Daniel Groelz

Non-formalin fixative versus formalin-fixed tissue: a comparison of histology and RNA quality.

Preanalytical handling of tissue samples can influence bioanalyte quality and ultimately outcome of analytical results. The aim of this study was to compare RNA quality, performance in real time RT PCR and histology of formalin-fixed tissue to that of tissue fixed and stabilized with a formalin-free fixative, the PAXgene Tissue System (PAXgene), in an animal model under highly controlled preanalytical conditions. Samples of rat liver, kidney, spleen, intestine, lung, heart muscle, brain, and stomach tissue were either fixed in formalin or fixed in PAXgene or fresh frozen in liquid nitrogen. RNA was extracted from all samples, examined for integrity in microcapillary electrophoresis, and used in a series of quantitative RT PCR assays with increasing amplicon length. Histology of paraffin-embedded samples was determined by staining with hematoxylin and eosin. Histology of all formalin-fixed and PAXgene fixed samples was comparable. RNA with acceptable integrity scores could be isolated from all embedded tissues, 4.0 to 7.2 for formalin and 6.4 to 7.7 for PAXgene, as compared to 8.0 to 9.2 for fresh frozen samples. While RNA with acceptable RINs (RNA integrity number) could be isolated from formalin-fixed samples, in microcapillary electrophoresis this RNA separated with a slower migration rate and displayed diffuse, less focused peaks for ribosomal RNA as compared to RNA from frozen or PAXgene fixed samples. Furthermore, RNA from formalin-fixed tissues exhibited inhibition in quantitative RT PCR assays which increased with increasing amplicon length, while RNA from PAXgene fixed samples did not show such inhibition. In conclusion, our results demonstrate that excluding other preanalytical factors, PAXgene Tissue System preserves histology similarly to formalin, but unlike formalin, does not chemically modify RNA. RNA purified from PAXgene fixed tissues is of high integrity and performs as well as RNA from fresh frozen tissue in RT PCR regardless of amplicon length.

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

Molecular diagnostics in personalized medicine increasingly relies on the combination of a variety of analytical technologies to characterize individual diseases and to select patients for targeted therapies. The gold standard for tissue‐based diagnostics is fixation in formalin and embedding in paraffin, which results in excellent preservation of morphology but negatively impacts on a variety of molecular assays. The formalin‐free, non‐cross‐linking PAXgene tissue system preserves morphology in a similar way to formalin, but also preserves biomolecules essentially in a similar way to cryopreservation, which markedly widens the spectrum, sensitivity and accuracy of molecular analytics. In this study, we have developed and tested a protocol for PAXgene‐fixed and paraffin‐embedded tissues for fluorescent in situ hybridization (FISH). The implementation of a 24‐h formalin postfixation step of slides from PAXgene‐fixed and paraffin‐embedded tissues allowed us to use the assays approved for formalin‐fixed and paraffin‐embedded tissues. The equivalence of the methodologies was demonstrated by FISH analysis of HER2 amplification in breast cancer cases. The 24‐h postfixation step of the slides used for FISH can be well integrated in the routine diagnostic workflow and allows the remaining PAXgene‐fixed and paraffin‐embedded tissue to be used for further molecular testing.

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

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.

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

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.

