A comparison of four commercial kits used for isolating circulating cell-free DNA: QuickGeneMINI8L (Kurabo), Maxwell RSC cfDNA Plasma Kit (Promega), cfKapture 21 Kit (MagBio), and QIAamp MinElute ccfDNA Kit (Qiagen)

A minimally-invasive alternative to surgical biopsies is a liquid biopsy (LB), a technique that has recently revolutionized the management of a number of tumors. One potential target biomarker of LB is cell-free DNA (cfDNA), which can act as a very sensitive indicator for certain tumors. Currently, clinical efforts are focused on increasing the quality of the cfDNA isolated for analysis. The present study compares the efficiency of isolation by four commercial kits: QuickGeneMINI8L (Kurabo), Maxwell RSC cfDNA Plasma Kit (Promega), cfKapture 21 Kit (MagBio), and QIAamp MinElute ccfDNA Kit (Qiagen). In each case, cfDNA was isolated from three plasma samples and one serum sample. Available method for the isolation give the ability to enrich optimal diagnostic quantity of cfDNA. cfDNA can be successfully separated using all investigated kits. The greatest efficiency was demonstrated by the QIAamp MinElute ccfDNA Kit (Qiagen) and cfKapture 21 (MagBio). Large amounts of cell-free DNA can be successfully isolated from small volumes of plasma

Dysregulation of microRNAs in triple-negative breast cancer

Objectives: Triple-negative breast cancer (TNBC) is an aggressive subtype of breast cancer with limited treatment options and poor prognosis. TNBC is usually diagnosed at a relatively young age and is characterized by high risk of developing metastases. Some epigenetic regulation of gene expression is associated with TNBC. Expression of microRNAs (miRNAs) can serve as a potential tool for identifying critical biomarkers in TNBC. The aim of our study is to examine expression of selected miRNAs in TNBC and to assess the relationship between miRNA expression and clinicopathological factors. Material and methods: Expression levels of 19 selected miRNAs were compared between cancerous and normal breast tissues by use of qPCR method. We have evaluated the relationship between the expression level of miRNAs and clinicopathological factors such as: age, tumor size and lymph node status. Results: We found that in TNBC tissues, when compared with normal breast tissues, the expression of miR-190a, miR- 136-5p and miR-126-5p was significantly reduced (p = 0.0041, p = 0.0007, p = 0.0007, respectively) whereas expression of miR-135b-5p and miR-182-5p was significantly increased (p = 0.0194, p = 0.0041, respectively). We found a linear trend for tumor size and expression of miR-126-5p (p = 0.0296) and miR-135b-5p (p = 0.0241). Conclusions: Our study confirms that miRNA expression profile is dysregulated in TNBC patients compared to healthy controls. MiR-190a, miR-136-5p, miR-126-5p, miR-135b-5p and miR-182-5p may be associated with development and progression of TNB

Association of microRNAs and pathologic response to preoperative chemotherapy in triple negative breast cancer: preliminary report

Triple negative breast cancer (TNBC) has caught the attention of oncologists worldwide because of poor prognosis and paucity of targeted therapies. Gene pathways have been widely studied, but less is known about epigenetic factors such as microRNAs (miRNAs) and their role in tailoring an individual systemic and surgical approach for breast cancer patients. The aim of the study was to examine selected miRNAs in TNBC core biopsies sampled before preoperative chemotherapy and the subsequent pathologic response in mastectomy or breast conservation specimens. Prior to treatment, core needle biopsies were collected from 11 female patients with inoperable locally advanced TNBC or large resectable tumors suitable for down-staging. In all 11 TNBC core biopsies we analyzed 19 miRNAs per sample: 512, 190, 200, 346, 148, 449, 203, 577, 93, 126, 423, 129, 193, 182, 136, 135, 191, 122 and 222 (miRCURY LNA™ Universal RT microRNA polymerase chain reaction Custom Pick & Mixpanels). The Wilcoxon signed-rank test was used to compare related samples. Ingenuity pathway analysis was used to evaluate potential functional significance of differentially expressed miRNAs. Statistical analysis showed that 3 of 19 miRNAs differed in relation to pathologic response i.e. good versus poor. These differences failed to reach statistical significance, although a trend was observed (p = 0.06). Among these miRNAs, we identified—miR-200b-3p, miR-190a and miR-512-5p. In summary, our results indicate that higher miR-200b-3p, higher miR-190a and lower miR-512-5p expression levels in core biopsies sampled from TNBC patients may be associated with better pathologic response to chemotherapy and the increased feasibility of breast conserving surgery in these patients. Although these results were from a small cohort, they provide an important basis for larger, prospective, multicenter studies to investigate the potential role of miRNAs in neoadjuvant setting

Reticular basement membrane thickness is associated with growth : and fibrosis-promoting airway transcriptome profile-study in asthma patients

