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Canine protothecosis is a rare disease caused by saprophytic unicellular achlorophyllous aerobic algae that are ubiquitous in the environment. We report a novel case of neurological and cardiological manifestations associated with disseminated protothecosis. An adult spayed female Boxer dog was presented with a 1‐week history of anorexia, progressive central vestibular signs, and a Grade III/VI systolic heart murmur. Magnetic resonance (MR) imaging revealed obstructive hydrocephalus at the level of the mesencephalic aqueduct, while echocardiography and elevated troponin levels suggested an infiltrative cardiomyopathy. No obvious cause was identified. Cerebrospinal fluid (CSF) collection was not performed due to associated procedural risks. Despite receiving symptomatic treatment and maintaining stability for 3 weeks, the dog eventually suffered cardiorespiratory arrest. Postmortem examination revealed disseminated protothecosis, predominantly affecting the heart and brain. We recommend that in cases where the cause of obstructive hydrocephalus is unclear, especially when CSF collection is not feasible, a comprehensive diagnostic method should be implemented. This includes meticulous investigations to identify infected tissues, followed by sampling and performing cytology/histology and culture tests to confirm the presence of the algal organism. Early diagnosis may allow early treatment, although long‐term prognosis remains largely unfavorable due to the absence of effective treatments. [ABSTRACT FROM AUTHOR]

The study evaluated expression profiles of few regulatory lncRNAs in oral squamous cell carcinoma and normal mucosa adjacent to oral cancer using paired fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues stored at a different duration of time (1–5 years) using real-time quantitative PCR. The quantity and quality of total RNA isolated from FFPE tissues was less compared with that of fresh frozen tissues, which resulted in a noncorrelation of quantification cycle values. Following normalization, the expression of lncRNAs in the paired tissues did not differ significantly. The differential expression of the lncRNAs in the study was consistent with The Cancer Genome Atlas head and neck squamous cell carcinoma database. The study findings demonstrate the possibility of performing accurate quantitative analysis of lncRNAs using short amplicons and standardized real-time quantitative PCR assays in oral squamous cell carcinoma FFPE samples.

The relatively recent developments in mass spectrometry (MS) have provided novel opportunities for this technology to impact modern medicine. One of those opportunities is in biomarker discovery and diagnostics. Key developments in sample preparation have enabled a greater range of clinical samples to be characterized at a deeper level using MS. While most of these developments have focused on blood, tissues have also been an important resource. Fresh tissues, however, are difficult to obtain for research purposes and require significant resources for long-term storage. There are millions of archived formalin-fixed paraffin-embedded (FFPE) tissues within pathology departments worldwide representing every possible tissue type including tumors that are rare or very small. Owing to the chemical technique used to preserve FFPE tissues, they were considered intractable to many newer proteomics techniques and primarily only useful for immunohistochemistry. In the past couple of decades, however, researchers have been able to develop methods to extract proteins from FFPE tissues in a form making them analyzable using state-of-the-art technologies such as MS and protein arrays. This review will discuss the history of these developments and provide examples of how they are currently being used to identify biomarkers and diagnose diseases such as cancer. [ABSTRACT FROM AUTHOR]

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol–chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol–chloroform DNA isolation protocol. [ABSTRACT FROM AUTHOR]

Background: Mass spectrometry imaging (MSI) derives spatial molecular distribution maps directly from clinical tissue specimens and thus bears great potential for assisting pathologists with diagnostic decisions or personalized treatments. Unfortunately, progress in translational MSI is often hindered by insufficient quality control and lack of reproducible data analysis. Raw data and analysis scripts are rarely publicly shared. Here, we demonstrate the application of the Galaxy MSI tool set for the reproducible analysis of a urothelial carcinoma dataset. Methods: Tryptic peptides were imaged in a cohort of 39 formalin-fixed, paraffin-embedded human urothelial cancer tissue cores with a MALDI-TOF/TOF device. The complete data analysis was performed in a fully transparent and reproducible manner on the European Galaxy Server. Annotations of tumor and stroma were performed by a pathologist and transferred to the MSI data to allow for supervised classifications of tumor vs. stroma tissue areas as well as for muscle-infiltrating and non-muscle infiltrating urothelial carcinomas. For putative peptide identifications, m/z features were matched to the MSiMass list. Results: Rigorous quality control in combination with careful pre-processing enabled reduction of m/z shifts and intensity batch effects. High classification accuracy was found for both, tumor vs. stroma and muscle-infiltrating vs. non-muscle infiltrating urothelial tumors. Some of the most discriminative m/z features for each condition could be assigned a putative identity: stromal tissue was characterized by collagen peptides and tumor tissue by histone peptides. Immunohistochemistry confirmed an increased histone H2A abundance in the tumor compared to the stroma tissues. The muscle-infiltration status was distinguished via MSI by peptides from intermediate filaments such as cytokeratin 7 in non-muscle infiltrating carcinomas and vimentin in muscle-infiltrating urothelial carcinomas, which was confirmed by immunohistochemistry. To make the study fully reproducible and to advocate the criteria of FAIR (findability, accessibility, interoperability, and reusability) research data, we share the raw data, spectra annotations as well as all Galaxy histories and workflows. Data are available via ProteomeXchange with identifier PXD026459 and Galaxy results via https://github.com/foellmelanie/Bladder%5fMSI%5fManuscript%5fGalaxy%5flinks. Conclusion: Here, we show that translational MSI data analysis in a fully transparent and reproducible manner is possible and we would like to encourage the community to join our efforts. [ABSTRACT FROM AUTHOR]

The relatively recent developments in mass spectrometry (MS) have provided novel opportunities for this technology to impact modern medicine. One of those opportunities is in biomarker discovery and diagnostics. Key developments in sample preparation have enabled a greater range of clinical samples to be characterized at a deeper level using MS. While most of these developments have focused on blood, tissues have also been an important resource. Fresh tissues, however, are difficult to obtain for research purposes and require significant resources for long-term storage. There are millions of archived formalin-fixed paraffin-embedded (FFPE) tissues within pathology departments worldwide representing every possible tissue type including tumors that are rare or very small. Owing to the chemical technique used to preserve FFPE tissues, they were considered intractable to many newer proteomics techniques and primarily only useful for immunohistochemistry. In the past couple of decades, however, researchers have been able to develop methods to extract proteins from FFPE tissues in a form making them analyzable using state-of-the-art technologies such as MS and protein arrays. This review will discuss the history of these developments and provide examples of how they are currently being used to identify biomarkers and diagnose diseases such as cancer.

