Formalin-fixed paraffin-embedded (FFPE) tissue represents a valuable source for translational cancer research. However, the widespread application of various downstream methods remains challenging. Here, we aimed to assess the feasibility of a genomic and gene expression analysis workflow using FFPE breast cancer (BC) tissue. We conducted a systematic literature review for the assessment of concordance between FFPE and fresh-frozen matched tissue samples derived from patients with BC for DNA and RNA downstream applications. The analytical performance of three different nucleic acid extraction kits on FFPE BC clinical samples was compared. We also applied a newly developed targeted DNA Next-Generation Sequencing (NGS) 370-gene panel and the nCounter BC360® platform on simultaneously extracted DNA and RNA, respectively, using FFPE tissue from a phase II clinical trial. Of the 3701 initial search results, 40 articles were included in the systematic review. High degree of concordance was observed in various downstream application platforms. Moreover, the performance of simultaneous DNA/RNA extraction kit was demonstrated with targeted DNA NGS and gene expression profiling. Exclusion of variants below 5% variant allele frequency was essential to overcome FFPE-induced artefacts. Targeted genomic analyses were feasible in simultaneously extracted DNA/RNA from FFPE material, providing insights for their implementation in clinical trials/cohorts.

Human papillomavirus (HPV) infections are an increasing cause of oropharyngeal squamous cell carcinomas (OPSCC). Integration of the viral genome into the host genome is suggested to affect carcinogenesis, however, the correlation with OPSCC patient prognosis is still unclear. Research on HPV integration is hampered by current integration detection technologies and their unsuitability for formalin-fixed paraffin-embedded (FFPE) tissues. This study aims to develop and validate a novel targeted proximity-ligation based sequencing method (targeted locus amplification/capture [TLA/TLC]) for HPV integration detection in cell lines and FFPE OPSCCs. For the identification of HPV integrations, TLA/TLC was applied to 7 cell lines and 27 FFPE OPSCCs. Following preprocessing steps, a polymerase chain reaction (PCR)-based HPV enrichment was performed on the cell lines and a capture-based HPV enrichment was performed on the FFPE tissues before paired-end sequencing. TLA was able to sequence up to hundreds of kb around the target, detecting exact HPV integration loci, structural variants, and chromosomal rearrangements. In all cell lines, one or more integration sites were identified, in accordance with detection of integrated papillomavirus sequences PCR data and the literature. TLC detected integrated HPV in 15/27 FFPE OPSCCs and identified simple and complex integration patterns. In general, TLA/TLC confirmed PCR data and detected additional integration sites. In conclusion TLA/TLC reliably and robustly detects HPV integration in cell lines and FFPE OPSCCs, enabling large, population-based studies on the clinical relevance of HPV integration. Furthermore, this approach might be valuable for clonality assessment of HPV-related tumors in clinical diagnostics.

Collagen is the most abundant protein in mammals and a major structural component of the extracellular matrix (ECM). Changes to ECM composition occur as a result of numerous physiological and pathophysiological causes, and a common means to evaluate these changes is the collagen 3 (Col3) to collagen 1 (Col1) ratio. Current methods to measure the Col3/1 ratio suffer from a lack of specificity and often under- or over-estimate collagen composition and quantity. This manuscript presents a targeted liquid chromatography tandem mass spectrometry (LC-MS/MS) method for quantification of Col3 and Col1 in FFPE tissues. Using surrogate peptides to generate calibration curves, Col3 and Col1 are readily quantified in FFPE tissue sections with high accuracy and precision. The method is applied to several tissue types from both human and reindeer sources, demonstrating its generalizability. In addition, the targeted LC-MS/MS method permits quantitation of the hydroxyprolinated form of Col3, which has significant implications for understanding not only the quantity of Col3 in tissue, but also understanding of the pathophysiology underlying many causes of ECM changes. This manuscript presents a straightforward, accurate, precise, and generalizable method for quantifying the Col3/1 ratio in a variety of tissue types and organisms.

