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A study published in Acta Neuropathologica explores the use of targeted whole-viral genome sequencing from formalin-fixed paraffin-embedded (FFPE) neuropathology specimens. The researchers conducted a proof-of-concept study using a viral nucleic acid enrichment protocol on 23 FFPE samples with confirmed viral infections. They found that the viral enrichment panel led to a significant enrichment of viral sequences, resulting in a high genome coverage and improved detection of viral integration sites. The study demonstrates that metagenomic sequencing of virus-enriched libraries from FFPE specimens is a valuable method for viral whole-genome sequencing, even in low-quality and low-biomass samples. [Extracted from the article]

Abstract 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. [ABSTRACT FROM AUTHOR]

Simple Summary: Pancreatic neuroendocrine tumors are rare, but their incidence is rising. Several grades exist, and distinguishing between these is pivotal for clinical management. Currently, the grades can only be differentiated by histological analysis requiring invasive sampling. MicroRNAs are short non-protein coding RNA molecules that were shown to be differentially expressed in a wide variety of tumors. Here, we examined whether microRNAs could be exploited to differentiate grade 1 and 2 pancreatic neuroendocrine tumors and established significantly differentially expressed microRNAs. Pancreatic neuroendocrine neoplasms pose a growing clinical challenge due to their rising incidence and variable prognosis. The current study aims to investigate microRNAs (miRNA; miR) as potential biomarkers for distinguishing between grade 1 (G1) and grade 2 (G2) pancreatic neuroendocrine tumors (PanNET). A total of 33 formalin-fixed, paraffin-embedded samples were analyzed, comprising 17 G1 and 16 G2 tumors. Initially, literature-based miRNAs were validated via real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR), confirming significant downregulation of miR-130b-3p and miR-106b in G2 samples. Through next-generation sequencing, we have identified and selected the top six miRNAs showing the highest difference between G1 and G2 tumors, which were further validated. RT-qPCR validation confirmed the downregulation of miR-30d-5p in G2 tumors. miRNA combinations were created to distinguish between the two PanNET grades. The highest diagnostic performance in distinguishing between G1 and G2 PanNETs by a machine learning algorithm was achieved when using the combination miR-106b + miR-130b-3p + miR-127-3p + miR-129-5p + miR-30d-5p. The ROC analysis resulted in a sensitivity of 83.33% and a specificity of 87.5%. The findings underscore the potential use of miRNAs as biomarkers for stratifying PanNET grades, though further research is warranted to enhance diagnostic accuracy and clinical utility. [ABSTRACT FROM AUTHOR]

Objective: To report the effect of fibrosis on yield of Deoxyribonucleic Acid extraction from Formalin-Fixed Paraffin-Embedded tissue. Study Design: Cross sectional study. Place and Duration of Study: Department of Molecular Pathology, Armed Forces Institute of Pathology, Rawalpindi Pakistan, Jul to Dec 2021. Methodology: Fifty-four Formalin-Fixed Paraffin-Embedded tissues having size <2cm containing fibrosis (Mild, Moderate, Severe) were included in the study. Formalin-Fixed Paraffin-Embedded tissues having tissue size > 2cm were excluded. Deoxyribonucleic Acid was extracted and quantified from Formalin-Fixed Paraffin-Embedded blocks. Results: Nine (17%) Formalin-Fixed Paraffin-Embedded tissues had no fibrosis, 28(51%) had Mild fibrosis, 9(17%) and 8(15%) had Moderate and Severe fibrosis. Measurement scale in 5 different stages for evaluation of our quantified results were used. For specimens with no fibrosis the DNA quantity fel on scale 1, 0(0%); on scale 2, 1(11%); on scale 3, 0(0%); on scale 4, 1(11%); and on scale 5, 7(78%). Deoxyribonucleic Acid yield in moderate fibrosis specimens fel on scale 1, 0(0%)' on scale 2, 7(78%); on scale 3, 0(0%); on scale 4, 1(11%); and on scale 5, 1(11%). Among severe fibrosis the Deoxyribonucleic Acid quantity fal on scale 1, 5(62%); on scale 2, 3(38%); on scale 3, 0(0%); on scale 4, 0(0%); and on scale 5, 0(0%). Conclusion: Formalin-Fixed Paraffin-Embedded tissues having severe fibrosis resulted in significantly lower Deoxyribonucleic Acid yield as compared to Formalin-Fixed Paraffin-Embedded tissues having no fibrosis. [ABSTRACT FROM AUTHOR]

