Abstract Circulating DNA can be released in the biological fluids by a physiological process and by different pathological conditions. The first reports detecting circulating DNA in human plasma date from the late 40s. Even when specific pathological conditions were analyzed, the clinical importance of circulating DNA remained unclear. After PCR introduction, genetic and epigenetic alterations in circulating DNA gained more prominence for understanding the mechanisms of cancer development and progression. Nowadays, the circulating DNA assays are highlighted for their clinical relevance for cancer screening in liquid biopsy. In this review, we described the landscape of studies on circulating DNA isolated from human plasma or serum and the molecular tools used to obtain these findings throughout the 20th century and the current application in cancer.

ABSTRACT CONTEXT: Complex karyotypes in acute myeloid leukemia (AML) are characterized by an overall low response rate with frequent relapses after clinical treatment. CASE REPORT: Here, we describe the case of a 61-year-old obese female with clinically diagnosed AML who presented a complex karyotype involving an uncommon abnormality: ring chromosome 11. Immunophenotypic analysis confirmed the diagnosis. Classical and molecular cytogenetic analyses, using GTG banding and FISH (fluorescence in situ hybridization), revealed the presence of complex structural rearrangement involving r(11), add(12)(p13), der(5) and der(13). CONCLUSIONS: Molecular cytogenetic analysis is suitable for better identification and characterization of chromosomal rearrangements in AML. Case reports like this, as well as population-based studies, are necessary for understanding the karyotypic changes that occur in humans.

Abstract Susceptibility to cancer ensues in individuals carrying malfunctioning DNA repair mechanisms. The impact of Single Nucleotide Polymorphisms (SNPs) in key DNA repair mechanisms on risk for prostate cancer was investigated in this case-control study. Samples consisted of 110 patients with confirmed prostate cancer and 200 unaffected men, from Rio de Janeiro, Brazil. XPD/Lys751Gln (rs13181), APEX1/Asp148Glu (rs1130409), and RAD51/G135C (rs1801320) SNPs were analyzed by PCR-RFLP. Allelic and genotypic frequencies were calculated and compared by Chi-Square test. The association between SNPs and clinical/epidemiological data was considered significant by Odds Ratio analysis, with IC95% and a p-value≤0.05. Only the XPD/Lys751Gln SNP significantly increased susceptibility to disease in southeastern Brazilian men, with p≤0.001 [OR=2.36 (1.46-3.84)], with no association with APEX1 or RAD51 SNPs. Combined XPD+RAD51 SNPs were highly associated with the disease, p≤0.005 [OR=3.40 (1.32-9.20)]. A Chi-Square significant association between XPD/Lys751Gln and Gleason score was also observed (OR=9.31; IC95%=1.19–428.0; p=0.022). Epidemiological inquiries revealed that exposure to pesticides significantly impacted the risk for prostate cancer in this population. DNA repair dysfunctions seem to prevail among workers exposed to chemical byproducts to cancer in this specific tissue. Non-invasive genotyping SNPs may help assessment of prostate cancer risk in environmentally exposed populations.

ABSTRACTPurpose:The aim of this study was to identify possible protein biomarkers and/or candidates for therapeutic targets in tissues of patients with SCCP, infected by HPV, applying one dimensional electrophoresis (1DE), followed by direct mass spectrometry (MS) analysis.Materials and Methods:Tissues from 10 HPV positive patients with SCCP and from 10 patients with HPV negative non-tumorous penile foreskins were analyzed applying 1D electrophoresis, followed by analysis with direct mass spectrometry (MS).Results:Sixty-three different proteins were identified in the first group and 50 in the second group. Recognition was possible for 28 proteins exclusively detected in Group 1 and 21 proteins presented only in Group 2.Conclusion:Some proteins in the first group are directly involved in the development of other types of cancer, and therefore, suitable for analysis. Complement C3 protein is a strong candidate for evaluating SCCP patients.

CONTEXTO E OBJETIVO: O vírus Epstein-Barr (EBV) é a causa mais comum de mononucleose infecciosa e também está associado com vários tumores humanos, tais como: linfoma de Burkitt, linfoma de Hodgkin, alguns casos de carcinoma gástrico e carcinoma nasofaríngeo, entre outras neoplasias. O objetivo deste estudo foi rastrear a presença de sequências de DNA-específcas do vírus EBV em biópsias de 75 gliomas primários usando reação em cadeia da polimerase (PCR), com confrmação por sequenciamento direto. TIPO DE ESTUDO E LOCAL: Estudo de prevalência sobre genética molecular de EBV em laboratório de patologia molecular de hospital universitário, e em laboratório de genética aplicada de instituição nacional. MÉTODOS: Foram obtidas 75 biópsias de gliomas primários e 6 de outros tumores do sistema nervoso central. Os tecidos foram imediatamente congelados para a extração posterior de DNA, através do método tradicional, usando a digestão por proteinase K e a extração pela mistura defenol-clorofórmio-álcool isolamílico. O DNA foi precipitado com etanol, ressuspendido em tampão e armazenado. As PCRs foram realizadas com iniciadores para amplificar a região BamM do EBV. Controles positivos e negativos foram adicionados a cada reação. Os produtos de PCR foram usados para sequenciamento direto para confrmação. RESULTADOS: As sequências virais foram positivas em 11/75 (14,7%) de nossas amostras. CONCLUSÃO: A prevalência de DNA de EBV foi de 11/75 (14.7%) na nossa coleção de gliomas. Mais estudos moleculares e epidemiológicos são necessários para esclarecer o possível papel do EBV na tumorigênese dos gliomas.