Gene expression is regulated by changes in chromatin architecture intrinsic to cellular differentiation and as an active response to environmental stimuli. Chromatin dynamics are a major driver of phenotypic diversity, regulation of development, and manifestation of disease. Remarkably, we know little about the evolutionary dynamics of chromatin reorganisation through time, data essential to characterise the impact of environmental stress during the ongoing biodiversity extinction crisis (20th-21st century). Linking the disparate fields of chromatin biology and museum science through their common use of the preservative formaldehyde (a constituent of formalin), we have generated historical chromatin profiles in museum specimens up to 117 years old. Historical chromatin profiles are reproducible, tissue-specific, sex-specific, and environmental condition-dependent in vertebrate specimens. Additionally, we show that over-fixation modulates differential chromatin accessibility to enable semi-quantitative estimates of relative gene expression in vertebrates and a yeast model. Our approach transforms formalin-fixed biological collections into an accurate, comprehensive, and global record of environmental impact on gene expression and phenotype.

Focal gene amplification, such as extrachromosomal DNA (ecDNA), plays an important role in cancer development and therapy resistance. While sequencing-based methodologies enable an unbiased identification of ecDNA, cytogenetic-based techniques, such as fluorescence in situ hybridization (FISH), remain time and cost-effective for identifying ecDNA in clinical specimens. The application of FISH in formalin-fixed paraffin-embedded (FFPE) tissue samples offers a unique avenue for detecting amplified genes, particularly when viable specimens are not available for karyotype examination. However, there is a lack of consensus procedures for this technique. This protocol provides comprehensive, fully optimized, step-by-step instructions for conducting FISH to detect gene amplification, including ecDNA, in FFPE tissue samples which present unique challenges that this protocol aims to overcome and standardize. By following this protocol, researchers can reproducibly acquire high-quality imaging data to assess gene amplification.

Most antibodies used in immunohistochemistry (IHC) have been developed by animal immunization. We wanted to explore naive antibody repertoires displayed on filamentous phages as a source of full-length antibodies for IHC on Formalin-Fixed and Paraffin-Embedded (FFPE) tissues. We used two isogenic mouse fibroblast cell lines that express or not human HER2 to generate positive and negative FFPE pseudo-tissue respectively. Using these pseudo-tissues and previously described approaches based on differential panning, we isolated very efficient antibody clones, but not against HER2. To optimize HER2 targeting and tissue specificity, we first performed 3-4 rounds of in vitro panning using recombinant HER2 extracellular domain (ECD) to enrich the phage library in HER2 binders, followed by one panning round using the two FFPE pseudo-tissues to retain binders for IHC conditions. We then analyzed the bound phages using next-generation sequencing to identify antibody sequences specifically associated with the HER2-positive pseudo-tissue. Using this approach, the top-ranked clone identified by sequencing was specific to the HER2-positive pseudo-tissue and showed a staining pattern similar to that of the antibody used for the clinical diagnosis of HER2-positive breast cancer. However, we could not optimize staining on other tissues, showing that specificity was restricted to the tissue used for selection and screening. Therefore, future optimized protocols must consider tissue diversity early during the selection by panning using a wide collection of tissue types.

Archived tumor specimens are routinely preserved by formalin fixation and paraffin embedding. Despite the conventional wisdom that proteomics might be ineffective due to the cross-linking and pre-analytical variables, these samples have utility for both discovery and targeted proteomics. Building on this capability, proteomics approaches can be used to maximize our understanding of cancer biology and clinical relevance by studying preserved tumor tissues annotated with the patients\' medical histories. Proteomics of formalin-fixed paraffin-embedded (FFPE) tissues also integrates with histological evaluation and molecular pathology strategies, so that additional collection of research biopsies or resected tumor aliquots is not needed. The acquisition of data from the same tumor sample also overcomes concerns about biological variation between samples due to intratumoral heterogeneity. However, the protein extraction and proteomics sample preparation from FFPE samples can be onerous, particularly for small (i.e., limited or precious) samples. Therefore, we provide a protocol for a recently introduced kit-based EasyPep method with benchmarking against a modified version of the well-established filter-aided sample preparation strategy using laser-capture microdissected lung adenocarcinoma tissues from a genetically engineered mouse model. This model system allows control over the tumor preparation and pre-analytical variables while also supporting the development of methods for spatial proteomics to examine intratumoral heterogeneity. Data are posted in ProteomeXchange (PXD045879).