Recovery and amplification of nucleic acids from archived formalin-fixed tissue samples is the most developing field in retrospective genetic studies. We compared different deparaffinization methods and DNA isolation techniques, and intergroup comparisons were performed to evaluate the effectiveness of different storing methods for archival OSCC samples based on obtained mean DNA quantity, quality, and PCR amplification of the P53 gene. The study comprised 75 archival histologically diagnosed OSCC samples which were divided into Group I: Formalin-fixed paraffin-embedded tissue blocks and Group II: Long-term formalin-fixed tissue. A comparison of different deparaffinization methods showed that xylene deparaffinization is an efficient method to obtain suitable DNA. Comparing different DNA isolation techniques illustrated that the conventional phenol–chloroform method gives better integrity to DNA in contrast with the kit method. Comparison between FFPET and long-term FFT samples demonstrated that samples fixed in formalin overnight and embedded in wax yield better quality and quantity DNA in comparison with long-term samples fixed in formalin. To obtain suitable integrity of DNA, tissue samples should be stored by fixing in formalin overnight followed by preparation of paraffin tissue blocks, deparaffinization by xylene, and subjecting them to the conventional phenol–chloroform DNA isolation protocol.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in biobanks and pathology archives are a vast but underutilized source for molecular studies on different diseases. Beyond being the "gold standard" for preservation of diagnostic human tissues, FFPE samples retain similar genetic information as matching blood samples, which could make FFPE samples an ideal resource for genomic analysis. However, research on this resource has been hindered by the perception that DNA extracted from FFPE samples is of poor quality. Here, we show that germline disease-predisposing variants and polygenic risk scores (PRS) can be identified from FFPE normal tissue (FFPE-NT) DNA with high accuracy. We optimized the performance of FFPE-NT DNA on a genome-wide array containing 657,675 variants. Via a series of testing and validation phases, we established a protocol for FFPE-NT genotyping with results comparable with blood genotyping. The median call rate of FFPE-NT samples in the validation phase was 99.85% (range 98.26%-99.94%) and median concordance with matching blood samples was 99.79% (range 98.85%-99.9%). We also demonstrated that a rare pathogenic PALB2 genetic variant predisposing to cancer can be correctly identified in FFPE-NT samples. We further imputed the FFPE-NT genotype data and calculated the FFPE-NT genome-wide PRS in 3 diseases and 4 disease risk variables. In all cases, FFPE-NT and matching blood PRS were highly concordant (all Pearson's r > 0.95). The ability to precisely genotype FFPE-NT on a genome-wide array enables translational genomics applications of archived FFPE-NT samples with the possibility to link to corresponding phenotypes and longitudinal health data., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Linking molecular and chemical changes to human disease states depends on the availability of appropriate clinical samples, mostly preserved as formalin‐fixed paraffin‐embedded (FFPE) specimens stored in tissue banks. Mass spectrometry imaging (MSI) enables the visualization of the spatiotemporal distribution of molecules in biological samples. However, MSI is not effective for imaging FFPE tissues because of the chemical modifications of analytes, including complex crosslinking between nucleophilic moieties. Here we used an MS‐compatible inorganic nucleophile, hydroxylamine hydrochloride, to chemically reverse inter‐ and intra‐crosslinks from endogenous molecules. The analyte restoration appears specific for formaldehyde‐reactive amino acids. This approach enabled the MSI‐assisted localization of pancreatic peptides expressed in the alpha, beta, and gamma cells. Pancreatic islet‐like distributions of islet hormones were observed in human FFPE tissues preserved for more than five years, demonstrating that samples from biobanks can effectively be investigated with MSI. [ABSTRACT FROM AUTHOR]

• Aberrant histone PTMs have been implicated with numerous pathologies. • Profiling histone PTMs in patient samples holds great promise. • Mass spectrometry (MS) is the method of choice for the analysis of histone PTMs. • We describe methods to extract histones from patient samples for MS-based PTM analysis. Aberrations in histone post-translational modifications (PTMs) have been implicated with the development of numerous pathologies, including cancer. Therefore, profiling histone PTMs in patient samples could provide information useful for the identification of epigenetic biomarkers, as well as for the discovery of potential novel targets. While antibody-based methods have been traditionally employed to analyze histone PTM in clinical samples, mass spectrometry (MS) can provide a more comprehensive, unbiased and quantitative view on histones and their PTMs. To combine the power of MS-based methods and the potential offered by histone PTM profiling of clinical samples, we have recently developed a series of methods for the extraction and enrichment of histones from different types of patient samples, including formalin-fixed paraffin-embedded tissues, fresh- and optimal cutting temperature-frozen tissues, and primary cells. Here, we provide a detailed description of these protocols, together with indications on the expected results and the most suitable workflow to be used downstream of each procedure. [ABSTRACT FROM AUTHOR]

Masson’s Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert’s Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections.

