KAPA HTP Library Preparation Kit（KK8234）: Sequence analysis of small input-volume formalin-fixed paraffin-embedded (FFPE) tissue samples in whole-exome sequencing (WES) using the SureSelect target enrichment system

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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Deciphering the Immune Microenvironment on A Single Archival Formalin-Fixed Paraffin-Embedded Tissue Section by An Immediately Implementable Multiplex Fluorescence Immunostaining Protocol

Cancers ◽

10.3390/cancers12092449 ◽

2020 ◽

Vol 12 (9) ◽

pp. 2449 ◽

Cited By ~ 2

Author(s):

Adrien Guillot ◽

Marlene S. Kohlhepp ◽

Alix Bruneau ◽

Felix Heymann ◽

Frank Tacke

Keyword(s):

Immune Cell ◽

Simple Procedure ◽

Parenchymal Cell ◽

Ffpe Tissue ◽

Immune Microenvironment ◽

Tissue Samples ◽

Tissue Organization ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

Technological breakthroughs have fundamentally changed our understanding on the complexity of the tumor microenvironment at the single-cell level. Characterizing the immune cell composition in relation to spatial distribution and histological changes may provide important diagnostic and therapeutic information. Immunostaining on formalin-fixed paraffin-embedded (FFPE) tissue samples represents a widespread and simple procedure, allowing the visualization of cellular distribution and processes, on preserved tissue structure. Recent advances in microscopy and molecular biology have made multiplexing accessible, yet technically challenging. We herein describe a novel, simple and cost-effective method for a reproducible and highly flexible multiplex immunostaining on archived FFPE tissue samples, which we optimized for solid organs (e.g., liver, intestine, lung, kidney) from mice and humans. Our protocol requires limited specific equipment and reagents, making multiplexing (>12 antibodies) immediately implementable to any histology laboratory routinely performing immunostaining. Using this method on single sections and combining it with automated whole-slide image analysis, we characterize the hepatic immune microenvironment in preclinical mouse models of liver fibrosis, steatohepatitis and hepatocellular carcinoma (HCC) and on human-patient samples with chronic liver diseases. The data provide useful insights into tissue organization and immune–parenchymal cell-to-cell interactions. It also highlights the profound macrophage heterogeneity in liver across premalignant conditions and HCC.

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Whole exome sequencing : a comparative study of formalin-fixed paraffin embedded versus frozen tumour tissues

10.5353/th_991044057352003414 ◽

2018 ◽

Author(s):

Wang-leuk, Jonathan Chan

Keyword(s):

Comparative Study ◽

Exome Sequencing ◽

Whole Exome Sequencing ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Formalin Fixed

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Caveolin-3 Null Mutation in Family with Barretts Esophagus and Esophageal Adenocarcinoma

10.1101/2021.05.15.444305 ◽

2021 ◽

Author(s):

Katherine S Garman ◽

Richard von Furstenberg ◽

Thomas C Becker ◽

Gary W Falk ◽

Joseph E Willis ◽

...

Keyword(s):

Exome Sequencing ◽

Whole Exome Sequencing ◽

Myoepithelial Cells ◽

Null Mutation ◽

High Grade ◽

Whole Exome ◽

Formalin Fixed Paraffin ◽

Formalin Fixed Paraffin Embedded ◽

Barretts Esophagus ◽

Formalin Fixed

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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