Tasnim Chagtai

Molecular profiling reveals frequent gain of MYCN and anaplasia-specific loss of 4q and 14q in wilms tumor

Anaplasia in Wilms tumor, a distinctive histology characterized by abnormal mitoses, is associated with poor patient outcome. While anaplastic tumors frequently harbour TP53 mutations, little is otherwise known about their molecular biology. We have used array comparative genomic hybridization (aCGH) and cDNA microarray expression profiling to compare anaplastic and favorable histology Wilms tumors to determine their common and differentiating features. In addition to changes on 17p, consistent with TP53 deletion, recurrent anaplasia‐specific genomic loss and under‐expression were noted in several other regions, most strikingly 4q and 14q. Further aberrations, including gain of 1q and loss of 16q were common to both histologies. Focal gain of MYCN, initially detected by high resolution aCGH profiling in 6/61 anaplastic samples, was confirmed in a significant proportion of both tumor types by a genomic quantitative PCR survey of over 400 tumors. Overall, these results are consistent with a model where anaplasia, rather than forming an entirely distinct molecular entity, arises from the general continuum of Wilms tumor by the acquisition of additional genomic changes at multiple loci.

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.

Gain of 1q As a Prognostic Biomarker in Wilms Tumors (WTs) Treated With Preoperative Chemotherapy in the International Society of Paediatric Oncology (SIOP) WT 2001 Trial: A SIOP Renal Tumours Biology Consortium Study

Purpose Wilms tumor (WT) is the most common pediatric renal tumor. Treatment planning under International Society of Paediatric Oncology (SIOP) protocols is based on staging and histologic assessment of response to preoperative chemotherapy. Despite high overall survival (OS), many relapses occur in patients without specific risk factors, and many successfully treated patients are exposed to treatments with significant risks of late effects. To investigate whether molecular biomarkers could improve risk stratification, we assessed 1q status and other potential copy number biomarkers in a large WT series. Materials and Methods WT nephrectomy samples from 586 SIOP WT 2001 patients were analyzed using a multiplex ligation-dependent probe amplification (MLPA) assay that measured the copy number of 1q and other regions of interest. Results One hundred sixty-seven (28%) of 586 WTs had 1q gain. Five-year event-free survival (EFS) was 75.0% in patients with 1q gain (95% CI, 68.5% to 82.0%) and 88.2% in patients without gain (95% CI, 85.0% to 91.4%). OS was 88.4% with gain (95% CI, 83.5% to 93.6%) and 94.4% without gain (95% CI, 92.1% to 96.7%). In univariable analysis, 1q gain was associated with poorer EFS (P < .001; hazard ratio, 2.33) and OS (P = .01; hazard ratio, 2.16). The association of 1q gain with poorer EFS retained significance in multivariable analysis adjusted for 1p and 16q loss, sex, stage, age, and histologic risk group. Gain of 1q remained associated with poorer EFS in tumor subsets limited to either intermediate-risk localized disease or nonanaplastic localized disease. Other notable aberrations associated with poorer EFS included MYCN gain and TP53 loss. Conclusion Gain of 1q is a potentially valuable prognostic biomarker in WT, in addition to histologic response to preoperative chemotherapy and tumor stage.

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.

Comparative methylome analysis identifies new tumour subtypes and biomarkers for transformation of nephrogenic rests into Wilms tumour

BackgroundWilms tumours (WTs) are characterised by several hallmarks that suggest epimutations such as aberrant DNA methylation are involved in tumour progression: loss of imprinting at 11p15, lack of recurrent mutations and formation of nephrogenic rests (NRs), which are lesions of retained undifferentiated embryonic tissue that can give rise to WTs.MethodsTo identify such epimutations, we performed a comprehensive methylome analysis on 20 matched trios of micro-dissected WTs, NRs and surrounding normal kidneys (NKs) using Illumina Infinium HumanMethylation450 Bead Chips and functionally validated findings using RNA sequencing.ResultsComparison of NRs with NK revealed prominent tissue biomarkers: 629 differentially methylated regions, of which 55% were hypermethylated and enriched for domains that are bivalent in embryonic stem cells and for genes expressed during development (P = 2.49 × 10-5). Comparison of WTs with NRs revealed two WT subgroups; group-2 WTs and NRs were epigenetically indistinguishable whereas group-1 WTs showed an increase in methylation variability, hypomethylation of renal development genes, hypermethylation and relative loss of expression of cell adhesion genes and known and potential new WT tumour suppressor genes (CASP8, H19, MIR195, RB1 and TSPAN32) and was strongly associated with bilateral disease (P = 0.032). Comparison of WTs and NRs to embryonic kidney highlighted the significance of polycomb target methylation in Wilms tumourigenesis.ConclusionsMethylation levels vary during cancer evolution. We have described biomarkers related to WT evolution from its precursor NRs which may be useful to differentiate between these tissues for patients with bilateral disease.

