Somatic TP 53 Mutations in Anaplastic Wilms Tumor

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 andmonitoring 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, matchedgermlineDNA,andcirculating tumorDNA (ctDNA) fromplasma, serum,andurinecollected throughout treatment. RESULTS:Mutant TP53wasdetectable inctDNAfromplasmaandserum inall patients.Wedidnotdetect 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 onepatient, ctDNA levels fell significantly followingnephrectomy.CONCLUSION:Wedemonstrate 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 TP53may have significant clinical impact on future risk stratification and surveillance. Translational Oncology (2018) 11, 1301–1306 www.transonc.com Trans la t iona l Onco logy Volume 11 Number 6 December 2018 pp. 1301–1306 1301 Address all correspondence to: Taryn D. Treger, UCL Great Ormond Street Institute of Child Health, London, United Kingdom or William Mifsud, Department of Pathology, Sidra Medicine, Doha, Qatar. E-mail:; t.treger@ucl.ac.uk Funding: T. T. and K. P. J. are supported in part by the National Institute of Health Research (NIHR) Great Ormond Street Hospital Biomedical Research Centre, and T. T. receives additional funding from the Francis Crick Institute. G. D. C. was supported by a Francis Crick Institute PhD studentship. The Francis Crick Institute receives its core funding from Cancer Research UK (FC010110), the UK Medical Research Council (FC010110), and the Wellcome Trust (FC010110). N. M. L. is a Winton Group Leader in recognition of the Winton Charitable Foundation's support towards the establishment of the Francis Crick Institute. N.M. L. is additionally funded by a Wellcome Trust Joint Investigator Award (103760/Z/14/Z) and the MRC eMedLab Medical Bioinformatics Infrastructure Award (MR/L016311/1). R. B. was funded by the Wellcome Trust (098521/B/12/Z). Ch. R. was funded by the Wellcome Trust Institutional Strategic Support Fund (105609/Z/14/Z). J. B is funded by Great Ormond Street Hospital Children's Charity (W1090). W. M. was funded by an NIHR Academic Clinical Lectureship. R.D.W. is funded byChildrenwithCancerUK (511184) andCancer Research UK (510445). This work was also supported by Great Ormond Street Hospital Children's Charity (W1090) and Cancer Research UK biomarker grant (C1188/A17297). The IMPORT study was supported in part by EU-FP7 project ENCCA (261474). Declarations of Interest: None. Present address: Department of Pediatrics, University of Cambridge, Cambridge, UK. Present Address: The Institute of Cancer Research, London, UK. Present Address: Department of Pathology, Sidra Medicine, Doha, Qatar. Received 18 April 2018; Revised 12 August 2018; Accepted 14 August 2018 © 2018 The Authors. Published by Elsevier Inc. on behalf of Neoplasia Press, Inc. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/). 1936-5233 https://doi.org/10.1016/j.tranon.2018.08.006 Introduction Wilms tumor (WT) or nephroblastoma is the most common childhood renal cancer, with 1 in 100,000 children diagnosed annually [1]. In Europe, children are treated with neoadjuvant chemotherapy prior to surgery as per The International Society of Pediatric Oncology Renal Tumors Study Group (SIOP RTSG) guidelines [2]. Conversely, in North America, children undergo immediate surgery prior to chemotherapy in accordance with the Children's Oncology Group (COG). For both groups, tumor histology and stage dictate the intensity of postoperative treatment, with chemotherapy and sometimes radiotherapy. Regardless of the protocol used, overall survival approaches 90% [3]. Despite this excellent prognosis, approximately 15% of patients relapse, and for the subgroup displaying high-risk histology, more than one in four patients need intensified treatment for disease recurrence [4]. Diffuse anaplasia (DAWT) is classified as high risk by both SIOP RTSG and COG. Needle biopsy rarely captures anaplasia, but it is found in 5%-10% of cases following surgical resection [5]. Approximately 60% of DAWTs have somatic mutations in the tumor suppressor gene TP53 and/or 17p loss. Although these mutations likely confer an increased risk of both relapse and mortality, particularly in advanced tumor stage, genetic testing of surgically resected tumors is not routinely performed [6,7]. At diagnosis, there are no radiological findings to clearly differentiate a WT from other renal tumors or to differentiate the subtype of WT. In most countries that follow the SIOP