not significant (p>0.05). <b>B.</b> Capillary sequencing of control germline DNA (Reference sequence) and patient's blood samples extracted at three time points, showing very low level of adenine (black arrow). The guanine (black peak) dropped approximately between 8–17% relative to control, as given by the software, which would not usually be classed as significant when looking for heterozygous germline mutations. <b>C and D.</b> Immunohistochemistry to show strong nuclear staining p53 in neuroblastoma (C) and sarcoma NOS (D).</p

Immunophenotyping of paediatric glioma cell lines.

<p>All lines were grown as monolayers and stained with a variety of glial and stem cell markers including glial fibrillary acidic protein (GFAP), S100, vimentin, synaptophysin, nestin and CD133. H&E – haematoxylin and eosin. All images original magnification ×400.</p

Methylation-specific multiplex ligation-dependent probe amplification of 39 genes in paediatric glioma cell lines.

<p>Values give percentage methylation at CpG islands in the genes promoter. DEL = gene deletion. n.d = not done.</p

Characteristics and immunophenotype of paediatric glioma cells grown as monolayers.

<p>The astrocytic nature of the cells was confirmed in culture by immunohistochemistry with a range of glial and stem cell markers. (+++) strongly positive; (++) moderately positive; (+) weakly positive; (−) negative.</p

Expression profiling of paediatric and adult glioblastoma cell lines.

<p>(A) Heatmap demonstrating hierarchical clustering of 93 differentially expressed genes between paediatric (SF188, KNS42, UW479) and adult (LN229, A172, U118MG, U87MG, SF268) high grade glioma cell lines. (B) Quantitative real-time (TaqMan) RT-PCR confirming differential expression of <i>CRKL</i>, <i>LYN</i>, <i>EPHA6</i> and <i>AXL</i>. Expression values are plotted relative to Universal Human Reference RNA. (C) Gene Set Enrichment Analysis highlighting co-ordinated differential expression of gene sets defined <i>a priori</i>. Enriched in paediatric high grade glioma cell lines - MORF_MSH2, GNF2_MLH1, GCM_RAD21, DNA_replication_reactome; enriched in adult lines IL6_SCAR_FIBRO_UP, CROONQUIST_IL6_RAS_UP, TGFBETA_C1_UP. Nominal p value<0.001.</p