Metabolomic Analysis of Plasma from Breast Cancer Patients Using Ultra-High-Performance Liquid Chromatography Coupled with Mass Spectrometry: An Untargeted Study.

Breast cancer (BC) is one of the leading causes of cancer mortality in women worldwide, and therefore, novel biomarkers for early disease detection are critically needed. We performed herein an untargeted plasma metabolomic profiling of 55 BC patients and 55 healthy controls (HC) using ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC/Q-TOF-MS). Pre-processed data revealed 2494 ions in total. Data matrices' paired t-tests revealed 792 ions (both positive and negative) which presented statistically significant changes (FDR < 0.05) in intensity levels between cases versus controls. Metabolites identified with putative names via MetaboQuest using MS/MS and mass-based approaches included amino acid esters (i.e., N-stearoyl tryptophan, L-arginine ethyl ester), dipeptides (ile-ser, met-his), nitrogenous bases (i.e., uracil derivatives), lipid metabolism-derived molecules (caproleic acid), and exogenous compounds from plants, drugs, or dietary supplements. LASSO regression selected 16 metabolites after several variables (TNM Stage, Grade, smoking status, menopausal status, and race) were adjusted. A predictive conditional logistic regression model on the 16 LASSO selected ions provided a high diagnostic performance with an area-under-the-curve (AUC) value of 0.9729 (95% CI 0.96−0.98) on all 55 samples. This study proves that BC possesses a specific metabolic signature that could be exploited as a novel metabolomics-based approach for BC detection and characterization. Future studies of large-scale cohorts are needed to validate these findings.

Germline variants in DNA repair genes are associated with young-onset head and neck cancer.

The genetic predisposition to head and neck carcinomas (HNSCC) and how the known risk factors (papillomavirus infection, alcohol, and tobacco consumption) contribute to the early-onset disease are barely explored. Although HNSCC at early onset is rare, its frequency is increasing in recent years. Germline and somatic variants were assessed to build a comprehensive genetic influence pattern in HNSCC predisposition and patient outcome. Whole-exome sequencing was performed in 45 oral and oropharynx carcinomas paired with normal samples of young adults (≤49 years). We found FANCG, CDKN2A, and TPP germline variants previously associated with HNSCC risk. At least one germline variant in DNA repair pathway genes was detected in 67% of cases. Germline and somatic variants (including copy number variations) in FAT1 gene were identified in 9 patients (20%) and 12 tumors (30%), respectively. Somatic variants were found in HNSCC associated genes, such as TP53, CDKN2A, and PIK3CA. To date, 55 of 521 cases from the large cohort of TCGA presented < 49 years old. A comparison between the somatic alterations of TCGA-HNSCC at early onset and our dataset revealed strong similarities. Protein-protein interaction analysis between somatic and germline altered genes revealed a central role of TP53. Altogether, germline alterations in DNA repair genes potentially contribute to an increased risk of developing HNSCC at early-onset, while FAT1 could impact the prognosis.

Penile Cancer-Derived Cells Molecularly Characterized as Models to Guide Targeted Therapies.

Penile cancer (PeCa) is a common disease in poor and developing countries, showing high morbidity rates. Despite the recent progress in understanding the molecular events involved in PeCa, the lack of well-characterized in vitro models precludes new advances in anticancer drug development. Here we describe the establishment of five human primary penile cancer-derived cell cultures, including two epithelial and three cancer-associated fibroblast (CAF) cells. Using high-throughput genomic approaches, we found that the epithelial PeCa derived- cells recapitulate the molecular alterations of their primary tumors and present the same deregulated signaling pathways. The differentially expressed genes and proteins identified are components of key oncogenic pathways, including EGFR and PI3K/AKT/mTOR. We showed that epithelial PeCa derived cells presented a good response to cisplatin, a common therapeutic approach used in PeCa patients. The growth of a PeCa-derived cell overexpressing EGFR was inhibited by EGFR inhibitors (cetuximab, gefitinib, and erlotinib). We also identified CAF signature markers in three PeCa-derived cells with fibroblast-like morphology, indicating that those cells are suitable models for PeCa microenvironment studies. We thus demonstrate the utility of PeCa cell models to dissect mechanisms that promote penile carcinogenesis, which are useful models to evaluate therapeutic approaches for the disease.

Locally advanced rectal cancer transcriptomic-based secretome analysis reveals novel biomarkers useful to identify patients according to neoadjuvant chemoradiotherapy response.