approach, preoperative chemotherapy is commenced without a confirmatory biopsy. Furthermore, biopsy provides limited diagnostic information in part due to the high degree of genetic intratumoral heterogeneity (ITH) found in WT [8,9]. In order to circumvent the issues of ITH and the limitations and risks related to the biopsy procedure, minimally invasive molecular biomarkers are urgently needed. Circulating tumor DNA (ctDNA), shed by tumor cells and detectable in a range of bodily fluids, is a promising candidate as it represents contributions from multiple tumor subclones [10]. However, efforts to characterize ctDNA in pediatric tumors have lagged behind the work achieved in adult cancers.One principal limitation in detection of ctDNA is its variable mutant allele fraction (MAF), among an abundant background of cell-free DNA released by nontumor cells. Droplet digital PCR (ddPCR) is an ideal tool for characterizing ctDNA as it provides a limit of detection comparable toMAF.This technique partitions a sample into 15,000-20,000 droplets, with PCR occurring in each droplet [11]. The number of DNA templates within each droplet is modeled by a Poisson distribution, and by quantifying the fraction of positive droplets, absolute DNA levels can be determined without extrapolation from a standard curve [12]. Due to the relative concentrations of targets and inhibitors in each droplet, ddPCR is more resistant to inhibition and more reproducible at low target concentrations than real-time quantitative PCR, making it attractive for diagnostic applications [13]. The purpose of this study was to test the feasibility of ctDNA detection in children with high-risk DAWT using plasma, serum, and urine collected throughout treatment. Materials and Methods Patients All patients with a diagnosis of WT were enrolled in the UK-wide Improving Population Outcomes for Renal Tumours of Childhood (IMPORT) study. Informed consent to undertake genetic testing of samples was obtained as part of the study, which was approved by the national research ethics committee (London Bridge REC 12/LO/ 0101). Normal kidney and multisampled tumor tissues were collected at surgery, while blood and urine were collected at up to five treatment time points: diagnosis, midchemotherapy, preoperative, postoperative, and end of treatment. Patients were treated with preoperative chemotherapy regimens according to stage as per SIOP WT 2001. Nonmetastatic cases (stages I-III) received vincristine/ actinomycin-D for 4 weeks, with doxorubicin added for metastatic disease (stage IV) for 6 weeks. Following nephrectomy, patients with focal or diffuse anaplasia were risk stratified to intermediateor high-risk subgroups, respectively, and postoperative treatment was further refined according to stage of disease at surgery. DNA Extraction All samples used for tumor DNA extraction were fresh-frozen specimens obtained at nephrectomy and stored at −80°C. Specimens underwent centralized histology review to confirm stage and histology, and those with tumor content of more than 50% were utilized for the study [14]. Tumor DNA and germline DNA from normal tissue were extracted by standard methods using either a standard detergent lysis/phenol-chloroform technique or the DNAeasy Blood and Tissue Kit (Qiagen). Germline DNA from cell fraction was extracted with the QIAamp DNA Blood Midi Kit (Qiagen). DNA was stored at −20°C. Extraction of ctDNA from 200 μl of plasma, serum, and urine supernatant was carried out with the Plasma/Serum Cell-free Circulating DNA Purification Mini Kit (Norgen) and the Circulating Nucleic Acid Kit (Qiagen) as per protocol. Elution volumes were 50 μl and 75 μl for the Norgen and Qiagen kits, respectively. Quantification and further quality control of tumor and germline DNA were undertaken with a NanoDrop spectrophotometer (Thermo Fisher Scientific), Qubit fluorometer (Thermo Fisher Scientific), and agarose gel. Sequencing of Tumor and Germline DNA For TP53mutational analysis, Sanger sequencing of tumor DNA was carried out by Great Ormond Street Hospital Genetics. Bidirectional sequencing was undertaken for all 11 exons of the gene. For the five patients with TP53 mutations, we performed Sanger sequencing on germline DNA. PCR amplification products were run on an agarose gel prior to clean-up with Illustra ExoStar 1-Step (GE Healthcare Life Sciences). Cleaned PCR products were sequenced using the BigDye Terminator v3.1 Cycle Sequencing Kit (Applied Biosys