Human papillomaviruses (HPVs) have been associated with a subset of head and neck squamous cell carcinomas (HNSCCs). The aim of this study was to determine the prevalence of HPV DNA in archived formalin‐fixed paraffin‐embedded tissue from patients with histologically confirmed HNSCCs in a South African cohort. A nested PCR was used for the detection of HPV DNA targeting the L1 gene. Positive samples were confirmed using an in‐house hemi‐nested PCR targeting the E6 gene and genotyped by sequence determination of amplicons. HPV DNA was detected in 57/780 (7.3%) samples, with the highest prevalence being in the sinonasal tract (16.0%) and oropharynx (10.8%). HPV16 was the most frequently detected type, being found in 26/57 (45.6%) positive samples. The prevalence of HPV DNA in HNSCCs found in this study was lower than that found in developed countries [ABSTRACT FROM AUTHOR]

Background: Diagnosis of extrapulmonary tuberculosis continues to be a challenge due to the complexity of the causative organism and the wide array of pathologic features seen in this infection. Xpert MTB/RIF can be used on fresh or frozen tissue specimens for diagnosis of tuberculosis with good results. However, there is little data on its use with formalin-fixed paraffin-embedded (FFPE) tissues. Objectives: The aim of this study was to demonstrate the potential utility of Xpert MTB/RIF and to compare its performance to Ziehl-Neelsen staining for the detection of Mycobacterium tuberculosis from FFPE tissues using histological features from haematoxylin and eosin staining as the gold standard. Methods: Eighty randomly selected archival FFPE tissues exhibiting histological features of tuberculosis were included in the study. After deparaffinisation and lysis, all the tissue specimens were subjected to the Xpert® MTB/RIF assay. The outcome measures were proportions of positively identified cases by each test. Results: Using histology as the gold standard, the sensitivity of Ziehl-Neelsen staining was 20.3% (95% confidence interval: 12% – 30.8%), and the sensitivity of the Xpert® MTB/RIF assay was 53.2% (95% confidence interval: 41.6% – 64.9%); the difference was statistically significant (p = 0.002). None of the cases tested positive for rifampicin resistance. Conclusion: With prior deparaffinisation and lysis, FFPE tissues are amenable to testing by Xpert® MTB/RIF assay. A validation study to determine the clinical utility, analytical optimisation and cost implications of this assay for FFPE tissues is recommended.

Introduction: This review is an update on recent progress in proteomic studies of formalin-fixed paraffin-embedded (FFPE) tissues, which open the opportunity to investigate diseases and research potential biomarkers, particularly when availability of fresh/frozen tissues is low. Areas covered: We described improvement of existing protocols or the new ones regarding deparaffinization and protein extraction of FFPE samples published from 2014 to today. Moreover, the growing interest to use FFPE tissues for mass spectrometry imaging approach is presented together with the search of post-translational modifications. Expert opinion: In the last few years, the number of papers using FFPE tissues in proteomic analysis is growing. The interest to apply proteomic analysis to FFPE tissues lies in the easy accessibility of a great number of samples from archives. Nevertheless, standardization in the approach among the different researchers is not achieved, making essentially incomparable the results obtained. This limit should be overcome. [ABSTRACT FROM AUTHOR]

The study evaluated expression profiles of few regulatory lncRNAs in oral squamous cell carcinoma and normal mucosa adjacent to oral cancer using paired fresh frozen and formalin-fixed paraffin-embedded (FFPE) tissues stored at a different duration of time (1-5 years) using real-time quantitative PCR. The quantity and quality of total RNA isolated from FFPE tissues was less compared with that of fresh frozen tissues, which resulted in a noncorrelation of quantification cycle values. Following normalization, the expression of lncRNAs in the paired tissues did not differ significantly. The differential expression of the lncRNAs in the study was consistent with The Cancer Genome Atlas head and neck squamous cell carcinoma database. The study findings demonstrate the possibility of performing accurate quantitative analysis of lncRNAs using short amplicons and standardized real-time quantitative PCR assays in oral squamous cell carcinoma FFPE samples.

Hepatocellular carcinoma (HCC) is the third leading cause of cancer-related death worldwide; however, the mutational properties of HCC-associated carcinogens remain largely uncharacterized. We hypothesized that mechanisms underlying chemical-induced HCC can be characterized by evaluating the mutational spectra of these tumors. To test this hypothesis, we performed exome sequencing of B6C3F1/N HCCs that arose either spontaneously in vehicle controls (n = 3) or due to chronic exposure to gingko biloba extract (GBE; n = 4) or methyleugenol (MEG; n = 3). Most archived tumor samples are available as formalin-fixed paraffin-embedded (FFPE) blocks, rather than fresh-frozen (FF) samples; hence, exome sequencing from paired FF and FFPE samples was compared. FF and FFPE samples showed 63% to 70% mutation concordance. Multiple known (e.g., Ctnnb1T41A, BrafV637E) and novel (e.g., Erbb4C559S, Card10A700V, and Klf11P358L) mutations in cancer-related genes were identified. The overall mutational burden was greater for MEG than for GBE or spontaneous HCC samples. To characterize the mutagenic mechanisms, we analyzed the mutational spectra in the HCCs according to their trinucleotide motifs. The MEG tumors clustered closest to Catalogue of Somatic Mutations in Cancer signatures 4 and 24, which are, respectively, associated with benzo(a)pyrene- and aflatoxin-induced HCCs in humans. These results establish a novel approach for classifying liver carcinogens and understanding the mechanisms of hepatocellular carcinogenesis. [ABSTRACT FROM AUTHOR]

The BIOMED-2 PCR protocol for targeting the IGH gene is widely employed for detecting clonality in B-cell malignancies. Unfortunately, the detection of clonality with this method is not very sensitive when paraffin sections are used as a DNA source. To increase the sensitivity, we devised a semi-nested modification of a JH consensus primer. The clonality detection rates of three assays were compared: the standard BIOMED-2, BIOMED-2 assay followed by BIOMED-2 re-amplification, and BIOMED-2 assay followed by semi-nested BIOMED-2. We tested more than 100 cases using paraffin-embedded tissues of various B-cell lymphomas, and found that the clonality detection rates with the above three assays were 63.9%, 79.6%, and 88.0%, respectively. While BIOMED-2 re-amplification was significantly more sensitive than the standard BIOMED-2, the semi-nested BIOMED-2 was significantly more sensitive than both the standard BIOMED-2 and BIOMED-2 re-amplification. An increase in sensitivity was observed in all lymphoma subtypes examined. In conclusion, tumor clonality may be detected in nearly 90% of B-cell lymphoma cases with semi-nested BIOMED-2. This ancillary assay may be useful when the standard BIOMED-2 fails to detect clonality in histopathologically suspected B-cell lymphomas. [ABSTRACT FROM AUTHOR]