Abstract 2745: Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA

Introduction: The current preanalytical workflows for circulating tumor DNA (ctDNA) analysis have limitations that affect the accurate detection and quantification of these plasma cancer biomarkers. Release of genomic DNA (gDNA) from white blood cells (WBCs) due to cell lysis or apoptosis during whole blood storage in EDTA tubes creates higher gDNA background levels, affecting the sensitivity of ctDNA assays. Current tubes that stabilize WBCs often contain crosslinking reagents, which have negative effects on sensitive downstream assays, including methylation-based assays. Using ctDNA assays, the new PAXgene® Blood ccfDNA System*, consisting of a blood collection tube with unique, non-crosslinking chemistry and an automated circulating cell-free DNA (ccfDNA) extraction kit, was evaluated in three research studies. Methods: Blood samples were collected into paired PAXgene and EDTA tubes and stored for 7 days at room temperature (RT). ccfDNA was isolated from plasma using the PAXgene kit or the QIAGEN QIAamp® Circulating Nucleic Acid Kit. Study 1: The ccfDNA from lung cancer patients was quantified by real-time PCR for the amount of the ERV sequence (as a measure of the total plasma DNA quantity) and after bisulfte treatment for mSHOX2 as a marker for ctDNA. Study 2: Blood from healthy donors was spiked with fragmented, fully-methylated CpGenome DNA. During storage, tubes were intermittently inverted to simulate tubes in transit. Subsequent to bisulfite conversion, PCR assays targeting ACTB and methylated BCAT1 and IKZF1 DNA were used to determine the yields of ccfDNA and ctDNA, respectively. Study 3: Restriction enzyme treated EGFR DNA containing exon 19 deletions and exon 20 and 21 substitutions (T790M, L858R) were spiked into healthy donors’ blood. ccfDNA was tested with the QIAGEN therascreen® EGFR Plasma RGQ PCR Kit*. Results: Both study 1 and 2 demonstrated constant levels of the methylation ctDNA markers, SHOX2, BCAT1 and IKZF1, over the investigated time course. There was no significant release of gDNA in the PAXgene tube whereas a significant release of gDNA was detected in EDTA samples. Likewise, study 3 showed constant EGFR Ct values in the PAXgene system with reliable mutation detection, whereas the high DNA concentration from the EDTA system resulted in false-positive callings. Conclusions: The new system allows researchers to accurately detect and quantify plasma cancer biomarkers from blood samples that have been stored in the tube for up to 7 days at RT. This includes challenging assays based on methylated ctDNA. The system provides the required assay sensitivity to allow the correct assay interpretation beyond the typical 3–6 hour storage limit for EDTA tubes. *For Research Use Only. Not for use in diagnostic procedures. Citation Format: Michael Fleischhacker, Bernd Schmidt, Rohan Baker, Susanne Pedersen, Natasha Cant, Maryam Zahedi-Nejad, Thorsten Voss, Andrea Ullius, Daniel Groelz. Evaluation of a new system for collection, stabilization, and purification of circulating tumor DNA [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2745. doi:10.1158/1538-7445.AM2017-2745

Pathogen Inactivating Properties and Increased Sensitivity in Molecular Diagnostics by PAXgene, a Novel Non-Crosslinking Tissue Fixative

Background Requirements on tissue fixatives are getting more demanding as molecular analysis becomes increasingly relevant for routine diagnostics. Buffered formaldehyde in pathology laboratories for tissue fixation is known to cause chemical modifications of biomolecules which affect molecular testing. A novel non-crosslinking tissue preservation technology, PAXgene Tissue (PAXgene), was developed to preserve the integrity of nucleic acids in a comparable way to cryopreservation and also to preserve morphological features comparable to those of formalin fixed samples. Methods Because of the excellent preservation of biomolecules by PAXgene we investigated its pathogen inactivation ability and biosafety in comparison to formalin by in-vitro testing of bacteria, human relevant fungi and human cytomegalovirus (CMV). Guidelines for testing disinfectants served as reference for inactivation assays. Furthermore, we tested the properties of PAXgene for detection of pathogens by PCR based assays. Results All microorganisms tested were similarly inactivated by PAXgene and formalin except Clostridium sporogenes, which remained viable in seven out of ten assays after PAXgene treatment and in three out of ten assays after formalin fixation. The findings suggest that similar biosafety measures can be applied for PAXgene and formalin fixed samples. Detection of pathogens in PCR-based diagnostics using two CMV assays resulted in a reduction of four to ten quantification cycles of PAXgene treated samples which is a remarkable increase of sensitivity. Conclusion PAXgene fixation might be superior to formalin fixation when molecular diagnostics and highly sensitive detection of pathogens is required in parallel to morphology assessment.

Abstract C98: Evaluation of the PAXgene tissue system: Preservation of morphology and gene expression in human melanoma.

Introduction: PreAnalytiX has recently developed the PAXgene® Tissue System, a formalin-free tissue fixation method for simultaneous preservation of tissue morphology and biomolecules. In this study, PAXgene Tissue was used to stabilize human melanoma specimens. Mirrored samples of melanoma were fixed with either neutral buffered formalin or PAXgene Tissue, or snap frozen in liquid nitrogen (LN2) and were compared for preservation of morphology, antigenicity, RNA and noncoding small RNAs. Materials and Methods: Three cases of melanoma were equally divided into two parts: one part was fixed with PAXgene Tissue reagents, the second part of cases 1 and 2 were fixed with formalin and the second part of case 3 was snap-frozen in LN2. Formalin and PAXgene Tissue fixed samples were processed and embedded in paraffin (resulting in FFPE and PFPE tissue respectively). Tissue sections were stained with HE 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C98.