Airway remodeling in asthma is characterized by reticular basement membrane (RBM) thickening, likely related to epithelial structural and functional changes. Gene expression profiling of the airway epithelium might identify genes involved in bronchial structural alterations. We analyzed bronchial wall geometry (computed tomography (CT)), RBM thickness (histology), and the bronchial epithelium transcriptome profile (gene expression array) in moderate to severe persistent (n = 21) vs. no persistent (n = 19) airflow limitation asthmatics. RBM thickness was similar in the two studied subgroups. Among the genes associated with increased RBM thickness, the most essential were those engaged in cell activation, proliferation, and growth (e.g., CDK20, TACC2, ORC5, and NEK5) and inhibiting apoptosis (e.g., higher mRNA expression of RFN34, BIRC3, NAA16, and lower of RNF13, MRPL37, CACNA1G). Additionally, RBM thickness correlated with the expression of genes encoding extracellular matrix (ECM) components (LAMA3, USH2A), involved in ECM remodeling (LTBP1), neovascularization (FGD5, HPRT1), nerve functioning (TPH1, PCDHGC4), oxidative stress adaptation (RIT1, HSP90AB1), epigenetic modifications (OLMALINC, DNMT3A), and the innate immune response (STAP1, OAS2). Cluster analysis revealed that genes linked with RBM thickness were also related to thicker bronchial walls in CT. Our study suggests that the pro-fibrotic profile in the airway epithelial cell transcriptome is associated with a thicker RBM, and thus, may contribute to asthma airway remodeling

cDNA sequencing improves the detection of P53 missense mutations in colorectal cancer

<p>Abstract</p> <p>Background</p> <p>Recently published data showed discrepancies beteween <it>P53 </it>cDNA and DNA sequencing in glioblastomas. We hypothesised that similar discrepancies may be observed in other human cancers.</p> <p>Methods</p> <p>To this end, we analyzed 23 colorectal cancers for <it>P53 </it>mutations and gene expression using both DNA and cDNA sequencing, real-time PCR and immunohistochemistry.</p> <p>Results</p> <p>We found <it>P53 </it>gene mutations in 16 cases (15 missense and 1 nonsense). Two of the 15 cases with missense mutations showed alterations based only on cDNA, and not DNA sequencing. Moreover, in 6 of the 15 cases with a cDNA mutation those mutations were difficult to detect in the DNA sequencing, so the results of DNA analysis alone could be misinterpreted if the cDNA sequencing results had not also been available. In all those 15 cases, we observed a higher ratio of the mutated to the wild type template by cDNA analysis, but not by the DNA analysis. Interestingly, a similar overexpression of <it>P53 </it>mRNA was present in samples with and without <it>P53 </it>mutations.</p> <p>Conclusion</p> <p>In terms of colorectal cancer, those discrepancies might be explained under three conditions: 1, overexpression of mutated <it>P53 </it>mRNA in cancer cells as compared with normal cells; 2, a higher content of cells without <it>P53 </it>mutation (normal cells and cells showing <it>K-RAS </it>and/or <it>APC </it>but not <it>P53 </it>mutation) in samples presenting <it>P53 </it>mutation; 3, heterozygous or hemizygous mutations of <it>P53 </it>gene. Additionally, for heterozygous mutations unknown mechanism(s) causing selective overproduction of mutated allele should also be considered. Our data offer new clues for studying discrepancy in <it>P53 </it>cDNA and DNA sequencing analysis.</p

Genetic risk factors of Alzheimer’s disease

Introduction. Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, which is a serious health problem for societies that live longer. Spontaneous dominant mutations and polymorphisms of selected genes play an important role in development of AD. Aim. Several polymorphisms in selected genes strongly associated with development of Alzheimer’s disease were highlighted in this review: APOE, CYP46, APP, PSEN1, PSEN2, UBQLN1, BACE1, PRND, APBB2, TOMM 40. These gene polymorphisms have a significant role in the development of Alzheimer’s disease and they have potential to be biomarkers. Researchers combine efforts to find significant polymorphisms that would ensure that a person is predisposed to the occurrence of disease symptoms. This topic is often taken up by scientists seeking to develop effective genetic tests for diagnosing AD. Material and methods. Analysis of literature from web of knowledge: Web of Science (all database), NCBI and PubMed. Results. We reviewed the selected important genes and polymorphisms which are most often associated with development of AD. Conclusion. It should be noted that nowadays scientists strive not to focus on only one polymorphism in the gene but on several polymorphisms in different genes concomitantly and above all on interactions between them to the diagnosis of this disease. Only this approach to AD will contribute to the creation of appropriate identification methods. Moreover, we should use the new generation tools - the platform for collecting data and personalized medicine

Genetic risk factors of Alzheimer’s disease

Introduction. Alzheimer’s disease (AD) is one of the most common neurodegenerative diseases, which is a serious health problem for societies that live longer. Spontaneous dominant mutations and polymorphisms of selected genes play an important role in development of AD. Aim. Several polymorphisms in selected genes strongly associated with development of Alzheimer’s disease were highlighted in this review: APOE, CYP46, APP, PSEN1, PSEN2, UBQLN1, BACE1, PRND, APBB2, TOMM 40. These gene polymorphisms have a significant role in the development of Alzheimer’s disease and they have potential to be biomarkers. Researchers combine efforts to find significant polymorphisms that would ensure that a person is predisposed to the occurrence of disease symptoms. This topic is often taken up by scientists seeking to develop effective genetic tests for diagnosing AD. Material and methods. Analysis of literature from web of knowledge: Web of Science (all database), NCBI and PubMed. Results. We reviewed the selected important genes and polymorphisms which are most often associated with development of AD. Conclusion. It should be noted that nowadays scientists strive not to focus on only one polymorphism in the gene but on several polymorphisms in different genes concomitantly and above all on interactions between them to the diagnosis of this disease. Only this approach to AD will contribute to the creation of appropriate identification methods. Moreover, we should use the new generation tools - the platform for collecting data and personalized medicine