NTRK, RET, BRAF, and ALK fusions in thyroid fine-needle aspirates (FNAs).

6083 Background: Receptor tyrosine kinase (RTK) fusions may be targeted by small molecule inhibitors to treat various advanced tumors, including thyroid cancer. Clinical trials have studied selective inhibitors of ALK, BRAF, NTRK and RET, leading to several FDA-approved therapies. The Afirma Genomic Sequencing Classifier (GSC) classifies cytologically indeterminate thyroid nodules as molecularly benign or suspicious. The Xpression Atlas reports 905 genomic variants and 235 fusion pairs on GSC Suspicious, Suspicious for Malignancy (SFM), and Malignant FNA samples at the time of diagnosis. Here we report the prevalence of these fusion genes in real-world clinical practice. Methods: We analyzed anonymized data from 50,644 consecutive Bethesda III-VI nodule FNA samples submitted to the Veracyte CLIA laboratory for molecular testing using whole transcriptome RNA sequencing (RNA-Seq). Gene pairs are listed alphabetically. Results: 32,080 Bethesda III/IV nodules were classified as GSC Benign and 278 were Parathyroid Classifier positive. No ALK, BRAF, NTRK1/3, or RET fusions were identified among these samples. Among 16,594 Bethesda III/IV GSC Suspicious FNAs, 3% (n = 529) were positive for ALK, BRAF, NTRK1/3 or RET fusions. Among the 1,692 Bethesda V/VI FNAs, the proportion of positive nodules was 8% (n = 135). Among these combined cohorts of Bethesda III/IV GSC Suspicious and Bethesda V/VI, the most common gene fusions observed for each of the 5 studied RTK genes was: ETV6/NTRK3 (n = 164, 72% of NTRK3 fusions), CCDC6/RET (n = 104, 55% of RET), BRAF/SND1 (n = 32, 20% of BRAF), ALK/STRN (n = 20, 37% of ALK), and NTRK1/TPM3 (n = 14, 50% of NTRK1). BRAF showed the highest diversity of fusions, with 80 gene partners. Different gene partners with RET, ALK, NTRK1, and NTRK3 numbered 25, 11, 9, and 5 , respectively . Conclusions: Whole-transcriptome RNA-seq on small sample thyroid FNA specimens can identify clinically relevant ALK, BRAF, NTRK, and RET fusions across Bethesda categories. The prevalence ranges from 3% in Bethesda III/IV Afirma GSC Suspicious specimens to 8% among Bethesda V/VI specimens. Future studies need to determine if detection of precision medicine candidates by pre-operative FNA can optimize initial treatment, predict response to treatment, and prioritize selective targeted therapy should systemic treatment be needed.[Table: see text]

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Moving towards a local testing solution for undetermined thyroid fine-needle aspirates: validation of a novel custom DNA-based NGS panel

Journal of Clinical Pathology ◽

10.1136/jclinpath-2021-207429 ◽

2021 ◽

pp. jclinpath-2021-207429

Author(s):

Roberta Sgariglia ◽

Mariantonia Nacchio ◽

Ilaria Migliatico ◽

Elena Vigliar ◽

Umberto Malapelle ◽

...

Keyword(s):