The metagenomic next-generation sequencing (mNGS) method is preferred for genotyping useful for the identification of organisms, illumination of metabolic pathways, and determination of microbiota. It can accurately obtain all the nucleic acid information in the test sample. Anthrax is one of the most important zoonotic diseases, infecting mainly herbivores and occasionally humans. The disease has four typical clinical forms, cutaneous, gastrointestinal, inhalation, and injection, all of which may result in sepsis or meningitis, with cutaneous being the most common form. Here, we report a case of cutaneous anthrax diagnosed by mNGS in a butcher. Histopathology of a skin biopsy revealed PAS-positive bacilli. Formalin-fixed paraffin-embedded (FFPE) tissue sample was confirmed the diagnosis of anthrax by mNGS. He was cured with intravenous penicillin. To our knowledge, this is the first case of cutaneous anthrax diagnosed by mNGS using FFPE tissue. mNGS is useful for identifying pathogens that are difficult to diagnose with conventional methods, and FFPE samples are simple to manage. Compared with traditional bacterial culture, which is difficult to cultivate and takes a long time, mNGS can quickly and accurately help us diagnose anthrax, so that anthrax can be controlled in a timely manner and prevent the outbreak of epidemic events. [ABSTRACT FROM AUTHOR]

Background: Pancreaticobiliary maljunction (PBM) is a known risk factor for biliary tract cancer. However, its association with carcinoma of the papilla of Vater (PVca) remains unknown. We report a case with PVca that was thought to be caused by the hyperplasia–dysplasia–carcinoma sequence, which is considered a mechanism underlying PBM-induced biliary tract cancer. Case presentation: A 70-year-old woman presented with white stool and had a history of cholecystectomy for the diagnosis of a non-dilated biliary tract with PBM. Esophagogastroduodenoscopy revealed a tumor in the papilla of Vater, and PVca was histologically proven by biopsy. We finally diagnosed her with PVca concurrent with non-biliary dilated PBM (cT1aN0M0, cStage IA, according to the Union for International Cancer Control, 8th edition), and subsequently performed subtotal stomach-preserving pancreaticoduodenectomy. Pathological findings of the resected specimen revealed no adenomas and dysplastic and hyperplastic mucosae in the common channel slightly upstream of the main tumor, suggesting a PBM related carcinogenic pathway with hyperplasia–dysplasia–carcinoma sequence. Immunostaining revealed positivity for CEA. CK7 positivity, CK20 negativity, and MUC2 negativity indicated that this PVca was of the pancreatobiliary type. Genetic mutations were exclusively detected in tumors and not in normal tissues, and bile ducts from formalin-fixed paraffin-embedded samples included mutated-ERBB2 (Mutant allele frequency, 81.95%). Moreover, of the cell-free deoxyribonucleic acid (cfDNA) extracted from liquid biopsy mutated-ERBB2 was considered the circulating-tumor deoxyribonucleic acid (ctDNA) of this tumor. Conclusions: Herein, we report the first case of PVca with PBM potentially caused by a "hyperplasia–dysplasia–carcinoma sequence" detected using immunostaining and next-generation sequencing. Careful follow-up is required if pancreaticobiliary reflux persists, considering the possible development of PVca. [ABSTRACT FROM AUTHOR]

ABSTRACT The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50–100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus-influenza virus co-infection.IMPORTANCEThis study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus-influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history.

