Mariana Maschietto

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

Purpose The presence of diffuse anaplasia in Wilms tumours (DAWT) is associated with TP53 mutations and poor outcome. As patients receive intensified treatment, we sought to identify whether TP53 mutational status confers additional prognostic information. Patients and Methods We studied 40 patients with DAWT with anaplasia in the tissue from which DNA was extracted and analysed for TP53 mutations and 17p loss. The majority of cases were profiled by copy number (n = 32) and gene expression (n = 36) arrays. TP53 mutational status was correlated with patient event-free and overall survival, genomic copy number instability and gene expression profiling. Results From the 40 cases, 22 (55%) had TP53 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 TP53 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 TP53 abnormalities. DAWT carrying TP53 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. Conclusion This study provides evidence that TP53 mutational analysis improves risk stratification in DAWT. This requires validation in an independent cohort before clinical use as a biomarker.

Intra-Tumor Genetic Heterogeneity in Wilms Tumor: Clonal Evolution and Clinical Implications

The evolution of pediatric solid tumors is poorly understood. There is conflicting evidence of intra-tumor genetic homogeneity vs. heterogeneity (ITGH) in a small number of studies in pediatric solid tumors. A number of copy number aberrations (CNA) are proposed as prognostic biomarkers to stratify patients, for example 1q + in Wilms tumor (WT); current clinical trials use only one sample per tumor to profile this genetic biomarker. We multisampled 20 WT cases and assessed genome-wide allele-specific CNA and loss of heterozygosity, and inferred tumor evolution, using Illumina CytoSNP12v2.1 arrays, a custom analysis pipeline, and the MEDICC algorithm. We found remarkable diversity of ITGH and evolutionary trajectories in WT. 1q + is heterogeneous in the majority of tumors with this change, with variable evolutionary timing. We estimate that at least three samples per tumor are needed to detect > 95% of cases with 1q +. In contrast, somatic 11p15 LOH is uniformly an early event in WT development. We find evidence of two separate tumor origins in unilateral disease with divergent histology, and in bilateral WT. We also show subclonal changes related to differential response to chemotherapy. Rational trial design to include biomarkers in risk stratification requires tumor multisampling and reliable delineation of ITGH and tumor evolution.

Is Wilms Tumor a Candidate Neoplasia for Treatment with WNT/β-Catenin Pathway Modulators?—A Report from the Renal Tumors Biology-Driven Drug Development Workshop

The European Network for Cancer Research in Children and Adolescents consortium organized a workshop in Rome, in June 2012, on “Biology-Driven Drug Development Renal Tumors Workshop” to discuss the current knowledge in pediatric renal cancers and to recommend directions for further research. Wilms tumor is the most common renal tumor of childhood and represents a success of pediatric oncology, with cure rates of more than 85% of cases. However, a substantial minority (∼25%) responds poorly to current therapies and requires “high-risk” treatment or relapse. Moreover, the successfully treated majority are vulnerable to the late effects of treatment, with nearly one quarter reporting severe chronic health conditions by 25 years of follow-up. Main purposes of this meeting were to advance our understanding on the molecular drivers in Wilms tumor, their heterogeneity and interdependencies; to provide updates on the clinical–pathologic associations with biomarkers; to identify eligible populations for targeted drugs; and to model opportunities to use preclinical model systems and prioritize targeted agents for early phase clinical trials. At least three different pathways are involved in Wilms tumor; this review represents the outcome of the workshop discussion on the WNT/β-catenin pathway in Wilms tumorigenesis. Mol Cancer Ther; 12(12); 2619–27. ©2013 AACR.

Biological Prognostic Factors in Wilms Tumors

The outcome is now good for most patients with Wilms tumor of the kidney. Over and above the gains in survival, the ability to progressively regulate the amount of chemotherapy and radiation so that groups of patients are receiving dosing adequate to achieve cure, but not more, has been made possible by the use of prognostic factors. We often now think of prognostic factors as molecular or biological findings, but factors used to predict outcome in patients with Wilms tumor – to thereby stratify therapy – include histology (favorable vs anaplastic), stage (using criteria such as lymph node involvement, local or intravascular tumor extension, and presence of metastatic disease), age at diagnosis, response to therapy, and now molecular or genetic changes (loss of heterozygosity (LOH) for chromosomes 1p and 16q).

Mutational spectrum of WTX, WT1, CTNNB1, APC and PLCG2 genes in Wilms tumor defined by massive parallel resequencing

Background The identification of molecular alterations that trigger Wilms tumor (WT) development is crucial to understanding the tumorigenesis of this malignancy. Currently, it is estimated that WTX and WT1 genomic losses together with CTNNB1 point mutations occur in about 30% of WTs. However, the majority of cases remain without any identified driver mutation. Results from a previous study by our group pointed to APC and PLCG2 as candidate genes altered in WT [1]. Given the advent of modern DNA sequencing technologies, it is now feasible to evaluate large genomic regions spanning complete genes (exons and introns), allowing the description of the mutation patterns occurring in tumor cells. Thus, the aim of this study was to identify point mutations and indels in the complete sequence of APC, CTNNB1, WT1, WTX and PLCG2 genes in order to characterize both the exonic mutational spectrum and the intronic nucleotide substitution pattern. Material and methods The complete genomic regions of the selected genes, spanning a total of 430 kb, were amplified by long-range PCR in 15 WTs and 3 non-neoplasic control samples, giving a total of 60 amplicons per sample (10 kb on average). The resulting amplicons were mixed at equimolar concentrations and, for each sample, the Ion PGM library preparation protocol was performed. The libraries of the 18 barcoded samples were combined in four sequencing pools that were individually submitted to an Ion PGM™ Sequencer run on an Ion 316™ Chip. Point mutation and indels not present in the non-neoplasic controls were selected for capillary sequencing validation. The validated

