Protocol for <scp>HER</scp>2 <scp>FISH</scp> determination on <scp>PAX</scp>gene‐fixed and paraffin‐embedded tissue in breast cancer

Oberauner-Wappis, Lisa; Loibner, Martina; Viertler, Christian; Groelz, Daniel; Wyrich, Ralf; Zatloukal, Kurt

doi:10.1111/iep.12185

Cited by 12 publications

(19 citation statements)

References 18 publications

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“…Evaluation of blinded, randomized, international ring trials with PFPE and matched FFPE cancer specimen came to the conclusion that the quality of histo- and cytomorphological features of PFPE specimen allows use of PAXgene Tissue for routine morphological diagnosis of colon [ 18 ] and breast cancer tissue (Viertler et al, in preparation). Further studies conducted outside SPIDIA confirmed compatibility of PAXgene Tissue treated tissues with commonly used methods such as immunohistochemistry (IHC) [ 19 – 21 ] and fluorescence in situ hybridization (FISH) [ 22 , 23 ] as well as with molecular biology applications including RT-qPCR [ 20 , 21 , 24 – 26 ], NGS technologies and genome-wide DNA methylation analysis [ 27 – 29 ] and metabolomic and proteomic analysis [ 30 ].…”

Section: Introductionmentioning

confidence: 99%

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

et al. 2018

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RNA and DNA analyses from paraffin-embedded tissues (PET) are an important diagnostic tool for characterization of a disease, exploring biomarkers and treatment options. Since nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissue are of limited use for molecular analyses due to chemical modifications of biomolecules alternate, formalin-free fixation reagents such as the PAXgene Tissue system are of evolving interest. Furthermore, biomedical research and biomarker development critically relies on using long-term stored PET from medical archives or biobanks to correlate molecular features with long-term disease outcomes. We therefore performed a comparative study to evaluate the effect of long term storage of FFPE and PAXgene Tissue-fixed and paraffin-embedded (PFPE) tissue at different temperatures on nucleic acid stability and usability in PCR. Matched FFPE and PFPE human tissues from routine clinical setting or rat tissues from a highly controlled animal model were stored at room temperature and 4°C, as well as in case of animal tissues frozen at -20°C and -80°C. RNA and DNA were extracted in intervals for up to nine years, and examined for integrity, and usability in quantitative RT-PCR (RT-qPCR) or PCR (qPCR) assays. PET storage at room temperature led to a degradation of nucleic acids which was slowed down by storage at 4°C and prevented by storage at -20°C or -80°C. Degradation was associated with an amplicon length depending decrease of RT-qPCR and qPCR efficiency. Storage at 4°C improved amplifiability in RT-qPCR and qPCR profoundly. Chemically unmodified nucleic acids from PFPE tissue performed superior compared to FFPE tissue, regardless of storage time and temperature in both human and rat tissues. In conclusion molecular analyses from PET can be greatly improved by using a non-crosslinking fixative and storage at lower temperatures such as 4°C, which should be considered in prospective clinical studies.

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Section: Introductionmentioning

confidence: 99%

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

et al. 2018

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“…The results demonstrate two key findings. First, a simple 24 h SBFS post-fixation step of sections cut from NCFPE tissues is sufficient to obtain equivalent results to those with FFPE in FISH analyses using FISH assays approved for FFPE tissues (see also reference 14 ). This protocol has the advantage of using FISH assays originally developed and approved for FFPE without the need for comprehensive revalidation (e.g., by optimizing the pre-hybridization conditions for non-cross-linked tissue).…”

Section: Discussionmentioning

confidence: 99%

“…SBFS is reported to penetrate tissue at an average rate of 1 mm per hour 22 to 5 mm in 2 h depending on the tissue type 23 24 . The observation that only prolonged post-fixation periods of more than 18 h could change the properties of NCFPE to FFPE sections, indicates that not the penetration of the fixative but the chemical reaction time is critical for achieving the desired results 1 14 .…”