The measurement of DNA adducts provides important information about human exposure to genotoxic chemicals and can be employed to elucidate mechanisms of DNA damage and repair. DNA adducts can serve as biomarkers for interspecies comparisons of the biologically effective dose of procarcinogens and permit extrapolation of genotoxicity data from animal studies for human risk assessment. One major challenge in DNA adduct biomarker research is the paucity of fresh frozen biopsy samples available for study. However, archived formalin-fixed paraffin-embedded (FFPE) tissues with clinical diagnosis of disease are often available. We have established robust methods to recover DNA free of crosslinks from FFPE tissues under mild conditions which permit quantitative measurements of DNA adducts by liquid chromatography-mass spectrometry. The technology is versatile and can be employed to screen for DNA adducts formed with a wide range of environmental and dietary carcinogens, some of which were retrieved from section-cuts of FFPE blocks stored at ambient temperature for up to nine years. The ability to retrospectively analyze FFPE tissues for DNA adducts for which there is clinical diagnosis of disease opens a previously untapped source of biospecimens for molecular epidemiology studies that seek to assess the causal role of environmental chemicals in cancer etiology.

Histopathology is the gold standard for fungal infection (FI) diagnosis, but it does not provide a genus and/or species identification. The objective of the present study was to develop targeted next-generation sequencing (NGS) on formalin-fixed tissue samples (FTs) to achieve a fungal integrated histomolecular diagnosis. Nucleic acid extraction was optimized on a first group of 30 FTs with Aspergillus fumigatus or Mucorales infection by macrodissecting the microscopically identified fungal-rich area and comparing Qiagen and Promega extraction methods through DNA amplification by A. fumigatus and Mucorales primers. Targeted NGS was developed on a second group of 74 FTs using three primer pairs (ITS-3/ITS-4, MITS-2A/MITS-2B, and 28S-12-F/28S-13-R) and two databases (UNITE and RefSeq). A prior fungal identification of this group was established on fresh tissues. Targeted NGS and Sanger sequencing results on FTs were compared. To be valid, the molecular identifications had to be compatible with the histopathological analysis. In the first group, the Qiagen method yielded a better extraction efficiency than the Promega method (100% and 86.7% of positive PCRs, respectively). In the second group, targeted NGS allowed fungal identification in 82.4% (61/74) of FTs using all primer pairs, in 73% (54/74) using ITS-3/ITS-4, in 68.9% (51/74) using MITS-2A/MITS-2B, and in 23% (17/74) using 28S-12-F/28S-13-R. The sensitivity varied according to the database used (81% [60/74] using UNITE compared to 50% [37/74] using RefSeq [ P = 0.000002]). The sensitivity of targeted NGS (82.4%) was higher than that of Sanger sequencing (45.9%; P < 0.00001). To conclude, fungal integrated histomolecular diagnosis using targeted NGS is suitable on FTs and improves fungal detection and identification.

Histone post-translational modifications (hPTMs) contribute to the regulation of gene expression and increasing evidence links them to the development of various pathologies, highlighting their potential as biomarkers for prognostic, diagnostic and therapeutic applications. Mass spectrometry (MS) has emerged as a powerful analytical tool for hPTM analysis, which has also been applied to the analysis of epigenetic aberrations in diseases. However, the potential offered by the MS-based hPTM analysis of clinical samples for epigenetic biomarker discovery has been left largely unexploited. This article summarizes the contribution of MS-based approaches to clinical epigenetics, with a special focus on the PAThology tissue analysis of Histones by Mass Spectrometry (PAT-H-MS) approach – which represents the first application of MS-based hPTM analysis to formalin-fixed paraffin-embedded clinical samples – discussing its strengths and limitations, as well as possible implementations. [ABSTRACT FROM PUBLISHER]

In this chapter, we describe the utility of fluorescence two-dimensional difference gel electrophoresis (2D-DIGE) as a proteomics platform for the global detection of expressed proteins in formalin-fixed paraffin-embedded (FFPE) tissues and its use for biomarker discovery/identification of proteins that may contribute to cancer development and progression. Formalin fixation and paraffin embedding of tissue is the standard processing methodology practiced in pathology laboratories worldwide, resulting in a highly stable form of tissue that is easily stored due to its inherent stability at room temperature. Consequently, FFPE tissues represent an attractive reservoir of clinical material for conducting retrospective protein biomarker analysis. A limitation for proteomics research in this type of clinical sample is the amount of viable protein that can be obtained from fixed tissues. Tissue biopsies are precious samples that can generally be acquired in very small amounts due to the invasive nature of the sample collection, mainly during surgery or biopsy. Subsequently, the amount of extracted protein can be, in many cases, very limited. The saturation 2D-DIGE technology has emerged as a useful method for protein analysis where only scarce amounts of protein are available. This approach can be adapted successfully to label low-level protein isolated from FFPE tissue., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Companion diagnostics, tests that purport to classify patients into 'responders' and 'non-responders' for a specified targeted therapy, demand methods that quantify the actual amount of the corresponding target molecule in tumors from these patients. Various methods are employed depending upon the nature of the target. Many of the candidate therapeutic agents target the abnormal expression of a protein, the detection of which lends itself to an immunohistochemistry (IHC) approach. This review focuses on IHC with formalin-fixed paraffin-embedded (FFPE) tissues for purely pragmatic reasons; first, the morphologic information pertaining to the tumor is of value and should not be discarded as in extraction type assays; second, FFPE tissues are mostly what we have to hand at the time that the diagnostic question is posed. During the four decades of employment of IHC involving the production of a variety of special stains used in the diagnosis or classification of tumors, we have acquired some bad habits, essentially when judging the IHC result via the perception of a 'good' stain that 'pleases the eye' of the user pathologist, and nothing more. This review takes, as its basic premise, the notion that IHC can be upgraded from its use as a qualitative special staining method to an accurate and reliable quantitative 'tissue-based immunologic assay'. If accomplished, this enhanced IHC assay would serve accurately to quantify proteins in tissue sections, analogous to the use of the ELISA (enzyme-linked immunosorbent assay) method in the clinical laboratory. The necessary steps for converting IHC to a tissue-based ELISA-like immunoassay of immediate practical use are reviewed with constructive suggestions for steps that can be (must be) taken to achieve the practical reality of quantitative in situ proteomics. [ABSTRACT FROM AUTHOR]

