CME Exam 3: Real-time Targeted Genome Profile Analysis of Pancreatic Ductal Adenocarcinomas Identifies Genetic Alterations that Might be Targeted With Existing Drugs or Used as Biomarkers

ABSTRACT Primary effusion lymphomas (PELs) are specifically associated with Kaposi's sarcoma-associated herpesvirus (KSHV) infection and most frequently occur in human immunodeficiency virus (HIV)-positive individuals as lymphomatous effusions in the serous cavities without a detectable solid tumor mass. Most PELs have concomitant Epstein-Barr virus (EBV) infection, suggesting that EBV is an important pathogenetic cofactor, although other as yet unidentified cofactors, such as cellular genetic alterations, are also likely to play a role. Lymphomatous effusions that lack KSHV also occur; these are frequently EBV associated in the setting of HIV infection. Here we used gene expression profile analysis to determine the viral impact on cellular gene expression and the pathogenesis of these lymphomatous effusions. Our results show that many genes, including cell cycle and signal transduction regulators, are differentially expressed between KSHV-positive PELs and KSHV-negative lymphomatous effusions and also between KSHV-positive, EBV-positive and KSHV-positive, EBV-negative PELs. Our results confirm that KSHV plays an important role in the pathogenesis of PELs, as its presence selects for a very distinct cellular gene expression category and a clearly different lymphoma type. Within the KSHV-positive PELs, the effect of EBV is more subtle but nevertheless clear.

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Expression and Functional Assessment of Some Featured Coding and Non-coding RNAs Encoded by 8q24 Chromosomal Region in CML Patients

Jentashapir Journal of Cellular and Molecular Biology ◽

10.5812/jjcmb.112986 ◽

2021 ◽

Vol In Press (In Press) ◽

Author(s):

Mina Zamani ◽

Hamid Galehdari ◽

Babak Bakhshinejad ◽

Mohammad-Reza Hajjari ◽

Ali-Mohammad Foroughmand

Keyword(s):

Real Time ◽

Chromosomal Region ◽

Correlation Coefficients ◽

Genetic Alterations ◽

Gene Expressions ◽

Altered Expression ◽

Critical Pathways ◽

Myc Oncogene ◽

Non Coding Rnas ◽

8Q24 Region

Background: The association between the human chromosomal 8q24 region and cancer development remains dim. The proto-oncogene MYC is known as the most prominent target of this chromosomal region. However, numerous cancer-associated genetic alterations in the region extend beyond the MYC locus. Accordingly, it is likely that the MYC oncogene is not the only target of these carcinogenesis-related alterations. Objectives: In the present study, the expression of MYC and the correlation between MYC and two non-coding RNAs, namely PVT1 (circular and linear forms) and CASC11, which are residents of the 8q24 region in the MYC neighborhood, were investigated in chronic myeloid leukemia (CML). Methods: Real-time polymerase chain reaction (PCR) was used to assess BCR-ABL transcripts and categorize positive and negative (normal) samples for CML. Afterward, real-time PCR was exploited to evaluate the expression of different genes, including MYC, linear PVT1, circular PVT (CircPVT1), CASC11, and ACTB in CML and normal samples. Results: We found that the expression of linear PVT1 is significantly increased in CML compared with normal samples. However, CircPVT1, CASC11, and MYC did not show significantly altered expression between CML and normal groups. The experimental and in silico analyses of the correlation coefficients of gene expressions suggested changes in the correlations between the gene expressions in CML compared with normal samples. We also assessed the miR-trapping potential of PVT1 and CASC11 and the possible effects of these interactions on signaling pathways. Our findings indicated that these lncRNAs could have a possible regulatory link with critical pathways associated with leukemogenesis. Conclusions: Our results indicate that non-coding genes surrounding MYC within the 8q24 region might have regulatory roles in CML carcinogenesis.