Local Level ◽

Needle Aspiration ◽

Molecular Testing ◽

Health Coverage ◽

Fine Needle ◽

Fine Needle Aspirates ◽

Gene Test ◽

Thyroid Fine Needle Aspiration ◽

Thyroid Fna ◽

Prohibitive Cost

AimsIn thyroid cytopathology, the undetermined diagnostic categories still pose diagnostic challenges. Although next-generation sequencing (NGS) is a promising technique for the molecular testing of thyroid fine-needle aspiration (FNA) specimens, access to such technology can be difficult because of its prohibitive cost and lack of reimbursement in countries with universal health coverage. To overcome these issues, we developed and validated a novel custom NGS panel, Nexthyro, specifically designed to target 264 clinically relevant mutations involved in thyroid tumourigenesis. Moreover, in this study, we compared its analytical performance with that of our previous molecular testing strategy.MethodsThe panel, which includes 15 genes (BRAF, EIF1AX, GNAS, HRAS, IDH1, KRAS, NF2, NRAS, PIK3CA, PPM1D, PTEN, RET, DICER1, CHEK2, TERT promoter), was validated with a cell-line derived reference standard and 72 FNA archival samples previously tested with the 7-gene test.ResultsNexthyro yielded 100% specificity and detected mutant alleles at levels as low as 2%. Moreover, in 5/72 (7%) FNAs, it detected more clinically relevant mutations in BRAF and RAS genes compared with the 7-gene test. Nexthyro also revealed better postsequencing metrics than the previously adopted commercial ‘generic’ NGS panel.ConclusionOur comparative analysis indicates that Nexthyro is a reliable NGS panel. The study also implies that a custom-based solution for routine thyroid FNA is sustainable at the local level, allowing patients with undetermined thyroid nodules affordable access to NGS.

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High heterogeneity undermines generalization of differential expression results in RNA-Seq analysis

Human Genomics ◽

10.1186/s40246-021-00308-5 ◽

2021 ◽

Vol 15 (1) ◽

Author(s):

Weitong Cui ◽

Huaru Xue ◽

Lei Wei ◽

Jinghua Jin ◽

Xuewen Tian ◽

...

Keyword(s):

Gene Expression ◽

Differential Expression ◽

Small Sample ◽

Differentially Expressed ◽

Cancer Type ◽

Rna Seq ◽

Sample Sizes ◽

Large Sample ◽

Expression Levels ◽

Gene Expression Levels

Abstract Background RNA sequencing (RNA-Seq) has been widely applied in oncology for monitoring transcriptome changes. However, the emerging problem that high variation of gene expression levels caused by tumor heterogeneity may affect the reproducibility of differential expression (DE) results has rarely been studied. Here, we investigated the reproducibility of DE results for any given number of biological replicates between 3 and 24 and explored why a great many differentially expressed genes (DEGs) were not reproducible. Results Our findings demonstrate that poor reproducibility of DE results exists not only for small sample sizes, but also for relatively large sample sizes. Quite a few of the DEGs detected are specific to the samples in use, rather than genuinely differentially expressed under different conditions. Poor reproducibility of DE results is mainly caused by high variation of gene expression levels for the same gene in different samples. Even though biological variation may account for much of the high variation of gene expression levels, the effect of outlier count data also needs to be treated seriously, as outlier data severely interfere with DE analysis. Conclusions High heterogeneity exists not only in tumor tissue samples of each cancer type studied, but also in normal samples. High heterogeneity leads to poor reproducibility of DEGs, undermining generalization of differential expression results. Therefore, it is necessary to use large sample sizes (at least 10 if possible) in RNA-Seq experimental designs to reduce the impact of biological variability and DE results should be interpreted cautiously unless soundly validated.

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184 Whole-transcriptome analysis by RNA-Seq for genetic diagnosis of Mendelian skin disorders in the context of consanguinity

Journal of Investigative Dermatology ◽

10.1016/j.jid.2021.02.204 ◽

2021 ◽

Vol 141 (5) ◽

pp. S32

Author(s):

L. Youssefian ◽

A. Saeidian ◽

P. Fortina ◽

A. South ◽

J. Uitto ◽

...

Keyword(s):

Transcriptome Analysis ◽

Genetic Diagnosis ◽

Rna Seq ◽

Skin Disorders ◽

Whole Transcriptome Analysis ◽

Whole Transcriptome

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Plasma IL-6 levels following corticosteroid therapy as an indicator of ICU length of stay in critically ill COVID-19 patients

Cell Death Discovery ◽

10.1038/s41420-021-00429-9 ◽

2021 ◽

Vol 7 (1) ◽

Author(s):

Samir Awasthi ◽

Tyler Wagner ◽

A. J. Venkatakrishnan ◽

Arjun Puranik ◽

Matthew Hurchik ◽

...

