Whole-Exome Sequencing (WES) Using Formalin-Fixed Paraffin Embedded (FFPE) Tissue

INTRODUCTION: Aggregation of Barretts esophagus (BE) and esophageal adenocarcinoma (EAC) in families has been termed familial Barretts esophagus (FBE). Analysis of single, large FBE families can enable the identification of genetic susceptibility to complex diseases such as BE and EAC. METHODS: Phenotypes of BE and EAC were ascertained in a large FBE family with 7 affected members: 4 men with EAC, 1 man with BE and high grade dysplasia, and 2 women with non-dysplastic BE by review of endoscopy and surgical pathology reports. Whole exome sequencing was performed on germline DNA from 3 affected members to identify variants in coding genes that segregated with disease. Formalin fixed paraffin embedded tissue from an affected family member as well as non-familial subjects with BE and EAC was examined with regular histology and immunohistochemistry. The CAV3 gene with a variant segregating in the family was further characterized in a porcine model of esophageal injury using immunofluorescence. RESULTS: Using a whole exome sequencing approach on an exceptional FBE family we identified a segregating nonsense mutation in the gene Caveolin-3 (CAV3). Histologic examination of a formalin fixed paraffin embedded (FFPE) esophagectomy specimen from an individual carrying the CAV3 null mutation revealed esophageal submucosal glands (ESMG) that showed acinar metaplasia with marked atypia and absence of myoepithelial cells, distinctly different from acinar metaplasia seen in ESMG of non-familial subjects with BE and high grade dysplasia. Immunofluorescence studies of ESMG in porcine esophagus revealed the presence of CAV3 in selected cells in a distribution that was consistent with myoepithelial cells. Experimental injury of the porcine esophagus using radiofrequency ablation revealed that CAV3 expression increased markedly within ESMGs, ESMG ductal epithelium, and overlying healing neosquamous epithelium 10 days after injury. CONCLUSIONS: We theorize that CAV3 expression, perhaps through myoepithelial cells within ESMGs, controls the differentiation and proliferation of squamous epithelial precursor cells in response to injury. Furthermore, the truncating nonsense CAV3 mutation discovered in a family disrupts normal squamous healing and the organization of ESMGs, making affected family members susceptible to the proliferation and development of metaplastic columnar BE and EAC.

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Faculty Opinions recommendation of Comparison of Accuracy of Whole-Exome Sequencing with Formalin-Fixed Paraffin-Embedded and Fresh Frozen Tissue Samples.

Faculty Opinions – Post-Publication Peer Review of the Biomedical Literature ◽

10.3410/f.725996494.793528011 ◽

2017 ◽

Author(s):

Alexandre Alanio

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Tissue Samples ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Frozen Tissue ◽

Formalin Fixed Paraffin Embedded ◽

Fresh Frozen ◽

Formalin Fixed

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Whole exome sequencing (WES) on formalin-fixed, paraffin-embedded (FFPE) tumor tissue in gastrointestinal stromal tumors (GIST)

BMC Genomics ◽

10.1186/s12864-015-1982-6 ◽

2015 ◽

Vol 16 (1) ◽

Cited By ~ 34

Author(s):

Annalisa Astolfi ◽

Milena Urbini ◽

Valentina Indio ◽

Margherita Nannini ◽

Chiara Giusy Genovese ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Gastrointestinal Stromal Tumors ◽

Tumor Tissue ◽

Stromal Tumors ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Ffpe Tumor ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Whole-exome sequencing and clinical interpretation of formalin-fixed, paraffin-embedded tumor samples to guide precision cancer medicine

Nature Medicine ◽

10.1038/nm.3559 ◽

2014 ◽

Vol 20 (6) ◽

pp. 682-688 ◽

Cited By ~ 340

Author(s):

Eliezer M Van Allen ◽

Nikhil Wagle ◽

Petar Stojanov ◽

Danielle L Perrin ◽

Kristian Cibulskis ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Clinical Interpretation ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Cancer Medicine ◽

Formalin Fixed

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Comparison of whole-exome sequencing of matched fresh and formalin fixed paraffin embedded melanoma tumours: implications for clinical decision making

Pathology ◽

10.1016/j.pathol.2016.01.001 ◽

2016 ◽

Vol 48 (3) ◽

pp. 261-266 ◽

Cited By ~ 21

Author(s):

Ricardo De Paoli-Iseppi ◽

Peter A. Johansson ◽

Alexander M. Menzies ◽

Kerith-Rae Dias ◽

Gulietta M. Pupo ◽

...

Keyword(s):

Decision Making ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Clinical Decision Making ◽

Clinical Decision ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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The Dutch National TissueArchive Portal enables efficient, consistent, and transparent procurement of diagnostic tissue samples for scientific use

Cell and Tissue Banking ◽

10.1007/s10561-021-09949-1 ◽

2021 ◽

Author(s):

Robin Verjans ◽

Annette H. Bruggink ◽

Robby Kibbelaar ◽

Jos Bart ◽

Aletta Debernardi ◽

...

Keyword(s):

The Netherlands ◽

Tissue Sample ◽

Ffpe Tissue ◽

Tissue Samples ◽

Internet Portal ◽

Formalin Fixed Paraffin ◽

Practical Support ◽

Formalin Fixed Paraffin Embedded ◽

The Rich ◽

Formalin Fixed

AbstractBiobanks play a crucial role in enabling biomedical research by facilitating scientific use of valuable human biomaterials. The PALGA foundation—a nationwide network and registry of histo- and cytopathology in the Netherlands—was established to promote the provision of data within and between pathology departments, and to make the resulting knowledge available for healthcare. Apart from the pathology data, we aimed to utilize PALGA’s nationwide network to find and access the rich wealth of Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples for scientific use. We implemented the Dutch National TissueArchive Portal (DNTP) to utilize PALGA’s nationwide network for requesting FFPE tissue samples. The DNTP consists of (1) a centrally organized internet portal to improve the assessing, processing, harmonization, and monitoring of the procurement process, while (2) dedicated HUB-employees provide practical support at peripheral pathology departments. Since incorporation of the DNTP, both the number of filed requests for FFPE tissue samples and the amount of HUB-mediated support increased 55 and 29% respectively. In line, the sample procurement duration time decreased significantly (− 47%). These findings indicate that implementation of the DNTP improved the frequency, efficiency, and transparency of FFPE tissue sample procurement for research in the Netherlands. To conclude, the need for biological resources is growing persistently to enable precision medicine. Here, we access PALGA’s national, pathology network by implementation of the DNTP to allow for efficient, consistent, and transparent exchange of FFPE tissue samples for research across the Netherlands.

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

Journal of Clinical Microbiology ◽

10.1128/jcm.01185-16 ◽

2016 ◽

Vol 54 (11) ◽

pp. 2798-2803 ◽

Cited By ~ 35

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.

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