Sandra Hing

Multiple congenital melanocytic nevi and neurocutaneous melanosis are caused by postzygotic mutations in codon 61 of NRAS

Congenital melanocytic nevi (CMN) can be associated with neurological abnormalities and an increased risk of melanoma. Mutations in NRAS, BRAF, and Tp53 have been described in individual CMN samples; however, their role in the pathogenesis of multiple CMN within the same subject and development of associated features has not been clear. We hypothesized that a single postzygotic mutation in NRAS could be responsible for multiple CMN in the same individual, as well as for melanocytic and nonmelanocytic central nervous system (CNS) lesions. From 15 patients, 55 samples with multiple CMN were sequenced after site-directed mutagenesis and enzymatic digestion of the wild-type allele. Oncogenic missense mutations in codon 61 of NRAS were found in affected neurological and cutaneous tissues of 12 out of 15 patients, but were absent from unaffected tissues and blood, consistent with NRAS mutation mosaicism. In 10 patients, the mutation was consistently c.181C>A, p.Q61K, and in 2 patients c.182A>G, p.Q61R. All 11 non-melanocytic and melanocytic CNS samples from 5 patients were mutation positive, despite NRAS rarely being reported as mutated in CNS tumors. Loss of heterozygosity was associated with the onset of melanoma in two cases, implying a multistep progression to malignancy. These results suggest that single postzygotic NRAS mutations are responsible for multiple CMN and associated neurological lesions in the majority of cases.

Array CGH profiling of favourable histology Wilms tumours reveals novel gains and losses associated with relapse

Despite the excellent survival of Wilms tumour patients treated with multimodality therapy, approximately 15% will suffer from tumour relapse, where response rates are markedly reduced. We have carried out microarray‐based comparative genomic hybridisation on a series of 76 Wilms tumour samples, enriched for cases which recurred, to identify changes in DNA copy number associated with clinical outcome. Using 1Mb‐spaced genome‐wide BAC arrays, the most significantly different genomic changes between favourable histology tumours that did (n = 37), and did not (n = 39), subsequently relapse were gains on 1q, and novel deletions at 12q24 and 18q21. Further relapse‐associated loci included losses at 1q32.1, 2q36.3‐2q37.1, and gain at 13q31. 1q gains correlated strongly with loss of 1p and/or 16q. In 3 of 11 cases with concurrent 1p−/1q+, a breakpoint was identified at 1p13. Multiple low‐level sub‐megabase gains along the length of 1q were identified using chromosome 1 tiling‐path arrays. One such recurrent region at 1q22‐q23.1 included candidate genes RAB25, NES, CRABP2, HDGF and NTRK1, which were screened for mRNA expression using quantitative RT‐PCR. These data provide a high‐resolution catalogue of genomic copy number changes in relapsing favourable histology Wilms tumours. Copyright

Genome complexity in acute lymphoblastic leukemia is revealed by array-based comparative genomic hybridization.

Chromosomal abnormalities are important for the classification and risk stratification of patients with acute lymphoblastic leukemia (ALL). However, approximately 30% of childhood and 50% of adult patients lack abnormalities with clinical relevance. Here, we describe the use of array-based comparative genomic hybridization (aCGH) to identify copy number alterations (CNA) in 58 ALL patients. CNA were identified in 83% of cases, and most frequently involved chromosomes 21 (n=42), 9 (n=21), 6 (n=16), 12 (n=11), 15 (n=11), 8 (n=10) and 17 (n=10). Deletions of 6q (del(6q)) were heterogeneous in size, in agreement with previous data, demonstrating the sensitivity of aCGH to measure CNA. Although 9p deletions showed considerable variability in both the extent and location, all encompassed the CDKN2A locus. Six patients showed del(12p), with a common region encompassing the ETV6 gene. Complex CNA were observed involving chromosomes 6 (n=2), 15 (n=2) and 21 (n=11) with multiple regions of loss and gain along each chromosome. Chromosome 21 CNA shared a common region of gain, with associated subtelomeric deletions. Other recurrent findings included dim(13q), dim(16q) and enh(17q). This is the first report of genome-wide detection of CNA in ALL patients using aCGH, and it has demonstrated a higher level of karyotype complexity than anticipated from conventional cytogenetic analysis.