Keyword(s):

Distress Syndrome ◽

Cell Types ◽

Specific Cell ◽

Cytokine Release ◽

Cytokine Release Syndrome ◽

Rna Seq ◽

Future Studies ◽

Icu Length Of Stay ◽

Potential Association ◽

Clinical Trial Results

AbstractIntensive care unit (ICU) admissions and mortality in severe COVID-19 patients are driven by “cytokine storms” and acute respiratory distress syndrome (ARDS). Interim clinical trial results suggest that the corticosteroid dexamethasone displays better 28-day survival in severe COVID-19 patients requiring ventilation or oxygen. In this study, 10 out of 16 patients (62.5%) that had an average plasma IL-6 value over 10 pg/mL post administration of corticosteroids also had worse outcomes (i.e., ICU stay >15 days or death), compared to 8 out of 41 patients (19.5%) who did not receive corticosteroids (p-value = 0.0024). Given this potential association between post-corticosteroid IL-6 levels and COVID-19 severity, we hypothesized that the glucocorticoid receptor (GR or NR3C1) may be coupled to IL-6 expression in specific cell types that govern cytokine release syndrome (CRS). Examining single-cell RNA-seq data from BALF of severe COVID-19 patients and nearly 2 million cells from a pan-tissue scan shows that alveolar macrophages, smooth muscle cells, and endothelial cells co-express NR3C1 and IL-6, motivating future studies on the links between the regulation of NR3C1 function and IL-6 levels.

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Variables affecting Corporate Governance in the Profitability of Banks in Pakistan

International Journal of Accounting and Financial Reporting ◽

10.5296/ijafr.v4i2.6080 ◽

2014 ◽

Vol 4 (2) ◽

pp. 66

Author(s):

Aisha Akram ◽

Muhammad Omair ◽

Huzaifa Ameen ◽

Zohaib Khan Babar ◽

Jawwad Hassan Jaskani

Keyword(s):

Corporate Governance ◽

Major Element ◽

Small Sample Size ◽

Profit Maximization ◽

Small Sample ◽

Effective Control ◽

Future Studies ◽

Business System ◽

Good Corporate Governance ◽

Executive Body

Purpose: Understanding the issue of Corporate Governance requires to think beyond the Profit maximization for the firm. Corporate governance is the effective control of the issue regarding top management. Corporate governance is the major element of the present international business system. This research therefore investigates the connection between corporate governance and financial institutions profitability in Pakistan. Methodology: For this purpose a questionnaire is being adapted and responses are gathered from the executive body in banks of all the three divisions of Southern Punjab. Findings: The research found that good corporate governance is necessary for the profitability in banks. Limitation: The limitation of the study is that we had a small sample size due to the time constraint but it provides a research framework for future studies.

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Impact of RNA Extraction and Target Capture Methods on RNA Sequencing Using Formalin-Fixed, Paraffin Embedded Tissues

10.1101/656736 ◽

2019 ◽

Author(s):

Christopher A. Hilker ◽

Aditya V. Bhagwate ◽

Jin Sung Jang ◽

Jeffrey G Meyer ◽

Asha A. Nair ◽

...

Keyword(s):

Gene Expression ◽

Fusion Gene ◽

Rna Extraction ◽

Extraction Methods ◽

Rna Seq ◽

Gene Detection ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Whole Transcriptome ◽

Formalin Fixed

AbstractFormalin fixed paraffin embedded (FFPE) tissues are commonly used biospecimen for clinical diagnosis. However, RNA degradation is extensive when isolated from FFPE blocks making it challenging for whole transcriptome profiling (RNA-seq). Here, we examined RNA isolation methods, quality metrics, and the performance of RNA-seq using different approaches with RNA isolated from FFPE and fresh frozen (FF) tissues. We evaluated FFPE RNA extraction methods using six different tissues and five different methods. The reproducibility and quality of the prepared libraries from these RNAs were assessed by RNA-seq. We next examined the performance and reproducibility of RNA-seq for gene expression profiling with FFPE and FF samples using targeted (Kinome capture) and whole transcriptome capture based sequencing. Finally, we assessed Agilent SureSelect All-Exon V6+UTR capture and the Illumina TruSeq RNA Access protocols for their ability to detect known gene fusions in FFPE RNA samples. Although the overall yield of RNA varied among extraction methods, gene expression profiles generated by RNA-seq were highly correlated (>90%) when the input RNA was of sufficient quality (≥DV200 30%) and quantity (≥ 100 ng). Using gene capture, we observed a linear relationship between gene expression levels for shared genes that were captured using either All-Exon or Kinome kits. Gene expression correlations between the two capture-based approaches were similar using RNA from FFPE and FF samples. However, TruSeq RNA Access protocol provided significantly higher exon and junction reads when compared to the SureSelect All-Exon capture kit and was more sensitive for fusion gene detection. Our study established pre and post library construction QC parameters that are essential to reproducible RNA-seq profiling using FFPE samples. We show that gene capture based NGS sequencing is an efficient and highly reproducible strategy for gene expression measurements as well as fusion gene detection.

