Gabriel de Souza Macedo

miRNA-21 and miRNA-34a Are Potential Minimally Invasive Biomarkers for the Diagnosis of Pancreatic Ductal Adenocarcinoma.

Objectives Pancreatic ductal adenocarcinoma (PDAC) is one of the most deadly cancers, and its diagnosis often requires invasive procedures. Deregulated miRNA expression has been described in patients with PDAC. In this study, we analyzed the expression levels of 6 miRNAs (miR-21, -34a, -155, -196a, -200b, and -376a involved in PDAC tumorigenesis) in serum and salivary samples to assess their potential role as circulating diagnostics biomarkers. Methods Serum and salivary samples were collected from patients with PDAC and healthy controls, and miRNA levels were quantified using qRT-PCR. Twenty-four patients with PDAC and 10 healthy controls were recruited. Results A significant difference between PDAC and healthy groups was observed for the expression of miR-21 and miR-34a (P < 0.001 and P = 0.001) in serum samples. Both miRNAs accurately discriminated between the 2 groups, with an area under the curve for miR-21 and miR-34a of 0.889 (P = 0.001) and 0.865 (P = 0.002), respectively. In general, the expression of miRNAs between salivary samples did not differ. Conclusions Serum miR-21 and miR-34a are potentially useful diagnostic biomarkers of PDAC. In addition, our results suggest that these miRNAs are not differentially expressed in saliva, making them unsuitable for use as noninvasive biomarkers for diagnostic purposes.

Increased Oxidative Damage in Carriers of the Germline TP53 p.R337H Mutation

Germline mutations in TP53 are the underlying defect of Li-Fraumeni Syndrome (LFS) and Li-Fraumeni-like (LFL) Syndrome, autosomal dominant disorders characterized by predisposition to multiple early onset cancers. In Brazil, a variant form of LFS/LFL is commonly detected because of the high prevalence of a founder mutation at codon 337 in TP53 (p.R337H). The p53 protein exerts multiple roles in the regulation of oxidative metabolism and cellular anti-oxidant defense systems. Herein, we analyzed the redox parameters in blood samples from p.R337H mutation carriers (C, n = 17) and non-carriers (NC, n = 17). We identified a significant increase in erythrocyte GPx activity and in plasma carbonyl content,an indicator of protein oxidative damage, in mutation carriers compared to non-carriers (P = 0.048 and P = 0.035, respectively). Mutation carriers also showed a four-fold increase in plasma malondialdehyde levels, indicating increased lipid peroxidation (NC = 40.20±0.71, C = 160.5±0.88, P<0.0001). Finally, carriers showed increased total antioxidant status but a decrease in plasma ascorbic acid content. The observed imbalance could be associated with deregulated cell bioenergetics and/or with increased inflammatory stress, two effects that may result from loss of wild-type p53 function. These findings provide the first evidence that oxidative damage occurs in carriers of a germline TP53 mutation, and these may have important implications regarding our understanding of the mechanisms responsible for germline TP53 p.R337H mutation-associated carcinogenesis.

Genetic Variations in the TP53 Pathway in Native Americans Strongly Suggest Adaptation to the High Altitudes of the Andes

The diversity of the five single nucleotide polymorphisms located in genes of the TP53 pathway (TP53, rs1042522; MDM2, rs2279744; MDM4, rs1563828; USP7, rs1529916; and LIF, rs929271) were studied in a total of 282 individuals belonging to Quechua, Aymara, Chivay, Cabanaconde, Yanke, Taquile, Amantani, Anapia, Uros, Guarani Ñandeva, and Guarani Kaiowá populations, characterized as Native American or as having a high level (> 90%) of Native American ancestry. In addition, published data pertaining to 100 persons from five other Native American populations (Surui, Karitiana, Maya, Pima, and Piapoco) were analyzed. The populations were classified as living in high altitude (≥ 2,500 m) or in lowlands (< 2,500 m). Our analyses revealed that alleles USP7-G, LIF-T, and MDM2-T showed significant evidence that they were selected for in relation to harsh environmental variables related to high altitudes. Our results show for the first time that alleles of classical TP53 network genes have been evolutionary co-opted for the successful human colonization of the Andes.

p53 signaling pathway polymorphisms, cancer risk and tumor phenotype in TP53 R337H mutation carriers