Archived patient-derived tissue specimens play a central role in understanding disease and developing therapies. To address specificity and sensitivity shortcomings of existing single-cell resolution proteoform analysis tools, we introduce a hybrid microfluidic platform (DropBlot) designed for proteoform analyses in chemically fixed single cells. DropBlot serially integrates droplet-based encapsulation and lysis of single fixed cells, with on-chip microwell-based antigen retrieval, with single-cell western blotting of target antigens. A water-in-oil droplet formulation withstands the harsh chemical (SDS, 6 M urea) and thermal conditions (98 °C, 1-2 hr) required for effective antigen retrieval, and supports analysis of retrieved protein targets by single-cell electrophoresis. We demonstrate protein-target retrieval from unfixed, paraformaldehyde-fixed (PFA), and methanol-fixed cells. Key protein targets (HER2, GAPDH, EpCAM, Vimentin) retrieved from PFA-fixed cells were resolved and immunoreactive. Relevant to biorepositories, DropBlot profiled targets retrieved from human-derived breast tumor specimens archived for six years, offering a workflow for single-cell protein-biomarker analysis of sparing biospecimens.

Osteosarcoma is a form of bone cancer that predominantly impacts osteoblasts, the cells responsible for creating fresh bone tissue. Typical indications include bone pain, inflammation, sensitivity, mobility constraints, and fractures. Utilising imaging techniques such as X-rays, MRI scans, and CT scans can provide insights into the size and location of the tumour. Additionally, a biopsy is employed to confirm the diagnosis. Analysing genes with distinct expression patterns unique to osteosarcoma can be valuable for early detection and the development of effective treatment approaches. In this research, we comprehensively examined the entire transcriptome and pinpointed genes with altered expression profiles specific to osteosarcoma. The study mainly aimed to identify the molecular fingerprint of osteosarcoma. In this study, we processed 90 FFPE samples from PathWest with an almost equal number of osteosarcoma and healthy tissues. RNA was extracted from Paraffin-embedded tissue; RNA was sequenced, the sequencing data was analysed, and gene expression was compared to the healthy samples of the same patients. Differentially expressed genes in osteosarcoma-derived samples were identified, and the functions of those genes were explored. This result was combined with our previous studies based on FFPE and fresh samples to perform a meta-analysis. We identified 1,500 identical differentially expressed genes in PathWest osteosarcoma samples compared to normal tissue samples of the same patients. Meta-analysis with combined fresh tissue samples identified 530 differentially expressed genes. IFITM5, MMP13, PANX3, and MAGEA6 were some of the most overexpressed genes in osteosarcoma samples, while SLC4A1, HBA1, HBB, AQP7 genes were some of the top downregulated genes. Through the meta-analysis, 530 differentially expressed genes were identified to be identical among FFPE (105 FFPE samples) and 36 fresh bone samples. Deconvolution analysis with single-cell RNAseq data confirmed the presence of specific cell clusters in FFPE samples. We propose these 530 DEGs as a molecular fingerprint of osteosarcoma.

In recent years, the development of spatial transcriptomic technologies has enabled us to gain an in-depth understanding of the spatial heterogeneity of gene expression in biological tissues. However, a simple and efficient tool is required to analyze multiple spatial targets, such as mRNAs, miRNAs, or genetic mutations, at high resolution in formalin-fixed paraffin-embedded (FFPE) tissue sections. In this study, we developed hydrogel pathological sectioning coupled with the previously reported Sampling Junior instrument (HPSJ) to assess the spatial heterogeneity of multiple targets in FFPE sections at a scale of 180 μm. The HPSJ platform was used to demonstrate the spatial heterogeneity of 9 ferroptosis-related genes (TFRC, NCOA4, FTH1, ACSL4, LPCAT3, ALOX12, SLC7A11, GLS2, and GPX4) and 2 miRNAs (miR-185-5p and miR522) in FFPE tissue samples from patients with triple-negative breast cancer (TNBC). The results validated the significant heterogeneity of ferroptosis-related mRNAs and miRNAs. In addition, HPSJ confirmed the spatial heterogeneity of the L858R mutation in 7 operation-sourced and 4 needle-biopsy-sourced FFPE samples from patients with lung adenocarcinoma (LUAD). The successful detection of clinical FFPE samples indicates that HPSJ is a precise, high-throughput, cost-effective, and universal platform for analyzing spatial heterogeneity, which is beneficial for elucidating the mechanisms underlying drug resistance and guiding the prescription of mutant-targeted drugs in patients with tumors.