Most patients with locally advanced rectal cancer (LARC) present incomplete pathological response (pIR) to neoadjuvant chemoradiotherapy (nCRT). Despite the efforts to predict treatment response using tumor-molecular features, as differentially expressed genes, no molecule has proved to be a strong biomarker. The tumor secretome analysis is a promising strategy for biomarkers identification, which can be assessed using transcriptomic data. We performed transcriptomic-based secretome analysis to select potentially secreted proteins using an in silico approach. The tumor expression profile of 28 LARC biopsies collected before nCRT was compared with normal rectal tissues (NT). The expression profile showed no significant differences between complete (pCR) and incomplete responders to nCRT. Genes with increased expression (pCR = 106 and pIR = 357) were used for secretome analysis based on public databases (Vesiclepedia, Human Cancer Secretome, and Plasma Proteome). Seventeen potentially secreted candidates (pCR = 1, pIR = 13 and 3 in both groups) were further investigated in two independent datasets (TCGA and GSE68204) confirming their over-expression in LARC and association with nCRT response (GSE68204). The expression of circulating amphiregulin and cMET proteins was confirmed in serum from 14 LARC patients. Future studies in liquid biopsies could confirm the utility of these proteins for personalized treatment in LARC patients.

Association of PDCD1 polymorphism to systemic lupus erythematosus and rheumatoid arthritis susceptibility / Associação entre o polimorfismo do gene PDCD1 e a susceptibilidade ao lúpus eritematoso sistêmico e à artrite reumatoide

ABSTRACT Objective: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p ≤ 0.05. Results: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. Conclusion: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.

RESUMO Objetivo: Este estudo teve como objetivo analisar a relação entre o polimorfismo do gene PDCD1 (programmed cell death 1) (PD1.3G/A - rs11568821) com características do lúpus eritematoso sistêmico (LES) e da artrite reumatoide (AR) em uma população do sul do Brasil. Métodos: A técnica de PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Lenght Polymorphism) foi utilizada para analisar amostras de 95 pacientes com LES e 87 com AR, assim como em 128 indivíduos do grupo controle de Santa Catarina, sul do Brasil. Foi analisada a probabilidade de equilíbrio de Hardy-Weinberg (EHW) e a odds ratio (OR), considerando um IC 95% e p ≤ 0,05. Resultados: As frequências alélicas PD1.3 A foram de 0,095 (LES), 0,115 (AR) e 0,078 (controles). Os genótipos do grupo controle estavam em EHW, enquanto aqueles dos pacientes com LES e AR não estavam. No entanto, não foi encontrada associação entre o polimorfismo PD1.3 e a suscetibilidade ao LES ou à AR, nem com dados clínicos ou epidemiológicos. Conclusão: Não foi encontrada associação significativa entre o polimorfismo PD1.3 e a susceptibilidade ao LES ou à AR nessa população do sul do Brasil.

Association of PDCD1 polymorphism to Systemic Lupus Erythematosus and Rheumatoid Arthritis susceptibility. / Associação entre o polimorfismo do gene PDCD1 e a susceptibilidade ao lúpus eritematoso sistêmico e à artrite reumatoide.

OBJECTIVE: This study aims to analyze the relationship of programmed cell death 1 (PDCD1) gene polymorphism (PD1.3G/A - rs11568821) with features of systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in a Southern Brazilian population. METHODS: Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) was performed in 95 SLE and 87 RA patients and 128 control group individuals from Santa Catarina, Southern Brazil. The Hardy-Weinberg Equilibrium (HWE) test, and odds ratio (OR) were analyzed, considering CI 95% and p≤0.05. RESULTS: The PD1.3A allele frequencies were 0.095 (SLE), 0.115 (RA) and 0.078 (controls). The genotypes of the control group were in HWE, while those of SLE and RA patients were not. However, we found no association between PD1.3 polymorphism and the SLE or RA susceptibility, nor clinical or epidemiological data. CONCLUSION: There was no significant association between PD1.3 polymorphism and SLE or RA susceptibility in this Southern Brazilian population.

Lack of association between interleukin-18 polymorphisms and rheumatoid arthritis.