Triple-negative breast cancer (TNBC) presents significant challenges to female health owing to the lack of therapeutic targets and its poor prognosis. In recent years, in the field of molecular pathology, there has been a growing focus on the role of intra-tumoral microbial communities and metabolic alterations in tumor cells. However, the precise mechanism through which microbiota and their metabolites influence TNBC remains unclear and warrants further investigation. In this study, we analyzed the microbial community composition in various subtypes of breast cancer through 16S rRNA MiSeq sequencing of formalin-fixed, paraffin-embedded (FFPE) tissue samples. Notably, Turicibacter, a microbe associated with cancer response, exhibited a significantly higher abundance in TNBC. Similarly, mass spectrometry-based metabolomic analysis revealed substantial differences in specific metabolites, such as nutriacholic, pregnanetriol, and cortol. Furthermore, we observed significant correlations between the intra-tumoral microbiome, clinicopathological characteristics, and human epidermal growth factor receptor-2 expression(HER2). Three microbial taxa (Cytophagaceae, Conexibacteraceae, and Flavobacteriaceae) were associated with tumor-infiltrating lymphocytes(TILs), which are indicative of antitumor immunity. This study creatively utilized FFPE tissue samples to assess intra-tumoral microbial communities and their related metabolic correlations, presenting avenues for the identification of novel diagnostic biomarkers, the development of therapeutic strategies, and the early clinical diagnosis of TNBC. [ABSTRACT FROM AUTHOR]

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-μm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were read by both pathologists, a total of 40 mismatches in the diagnosis were present. The median tCRM scores for AR, BL, and stable diagnoses were 4.87, 1.85, and 1.27, respectively, with an overall significant difference among all histologic groups (Kruskal-Wallis, p < 0.0001). There were significant differences in tCRM scores between pathologists both finding inflammation vs. disagreement (p = 0.003), and both finding inflammation vs. both finding no inflammation (p < 0.001), along with overall significance between all scores (Kruskal-Wallis, p < 0.001). A logistic regression model predicting graft inflammation using various clinical predictor variables and tCRM revealed the tCRM score as the only significant predictor of graft inflammation (OR: 1.90, 95% CI: 1.40-2.68, p < 0.0001). Accurate, quantitative, and unbiased assessment of rejection of the clinical sample is critical. Given the discrepant diagnoses between pathologists on the same samples, individuals could utilize the tCRM score as a tiebreaker in unclear situations. We propose that the tCRM quantitative score can provide unbiased quantification of graft inflammation, and its rapid evaluation by PCR on the FFPE shave can become a critical adjunct to help drive clinical decision making and immunosuppression delivery.

BackgroundMedian overall survival (OS) for women with high-grade serous ovarian cancer (HGSOC) is ∼4 years, yet survival varies widely between patients. There are no well-established, gene expression signatures associated with prognosis. The aim of this study was to develop a robust prognostic signature for OS in patients with HGSOC.Patients and methodsExpression of 513 genes, selected from a meta-analysis of 1455 tumours and other candidates, was measured using NanoString technology from formalin-fixed paraffin-embedded tumour tissue collected from 3769 women with HGSOC from multiple studies. Elastic net regularization for survival analysis was applied to develop a prognostic model for 5-year OS, trained on 2702 tumours from 15 studies and evaluated on an independent set of 1067 tumours from six studies.ResultsExpression levels of 276 genes were associated with OS (false discovery rate < 0.05) in covariate-adjusted single-gene analyses. The top five genes were TAP1, ZFHX4, CXCL9, FBN1 and PTGER3 (P < 0.001). The best performing prognostic signature included 101 genes enriched in pathways with treatment implications. Each gain of one standard deviation in the gene expression score conferred a greater than twofold increase in risk of death [hazard ratio (HR) 2.35, 95% confidence interval (CI) 2.02-2.71; P < 0.001]. Median survival [HR (95% CI)] by gene expression score quintile was 9.5 (8.3 to -), 5.4 (4.6-7.0), 3.8 (3.3-4.6), 3.2 (2.9-3.7) and 2.3 (2.1-2.6) years.ConclusionThe OTTA-SPOT (Ovarian Tumor Tissue Analysis consortium - Stratified Prognosis of Ovarian Tumours) gene expression signature may improve risk stratification in clinical trials by identifying patients who are least likely to achieve 5-year survival. The identified novel genes associated with the outcome may also yield opportunities for the development of targeted therapeutic approaches.