CONTEXT AND OBJECTIVE: The Epstein-Barr virus (EBV) is the most common cause of infectious mononucleosis and is also associated with several human tumors, including Burkitt's lymphoma, Hodgkin's lymphoma, some cases of gastric carcinoma and nasopharyngeal carcinoma, among other neoplasms. The aim of this study was to screen 75 primary gliomas for the presence of specific EBV DNA sequences by means of the polymerase chain reaction (PCR), with confirmation by direct sequencing. DESIGN AND SETTING: Prevalence study on EBV molecular genetics at a molecular pathology laboratory in a university hospital and at an applied genetics laboratory in a national institution. METHODS: A total of 75 primary glioma biopsies and 6 others from other tumors from the central nervous system were obtained. The tissues were immediately frozen for subsequent DNA extraction by means of traditional methods using proteinase K digestion and extraction with a phenol-chloroform-isoamyl alcohol mixture. DNA was precipitated with ethanol, resuspended in buffer and stored. The PCRs were carried out using primers for amplification of the EBV BamM region. Positive and negative controls were added to each reaction. The PCR products were used for direct sequencing for confirmation. RESULTS: The viral sequences were positive in 11/75 (14.7%) of our samples. CONCLUSION: The prevalence of EBV DNA was 11/75 (14.7%) in our glioma collection. Further molecular and epidemiological studies are needed to establish the possible role played by EBV in the tumorigenesis of gliomas.

Objective To evaluate the influence of polymorphisms in GSTA1, GSTM1, GSTT1, and GSTP1 in the risk of developing Prostate Cancer (PCa) in a population of Rio de Janeiro and compare the distribution of allele and genotype frequencies of the polymorphisms analyzed in the present study population with other regions in the country and different ethnic groups. Materials and Methods We analyzed a sample of the Brazilian population, comprising 196 patients with PCa treated by the urology services of the Brazilian National Cancer Institute (INCA) and Mario Kroeff Hospital (HMK), and 208 male blood donors from the Clementino Fraga Filho Hospital, Federal University of Rio de Janeiro (UFRJ). The polymorphisms were determined in DNA, extracted from peripheral blood leucocytes using the Polymerase Chain Reaction and Restriction Fragment Length Polymorphism (PCR-RFLP). Results Our results showed that the distribution of polymorphisms can vary significantly according to the Brazilian region and ethnic groups. The distribution of allele and genotype frequencies of the polymorphism GSTA1 was statistically different between cases and controls. Genotypes (A / B + B / B) were associated with protection (OR = 0.61, 95 % CI = 0.40-0.92) for PCa in comparison to genotype A / A. Conclusion The distribution of genotype frequencies of the polymorphism GSTA1 was statistically different between the case and control groups (p = 0.023), and the presence of genotypes A / B and B / B suggests a protective role against the risk of PCa compared to genotype A / A. This is the first study that reports the genotypic frequency of this polymorphism and its association with PCa in a Brazilian population sample.

Purpose To screen proteins/peptides in urine of Renal Cell Carcinoma (RCC) patients by SELDI-TOF (Surface Enhanced Laser Desorption Ionization - Time of Flight) in search of possible biomarkers. Material and Methods Sixty-one urines samples from Clear Cell RCC and Papillary RCC were compared to 29 samples of control urine on CM10 chip. Mass analysis was performed in a ProteinChip Reader PCS 4,000 (Ciphergen Biosystems, Fremont, CA) with the software Ciphergen Express 3.0. All chips were read at low and at high laser energy. For statistical analysis the urine samples were clustered according to the histological classification (Clear Cell and Papillary Carcinoma). For identification urine was loaded on a SDS PAGE gel and bands of most interest were excised, trypsinized and identified by MS/MS. Databank searches were performed in Swiss-Prot database using the MASCOT search algorithm and in Profound. Results Proteins that were identified from urine of controls included immunoglobulin light chains, albumin, secreted and transmembrane 1 precursor (protein K12), mannan-binding lectin-associated serine protease-2 (MASP-2) and vitelline membrane outer layer 1 isoform 1. Identification of immunoglobulins and isoforms of albumin are quite common by proteomics and therefore cannot be considered as possible molecular markers. K12 and MASP-2 play important physiological roles, while vitellite membrane outer layer 1 role is unknown since it was never purified in humans. Conclusions The down expression of Protein K-12 and MASP-2 make them good candidates for RCC urine marker and should be validated in a bigger cohort including the other less common histological RCC subtypes.