Breast cancer is a complex and heterogeneous disease. Signaling by estrogen receptor (ER), progesterone receptor (PR), and/or human EGF-like receptor 2 (HER2) is a main driver in the development and progression of a large majority of breast tumors. Molecular characterization of primary tumors has identified major subtypes that correlate with ER/PR/HER2 status, and also subgroup divisions that indicate other molecular and cellular features of the tumors. While some of these research findings have been incorporated into clinical practice, several challenges remain to improve breast cancer management and patient survival, for which the integration of novel biomarkers into current practice should be beneficial. microRNAs (miRNAs) are a class of short non-coding regulatory RNAs with an etiological contribution to breast carcinogenesis. miRNA-based diagnostic and therapeutic applications are rapidly emerging as novel potential approaches to manage and treat breast cancer. Rapid technological development enables specific and sensitive detection of individual miRNAs or the entire miRNome in tissues, blood, and other biological specimens from breast cancer patients. This review focuses on recent miRNA research and its potential to address unmet clinical needs and challenges. The four sections presented discuss miRNA findings in the context of the following clinical challenges: biomarkers for early detection; prognostic and predictive biomarkers for treatment decisions using targeted therapies against ER and HER2; diagnostic and prognostic biomarkers for subgrouping of triple-negative breast cancer, for which there are currently no targeted therapies; and biomarkers for monitoring and characterization of metastatic breast cancer. The review concludes with a critical analysis of the current state of miRNA breast cancer research and the need for further studies using large patient cohorts under well-controlled conditions before considering the clinical implementation of miRNA biomarkers. [ABSTRACT FROM AUTHOR]

The identification of clinically relevant biomarkers represents an important challenge in oncology. This problem can be addressed with biomarker discovery and verification studies performed directly in tumor samples using formalin-fixed paraffin-embedded (FFPE) tissues. However, reliably measuring proteins in FFPE samples remains challenging. Here, we demonstrate the use of liquid chromatography coupled to multiple reaction monitoring mass spectrometry (LC-MRM/MS) as an effective technique for such applications. An LC-MRM/MS method was developed to simultaneously quantify hundreds of peptides extracted from FFPE samples and was applied to the targeted measurement of 200 proteins in 48 triple-negative, 19 HER2-overexpressing, and 20 luminal A breast tumors. Quantitative information was obtained for 185 proteins, including known markers of breast cancer such as HER2, hormone receptors, Ki-67, or inflammation-related proteins. LC-MRM/MS results for these proteins matched immunohistochemistry or chromogenic in situ hybridization data. In addition, comparison of our results with data from the literature showed that several proteins representing potential biomarkers were identified as differentially expressed in triple-negative breast cancer samples. These results indicate that LC-MRM/MS assays can reliably measure large sets of proteins using the analysis of surrogate peptides extracted from FFPE samples. This approach allows to simultaneously quantify the expression of target proteins from various pathways in tumor samples. LC-MRM/MS is thus a powerful tool for the relative quantification of proteins in FFPE tissues and for biomarker discovery., Competing Interests: Conflict of interest The authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

miRNAs are short, non-coding, regulatory RNAs that exert cell type-dependent, context-dependent, transcriptome-wide gene expression control under physiological and pathological conditions. Tissue slide-based assays provide qualitative (tumor compartment) and semi-quantitative (expression levels) information about altered miRNA expression at single-cell resolution in clinical tumor specimens. Reviewed here are key technological advances in the last 5 years that have led to implementation of fully automated, robust and reproducible tissue slide-based assays for in situ miRNA detection on US FDA-approved instruments; recent tissue slide-based discovery studies that suggest potential clinical applications of specific miRNAs in cancer medicine are highlighted; and the challenges in bringing tissue slide-based miRNA assays into the clinic are discussed, including clinical validation, biomarker performance, biomarker space and integration with other biomarkers. [ABSTRACT FROM AUTHOR]

Abstract: Background: Formalin fixation, duration of tissue storage and tissue enrichment techniques can affect DNA methylation yield but these effects have not been quantitatively measured. The aim is to investigate the relative impact of these conditions on DNA methylation in rectal cancer. Methods: 10 rectal cancers with matched undissected fresh frozen tissues, laser capture microdissected (LCM) formalin-fixed paraffin-embedded (FFPE) tissues, manual macrodissected FFPE tissues, adjacent normal mucosa and stromal tissues were analysed for APC and LINE-1 methylation using bisulphite pyrosequencing. Results: FFPE cancer tissues, which had been stored for at least 4years showed similar APC and LINE-1 methylation changes to matched fresh frozen cancer tissues. Laser capture microdissection did not increase the degree of methylation detected compared to manual macrodissection. Analysis of stromal tissues showed that they had undergone significant methylation changes compared to adjacent macroscopically normal mucosa, but not to the same extent as cancer tissues. Conclusion: Reliable DNA methylation results can be obtained from FFPE rectal cancer tissues, which have been in long-term storage. Because only minor differences in methylation between macrodissected and LCM cancer tissues were found, our results do not support the routine use of LCM to enrich for cancer cells for DNA methylation studies. [Copyright &y& Elsevier]

The article discusses a study on the use of polymerase chain reaction (PCR) for the detection of Marek's disease virus (MDV) and reticuloendotheliosis (RE) virus in formalin-fixed, paraffin-embedded (FFPE) tumorous tissues. It outlines the methods used in the study that included tissue samples and inoculation and deoxyribonucleic acid (DNA) extraction. Results show the detection of MDV-infected tissues after being fixed in buffered formalin, and after vaccination with Rispens strain of MDV.

