Systematic evaluation of PAXgene® tissue fixation for the histopathological and molecular study of lung cancer

Abstract

Whilst adequate for most existing pathological tests, formalin is generally considered a poor DNA preservative and use of alternative fixatives may prove advantageous for molecular testing of tumour material; an increasingly common approach to identify targetable driver mutations in lung cancer patients. We collected paired PAXgene® tissue‐fixed and formalin‐fixed samples of block‐sized tumour and lung parenchyma, Temno‐needle core tumour biopsies and fine needle tumour aspirates (FNAs) from non‐small cell lung cancer resection specimens. Traditionally processed formalin fixed paraffin wax embedded (FFPE) samples were compared to paired PAXgene® tissue fixed paraffin‐embedded (PFPE) samples. We evaluated suitability for common laboratory tests (H&E staining and immunohistochemistry) and performance for downstream molecular investigations relevant to lung cancer, including RT‐PCR and next generation DNA sequencing (NGS). Adequate and comparable H&E staining was seen in all sample types and nuclear staining was preferable in PAXgene® fixed Temno tumour biopsies and tumour FNA samples. Immunohistochemical staining was broadly comparable. PFPE samples enabled greater yields of less‐fragmented DNA than FFPE comparators. PFPE samples were also superior for PCR and NGS performance, both in terms of quality control metrics and for variant calling. Critically we identified a greater number of genetic variants in the epidermal growth factor receptor gene when using PFPE samples and the Ingenuity® Variant Analysis pipeline. In summary, PFPE samples are adequate for histopathological diagnosis and suitable for the majority of existing laboratory tests. PAXgene® fixation is superior for DNA and RNA integrity, particularly in low‐yield samples and facilitates improved NGS performance, including the detection of actionable lung cancer mutations for precision medicine in lung cancer samples.

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology

“…However, other QC metrics were not evaluated in that study. Southwood et al, applying the Qiagen sequencing platform on PFPE lung tissues, reported superior NGS performance, both in terms of QC metrics and for variant calling with higher unique molecular identifier (UMI) reads and deeper mean depth of coverage than those of FFPE tissues [ 9 ]. We did not observe PFPE tissues exhibiting more mapped reads nor deeper mean depth of coverage using the Ion Torrent platform.…”

“…A recently developed methanol and acetic acid-based fixative, PAXgene (PAXgene Tissue Fix Container, Preanalytix GmbH, Hombrechtikon, Switzerland), works via a non-crosslinking mechanism. Studies involving multiple types of cancer have demonstrated that PAXgene-fixed, paraffin-embedded (PFPE) tissue yields adequate histomorphology in the majority of cases (i.e., comparable to FFPE, far better than frozen tissue) [ 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. These studies also suggested PFPE is associated with improved DNA and RNA yield and quality compared to FFPE [ 6 , 7 , 8 , 9 , 10 , 11 ].…”

“…Studies involving multiple types of cancer have demonstrated that PAXgene-fixed, paraffin-embedded (PFPE) tissue yields adequate histomorphology in the majority of cases (i.e., comparable to FFPE, far better than frozen tissue) [ 4 , 5 , 6 , 7 , 8 , 9 , 10 ]. These studies also suggested PFPE is associated with improved DNA and RNA yield and quality compared to FFPE [ 6 , 7 , 8 , 9 , 10 , 11 ]. In addition, three cancer morphology ring studies under the SPIDIA consortium (standardization and improvement of generic pre-analytical tools and procedures for in vitro diagnostics) focusing on colon, breast and prostate have been executed and demonstrated promising advantages using the PAXgene fixative in routine pathology in comparison to formalin fixation [ 4 , 8 , 12 ].…”

Comprehensive Evaluation of PAXgene Fixation on Oral Cancer Tissues Using Routine Histology, Immunohistochemistry, and FTIR Microspectroscopy

“…Further, prior studies have shown PAXgene fixation is superior in preserving RNA integrity, particularly in low-yield samples for molecular experiments [3,5,25]. It also was reported that the PAXgene-fixed RNA has better RT-PCR efficiency than NBF fixation and was comparable to snap-frozen samples, irrespective of the length of the mRNA, in a wide variety of cancerous tissues, including squamous cell carcinoma [5,25,26]. Further in-depth comparative studies are warranted in oral tissue to understand the effect of PAXgene fixation on RNA integrity and downstream analysis such as RT-PCR and RNA sequencing.…”

Diagnostic and prognostic biomarkers in oligometastatic non-small cell lung cancer: a literature review

“…Especially during the natural history of longlasting cancers, the possible contribution of core-needles as repeat biopsies is debated. Traditionally, processed formalin-fixed paraffin-embedded (FFPE) samples have been compared to paired PAXgene ® tissue fixed paraffinembedded (PFPE) tissues, which might be superior for DNA and RNA integrity, particularly in low-yield samples (15). Cytology and micro-histological approaches might work in a complementary fashion (16), however, when neoplastic sites are not accessible or clinicians have not the possibility to apply an interventional procedure with the requested advantage, the recourse to liquid biopsy is completely justified.…”