Abstract A1-67: Prognostic significance of copy number aberrations in Wilms tumor

Background and Purpose: Treatment of Wilms tumor (WT) patients under International Society of Paediatric Oncology (SIOP) protocols is currently stratified by staging and histopathology at nephrectomy after neoadjuvant chemotherapy. However, most relapses occur in cases without specific histopathological risk factors, and there is a clinical need for better prognostic biomarkers. Combined loss of heterozygosity (LOH) of 1p and 16q has recently been introduced in the US as an adverse prognostic indicator, while previous work in our and other laboratories suggests that 1q gain may have a similar association with poor outcome. To examine the clinical significance of 1q gain and assess its potential as a WT biomarker, we developed a simple, effective assay that measures its genomic copy number together with that of several other loci of interest, and applied it to a large tumor series. Methods: 686 frozen tumor samples from the SIOP WT 2001 trial (from a total of 7 countries) were assayed using a rapid multiplex ligation-dependent probe amplification (MLPA) assay that was developed and optimized in association with MRC-Holland b.v. to assess the copy number status of 1p, 1q, 16q, WT1, WTX, TP53, MYCN and FBXW7. Analyses were conducted in 3 laboratories, with exchange of a blinded quality assurance sample set. Results: 1q gain was present in 28% (190/686) of the cases. The 5- year Event Free Survival (EFS) rate was 72.6% (95% Confidence Interval (CI), 66.3%-85%) for those with 1q gain and 86.4% (95% CI, 83.4%-89.6%) for those who lacked 1q gain (p= Conclusion: Gain of 1q is a potential adverse biomarker for WT. Its association with high risk histological features after pre-operative chemotherapy and independent impact on survival require assessment in a larger number of patients before consideration for clinical use. Citation Format: Tasnim Chagtai, Christina Zill, Linda Dainese, Jenny Wegert, Mariana Maschietto, Gordan Vujanic, Neil Sebire, Ivo Leuschner, Peter Ambros, Maureen O9Sullivan, Christophe Bergeron, David Gisselsson, Marcel Kool, Marry van den Heuvel-Eibrink, Norbert Graf, Harm van Tinteren, Aurore Coulomb, Manfred Gessler, Richard Williams, Kathy Pritchard-Jones. Prognostic significance of copy number aberrations in Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Translation of the Cancer Genome; Feb 7-9, 2015; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(22 Suppl 1):Abstract nr A1-67.

Abstract 1837: Clinical diversity and tumor spectrum in Xeroderma Pigmentosum Brazilian patients

Xeroderma Pigmentosum (XP) is a rare autossomic recessive hereditary disorder in which normal ability to repair DNA damage caused by ultraviolet light (UV) is deficient due to the presence of mutations in nucleotide excision repair pathway genes (XPA, ERCC3, XPC, ERCC2, DDB, ERCC4 and ERCC5) or in POLH gene, responsible for the replication of damaged DNA on the leading strand. These different genetic alteration result in distict phenotypes classifified into eight genetic complementation sub groups, XP-A to XP-G and a variant group XP-V. Clinical characteristics of the syndrome include skin poikiloderma and eye hypersensibility to UV radiation. Carriers have an increased risk of early onset multiple cutaneous, mucocutaneous and ophthalmologic malignancies when compared with normal population. Occasionally neurological impairment is observed in certain variations of the syndrome. Given the paucity of specific literature data about the incidence and clinical profile of the disease in Brazilian population, this study was conducted to describe clinical diversity and tumor spectrum of XP syndrome in Brazilian population. Twenty-nine XP patients from 19 non-related families from Hospital A.C. Camargo (Sao Paulo, Brazil), Hospital Universitario Alcides Carneiro (Campina Grande, Paraiba) and Hospital de Clinicas de Porto Alegre (Rio Grande do Sul, Brazil). Data collection was performed after genetic counseling and signed informed consent. Clinical characteristics were obtained through medical ascertainment. The median age was 24 (4 to 61 years) in a predominant female population (19/29). Basal cell carcinomas (BCC) were the most frequent neoplasm, present in 86,20% (25/29) of all patients, with the occurrence of up to 60 BCCs in a same patient. Squamous cell carcinomas and melanoma were present in 66,7% and 37,5% respectively. Six patients developed the first malignant tumor before the age of six and four patients developed the first tumor in early adulthood. One male patient developed a diffuse gastric cancer at 50 years old. Visual impairment occurred in 45,83% of the patient. Despite the unknown frequency of affected patients, the Brazilian population of XP cases seems to be underestimated. Genetic counseling is fundamental to provide information to carriers and family members and enable preventive measurements. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1837. doi:10.1158/1538-7445.AM2011-1837