Masson's Trichrome Staining (MTS) is a useful tool for analyzing fibrosis in a plethora of disease pathologies by differential staining of tissue components. It is used to identify collagen fibers in different tissues like heart, lung, skin, and muscles. Especially in cardiac fibrosis, MTS stains the collagen fibers (blue color), which helps in the distinction of scar area versus the healthy area (red color). However, there are several challenges to stain both paraffin-embedded sections and frozen (cryosections) using a single protocol. Therefore, the goal of this study was to develop a simple short protocol to assess cardiac fibrosis in both paraffin-embedded and cryo heart sections. MTS uses three different stains, i.e., Weigert's Iron Hematoxylin, Biebrich scarlet-acid fuchsin, and aniline blue to detect nuclei, cytoplasm, and collagen, respectively. In this study, we developed a simple short protocol that can be adapted by any lab to easily assess cardiac fibrosis in paraffin and frozen heart sections. Furthermore, we have addressed the challenges that are commonly faced during the immunostaining process and troubleshooting techniques. Overall, we have successfully developed a simple one-step protocol to assess myocardial fibrosis in paraffin-embedded and frozen cryosections. [ABSTRACT FROM AUTHOR]

Abstract: The use of high throughput sequencing (HTS) technologies in biomedicine is expanding in a variety of fields in recent years. The 454 system is an HTS platform that is ideally suited to characterize B cell receptor (BCR) repertoires by sequencing of immunoglobulin (Ig) genes, as it is able to sequence stretches of several hundred nucleotides. Most studies that used this platform for antibody repertoire analyses have started from fresh or frozen tissues or peripheral blood samples, and rely on starting with optimal quality DNA. In this paper we demonstrate that BCR repertoire analysis can be done using DNA from formalin-fixed paraffin-embedded (FFPE) human tissue samples. The heterogeneity of BCR repertoires we obtained confirms the plausibility of HTS of DNA from FFPE specimens. The establishment of experimental protocols and computational tools that enable sequence data analysis from the low quality DNA of FFPE tissues is important for enabling research, as it would enable the use of the rich source of preserved samples in clinical biobanks and biopsy archives. [Copyright &y& Elsevier]

Abstract: As emerging novel DNA-based methodologies are adopted, nucleic acid-based assays depend critically on the quality and quantity of extracted DNA. Formalin-fixed, paraffin embedded (FFPE) tissue samples provide an invaluable resource for subsequent molecular studies of clinical phenotypes, but high-quality DNA extraction from archival FFPE tissue specimens remains complex and time-consuming. To address this challenge, we have developed a reliable rapid DNA extraction method for FFPE tissue specimens. It is based on deparaffinization at high temperature coupled with relieving crosslink in a pressure cooker. The DNA yield by this rapid method resulted in an average 1.8-fold increase in comparison with the commercial kit and OD 260/280 ratios between 1.87 and 1.95. The DNA obtained by the rapid method was suitable for methylation analyses in colon cancer patients. These data suggest that this new DNA extraction method coupled with methylation-specific polymerase chain reaction can be used for epigenetic studies with the advantages of rapidity and high quality and may contribute to the development of biomarkers in clinical studies. [Copyright &y& Elsevier]

Background. To date, little proteomic information has been available from the glomeruli of diabetic patients, possibly due to the clinical limitations of renal biopsy in diabetic patients and insufficient quantities of such specimens for proteome analysis. The purpose of the present study was to identify altered protein expression profiles in diabetic glomeruli using formalin-fixed paraffin-embedded (FFPE) kidney tissues from diabetic patients. Methods. Glomeruli were laser microdissected from FFPE autopsy kidney tissues from 10 patients with diabetic nephropathy and 10 non-diabetic control patients and underwent proteome analysis using QSTAR Elite liquid chromatography with tandem mass spectrometry and iTRAQ technology. Immunohistochemical analysis was performed on 93 autopsy samples from diabetic patients with and without nephropathy (n = 45 and n = 48, respectively). Results. Thirty-one renal and urological disease-related proteins displayed a differential abundance in glomerular samples from patients with diabetic nephropathy compared with non-diabetic control patients. Among them, we found that nephronectin, which functions in the assembly of extracellular matrix, showed clearly positive immunoreactivity in diabetic glomeruli. The numerical fraction of nephronectin-positive glomerular cross sections was increased significantly in diabetic patients with nephropathy compared to those without nephropathy (32.1 ± 31.5 versus 4.14 ± 5.65%, P < 0.0001). Furthermore, there was a significant positive correlation between this numerical fraction of nephronectin-positive glomerular cross sections and the glomerular sclerosis index (ρ = 0.881, P < 0.0001, n = 93). Conclusion. The present study demonstrated, for the first time, that nephronectin may be associated with the development of diabetic glomerulosclerosis and that proteome analysis with FFPE kidney tissues from diabetic patients with nephropathy is useful in understanding diabetic nephropathy. [ABSTRACT FROM PUBLISHER]

