Circulating mRNA signature as a marker for high-risk prostate cancer.

Prostate cancer (PCa) is the second most common cancer in men. The indolent course of the disease makes the treatment choice a challenge for physicians and patients. In this study, a minimally invasive method was used to evaluate the potential of molecular markers in identifying patients with aggressive disease. Cell-free plasma samples from 60 PCa patients collected before radical prostatectomy were used to evaluate the levels of expression of eight genes (AMACR, BCL2, NKX3-1, GOLM1, OR51E2, PCA3, SIM2 and TRPM8) by quantitative real-time PCR. Overexpression of AMACR, GOLM1, TRPM8 and NKX3-1 genes was significantly associated with aggressive disease characteristics, including extracapsular extension, tumor stage and vesicular seminal invasion. A trio of genes (GOLM1, NKX3-1 and TRPM8) was able to identify high-risk PCa cases (85% of sensitivity and 58% of specificity), yielding a better overall performance compared with the biopsy Gleason score and prostate-specific antigen, routinely used in the clinical practice. Although more studies are required, these circulating markers have the potential to be used as an additional test to improve the diagnosis and treatment decision of high-risk PCa patients.

Effect of UGT1A1, UGT1A3, DIO1 and DIO2 polymorphisms on L-thyroxine doses required for TSH suppression in patients with differentiated thyroid cancer.

AIM: To evaluate the impact of genetic polymorphisms in uridine 5'-glucuronosylytansferases UGT1A1 and UGT1A3 and iodothyronine-deiodinases types 1 and 2 on levothyroxine (T4 ; 3,5,3',5'-triiodo-L-thyronine) dose requirement for suppression of thyrotropin (TSH) secretion in patients with differentiated thyroid cancer (DTC). METHODS: Patients (n = 268) submitted to total thyroidectomy and ablation by (131) I, under T4 therapy for at least 6 months were recruited in three public institutions in Brazil. Multivariate regression modelling was applied to assess the association of T4 dosing with polymorphisms in UGT1A1 (rs8175347), UGT1A3 (rs3806596 and rs1983023), DIO1 (rs11206244 and rs2235544) and DIO2 (rs225014 and rs12885300), demographic and clinical variables. RESULTS: A regression model including UGT1A haplotypes, age, gender, body weight and serum TSH concentration accounted for 39% of the inter-individual variation in the T4 dosage. The association of T4 dose with UGT1A haplotype is attributed to reduced UGT1A1 expression and T4 glucuronidation in liver of carriers of low expression UGT1A1 rs8175347 alleles. The DIO1 and DIO2 genotypes had no influence of T4 dosage. CONCLUSION: UGT1A haplotypes associate with T4 dosage in DTC patients, but the effect accounts for only 2% of the total variability and recommendation of pre-emptive UGT1A genotyping is not warranted.

Circulating mRNAs and miRNAs as candidate markers for the diagnosis and prognosis of prostate cancer.

Circulating nucleic acids are found in free form in body fluids and may serve as minimally invasive tools for cancer diagnosis and prognosis. Only a few studies have investigated the potential application of circulating mRNAs and microRNAs (miRNAs) in prostate cancer (PCa). The Cancer Genome Atlas (TCGA) database was used for an in silico analysis to identify circulating mRNA and miRNA as potential markers of PCa. A total of 2,267 genes and 49 miRNAs were differentially expressed between normal and tumor samples. The prediction analyses of target genes and integrative analysis of mRNA and miRNA expression revealed eleven genes and eight miRNAs which were validated by RT-qPCR in plasma samples from 102 untreated PCa patients and 50 cancer-free individuals. Two genes, OR51E2 and SIM2, and two miRNAs, miR-200c and miR-200b, showed significant association with PCa. Expression levels of these transcripts distinguished PCa patients from controls (67% sensitivity and 75% specificity). PCa patients and controls with prostate-specific antigen (PSA) ≤ 4.0 ng/mL were discriminated based on OR51E2 and SIM2 expression levels. The miR-200c expression showed association with Gleason score and miR-200b, with bone metastasis, bilateral tumor, and PSA > 10.0 ng/mL. The combination of circulating mRNA and miRNA was useful for the diagnosis and prognosis of PCa.

PHF21B as a candidate tumor suppressor gene in head and neck squamous cell carcinomas.