Bilateral Wilms Tumor with TP53-Related Anaplasia

Wilms tumor (WT) with diffuse anaplasia has an unfavorable prognosis and is often (>70%) associated with mutations in the TP53 gene. Although most WTs are unilateral, 5–10% are bilateral, and they are almost always present with nephrogenic rests. The latter are considered a precursor of WT. Two cases of bilateral WTs with nephroblastomatosis, in which anaplastic changes were detected over a period of time, were analyzed using clinical, radiological, histopathological, and molecular-genetic data. TP53 was analyzed by direct sequencing of its full coding sequence and intron-exon boundaries in 11 fragments. DNA was extracted from paraffin-embedded or frozen specimens. High-resolution genomic copy number profiling was carried out by UCL Genomics on the Affymetrix Human Mapping 250K Nsp or Genome-Wide Human SNP Array 6.0 platform. Both cases demonstrated a strong association between the appearance of anaplastic clones and TP53 mutations. Synchronous ganglioneuroma was diagnosed in one case. Our cases are unique as they represent a long disease history and demonstrate the difficulties in managing rare cases of bilateral WT with anaplasia. These cases also emphasize the practical importance of modern molecular-genetic techniques and their clinical application. Moreover, they highlight the issue of the adequate sampling needed in order to gather comprehensive, efficient, and sufficient information about genetic events in a single tumor.

Somatic TP53 Mutations Are Detectable in Circulating Tumor DNA from Children with Anaplastic Wilms Tumors

BACKGROUND: Diffuse anaplastic Wilms tumor (DAWT) is a rare, high-risk subtype that is often missed on diagnostic needle biopsy. Somatic mutations in TP53 are associated with the development of anaplasia and with poorer survival, particularly in advanced-stage disease. Early identification of DAWT harboring TP53 abnormalities could improve risk stratification of initial therapy and monitoring for recurrence. METHODS: Droplet digital polymerase chain reaction (ddPCR) was used to evaluate 21 samples from 4 patients with DAWT. For each patient, we assessed TP53 status in frozen tumor, matched germline DNA, and circulating tumor DNA (ctDNA) from plasma, serum, and urine collected throughout treatment. RESULTS: Mutant TP53 was detectable in ctDNA from plasma and serum in all patients. We did not detect variant TP53 in the same volume (200 μl) of urine. One patient displayed heterogeneity of TP53 in the tumor despite both histological sections displaying anaplasia. Concentration of ctDNA from plasma/serum taken prenephrectomy varied significantly between patients, ranging from 0.44 (0.05-0.90) to 125.25 (109.75-140.25) copies/μl. We observed variation in ctDNA throughout treatment, and in all but one patient, ctDNA levels fell significantly following nephrectomy. CONCLUSION: We demonstrate for the first time that ddPCR is an effective method for detection of mutant TP53 in ctDNA from children with DAWT even when there is intratumoral somatic heterogeneity. This should be further explored in a larger cohort of patients, as early detection of circulating variant TP53 may have significant clinical impact on future risk stratification and surveillance.