Abstract: The use of radioactive in situ hybridization (ISH) to quantitatively determine low-to-moderate abundant mRNA expression in formalin-fixed, paraffin-embedded archival post-mortem human brain tissue is often limited by non-specific-deposits, visible as speckles. In the present study, optimal hybridization conditions were achieved for quantifying the mRNA expression of histidine decarboxylase (HDC) by a number of alterations in a routine protocol, which included (1) during purification of the oligo-probes, glycogen was omitted as a carrier for precipitation, (2) after precipitation, the labeled probe contained within the pellet was first dissolved in water instead of in hybridization buffer (HBF), (3) during hybridization, the dithiothreitol (DTT) concentration was increased from 200 to 800mM in HBF, and (4) stringencies during hybridization and post-hybridization washes were increased by increasing the temperature. The effect of the adjustment was quantified on adjacent sections from 18 subjects (9 with Parkinson’s disease and 9 controls), by comparing the data from the standard and new protocol. The results showed that the improved protocol brought about significantly clearer background with higher signal-to-noise ratios (p =0.001). We propose that this protocol is also applicable for detection of other lower-abundant genes in human brain tissue and probably in other tissues as well. In the present study, this is not only illustrated for HDC ISH, but also for corticotrophin-releasing hormone mRNA expression in the hypothalamic paraventricular nucleus. [Copyright &y& Elsevier]

Abstract: The etiology of mycosis fungoides (MF) remains to be determined. Several studies have proposed a viral etiology with controversial results. In this case–control study we investigated the presence of Epstein–Barr virus (EBV) and the debated presence of Human T-cell lymphotrophic virus I (HTLV-I) sequences, by polymerase chain reaction on nucleic acid extracts from formalin-fixed paraffin-embedded skin biopsies. Moreover, by a multivariate approach we analyzed in the same case–control study also the contribution of two previously examined pathogens: Hepatitis C virus (HCV) and Borrelia burgdorferi (Bb). Significant differences in the frequency of infectious agents in cases and controls were detected for Bb, HTLV-I and EBV. In MF patients we found the concurrent presence of two or three of these pathogen sequences in 21 out of 83 cases, but only in 1 out of 83 healthy controls. Our results suggest that the persistence of multiple infectious agents may cause a long-term antigenic stimulation contributing to the malignant transformation of T lymphocytes, especially when associated with HTLV-I like sequences. However, these infectious agents do not seem to have effects on disease progression. [Copyright &y& Elsevier]

Summary: Despite the advances in cancer treatment, the 5-year survival rate for oral cancer has not changed significantly for the past 40years and still remains among the worst of all anatomic sites. Gene expression microarrays have been used successfully in the identification of genetic alterations in cancer development, however, these have hitherto been limited by the need for specimens with good quality intact RNA. Here, we demonstrated the use of formalin-fixed paraffin-embedded tissues in microarray experiments to identify genes differentially expressed between cancerous and normal oral tissues. Forty-three tissue samples were macrodissected and gene expression analyses were conducted using the Illumina DASL assay. We report RNA yield of 2.4 and 0.8μg/mm3 from tumour and normal tissues, respectively and this correlated directly with the tissue volume used for RNA extraction. Using unsupervised hierarchical clustering, distinct gene expression profiles for tumour and normal samples could be generated, and differentially expressed genes could be identified. The majority of these genes were involved in regulation of apoptosis and cell cycle, metastasis and cell adhesion including BCL2A1, BIRC5, MMP1, MMP9 and ITGB4. Representative genes were further validated in independent samples suggesting that these genes may be directly associated with oral cancer development. The ability to conduct microarrays on formalin-fixed paraffin-embedded specimens represents a significant advancement that could open up avenues for finding genes that could be used as prognostication and predictive tools for cancer. [Copyright &y& Elsevier]

Abstract: A preceding paper suggested 81 candidates of stage-specifically expressed proteins for either stage IA or IIIA by global shotgun proteomics and spectral counting. Six proteins, a subset of these proteins, were chosen for a further verification study since they are potentially soluble and/or secretory, which nature is convenient for detecting them in blood in clinical practice. The multiple-reaction monitoring (MRM) quantitative analysis suggested that napsin-A and anterior gradient protein 2 homolog (hAG-2) out of the 6 candidates would be useful for determining stage IA or IIIA and are related to metastasis. In the study we noted that stage IIIA patients with better outcome showed napsin-A profiles similar to that of stage IA patients. We therefore examined 14 additional patients for analysis, which contained the IA-stage patients of poorer outcome and the IIIA-stage patients of better outcome. The MRM analysis of napsin-A for all patients suggests that napsin-A contents correlate with better outcome in stage IA. This and discovery studies demonstrate that direct isolation of tumor cells alone by laser microdissection (LMD) greatly reduces complexity on comprehensive analyses, and that MRM mass spectrometry using the endogenous internal standard is a feasible technology for quantitative verification of target proteins in formalin-fixed paraffin embedded (FFPE) tissues. [Copyright &y& Elsevier]

Abstract: Objectives: 5382insC frameshift mutation along with 5331G>A (G1738R) missense mutation, both found in exon 20 of the BRCA1 gene, are relatively frequent among the Greek breast and ovarian cancer population (46%). Our goal was to develop a novel, reliable and rapid genotyping/scanning method for mutation detection of the exon 20 of the BRCA1 gene, using high-resolution melting curve analysis. Design and methods: The developed methodology was based on real-time PCR and high-resolution melting curve analysis in the presence of LCGreen I dye. Two amplicons on the exon 20 of BRCA1 gene were designed (157 bp and 100 bp), one flanking the exon''s boundaries, and one embracing the 5382insC mutation. Our methodology was first optimized and validated by using genomic DNA samples with the 5382insC and 5331G>A (G1738R) mutations and wild-type. In total, the developed methodology was applied on 90 peripheral blood and 127 formalin-fixed paraffin-embedded breast tissue samples. Results: Sensitivity studies with gDNA isolated from peripheral blood showed that mutated DNA could be reliably detected in the presence of wild-type DNA at 5% and 0.5% ratio with the larger and the smaller amplicon, respectively. By using the developed methodology we successfully identified 5382insC, 5331G>A and 5370C>T (R1751X) mutations, in genomic DNA isolated from peripheral blood samples and 5382insC mutation in two breast tumors, as verified by DNA sequencing. Conclusions: The combination of real-time PCR and high-resolution melting curve analysis provides a cost-efficient, simple and rapid approach to successfully scan exon 20 of BRCA1 gene for these clinically important and frequent mutations. [Copyright &y& Elsevier]