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Sequential transformation of 4-androstenedione into dihydrotestosterone in prostate carcinoma (DU-145) cells indicates that 4-androstenedione and not testosterone is the substrate of 5α-reductase

Hormone Molecular Biology and Clinical Investigation ◽

10.1515/hmbci.2010.009 ◽

2010 ◽

Vol 1 (2) ◽

Cited By ~ 2

Author(s):

Melanie Samson ◽

Fernand Labrie ◽

Van Luu-The

Keyword(s):

Real Time ◽

Prostate Carcinoma ◽

Real Time Pcr ◽

Profile Analysis ◽

Reductase Activity ◽

Hydroxysteroid Dehydrogenase ◽

Pcr Analysis ◽

No Production ◽

Reduction Step ◽

Type 3

Abstract: Although it is well recognized that 5α-reductases possess higher affinity for 4-androstenedione than testosterone, and the affinity of 4-androstenedione is higher for 5α-reductases than 17β-hydroxysteroid dehydrogenases, it is generally believed that dihydrotestosterone is necessarily produced by the transformation of testosterone into dihydrotestosterone, suggesting that the step catalyzed by 17β-hydroxysteroid dehydrogenase precedes the step catalyzed by 5α-reductase. This interpretation is in contradiction with the enzymatic kinetic law that suggests that the 5α-reduction step that catalyzes the transformation of 4-dione into 5α-androstane-3,17-dione precedes the 17keto-reduction step.: To verify which of these two pathways is operative, we quantified mRNA expression levels of steroidogenic enzymes in prostate carcinoma DU-145 cells by real-time PCR and determined the metabolites produced after incubation with [: Real-time PCR analysis strongly suggests that the new type 3 5α-reductase is responsible for 5α-reductase activity in DU-145 cells. Steroid profile analysis shows that in the absence of inhibitor 5α-androstanedione is first produced, followed by the production of androsterone and dihydrotestosterone. The concentration of testosterone was not detectable. In the presence of Finasteride, an inhibitor of 5α-reductase, there was no transformation of 4-androstenedione and also there was no production of testosterone. The present data clearly indicate that the biosynthesis of dihydrotestosterone in DU-145 cells does not require testosterone as intermediate, and the step catalyzed by 5α-reductase precedes the step catalyzed by 17β-hydroxysteroid dehydrogenase.

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Implementation of an NGS panel for clinical practice in paraffin-embedded tissue samples from locally advanced and metastatic melanoma patients

10.37349/etat.2020.00006 ◽

2020 ◽

Vol 1 (2) ◽

pp. 101-108

Author(s):

Paola Castillo ◽

Marta Marginet ◽

Pedro Jares ◽

Mireia García ◽

Elena Gonzalvo ◽

...

Keyword(s):

Polymerase Chain Reaction ◽

Clinical Practice ◽

Metastatic Melanoma ◽

Real Time ◽

Genetic Alterations ◽

Advanced Melanoma ◽

Chain Reaction ◽

Number Of Patients ◽

Targeted Ngs ◽

Polymerase Chain

Aim: Single biomarker diagnostic test of BRAFV600 locus in metastatic melanoma is mandatory for treatment decision; however, multiple-gene based techniques, such as targeted next-generation sequencing (NGS) are being used to maximize the number of patients that can benefit from a targeted therapy. The main objective of this study is to investigate whether an NGS panel could be adopted in routine clinical care for advanced melanoma. Methods: Patients diagnosed with advanced melanoma at our center from 2017 to 2019 were included. Presence of genetic alterations was performed using two methods: real-time polymerase chain reaction-based Idylla test (Biocartis) and NGS with the oncomine solid tumor DNA kit (Thermo Fisher Scientific). Total genomic DNA was extracted from formalin-fixed and paraffin embedded samples for sequencing. Results: A total of 155 samples were evaluated for molecular analysis but 40 samples (25.8%) were inadequate for sequencing. The clinical utility of BRAFV600 real-time polymerase chain reaction and targeted-NGS was compared in 29 samples and a very good concordance was observed (Kappa = 0.89, 95% confidence interval 0.68 ± 1.05). An oncogenic mutation by NGS was found in 75 samples (65%) – 53% of whom were candidates for personalized therapies. The most prevalent mutated genes were BRAF (39%), TP53 (23%), and NRAS (14%). Other genes identified at lower incidence (< 5%) were: PIK3CA, ERBB4, CTNNB1, STK11, FGFR1, SMAD4, KRAS, FGFR3, PTEN and AKT. Co-occurrence of oncogenic mutations was detected in 40% of the samples. Among the mutations identified, TP53 was significantly more prevalent in men (men 31.8% versus women 12.2%, P = 0.03) and NRAS in women (men 9.1% versus women 24.4%, P = 0.03). Conclusions: Targeted-NGS testing is a feasible technique to implement in the routine clinical practice. Based on our results, NGS has provided more information on target-genes than RT-PCR technique, maximizing the benefit for patients with advanced melanoma.