Long-term kidney transplant (KT) allograft outcomes have not improved as expected despite a better understanding of rejection and improved immunosuppression. Previous work had validated a computed rejection score, the tissue common rejection module (tCRM), measured by amplification-based assessment of 11 genes from formalin-fixed paraffin-embedded (FFPE) biopsy specimens, which allows for quantitative, unbiased assessment of immune injury. We applied tCRM in a prospective trial of 124 KT recipients, and contrasted assessment by tCRM and histology reads from 2 independent pathologists on protocol and cause biopsies post-transplant. Four 10-μm shaves from FFPE biopsy specimens were used for RNA extraction and amplification by qPCR of the 11 tCRM genes, from which the tCRM score was calculated. Biopsy diagnoses of either acute rejection (AR) or borderline rejection (BL) were considered to have inflammation present, while stable biopsies had no inflammation. Of the 77 biopsies that were read by both pathologists, a total of 40 mismatches in the diagnosis were present. The median tCRM scores for AR, BL, and stable diagnoses were 4.87, 1.85, and 1.27, respectively, with an overall significant difference among all histologic groups (Kruskal-Wallis, p < 0.0001). There were significant differences in tCRM scores between pathologists both finding inflammation vs. disagreement (p = 0.003), and both finding inflammation vs. both finding no inflammation (p < 0.001), along with overall significance between all scores (Kruskal-Wallis, p < 0.001). A logistic regression model predicting graft inflammation using various clinical predictor variables and tCRM revealed the tCRM score as the only significant predictor of graft inflammation (OR: 1.90, 95% CI: 1.40-2.68, p < 0.0001). Accurate, quantitative, and unbiased assessment of rejection of the clinical sample is critical. Given the discrepant diagnoses between pathologists on the same samples, individuals could utilize the tCRM score as a tiebreaker in unclear situations. We propose that the tCRM quantitative score can provide unbiased quantification of graft inflammation, and its rapid evaluation by PCR on the FFPE shave can become a critical adjunct to help drive clinical decision making and immunosuppression delivery.

Microbiome research has enormous potential in cancer research and the use of formalin-fixed paraffin-embedded (FFPE) tissues could offer many advantages. The tumor microenvironment represents a suitable niche for specific microbes and evidence proves the presence of an endogenous tumor microbiota, here referred to as oncobiota. Awareness of the oncobiota role in tumorigenesis could have a large influence on cancer care, in terms of diagnosis, prevention, and treatment. Moreover, understanding the microbial-related tumor microenvironment, and its influence on tumor immune response and cancer cells will help define important pathogenetic mechanisms in cancer starting or progression. Routine collection of histopathological FFPE samples provides a large availability of specimens essential for affordable and impactful retrospective analyses and for getting robust statistical results. The FFPE tissues are common in the analysis of tumor biopsies including the tumor microbiota characterization which has an important role in the modulation of our immune system and consequently of tumor cells. However, the microbiota analysis starting from FFPE tissues presents methodological pitfalls and limits that may negatively affect the oncobiota research. After examining the methodological and analytical difficulties of this approach, this work seeks to offer workable solutions to promote that research area.

