1,720,959 research outputs found
Higher random oligo concentration improves reverse transcription yield of cDNA from bioptic tissues and quantitative RT-PCR reliability.
No full textReal time quantitative reverse transcription-PCR (qRT-PCR) is the most sensitive technique for detection and quantification of mRNA targets. Reliable quantification of gene expression in formalin-fixed, paraffin- embedded tissues (FFPE), however, has been subjected to serious limitations so far, mainly due to the fragmentation of RNA transcripts. We tried to improve the sensitivity and reliability of mRNA quantification in FFPE by boosting the reverse transcription (RT) step, that is neglected in most of the protocol analysis, but that represents the first confounding event in a quantitative analysis. For this purpose, we compared yield, reproducibility and linearity of RTs performed with random hexamers, random pentadecamers, or a mixture of antisense specific primers in presence of either Moloney murine leukemia virus (MmLV) or the avian myeloblastosis virus (AMV) enzymes. Random primers were tested at two concentrations, 0.14 and 3.35 nmol/reaction. Our qRT-PCR results indicate an
Management of stage II colon cancer - the use of molecular biomarkers for adjuvant therapy decision
No full textBackground: There is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy. Methods: We examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), beta-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded. Results: The presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05). Conclusions: Patients' characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer
Thymidilate synthase expression predicts longer survival in patients with stage II colon cancer treated with 5-flurouracil independently of microsatellite instability
No full textBACKGROUND: 5-Fluorouracil (5-FU) is the most commonly used therapeutic agent for colon cancer treatment. Several studies have evaluated in patients with colon cancer, either the role of genes involved in the 5-FU pathway, such as thymidylate synthase (TS), thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) or the role of microsatellite instability (MSI) as prognostic or predictive markers for adjuvant chemotherapy efficacy, with discordant results. In this study we investigated the combined effect of TS, TP, DPD mRNA expression and MSI status in primary tumors of patients with colon cancer, all treated with 5-FU adjuvant therapy. METHODS: TS, TP and DPD expression levels were investigated by real-time quantitative RT-PCR on RNA extracts from formalin-fixed and paraffin-embedded tissues of 55 patients with colon adenocarcinoma. In the same case study MSI status was assessed on DNA extracts. RESULTS: A higher TS expression was significantly associated with a longer survival for patients with cancers of stage II (P < 0.01), but not for those with stage III (P = 0.68). In addition, in multivariate analysis, a higher TS expression was significantly associated with a decreased risk of death (HR 0.13, 95% CI 0.03-0.59, P < 0.01), while the MSI status did not have effects on patients' survival. CONCLUSIONS: This retrospective investigation suggests that TS gene expression at mRNA level can be a useful marker of better survival in patients (especially of those with cancers of stage II) receiving 5-FU adjuvant chemotherapy, independently of the MSI status
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Variations on the Author
Full text link“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Full text linkWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Reverse translational research in colorectal cancer
Full text link2010/2011Background. A major clinical problem in oncology is that patients with tumors of the same stage and histological type often do not have the same prognosis and they do not show the same response to the therapeutic treatments. This is a major concern in colorectal cancer (CRC), because of its high incidence and impact on public health.
Aim. The main goal of the present work is to identify a set of markers of prognosis and therapy efficacy in CRC patients using a reverse translational research approach. In the first part of the work, we focused on the selection criteria to submit stage II colon cancer patients to 5-FU based adjuvant therapy, while in the second part we searched for new molecular targets to predict the response to cetuximab or panitumumab in the metastatic setting.
Methods. To the first purpose, we evaluated the mRNA expression levels of thymidylate synthase (TS) by qRT-PCR and IHC, the MMR (mismatch repair system) status by immunohistochemistry (IHC), the CIMP (CpG island methylator phenotype) status by methylation specific PCR (MSP) and the expression of p53, β-catenin, GSK3β and CD8 by IHC on a retrospective case study comprising 60 stage II colon cancer patients submitted to 5-FU adjuvant therapy and 60 colon cancer patients who did not receive therapy. Results of molecular analyses were compared with clinical-pathological factors and histological features of the cancers. To the second purpose, we evaluated KRAS mutational status by direct sequencing and an alteration at the DNA level in one of the genes belonging to the EGFR pathway in a retrospective case study comprising 98 recurrent CRC patients treated with cetuximab or panitumumab and 65 recurrent patients who did not receive treatment. mRNA expression levels of the EGFR-pathway gene were evaluated by qRT-PCR. Information about the gene and the method for alteration assessment cannot however be disclosed because of patent pending.
Results. In the first part of the study, we found a better survival in patients with a high mRNA expression level of TS and treated with 5-FU (87% survival for TS high versus 60% for TS low at 5 years of follow up) while an opposite behaviour was displayed by patients who did not received any therapy (83% survival for low TS versus 60% for high TS expressors at five years of follow up). These results were independent from clinical-pathological and the other examined molecular factors. Molecular parameters and clinical-pathological variables did not show any correlation with patients’ survival, with the exception of the presence of tumoral CD8+ infiltrating lymphocytes, which was a good prognostic factor (84% survival for patients with tumor infiltrated versus 63% for those without infiltration at 5 years of follow up), independently of 5-FU treatment. In the second part of the study we found, as expected, an advantage in progression free survival in patients having a wild type K-RAS, but a greater advantage in patients showing one of the types related to the alteration we evaluated (81% survival for patients with the type 1 alteration versus 51% for KRAS wild type patients at 6 months of follow up). The effect of such alteration was specifically related to the treatment with monoclonal antibodies because it was lost in the group of 65 recurrent patients without cetuximab/panitumumab treatment. We also found that higher mRNA levels of the EGFR pathway gene were predictive of better benefit from the therapy with monoclonal antibodies and this effect was shown to be cumulative with a wild type KRAS status.
Conclusions. As far it concerns the first part of the study, we therefore identified mRNA expression level of thymidylate synthase (TS), the 5-FU main cellular target, as the most important discriminator in the decision of which stage II colon cancer patients should be submitted to adjuvant 5-FU therapy. This evidence confirms the results obtained previously with a different training set of patients. With respect to the second part of the study, we identified an alteration at the DNA level in one of the genes belonging to the EGFR pathway as a new biomarker of cetuximab/panitumumab treatment efficacy in recurrent CRC patients.XXIV Cicl
Dispelling the Myths Behind First-author Citation Counts
Full text linkWe conducted a full-scale evaluative citation analysis study of scholars in the XML research field to explore just how different from each other author rankings resulting from different citation counting methods actually are, and to demonstrate the capability of emerging data and tools on the Web in supporting more realistic citation counting methods. Our results contest some common arguments for the continued
use of first-author citation counts in the evaluation of scholars, such as high correlations between author rankings by first-author citation counts and other citation
counting methods, and high costs of using more realistic citation counting methods that are not well-supported by the ISI databases. It is argued that increasingly available digital full text research papers make it possible for citation analysis studies to go beyond what the ISI databases have directly supported and to employ more
sophisticated methods
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