Section: Discussionmentioning

confidence: 99%

“…In a previous study, we showed that NCFPE tissues cannot be used for FISH assays which have been approved for FFPE tissue. However, tests of time series of different post fixation procedures of NCFPE tissues with 4 % buffered formaldehyde showed that post fixation times of 18 h or longer of NCFPE tissue sections achieved equivalent results to those with FFPE tissues 14 .…”

Section: Introductionmentioning

confidence: 95%

“…One particular application in cancer diagnostics is FISH detecting a comprehensive range of chromosomal alterations, such as translocations, submicroscopic deletions and amplifications 14 . For example, twenty percent of invasive breast cancer tumors show amplification of the human epidermal growth factor receptor 2 (HER2) gene 15 16 17 , which is associated with poor prognosis 18 19 .…”

Section: Introductionmentioning

confidence: 99%

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Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Loibner¹,

Oberauner-Wappis²,

Viertler

et al. 2017

JoVE

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Morphologic assessment of formalin-fixed, paraffin-embedded (FFPE) tissue samples has been the gold standard for cancer diagnostics for decades due to its excellent preservation of morphology. Personalized medicine increasingly provides individually adapted and targeted therapies for characterized individual diseases enabled by combined morphological and molecular analytical technologies and diagnostics. Performance of morphologic and molecular assays from the same FFPE specimen is challenging because of the negative impact of formalin due to chemical modification and cross-linking of nucleic acids and proteins. A non-cross-linking, formalin-free tissue fixative has been recently developed to fulfil both requirements, i.e., to preserve morphology like FFPE and biomolecules like cryo-preservation. Since FISH is often required in combination with histopathology and molecular diagnostics, we tested the applicability of FISH protocols on tissues treated with this new fixative. We found that formalin post-fixation of histological sections of non-cross-linking, formalin-free and paraffin-embedded (NCFPE) breast cancer tissue generated equivalent results to those with FFPE tissue in human epidermal growth factor receptor 2 (HER2) FISH analysis. This protocol describes how a FISH assay originally developed and validated for FFPE tissue can be used for NCFPE tissues by a simple post-fixation step of histological sections.

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Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

Meecham

Miranda

Morris

et al. 2021

Histochem Cell Biol

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Formalin is the principal tissue fixative used worldwide for clinical and research purposes. Despite optimal preservation of morphology, its preservation of DNA and RNA is poor. As clinical diagnostics increasingly incorporates molecular-based analysis, the requirement for maintaining nucleic acid quality is of increasing importance. Here we assess an alternative non-formalin-based tissue fixation method, PAXgene Tissue system, with the aim of better preserving nucleic acids, while maintaining the quality of the tissue to be used for vital existing diagnostic techniques. In this study, these criteria are assessed in a clinically representative setting. In total, 203 paired PAXgene Tissue and formalin-fixed samples were obtained. Blind-scored haematoxylin and eosin (H&E) sections showed comparable and acceptable staining. Immunohistochemistry (IHC) staining was suboptimal using existing protocols but improved with minor method adjustment and optimisation. Quality of DNA and RNA was significantly improved by PAXgene tissue fixation [RIN 2.8 versus 3.8 (p < 0.01), DIN 5.68 versus 6.77 (p < 0.001)], which translated into improved performance on qPCR assay. These results demonstrate the potential of PAXgene Tissue to be used routinely in place of formalin, maintaining adequate histological staining and significantly improving the preservation of biological molecules in the genomic era.

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Protocol for HER2 FISH determination on PAXgene‐fixed and paraffin‐embedded tissue in breast cancer

Cited by 12 publications

References 18 publications

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

Impact of storage conditions on the quality of nucleic acids in paraffin embedded tissues

Protocol for HER2 FISH Using a Non-cross-linking, Formalin-free Tissue Fixative to Combine Advantages of Cryo-preservation and Formalin Fixation

Alternative tissue fixation for combined histopathological and molecular analysis in a clinically representative setting

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