Background: Organising pneumonia (OP) is reported in patients with haematologic malignancy suspected of having invasive mould disease, yet little is known about this relationship. Objective: To investigate molecular evidence of invasive mould pneumonia in paraffin‐embedded lung tissues from histologically diagnosed OP patients with suspected invasive mould pneumonia. Patients/Methods: Patients with haematologic malignancy suspected to have invasive pulmonary mould disease who underwent lung biopsy at a tertiary hospital, Seoul, South Korea, between 2008 and 2020, were retrospectively reviewed. To find molecular evidence of fungal infection, PCR assay was used to detect Aspergillus‐ and Mucorales‐specific DNA within OP lung tissue sections. Results: Forty‐seven patients with suspected invasive mould pneumonia underwent lung biopsy and 15 (32%) were histologically diagnosed as OP without any evidence of fungal hyphae. Of these 15 patients, 3 (20%) received allogenic haematopoietic stem cell transplantation prior to developing OP. Before biopsy, 2 and 13 patients had probably and possible invasive mould disease, respectively. The median antifungal treatment length was 81 [8–114] days, and the median steroid treatment dosage was 0.35 mg/kg/day for 36 days (methylprednisolone equivalent doses), respectively. After biopsy, three patients with possible invasive mould infection revealed probable invasive pulmonary aspergillosis. From the 15 paraffin‐embedded lung tissues, 6 (40%) exhibited positive PCR assay results for detecting Aspergillus‐ and Mucorales‐specific DNA. Conclusions: More than one third of OP cases in patients with suspected invasive mould pneumonia exhibited molecular evidence of invasive mould infection by fungus‐specific PCR in lung tissues, likely associated with concurrent or prior fungal infection. [ABSTRACT FROM AUTHOR]

BackgroundInvestigations in human disease pathogenesis have been hampered due to paucity of access to fresh-frozen tissues (FFT) for use in global, data-driven methodologies. As an alternative, formalin-fixed, paraffin-embedded (FFPE) tissues are readily available in pathology banks. However, the use of formalin for fixation can lead to the loss of proteins that appear during inflammation, thus introducing an inherent sample bias. To address this, we compared FF and FFPE tissue proteomics to determine whether FFPE-tissue can be used effectively in inflammatory diseases.MethodsAdjacent kidney slices from lupus nephritic mice were processed as FFPE or FFTs. Their tissue lysates were run together using proteomics workflow involving filter-aided sample preparation, in-solution dimethyl isotope labeling, StageTip fractionation, and nano-LC MS/MS through an Orbitrap XL MS.ResultsWe report a >97% concordance in protein identification between adjacent FFPE and FFTs in murine lupus nephritic kidneys. Specifically, proteins representing pathways, namely, 'systemic lupus erythematosus', 'interferon-α', 'TGF-β', and 'extracellular matrix', were reproducibly quantified between FFPE and FFTs. However, 12%-29% proteins were quantified differently in FFPE compared to FFTs, but the differences were consistent across experiments. In particular, certain proteins represented in pathways, including 'inflammatory response' and 'innate immune system' were quantified less in FFPE than in FFTs. In a pilot study of human FFPE tissues, we identified proteins relevant to pathogenesis in lupus nephritic kidney biopsies compared to control kidneys.ConclusionThis is the first report of lupus nephritis kidney proteomics using FFPE tissue. We concluded that archived FFPE tissues can be reliably used for proteomic analyses in inflammatory diseases, with a caveat that certain proteins related to immunity and inflammation may be quantified less in FFPE than in FFTs.

Traumatic, inherited, and age-related degenerative diseases of the retina, such as retinal detachment, retinitis pigmentosa, and age-related macular degeneration, are characterized by the irreversible loss of retinal neurons. While current treatments aim to prevent neuronal degeneration, there are no available treatments to restore neurons after loss. Cultured murine neuroretinal tissue explants model retinal injury and offer a high throughput approach to identify experimental interventions capable of regenerating neurons. Formalin-fixed paraffin-embedded (FFPE) preparations of murine neuroretinal explants can be used to identify cells throughout the retinal layers to provide information on proliferation and activity following exposure to therapeutics. However, retinal explants are friable, particularly after ex vivo culture, sample handling and FFPE processing steps can result in tissue loss and damage. Friability also prohibits bisecting samples post-culture to display more than one region of interest for analysis. We developed a sample handling and embedding technique for cultured murine neuroretinal explants using HistogelTM in combination with a post-processing trimming step that eliminates tissue loss, increases cross-sectional retinal representation, and captures proximal and central retina on one slide to facilitate analysis of explants subjected to neurotrophic compounds. [ABSTRACT FROM AUTHOR]