Abstract A1-59: Multiple mechanisms of MYCN dysregulation in Wilms tumor

Background and Purpose: Wilms tumor (WT) is the commonest pediatric renal malignancy. A significant minority of cases have mutations in known genes or recurrent copy number aberrations, but few of these have prognostic value. We have previously observed MYCN copy number gain in WT, an aberration associated with poor outcome in several other childhood cancers. We therefore set out to assess the prognostic significance of MYCN gain in a large WT series, and to explore other mechanisms by which the function of this gene may be dysregulated. Methods: MYCN copy number was measured in 293 tumours by Multiplex ligation-dependent probe amplification. Whole exome sequencing (Agilent SureSelect V4+UTR/Illumina HiSeq) was carried out on 51 WT tumour/germline pairs. Sanger sequencing was used to confirm variants of interest and to assess an independent panel of 168 WTs. 65 WTs were profiled on Affymetrix SNP arrays (250K Nsp or SNP 6.0), Illumina HumanMethylation450 arrays, and Illumina HumanHT-12 expression arrays, and additional selected tumors on Illumina HumanCytoSNP-12 arrays. Results: MYCN gain is associated with anaplasia and with poorer relapse-free and overall survival, independent of histology. A somatic p.P44L (c.131C>t) mutation was detected in 3/45 exomes that gave informative data at this position, and in 5/168 additional tumors screened for this variant, an overall frequency of 3.8%. MYCN overexpression in high risk tumors was associated with hypomethylation at specific loci. We observed parallel evolution of genomic copy number gain and point mutation of MYCN in the contralateral tumors of a remarkable bilateral case in which independent contralateral mutations of TP53 also evolved over time. In a second bilateral case, we detected MYCN gain as a germline aberration. Conclusions: MYCN gain is associated with poor outcome and anaplastic histology in WT. MYCN also appears to be disrupted by other mechanisms in WT, including DNA hypomethylation and a recurrent potential gain of function mutation, P44L. The discovery of a germline aberration suggests that MYCN dysregulation may contribute to tumor predisposition. Citation Format: Richard D. Williams, Tasnim Chagtai, Marisa Alcaide-German, John Apps, Jenny Wegert, Sergey Popov, Gordan Vujanic, Harm van Tinteren, Marry M. van den Heuvel-Eibrink, Marcel Kool, David Gisselsson, Norbert Graf, Manfred Gessler, Kathy Pritchard-Jones. Multiple mechanisms of MYCN dysregulation 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 A1-59.

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 3829: TP53 mutation status defines two distinct classes of diffuse anaplastic Wilms tumor.

Wilms tumor (WT) is a paediatric renal tumor with a relapse rate of 10 to 15%. Tumor stage and the histological presence of diffuse anaplasia (DA) are the most important adverse prognostic factors in WT. Previous studies have described TP53 mutations at a frequency of up to 75% in DA WT cases, however only small cohorts were analysed. The aim of this study was to evaluate the frequency of TP53 mutations in a large group of DA WT and correlate these data with tumor and patient characteristics. DA WTs from 61 patients were analysed for TP53 mutations using Sanger sequencing. A paediatric pathologist confirmed the presence of anaplastic cells in the specimen used for DNA extraction. Sequence alterations were classified as damaging or neutral mutations with reference to databases of known mutations and functional prediction algorithms (SIFT, PolyPhen2). Loss of 17p, where TP53 is located, was evaluated using CGH or SNP arrays and gene expression profiling was carried out using cDNA microarrays. Kaplan-Meier Survival analysis and Breslow9s test were used to compare TP53 alterations with clinico-pathological characteristics. Of the 61 cases, 29 (47.5%) had at least one damaging TP53 mutation and 26 of these also had TP53 loss. Most of the cases (20 out of 29, 68.9%) presented TP53 mutations located within exons 5 to 8. These exons comprise the DNA-binding domain where the hotspots are located. The other 32 DA WT cases did not harbour mutations but six had 17p loss. Patients with DA WT that carry TP53 mutations and/or 17p loss had a worse response to treatment than those patients that had wild type TP53 (p The CGH profiles showed an increase in the number of gains and losses seen in DA WT with TP53 mutations, compared to those without, suggesting a more unstable genome. Gene expression profiling revealed 71 differentially expressed genes between DA WT with and without mutations. These genes may be associated with the anaplastic phenotype, but their role in the pathogenesis of WT remains to be confirmed. Evaluating a large series of anaplastic WT, we found that the frequency of TP53 mutations in DA WT was lower than previously reported. However, samples with apparent wild type TP53 will be further evaluated by deep-sequencing to determine if mutations are present in a minority cell population. The relatively high frequency of wild type TP53 in DA WT suggests that TP53 mutations are neither necessary nor sufficient to generate anaplasia. Grants: Cancer Research UK; EU FP7 P-medicine, Sophie Barringer Trust and GOSHCC. Citation Format: Mariana Maschietto, Tasnim Chagtai, Sergey D. Popov, Neil Sebire, Gordan Vujanic, Sandra Hing, Paul Grundy, Jeffrey S. Dome, Kathy Pritchard-Jones, Richard D. Williams. TP53 mutation status defines two distinct classes of diffuse anaplastic Wilms tumor. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3829. doi:10.1158/1538-7445.AM2013-3829