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Rapid and Quantitative Assessment of Cell Quality, Identity, and Functionality for Cell-Based Assays Using Real-Time Cellular Analysis

CrossRef Listing of Deleted DOIs ◽

10.1177/1087057110397359 ◽

2011 ◽

Vol 16 (3) ◽

pp. 313-322 ◽

Cited By ~ 30

Author(s):

Jeffrey T. Irelan ◽

Meng-Jou Wu ◽

Jonathan Morgan ◽

Ning Ke ◽

Biao Xi ◽

...

Keyword(s):

Quality Control ◽

Cell Line ◽

Real Time ◽

Cell Lines ◽

Quantitative Measure ◽

Genetic Alterations ◽

Cellular Analysis ◽

Identical Cell ◽

Reproducible Method ◽

Impedance Profile

Strict quality control of cells is required for the standardization and interpretation of results in all areas of cell-based research, especially in drug discovery. Real-time cellular analysis using electrical impedance as a readout offers a rapid and highly reproducible method for quality control as it provides a quantitative measure of overall cell morphology and growth. In a case study, the authors demonstrate that samples of a single cell line obtained from several different labs show clear differences in their impedance profiles when compared with the corresponding standard cell line. A number of kinetic parameters were derived from the impedance profiles and used to quantify the differences among these cell lines. Our findings indicate that this methodology can detect cell line differences including mix-ups or contaminations, genetic alterations, and potential epigenetic changes occurring during passaging, all of which can occur in the time scale of a screening campaign. Finally, we provide evidence that these impedance profile differences can be predictive of different outcomes in cell-based functional assays for the effects of small molecules on otherwise seemingly identical cell lines.

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Genetic alterations of SUGP1 mimic mutant-SF3B1 splice pattern in lung adenocarcinoma and other cancers

Oncogene ◽

10.1038/s41388-020-01507-5 ◽

2020 ◽

Vol 40 (1) ◽

pp. 85-96

Author(s):

Samar Alsafadi ◽

Stephane Dayot ◽

Malcy Tarin ◽

Alexandre Houy ◽

Dorine Bellanger ◽

...

Keyword(s):

Lung Adenocarcinoma ◽

Profile Analysis ◽

Genetic Alterations ◽

Splicing Factor ◽

Definitive Evidence ◽

Splicing Pattern ◽

Comprehensive Determination ◽

Lung Adenocarcinomas ◽

Site Recognition

AbstractGenes involved in 3′-splice site recognition during mRNA splicing constitute an emerging class of oncogenes. SF3B1 is the most frequently mutated splicing factor in cancer, and SF3B1 mutants corrupt branchpoint recognition leading to usage of cryptic 3′-splice sites and subsequent aberrant junctions. For a comprehensive determination of alterations leading to this splicing pattern, we performed a pan-TCGA screening for SF3B1-specific aberrant acceptor usage. While the most of aberrant 3′-splice patterns were explained by SF3B1 mutations, we also detected nine SF3B1 wild-type tumors (including five lung adenocarcinomas). Genomic profile analysis of these tumors identified somatic mutations combined with loss-of-heterozygosity in the splicing factor SUGP1 in five of these cases. Modeling of SUGP1 loss and mutations in cell lines showed that both alterations induced mutant-SF3B1-like aberrant splicing. Our study provides definitive evidence that genetic alterations of SUGP1 genocopy SF3B1 mutations in lung adenocarcinoma and other cancers.

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