Prognostic classification of relapsing favorable histology Wilms tumor using cDNA microarray expression profiling and support vector machines

Treatment of Wilms tumor has a high success rate, with some 85% of patients achieving long‐term survival. However, late effects of treatment and management of relapse remain significant clinical problems. If accurate prognostic methods were available, effective risk‐adapted therapies could be tailored to individual patients at diagnosis. Few molecular prognostic markers for Wilms tumor are currently defined, though previous studies have linked allele loss on 1p or 16q, genomic gain of 1q, and overexpression from 1q with an increased risk of relapse. To identify specific patterns of gene expression that are predictive of relapse, we used high‐density (30 k) cDNA microarrays to analyze RNA samples from 27 favorable histology Wilms tumors taken from primary nephrectomies at the time of initial diagnosis. Thirteen of these tumors relapsed within 2 years. Genes differentially expressed between the relapsing and nonrelapsing tumor classes were identified by statistical scoring (t test). These genes encode proteins with diverse molecular functions, including transcription factors, developmental regulators, apoptotic factors, and signaling molecules. Use of a support vector machine classifier, feature selection, and test evaluation using cross‐validation led to identification of a generalizable expression signature, a small subset of genes whose expression potentially can be used to predict tumor outcome in new samples. Similar methods were used to identify genes that are differentially expressed between tumors with and without genomic 1q gain. This set of discriminators was highly enriched in genes on 1q, indicating close agreement between data obtained from expression profiling with data from genomic copy number analyses.

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.

Expression profile of wild-type ETV6 in childhood acute leukaemia

Summary. Comparative expression analysis of wild‐typeETV6 in the disease state showed an absence of expression in ETV6–CBFA2 acute lymphoblastic leukaemia (ALL) when compared with non‐ETV6–CBFA2 ALL and acute myeloid leukaemia. Fluorescent in‐situ hybridization and loss of heterozygosity studies showed that 73% of the ETV6–CBFA2 samples had a fully or partially deleted second ETV6 allele, explaining the lack of wild‐type expression in these patients. Although the second ETV6 allele was identified in the remaining patients, no ETV6 expression was detected. These observations support the hypothesis that loss of ETV6 expression is a critical secondary event for leukaemogenesis in ETV6–CBFA2 ALL.

Clinical features of molecular pathology of solid tumours in childhood

The outlook for children with cancer has improved substantially over the past 20 years, with over three-quarters of children now surviving in the long term. Better use of existing cytotoxic drugs and supportive care have made large contributions, but some of the improvement in survival is due to a greater knowledge of childhood cancer at the cellular and molecular levels. As in leukaemias, several childhood solid tumours carry balanced chromosomal translocations, resulting in fusion genes that encode chimeric proteins with new oncogenic properties. Many of these fusion genes, and other genetic aberrations are tumour specific and are related to outcome. Tumour biology now plays an important part in identifying appropriate treatment through more accurate diagnoses and new risk stratifications based on molecular markers.

Genetic heterogeneity for SMARCB1, H3F3A and BRAF in a malignant childhood brain tumour: genetic-pathological correlation.

Intra-tumour heterogeneity is an important diagnostic, therapeutic and prognostic challenge. Its extent and mechanism in brain tumours is incompletely understood[1]. We describe a malignant tumour with unique pathological and genetic features. Most notably the tumour contained mutations in the SMARCB1 gene (typically associated with Atypical Teratoid/Rhabdoid Tumours[2]), the H3F3A gene (typically associated with high grade glioma in children[3]) and the BRAF gene. Furthermore, there was marked heterogeneity in mutation load between different parts of the tumour. This heterogeneity has implications both for the evolution of the tumour and for its diagnosis.

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