PurposeWe attempted to detect, for the first time in a Brazilian cohort, differences in protein expression between clear-cell renal cell carcinoma (ccRCC) and their normal adjacent tissues, aiming to identify biomarkers and/or therapeutic target candidates for this disease.Material and MethodsTwenty-four ccRCC and adjacent normal tissues were collected after surgery and their protein extracts were quantified, pooled and separated by two-dimensional polyacrylamide gel electrophoresis (2DE), followed by statistical analysis of the stained gels. Spots of interest were excised from the gels, digested with trypsin and identified by MALDI-TOF-TOF mass spectrometry.ResultsTwenty-six differential spots were detected between the two classes of tissues, among which twenty were identified by mass spectrometry and sixteen were found to be non-redundant. Eleven proteins were either underexpressed or undetected in the ccRCC extracts, such as prohibitin and peroxiredoxin-3, whereas five were found to be overexpressed or exclusively detected in the ccRCC extract, including αβ crystalin and heat shock protein 27.CONCLUSIONSSeveral proteins were detected at differential levels when compared to normal adjacent tissues, and, moreover, many have been previously described by their relationship with RCC. Therefore, this work corroborates previous reports on the search for biomarkers for ccRCC, as well as it points out new candidates that may be validated in future studies.

PurposeTo investigate the use of ClinProt technique to identify cancer markers in plasma of patients suffering from squamous cell carcinoma of the penis (SCCP).Materials and MethodsPlasma of 36 healthy subjects and 25 patients with penile carcinoma who underwent surgical treatment between June 2010 and June 2011 was collected and analyzed by the ClinProt/MALDI/ToF technique. Then the peptides were identified from the C8 MB eluted fraction of patients' and control subjects' plasma by LIFT MS/MS.ResultsA cluster of 2 peptides (A=m/z 1897.22 ± 9 Da and B=m/z 2021.99 ± 9 Da) was able to discriminate patients from control subjects. Cross validation analysis using the whole casuistic showed 62.5% and 86.76% sensitivity and specificity, respectively. The cluster also showed very high sensitivity (100%) and specificity (97%) for SCCP patients that died due to the disease. Furthermore, patients with lymph node involvement presented sensitivity and specificity of 80% and 97%, respectively. These two peptides were identified by the proteomic approach based on a MALDI-TOF/TOF as fragments of C3 (m/z 1896.17) and C4a/b (m/z 2021.26) complement proteins.ConclusionsThe results showed that as the disease progresses, the fragments C3 and C4 A/B are less expressed in comparison with healthy subjects. These results may be useful as prognostic tools.

Members of the Herpesviridae family have been implicated in a number of tumours in humans. At least 75% of the human population has had contact with cytomegalovirus (HCMV). In this work, we screened 75 Brazilian glioma biopsies for the presence of HCMV DNA sequences. HCMV DNA was detected in 36% (27/75) of the biopsies. It is possible that HCMV could be a co-factor in the evolution of brain tumours.

Penile cancer is a potentially mutilating disease. Although its occurrence is relatively rare worldwide, penile cancer rates can be high in developing countries. A few studies have been conducted on the involvement of human papillomavirus (HPV) in penile carcinoma, which have found HPV present in 30-70% of penile malignant lesions, with a higher prevalence of HPV 16 and 18. It has been assumed that cofactors, such as Epstein-Barr virus (EBV) infections, may play a role in the progression of penile neoplasia. The aim of this study was to determine HPV and EBV prevalence in 135 penile malignant lesions from Brazilian men through the use of MY09/11 polymerase chain reaction (PCR), type-specific PCR and restriction fragment length polymorphism analysis. HPV prevalence among the men tested was 60.7%. Of the men who tested positive, 27 presented with HPV 16 (29.7%), five with HPV 18 (5.5%), 21 with HPV 45 (23.1%) and nine with HPV 6 (9.9%). Seven mixed infections were detected (9.2%), while 11 cases remained untyped (13.4%). Regarding EBV positivity, 46.7% of the samples contained EBV DNA with EBV-1 as the most prevalent type (74.6%). More than 23% of the men were co-infected with both HPV and EBV, while 35% presented exclusively with HPV DNA and 20% presented only with EBV DNA. Penile carcinoma aetiology has not been fully elucidated and the role of HPV and EBV infections individually or synergistically is still controversial. Hence, more studies are needed to determine their possible role in carcinogenesis.