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TP53 protein is one of the most important and studied human tumor suppressors. Whereas TP53 drives the malignant transformation and is the most frequently mutated gene in human cancers, the location and the type of its mutations for specific tumor types in ethnically admixed populations have not been evaluated. In this study we employed an amplicon panel to capture and to deep-sequence all the exons of TP53 in a series of 29 tumor biopsy samples derived from Brazilian individuals diagnosed with gastric adenocarcinomas (GACs), which were compared to Asian and Europeans. TP53 sequences were obtained from Brazilian samples with the Ion Torrent AmpliSeq TP53-panel and determined in the Ion PGM Torrent and Ion Proton platforms. We generated a mean of 7.25 million reads per patient, resulting in a median coverage >13,000X. All samples were covered above 6,400X. The mutations observed here were compared to those described in patients from different ethnic groups obtained from TCGA composed for 103 Asians patients (Japan, Hong-Kong, South Korea, Vietnam) and 78 cases of Europeans. The Asian and European populations showed higher prevalence of intestinal type GAC (53.7% and 61.6%, respectively) in contrast to our cohort, in which 69% of the patients had diffuse-type GACs (pA p.Arg282Trp in the DNA binding domain in exon 8, was found in all three sample groups studied, suggesting a possible role for this mutation in GAC pathogenesis. Although TP53 is the most frequently mutated gene in GACs we found no specific patterns that could explain the differences in the clinical behavior of this tumor between Asian and non-Asian patients. Further analyses are being performed trying to correlate mutations in TP53 and other genes with the different clinical outcomes observed in GACs from Asians and non-Asians, and in a setting of high genetic admixture, as observed in Brazil. Citation Format: Melissa Pool Pizzi, Helano Carioca Freitas, Maria Galli Amorim, Bruna Durães de Figueiredo Barros, Frederico Omar Gleber-Netto, Ana Flávia Mattos Costa, Maria Dirlei Begnami, Adriane Graicer Pelosof, Diana Noronha Nunes, Emmanuel Dias-Neto. Gastric adenocarcinoma TP53 mutations in an ethnically admixed population. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3697.

Detection of tumor DNA in urine has been shown as viable method of diagnosis not only for urinary tract (e.g., kidney and bladder) cancers but also for other types, e.g. prostate. Applications of such method, resemble those which use plasma DNA and include cancer detection, monitoring of tumor growth or recurrence and response to chemotherapy or radiation therapy. As far as we know, there is a lack of studies applying urine DNA detection in Wilms tumor (WT), an embryonary kidney cancer type. Here we show evidences of detection for two somatic variants in the urine DNA of one WT patient. By using NGS exome sequencing of tumor tissue and leukocytes samples of the same patient we were able to find new somatic variants: a frame-shift indel in TNRC18 and a misssense SNV in INTS1 gene. Target sequencing applied to the DNA from the patient's urine revealed the presence of these two somatic variants. More interestingly, both variants could not be found in the urine DNA of this patient after treatment (chemotherapy and nephrectomy). Additionally, these genes are poorly characterized in WT contributing for the comprehension of the cellular processes that are operating in tumorigenesis of WT. Altogether, our findings contribute with the mutational repertoire of WT and reveals the potential of using urine DNA sequencing as a noninvasive cancer screening approach. Citation Format: Ana C. K. Miguez, Rodrigo F. Ramalho, Elisa N. Ferreira, Bruna D. F. Barros, Claudia A. A. de Paula, Renan Valieris, Louise D. C. Mota, Jorge E. Souza, Isabela W. Cunha, Cecília L. Costa, Sandro J. de Souza, Dirce M. Carraro. Urine as a potential liquid biopsy for detecting tumor DNA in Wilms tumor patient: Detection of somatic mutations in urine opens perspectives of monitoring chemotherapy response in WT patients. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr A32.

Background: Wilms Tumor (WT) is the most common pediatric kidney cancer and in approximately 2% of cases presents a familial predisposition. In both sporadic and inherited WT cases, several genes have been identified as being somatically mutated. The most frequent WT mutated genes are WT1, CTNNB1 and WTX, that together account for approximately 30% of cases. In addition, mutation in other genes such as TP53, IGF2, DIS3L2, FBXW7, MYCN, and DICER have also been described in few cases. However, up to 60-70% of Wilms tumors remain without any associated driver mutation. In this sense, the aim of this project was to identify novel somatic and de novo alterations possibly associated with WT tumorigenesis through exome sequencing. Methods: The study design consisted of performing exome sequencing of 4 samples related to a sporadic WT patient: patient blood and tumor samples, in addition to mother's and father's blood samples. Coding regions of genomic DNA were enriched using SureSelect Human All Exon 50Mb Kit (Agilent). Libraries construction and sequencing at SOLiD 4 and 5500xl were performed following the manufacturer's instructions. Resulting sequences were mapped to a reference genome with Bioscope and NovoalignCS software. Sequence variants were identified with SAMtools and filtered if present on dbSNP database. Initially a small set of de novo and somatic mutations were selected to be validated by sanger sequencing. Results: From 84 unknown de novo alterations, we selected 11 for validation according to the following criteria: all loss of function variants (1 splice site and 2 nonsenses; 8 missense variants that were classified as pathogenic in the prediction programs SIFT and/or Polyphen2 and were not at highly repetitive or homologous regions of the genome. From these 11 candidates, only one was confirmed to be a de novo mutation, while the remaining ten were either false positive or inherited variants. Regarding somatic alterations, 8 candidate mutations were identified and selected for validation. After sanger sequencing, only one was validated as a somatic mutation, one variant was also present in patient's blood and 6 alterations were not confirmed in the tumor, suggesting it to be sequencing artifacts. The gene affected by the validated somatic mutation encodes an important protein involved in RNA regulation and cell differentiation, and was not described to be mutated in any tumor type until now. Remarkably, this somatic mutation affects a highly conserved amino acid of this gene and this residue is a known binding site for a magnesium cofactor that is essential for the protein activity. The presence of this mutation was further evaluated in a cohort of 97 Wilms tumor samples and found to be present in 10% of cases. Preliminary functional studies of HEK293 cells transfected with an expression vector of this mutation demonstrated an impaired activity of the mutated protein. Conclusion: In this study we were able to identify one de novo and one somatic novel mutations in Wilms tumor. Initial results from our group suggest that this somatic mutation represents a driver and frequent event in this type of tumor. Citation Format: Giovana T. Torrezan, Elisa N. Ferreira, Mayra T M Castro, Adriana M. Nakahata, Pedro A. Galante, Daniel T. Ohara, Bruna D. Barros, Mariana Maschietto, Isabela W. Cunha, Cecilia M L Costa, Beatriz D. Camargo, Dirce M. Carraro. Identification of somatic and de novo mutations by exome sequencing in sporadic Wilms tumor. [abstract]. In: Proceedings of the AACR Special Conference on Pediatric Cancer at the Crossroads: Translating Discovery into Improved Outcomes; Nov 3-6, 2013; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2013;74(20 Suppl):Abstract nr A29.