OBJECTIVE: To assess the association of the polymorphisms of the interleukin-18 (IL-18) gene with rheumatoid arthritis (RA) and with risk factors for cardiovascular diseases (CVD). METHODS: This sample comprised 97 patients with RA and 151 healthy controls. In the patients, risk factors for CVD were analyzed, such as cholesterol levels, arterial hypertension, smoking habit, C-reactive protein (CRP) level, and rheumatoid factor. DNA was extracted and the single nucleotide polymorphisms (SNP) at the -607C/A and -137G/C positions of the IL-18 gene were assessed in both groups. The Hardy-Weinberg equilibrium (HWE) was calculated and the odds ratio (OR) test performed, considering a 95% CI and P < 0.05. RESULTS: The frequencies of the -607A allele in patients with RA and in controls were 0,443 and 0.424, respectively, and of the -137C allele, 0.304 and 0.291, respectively. The genotype frequencies were in HWE, except for controls in the -137 locus (P = 0.006). Association of the polymorphisms of the IL-18 gene was found with neither RA nor risk factors for CVD, including cholesterol level and CRP (P > 0.05). In addition, more smokers were found among patients with RA as compared with controls (OR = 1.691; P = 0.088), and the CRP levels were slightly higher in patients who smoked than in patients who did not (OR = 2.673; P = 0.061). CONCLUSIONS: In this sample of patients with RA in the South of Brazil, association of the polymorphisms of the IL-18 gene was observed with neither RA nor risk factors for CVD.

Ausência de associação entre os polimorfismos do gene interleucina-18 e artrite reumatoide / Lack of association between interleukin-18 polymorphisms and rheumatoid arthritis

OBJETIVO: Analisar a associação dos polimorfismos do gene interleucina-18 (IL-18) com artrite reumatoide (AR) e com fatores de risco de doenças cardiovasculares (DCV). MÉTODOS: A amostra foi constituída por 97 pacientes com AR e 151 controles saudáveis. Nos primeiros, foram analisados fatores de risco de DCV, tais como níveis do colesterol, hipertensão arterial, tabagismo e fator reumatoide, bem como o nível da proteína C-reativa (CRP). O DNA foi extraído e foram analisados os polimorfismos de nucleotídeo único (SNP) nas posições -607C/A e -137G/C do gene IL-18 em ambos os grupos. O equilíbrio de Hardy-Weinberg (EHW) e o odds ratio (OR) foram realizados, considerando IC 95% e P < 0,05. RESULTADOS: As frequências do alelo -607A nos pacientes com AR e nos controles foram de 0,443 e 0,424 e do alelo -137C foram de 0,304 e 0,291, respectivamente. As frequências do genótipo estavam em EHW, exceto em controles no locus -137 (P = 0,006). Não foi encontrada associação dos polimorfismos do gene IL-18 com AR, nem com fatores de risco de DCV, incluindo o nível do colesterol e de CRP (P > 0,05). Além disso, observaram-se mais indivíduos fumantes entre pacientes com AR em comparação aos controles (OR = 1,691; P = 0,088), e os níveis de CRP eram ligeiramente mais elevados em pacientes fumantes quando comparados aos de pacientes não fumantes (OR = 2,673; P = 0,061). CONCLUSÕES: Ao analisar uma amostra de pacientes com AR no sul do Brasil, não foi encontrada associação dos polimorfismos do gene IL-18 com AR, nem com os fatores de risco de DCV.

OBJECTIVE: To assess the association of the polymorphisms of the interleukin-18 (IL-18) gene with rheumatoid arthritis (RA) and with risk factors for cardiovascular diseases (CVD). METHODS: This sample comprised 97 patients with RA and 151 healthy controls. In the patients, risk factors for CVD were analyzed, such as cholesterol levels, arterial hypertension, smoking habit, C-reactive protein (CRP) level, and rheumatoid factor. DNA was extracted and the single nucleotide polymorphisms (SNP) at the -607C/A and -137G/C positions of the IL-18 gene were assessed in both groups. The Hardy-Weinberg equilibrium (HWE) was calculated and the odds ratio (OR) test performed, considering a 95% CI and P < 0.05. RESULTS: The frequencies of the -607A allele in patients with RA and in controls were 0,443 and 0.424, respectively, and of the -137C allele, 0.304 and 0.291, respectively. The genotype frequencies were in HWE, except for controls in the -137 locus (P = 0.006). Association of the polymorphisms of the IL-18 gene was found with neither RA nor risk factors for CVD, including cholesterol level and CRP (P > 0.05). In addition, more smokers were found among patients with RA as compared with controls (OR = 1.691; P = 0.088), and the CRP levels were slightly higher in patients who smoked than in patients who did not (OR = 2.673; P = 0.061). CONCLUSIONS: In this sample of patients with RA in the South of Brazil, association of the polymorphisms of the IL-18 gene was observed with neither RA nor risk factors for CVD.