scholarly journals Approaches to Gene Mutation Analysis Using Formalin-Fixed Paraffin-Embedded Adrenal Tumor Tissue From Patients With Primary Aldosteronism

2021 ◽  
Vol 12 ◽  
Author(s):  
Kazutaka Nanba ◽  
William E. Rainey ◽  
Aaron M. Udager
Keyword(s):  
Primary Aldosteronism ◽  
Zona Glomerulosa ◽  
Genetic Profiling ◽  
Tissue Sections ◽  
Whole Exome ◽  
Formalin Fixed Paraffin ◽  
Aldosterone Production ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Material ◽  
Formalin Fixed

Aldosterone production is physiologically under the control of circulating potassium and angiotensin II as well as adrenocorticotropic hormone and other secretagogues such as serotonin. The adrenal’s capacity to produce aldosterone relies heavily on the expression of a single enzyme, aldosterone synthase (CYP11B2). This enzyme carries out the final reactions in the synthesis of aldosterone and is expressed almost solely in the adrenal zona glomerulosa. From a disease standpoint, primary aldosteronism (PA) is the most common of all adrenal disorders. PA results from renin-independent adrenal expression of CYP11B2 and production of aldosterone. The major causes of PA are adrenal aldosterone-producing adenomas (APA) and adrenal idiopathic hyperaldosteronism. Our understanding of the genetic causes of APA has significantly improved through comprehensive genetic profiling with next-generation sequencing. Whole-exome sequencing has led to the discovery of mutations in six genes that cause renin-independent aldosterone production and thus PA. To facilitate broad-based prospective and retrospective studies of APA, recent technologic advancements have allowed the determination of tumor mutation status using formalin-fixed paraffin-embedded (FFPE) tissue sections. This approach has the advantages of providing ready access to archival samples and allowing CYP11B2 immunohistochemistry-guided capture of the exact tissue responsible for inappropriate aldosterone synthesis. Herein we review the methods and approaches that facilitate the use of adrenal FFPE material for DNA capture, sequencing, and mutation determination.

scholarly journals Discrimination of Aspergillosis, Mucormycosis, Fusariosis, and Scedosporiosis in Formalin-Fixed Paraffin-Embedded Tissue Specimens by Use of Multiple Real-Time Quantitative PCR Assays

2016 ◽  
Vol 54 (11) ◽  
pp. 2798-2803 ◽  
Author(s):  
Elham Salehi ◽  
Mohammad T. Hedayati ◽  
Jan Zoll ◽  
Haleh Rafati ◽  
Maryam Ghasemi ◽  
...  
Keyword(s):  
Fusarium Oxysporum ◽  
Aspergillus Flavus ◽  
Ffpe Tissue ◽  
Content Type ◽  
Real Time Quantitative Pcr ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Tissue Specimens ◽  
Formalin Fixed

In a retrospective multicenter study, 102 formalin-fixed paraffin-embedded (FFPE) tissue specimens with histopathology results were tested. Two 4- to 5-μm FFPE tissue sections from each specimen were digested with proteinase K, followed by automated nucleic acid extraction. Multiple real-time quantitative PCR (qPCR) assays targeting the internal transcribed spacer 2 (ITS2) region of ribosomal DNA, using fluorescently labeled primers, was performed to identify clinically important genera and species of Aspergillus , Fusarium , Scedosporium , and the Mucormycetes . The molecular identification was correlated with results from histological examination. One of the main findings of our study was the high sensitivity of the automated DNA extraction method, which was estimated to be 94%. The qPCR procedure that was evaluated identified a range of fungal genera/species, including Aspergillus fumigatus , Aspergillus flavus , Aspergillus terreus , Aspergillus niger , Fusarium oxysporum , Fusarium solani , Scedosporium apiospermum , Rhizopus oryzae , Rhizopus microsporus , Mucor spp., and Syncephalastrum . Fusarium oxysporum and F. solani DNA was amplified from five specimens from patients initially diagnosed by histopathology as having aspergillosis. Aspergillus flavus , S. apiospermum , and Syncephalastrum were detected from histopathological mucormycosis samples. In addition, examination of four samples from patients suspected of having concomitant aspergillosis and mucormycosis infections resulted in the identification of two A. flavus isolates, one Mucor isolate, and only one sample having both R. oryzae and A. flavus . Our results indicate that histopathological features of molds may be easily confused in tissue sections. The qPCR assay used in this study is a reliable tool for the rapid and accurate identification of fungal pathogens to the genus and species levels directly from FFPE tissues.

scholarly journals Quantitative radioimmunohistochemical measurements of p185(erbB-2) in frozen tissue sections.

1996 ◽  
Vol 44 (11) ◽  
pp. 1251-1259 ◽  
Author(s):  
J R Reeves ◽  
J J Going ◽  
G Smith ◽  
T G Cooke ◽  
B W Ozanne ◽  
...  
Keyword(s):  
Tumor Biology ◽  
Breast Carcinomas ◽  
Tissue Sections ◽  
Biopsy Specimens ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Interstudy Variability ◽  
High Degree ◽  
The Relationship ◽  
Formalin Fixed

The relationship between expression of the c-erbB-2 proto-oncogene and the biology of breast cancer has been investigated widely, most studies using immunohistochemistry in formalin-fixed, paraffin-embedded tissues. This technique is at best semiquantitative and there is a high degree of interstudy variability because of its subjective nature and poor methodological standardization. The relationship between the levels of expression and biology can be examined thoroughly only with an accurately quantitative technique. We have developed a radioimmunohistochemical assay to measure p185(erbB-2) in tissue biopsy specimens. The method involves incubating frozen sections with 125I-labeled monoclonal antibody, microautoradiograpy, and grain counting with image analysis. Sections of cell pellets with known c-erbB-2 levels are processed with each batch of samples as internal calibration standards. We have quantified c-erbB-2 expression in 60 breast carcinomas and compared the results with conventional immunohistochemistry. Radioimmunohistochemistry measured receptor levels throughout the range of expression in breast carcinomas, whereas conventional immunohistochemistry detected the protein only in the highest expressing tumors. The quantitative, objective data produced by radioimmunohistochemistry allow a more thorough evaluation of the relationship between c-erbB-2 expression and tumor biology. This technique may have applications in other fields where quantitative data is required and relevant monoclonal antibodies are available.

Application of in situ hybridization with a novel phenytoin-labeled probe to conventional formalin-fixed, paraffin-embedded tissue sections

1995 ◽  
Vol 52 (3) ◽  
pp. 309-316 ◽  
Author(s):  
Yoshito Eizuru ◽  
Yoichi Minamishima ◽  
Tadashi Matsumoto ◽  
Toshinari Hamakado ◽  
Mikio Mizukoshi ◽  
...  
Keyword(s):  
In Situ Hybridization ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Labeled Probe ◽  
Formalin Fixed

scholarly journals Multi-center real-world comparison of the fully automated Idylla™ microsatellite instability assay with routine molecular methods and immunohistochemistry on formalin-fixed paraffin-embedded tissue of colorectal cancer

2020 ◽  
Author(s):  
Ana Velasco ◽  
Fatma Tokat ◽  
Jesper Bonde ◽  
Nicola Trim ◽  
Elisabeth Bauer ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Microsatellite Instability ◽  
Real World ◽  
Diagnostic Testing ◽  
Molecular Methods ◽  
Tissue Sections ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
High Concordance ◽  
Formalin Fixed

Abstract Microsatellite instability (MSI) is present in 15–20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.

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