Li-Fraumeni and Li-Fraumeni-like syndrome (LFS/LFL) are clinically heterogeneous cancer predisposition syndromes characterized by diagnosis of early-onset and often multiple cancers with variable tumor patterns and incomplete penetrance. To date, the genetic modifiers described in LFS/LFL have been shown to map to either TP53 or its main negative regulator, MDM2. Additionally, all studies were focused on families with different TP53 germline mutations. Hence, in this study we explored the effect of the most studied polymorphisms of p53 pathway genes on clinical manifestations of individuals carrying the founder TP53 mutation R337H (n = 136) and controls (n = 186). Cancer-affected carriers had been diagnosed either with adrenocortical carcinoma (ACC, n = 29) or breast cancer (BC, n = 43). Allelic discrimation using TaqMan assay was used for genotyping MDM2 SNP 309 (rs2279744) as well as MDM4 (rs1563828) and USP7 (rs1529916) polymorphisms. We found significantly higher MDM2 SNP 309 GG genotype and G allele frequencies in the LFS cohort than in controls. Furthermore, median age at first diagnosis was earlier in MDM2 SNP309 GG carriers when compared to other genotypes for both cancers (ACC: age 1 vs. 2 years; BC: age 35 vs. 43 years, respectively), although not statistically different. The allelic and genotypic frequencies for all SNPs did not differ between cancer affected and unaffected carriers, neither between patients with ACC or BC. In conclusion, our results suggest that MDM2 SNP 309 may contribute to the LFL phenotype and also to an earlier age at diagnosis of ACC and BC cancer in carriers of the R337H founder mutation.

Comparison of multiple genotyping methods for the identification of the cancer predisposing founder mutation p.R337H in TP53

Abstract Germline mutations in the TP53 gene are associated with Li-Fraumeni and Li-Fraumeni-Like Syndromes, characterized by increased predisposition to early-onset cancers. In Brazil, the prevalence of the TP53-p.R337H germline mutation is exceedingly high in the general population and in cancer-affected patients, probably as result of a founder effect. Several genotyping methods are used for the molecular diagnosis of LFS/LFL, however Sanger sequencing is still considered the gold standard. We compared performance, cost and turnaround time of Sanger sequencing, PCR-RFLP, TaqMan-PCR and HRM in the p.R337H genotyping. The performance was determined by analysis of 95 genomic DNA samples and results were 100% concordant for all methods. Sequencing was the most expensive method followed by TaqMan-PCR, PCR-RFLP and HRM. The overall cost of HRM increased with the prevalence of positive samples, since confirmatory sequencing must be performed when a sample shows an abnormal melting profile, but remained lower than all other methods when the mutation prevalence was less than 2.5%. Sequencing had the highest throughput and the longest turnaround time, while TaqMan-PCR showed the lowest turnaround and hands-on times. All methodologies studied are suitable for the detection of p.R337H and the choice will depend on the application and clinical scenario.

Abstract 804: Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Background: Li-Fraumeni Syndrome (LFS) is a cancer predisposition syndrome associated with TP53 germline mutations and characterized by increased risk to multiple early-onset cancers. Studies in families from Southern and Southeastern Brazil have identified a germline founder mutation in the TP53 gene, the p.R337H, in high population prevalence (∼0.3%). Unlike the majority of the mutations in TP53, which are missense mutations located in the DNA-binding domain (DBD) of the protein, the TP53 p.R337H is located in the oligomerization domain (OD). Recently, we identified a new rare germline variant, the rs78378222 (A>C), in the 3′ UTR of the gene in 7/130 of the patients who tested negative for coding germline TP53 mutations. Based on the broad spectrum of p53 functions, which include metabolism and DNA damage response regulation, in the present study we aimed to characterize the functional impact of germline TP53 mutations identified in Brazilian families. Methods: Primary fibroblasts from p.R337H/WT (n = 2), p.R337H/p.R337H (n = 1), DBD mutations (n = 2), rs78378222[C] patients and from WT p53 controls (n = 2) were included. The DNA damage induction was performed using UVB (0.2W/m2) and ionizing irradiation (IR) (1 Gy). Results: First, we observed that all p53-mutated fibroblasts appeared to have lower p53 levels but higher levels of reactive oxygen species (ROS) and mitochondrial biomass compared to controls. Fibroblasts homozygous to p.R337H mutation presented more pronounced ROS production and, notably, higher expression of two antioxidant proteins, SOD2 and GPX1. On the other hand, the results of high-resolution respirometry showed higher oxygen consume in DBD-mutated fibroblasts. Second, using a specific multiplexed enzymatic DNA repair assay on biochip, we simultaneously investigated several DNA repair pathways. Overall, we observed a high DNA repair activation profile in p.R337H/p.R337H and poor DNA repair capacity in DBD-mutated fibroblasts. Interestingly, all p53-mutated fibroblasts showed higher repair activity of apurinic/pyrimidinic (AP) sites, a DNA lesion caused mainly by ROS attacks. Conclusions: Our data indicate important alterations in both metabolism and DNA damage response in normal cells (fibroblasts) from LFS patients, suggesting haploinsufficiency mechanism associated to these mutations. We were also able to demonstrate distinct phenotypes according to mutational status, which may explain, at least in part, the clinical variability in LFS. Citation Format: Gabriel Macedo, Sylvie Sauvaigo, Michele Silva Alves, Sylvain Caillat, Igor Araujo Vieira, Fernanda Timm, Cristina Brinckmann Oliveira Netto, Mauro Antonio Alves Castro, Ivi Bristot, Angela Fachel, Fabio Klamt, Patricia Ashton-Prolla. Energetic metabolism and DNA damage response in fibroblasts from Li-Fraumeni syndrome patients: new insights into the molecular mechanisms of the disease. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 804. doi:10.1158/1538-7445.AM2015-804