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P0453BIOSIMILAR RITUXIMAB AND BRAND RITUXIMAB: RELAPSES AND PROTEINURIA IN THE GLOMERULAR PRYMARY DISEASES

Nephrology Dialysis Transplantation ◽

10.1093/ndt/gfaa142.p0453 ◽

2020 ◽

Vol 35 (Supplement_3) ◽

Author(s):

Roxana Bury ◽

Irene Agraz ◽

Maria Jose Soler Romeo ◽

Clara Garcia Carro ◽

Eugenia Espinel ◽

...

Keyword(s):

Lymphocyte Count ◽

Leucocyte Count ◽

Statistical Significance ◽

Laboratory Data ◽

Small Sample ◽

Response To Treatment ◽

Glomerular Diseases ◽

Non Hodgkin Lymphoma ◽

Before And After ◽

Primary Glomerular Diseases

Abstract Background and Aims Biosimilar drugs need to prove similar effectiveness to the brand name drug, in order to have their authorization from regulatory agencies. Rituximab initially developed as a treatment for non-Hodgkin lymphoma, is used as a therapeutic alternative to several autoimmune diseases, including primary glomerular diseases. The objective of this study is to observe if there were differences in terms of effects measured in proteinuria and relapse episodes between rituximab brand versus biosimilar in primary glomerular diseases Method This is a retrospective descriptive study that included patients receiving rituximab brand or biosimilar for the first time between March 2018 to March 2019, collecting information from the reported medical records with primary glomerulopathy were included. The collected laboratory data included creatinine, proteinuria, leukocyte and lymphocyte count before (0-60 days before) and after (0-60 days after) administration of rituximab. Results A total of 19 patients with primary glomerulopathy were included. Six patients (59-years-old (26-74); 50% female) with baseline 6.52±2.00x10^9/L leucocyte count, 2.28±1.10x10^9/L lymphocyte count, 1.63±1.04 mg/dL creatinine and 6.84±3.36g/24h proteinuria, were treated with biosimilar-rituximab. Thirteen patients (58-years-old (25-81); 30% female) with baseline 9.80±4.62x10^9/L leucocyte count, 1.92±1.13 x10^9/L lymphocyte count, 1.61±0.85 mg/dL creatinine and 5.81±4.55 g/24h proteinuria, were treated with brand-rituximab. After the rituximab administration, these values were 6.13±1.94x10^9/L leucocyte count, 1.30±0.59x10^9/L lymphocyte count, 1.16±1.19 mg/dL creatinine, 3.29±0.58g/24h and proteinuria for the biosimilar group; and 8.77±3.78x10^9/L leucocyte count, 1.67±1.13x10^9/L lymphocyte count, 1.56±1.19 mg/dL creatinine and 3.36±2.20g/24h proteinuria for the brand group. After rituximab administration CD19+ lymphocytes become negative in both groups (5/5 for the biosimilar group; 6/6 for the brand group). There were 2 total remissions, 1 partial remission and 3 without response with the biosimilar and 1 total remission, 5 partial remissions and 7 without response with the rituximab brand. Biosimilar was well tolerated in 6/6 patients and Rituximab brand infection did not develop was well tolerated in 11/13 patients and 4/13 patients showed an episode of infection. No statistically significant results were observed for the response to treatment between both groups. Conclusion The biosimilar shows a similar profile in terms of proteinuria and remissions against rituximab mark in the reported follow-up period, however our study is limited since it has a small sample, so a larger study is necessary to demonstrate these results with statistical significance

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