A significant association between DNA losses on 22q13.31 and head and neck squamous cell carcinomas (HNSCC) was previously reported by our group. Our data indicated that PHF21B gene, mapped on 22q13.31 and encoding a protein with function of chromatin-mediated transcriptional regulation, might be a putative tumor suppressor gene. To test this hypothesis, gene copy number was assessed in 75 HNSCC and 49 matched peripheral blood samples. PHF21B losses were detected in 43 tumors and were significantly associated with patients with familial history of cancer (P < 0.0001); i.e., 36/43 cases showed a positive family history of cancer and 22/36 had first-degree relatives with cancer (P = 0.049). In attempt to investigate other mechanisms for PHF21B loss of function, DNA sequencing was performed and no mutations were detected. We next evaluated the gene expression levels after inhibition of DNA methylation in nine HNSCC and breast carcinoma cell lines. Additionally, PHF21B expression levels were evaluated in colon cancer HCT116 cells as well as in its counterpart DKO (double knockout of DNMT1 and DNMT3B). The higher expression levels of PHF21B gene detected in DKO cells were inversely correlated with the DNA methylation. Further, DNA methylation in the specific promoter-associated CpG Island was investigated. Interestingly, gene hypermethylation was detected in 13/37 tumors: 5/13 HNSCC cases had family history of cancer in first-degree relatives and 8/13 showed both, DNA methylation and PHF21B losses in the tumor sample. One patient had PHF21B loss in the peripheral blood cells and PHF21B methylation in the tumor sample. Additionally, overexpression of PHF21B in cell lines drastically reduces clonogenic and migratory abilities. These data suggest that PHF21B is a novel tumor suppressor gene that can be inactivated by genetic and epigenetic mechanisms in the human cancer.

An integrative genomic and transcriptomic analysis reveals potential targets associated with cell proliferation in uterine leiomyomas.

BACKGROUND: Uterine Leiomyomas (ULs) are the most common benign tumours affecting women of reproductive age. ULs represent a major problem in public health, as they are the main indication for hysterectomy. Approximately 40-50% of ULs have non-random cytogenetic abnormalities, and half of ULs may have copy number alterations (CNAs). Gene expression microarrays studies have demonstrated that cell proliferation genes act in response to growth factors and steroids. However, only a few genes mapping to CNAs regions were found to be associated with ULs. METHODOLOGY: We applied an integrative analysis using genomic and transcriptomic data to identify the pathways and molecular markers associated with ULs. Fifty-one fresh frozen specimens were evaluated by array CGH (JISTIC) and gene expression microarrays (SAM). The CONEXIC algorithm was applied to integrate the data. PRINCIPAL FINDINGS: The integrated analysis identified the top 30 significant genes (P<0.01), which comprised genes associated with cancer, whereas the protein-protein interaction analysis indicated a strong association between FANCA and BRCA1. Functional in silico analysis revealed target molecules for drugs involved in cell proliferation, including FGFR1 and IGFBP5. Transcriptional and protein analyses showed that FGFR1 (P = 0.006 and P<0.01, respectively) and IGFBP5 (P = 0.0002 and P = 0.006, respectively) were up-regulated in the tumours when compared with the adjacent normal myometrium. CONCLUSIONS: The integrative genomic and transcriptomic approach indicated that FGFR1 and IGFBP5 amplification, as well as the consequent up-regulation of the protein products, plays an important role in the aetiology of ULs and thus provides data for potential drug therapies development to target genes associated with cellular proliferation in ULs.

Genomic signatures predict poor outcome in undifferentiated pleomorphic sarcomas and leiomyosarcomas.

Undifferentiated high-grade pleomorphic sarcomas (UPSs) display aggressive clinical behavior and frequently develop local recurrence and distant metastasis. Because these sarcomas often share similar morphological patterns with other tumors, particularly leiomyosarcomas (LMSs), classification by exclusion is frequently used. In this study, array-based comparative genomic hybridization (array CGH) was used to analyze 20 UPS and 17 LMS samples from untreated patients. The LMS samples presented a lower frequency of genomic alterations compared with the UPS samples. The most frequently altered UPS regions involved gains at 20q13.33 and 7q22.1 and losses at 3p26.3. Gains at 8q24.3 and 19q13.12 and losses at 9p21.3 were frequently detected in the LMS samples. Of these regions, gains at 1q21.3, 11q12.2-q12.3, 16p11.2, and 19q13.12 were significantly associated with reduced overall survival times in LMS patients. A multivariate analysis revealed that gains at 1q21.3 were an independent prognostic marker of shorter survival times in LMS patients (HR = 13.76; P = 0.019). Although the copy number profiles of the UPS and LMS samples could not be distinguished using unsupervised hierarchical clustering analysis, one of the three clusters presented cases associated with poor prognostic outcome (P = 0.022). A relative copy number analysis for the ARNT, SLC27A3, and PBXIP1 genes was performed using quantitative real-time PCR in 11 LMS and 16 UPS samples. Gains at 1q21-q22 were observed in both tumor types, particularly in the UPS samples. These findings provide strong evidence for the existence of a genomic signature to predict poor outcome in a subset of UPS and LMS patients.