Formalin-fixed, paraffin-embedded tissues are widely available for gene expression analysis using TaqMan® PCR. Five methods, including 4 commercial kits, for recovering RNA from paraffin-embedded renal tumor tissue were compared. The MasterPure™ kit from Epicentre produced the highest RNA yield. However, the difference in RNA yield between the kit from Epicenter and Invitrogen's TRIzol method was not significant. Using the top 3 RNA isolation methods, the manufacturers' protocols were modified to include an overnight Proteinase K digestion. Overnight protein digestion resulted in a significant increase in RNA yield. To optimize the reverse transcription reaction, conventional reverse transcription with random oligonucleotide primers was compared to reverse transcription using primers specific for genes of interest. Reverse transcription using gene-specific primers significantly increased the quantity of cDNA detectable by TaqMan® PCR. Therefore, expression profiling of formalin-fixed, paraffin-embedded tissue using TaqMan® qPCR can be optimized by using the MasterPure™ RNA isolation kit modified to include an overnight Proteinase K digestion and gene-specific primers during the reverse transcription. (Journal of Biomolecular Screening 2010:80-85) [ABSTRACT FROM AUTHOR]

Abstract: Formalin-fixed paraffin-embedded tissues (FFPET) represent the largest source of archival biological material available for genomic studies. In this work we present an advanced protocol for extraction of high quality DNA from FFPET that can be applied in several molecular studies. Although cat mammary tumours (CMT) are the third most frequent tumour in cats the recovery of significant number of samples for molecular studies are in some way restricted to FFPET samples. We were able to obtain high quality DNA from FFPET of thirty six CMT that were subjected to pre-fixation and fixation processes routinely used in the veterinary hospitals. The quality of DNA obtained was tested by PCR amplification using six sets of primers that amplify single-copy fragments. The DNA fragments obtained were further sequenced. This protocol was able to provide FFPET gDNA that can be amplified and sequenced for larger fragments up to 1182bp. [Copyright &y& Elsevier]

Abstract: The aim of our study was to establish a new PCR protocol for the detection and discrimination of Echinococcus granulosus complex on one hand and Echinococcus multilocularis in formalin-fixed, paraffin-embedded tissues (FFPTs) on the other. The target sequences for all PCRs are located on a 471bp segment of the mitochondrial ND1 gene, the fragment sizes of the amplification products are 295bp (for the sheep strain of E. granulosus), 204bp (for the pig strain of E. granulosus) and 252bp (for E. multilocularis), respectively. In total, 80 FFPTs from patients with histologically confirmed echinococcosis (76 with E. granulosus and four with E. multilocularis) operated on in Austrian hospitals between 1978 and 2005 were examined. In 68 (85%) samples, we were able to detect specific DNA fragments with our newly established PCR protocols. Thirty-eight (47.5%) of 80 clinical samples were identified as the G1 strain, 26 (32.5%) as the G5, 6 or 7 strains and four (5%) as E. multilocularis. The specificity of all three PCRs was 100%; for the discrimination between G6 and G7 strains, sequencing of an additional 234bp PCR fragment was necessary and showed that three out of 26 G5, 6 or 7 PCR-positive patients were infected with E. granulosus genotype G6 (the camel strain). [Copyright &y& Elsevier]

Abstract: The aim of this study was to evaluate the application of PCR technique for the detection of BoHV-5 in routinely formalin-fixed, paraffin-embedded brain tissues in 20 naturally infected calves affected by fatal meningoencephalitis. Brains were divided into two halves, one kept fresh for virus isolation and PCR assay, targeting the glycoprotein C gene from BoHV-5 genome. The other half brain, corresponding to posterior cortex region, was submitted to formalin fixation and embedded into paraffin blocks for microscopic evaluation and total DNA isolation. Most of the slides showed severe multifocal non-supurative encephalitis with neuronal degeneration, neurophagia, and no acidophilic intranuclear inclusions could be found in neurons and glial. The 20 fresh samples were confirmed, by virus isolation and PCR assay, as having the BoHV-5 virus and, respective glicoprotein C sequence, while 15 of 20 formalin-fixed, paraffin-embedded samples were considered positive for the same analysis. The results revealed the first description of PCR efficiency, applied to formalin-fixed, paraffin-embedded brain collected from naturally infected calves, improving the detection of BoHV-5 from archival samples in South America. [Copyright &y& Elsevier]

To examine matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinases (TIMP) mRNA levels in archival breast cancer biopsies, we employed microdissection to separate tumour tissue from the surrounding breast tissue, or stroma and RT-PCR to determine gross qualitative and small quantitative differences in the patterns of expression. In this study, a significant correlation (p < 0.05, by Mann-Whitney U analysis) between TIMP-2 expression and lymph node involvement was identified, while MMP-11 and TIMP-1 expression patterning also significantly (p < 0.05) differed between those tumours showing calcification and those that did not. When compared by Spearmans' ρ correlation analysis, a significant association (p < 0.05, ρ = 0.404) was identified in the pattern of MMP-2 and MMP-9 gene expression. In this study, the use of microdissection and a systematic strategy of RT-PCR analysis have allowed us to investigate localized MMP and MMP inhibitor expression within breast tumours. We have identified patterns of gene expression that may further reveal aspects of breast carcinogenesis, and a robust method for examining changes in clinically important genes using archival biopsies and across stroma-tumour boundaries. Copyright © 2003 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]