Expressão gênica global em carcinomas papilíferos de tireoide: busca de marcadores diagnósticos, prognósticos e alvos terapêuticos / Global gene expression in papillary thyroid carcinoma: search for diagnostic, prognostic markers and therapeutic targets

Contexto. O carcinoma papilífero de tireoide (CPT) é a neoplasia maligna endócrina mais frequente, apresentando um evidente aumento de incidência nos últimos anos. Embora o comportamento dos CPT é geralmente indolente, uma parcela dos pacientes apresenta doença agressiva, para os quais não restam opções terapêuticas eficazes. Apesar da mutação em BRAF ser frequentemente descrita como um fator de pior prognóstico, até o presente, nenhum marcador molecular é usado na rotina clínica. Objetivos. Identificar marcadores diagnósticos, prognósticos e possíveis candidatos à intervenção terapêutica em CPT pela análise de expressão gênica global. Métodos. Foram incluídos 250 casos de CPT tratados com tireoidectomia total e radioiodoterapia. A mutação BRAFV600E foi detectada e quantificada por pirosequenciamento (225 CPT). Experimentos de microarray para análise de expressão gênica foram conduzidos em 61 CPT e 13 amostras de tireoide não neoplásica adjacente ao tumor (TN). O perfil de expressão do CPT foi comparado com o TN e de acordo com status de mutação em BRAF, acometimento linfonodal e evolução clínica. Os resultados foram comparados com os depositados em bancos de dados públicos. Foram também realizadas análises moleculares in silico e confirmação dos achados para um subconjunto de genes. Trinta e nove transcritos alvos e cinco referências foram selecionados para confirmação por RT-qPCR em 206 CPT, 33 TN e 27 lesões benignas de tireoide (LBT). Resultados. Foram identificados 589 transcritos diferencialmente expressos na comparação entre CPT e TN, onde GABRB2 foi ranqueado como o mais frequentemente alterado pela meta-análise. Os resultados foram coletivamente interpretados por análises in silico, mostrando evidências de uma possível perda de sensibilidade à tiroxina e alteração do metabolismo de ácido retinóico (AR) como resultado da ativação de EZH2 e de histona deacetilases (HDAC). Vinte e oito transcritos foram confirmados como diferencialmente expressos por RT-qPCR. A combinação da expressão de CLDN10, HMGA2 e LAMB3 foi capaz de discriminar todas as amostras de CPT das normais/benignas em um grupo de treinamento (86 CPT, 23 TN e oito LBT). O algoritmo diagnóstico foi validado em um grupo independente (120 CPT, 10 TN e 19 LBT), alcançando uma área abaixo da curva (AUC) de 0,99 com 97,5% de sensibilidade e 90,3% de valor preditivo negativo (VPN). Maiores escores desse algoritmo foram associados com acometimento linfonodal. O perfil de expressão também foi comparado de acordo com a mutação em BRAF (presente em 62,5% dos casos) e associado com a presença de extensão extra-tireoidiana. Uma análise de agrupamento hierárquico não supervisionado revelou que a frequência de alelos BRAFV600E é um importante fator na variabilidade transcricional dos CPT. Foram identificados 188 genes diferencialmente expressos em tumores portadores da mutação (confirmados pela comparação com bancos de dados externos), destacando uma possível modulação da via da neuregulina. Onze de 12 transcritos, incluindo ERBB3, foram confirmados por RT-qPCR. Para a busca de marcadores prognósticos, foram comparados grupos de acordo com a evolução clínica (recidiva como desfecho) e status linfonodal, encontrando 28 e 32 genes, respectivamente. Entre os 12 genes selecionados para confirmação, a redução de expressão de GADD45B foi associada com melhor evolução clínica e linfonodos cervicais livres de lesão ao diagnóstico. Conclusões. Em conjunto, esses resultados ajudam a compreender a biologia do CPT, revelando EZH2 e HDAC como possíveis alvos terapêuticos. Além do mais, os resultados fornecem uma promissora ferramenta diagnóstica com implicações prognósticas. Foi evidenciada a possível participação da via da neuregulina em CPT com a mutação em BRAF e GADD45B como um marcador útil para melhor estratificar pacientes com CPT de acordo com o risco de recidiva