Corneal transplantation is one of the most common forms of tissue transplantation worldwide. Donor corneal tissue used in transplantation is provided by eye banks, which store the tissue in culture medium after procurement. To date, the effects of cell culture on human corneal tissue have not been fully elucidated. Using the 3′ RNA sequencing method for massive analysis of cDNA ends (MACE), we show that cultivation of corneal tissue leads to significant changes in a variety of molecular processes in human corneal tissue that go well beyond aspects of previously known culture effects. Functionally grouped network analysis revealed nine major groups of biological processes that were affected by corneal organ culture, among them keratinization, hypoxia, and angiogenesis, with genes from each group being affected by culture time. A cell type deconvolution analysis revealed significant modulations of the corneal immune cell profile in a time dependent manner. The results suggest that current culture conditions should be further refined and that prolonged cultivation may be detrimental. Recently, we showed that MACE enables transcriptional profiling of formalin-fixed and paraffin-embedded (FFPE) conjunctival tissue with high accuracy even after more than 10 years of storage. Here we demonstrate that MACE provides comparable results for native and FFPE corneal tissue, confirming that the technology is suitable for transcriptome analysis of a wide range of archived diseased corneal samples stored in histological archives. Finally, our data underscore the feasibility of bioinformatics cell-type enrichment analysis in bulk RNA-seq data to profile immune cell composition in fixed and archived corneal tissue samples, for which RNA-seq analysis of individual cells is often not possible. [ABSTRACT FROM AUTHOR]

Quantification of epidermal growth factor receptor (EGFR) mutations is important for the prediction of tyrosine kinase inhibitor (TKI) efficacy in patients with non-small cell lung cancer (NSCLC). However, clinicians lack a sensitive and convenient method to quantify EGFR mutant abundance. The present study introduces a novel method, namely amplification refractory mutation system (ARMS)-Plus, for the quantitative analysis of EGFR exon 19 deletion (19Del), L858R and T790M mutations. Formalin-fixed paraffin-embedded tumor samples were collected from 77 patients with lung adenocarcinoma. DNA was extracted and analyzed for EGFR mutations using ARMS-Plus. The performance of ARMS-Plus was then compared with that of conventional ARMS-polymerase chain reaction (ARMS-PCR) and droplet digital PCR (ddPCR). The results demonstrated that the concordance rate of EGFR mutation testing between ARMS-Plus and ddPCR was 98.7% (76/77, Kappa=0.9739). 19Del and L858R mutations were detected in 23 and 12 patients, respectively. There was a significant difference between ARMS-Plus and ddPCR in the evaluation of 19Del mutant abundance (P=0.0002); however, not in that of L858R mutant abundance (P=0.7334). The ARMS-Plus results in L858R mutant abundance were concordant with that of ddPCR (R2=0.8081). These results indicated that the sensitivity and specificity of ARMS-Plus in identifying EGFR mutations were similar to that of ddPCR. For quantitative analysis, the results of ARMS-Plus in evaluating L858R mutant abundance revealed a positive correlation with the ddPCR results. Thus, ARMS-Plus provides an alternative method, which is reliable and cost-effective, to quantify EGFR mutations and thereby, aid treatment decisions in patients with lung adenocarcinoma.

Background: The detection of acid-fast bacilli in extrapulmonary tissue samples is challenging due to its paucibacillary nature. The present study assessed the utility of immunohistochemistry (IHC) using anti-Mycobacterium tuberculosis antibody (ab905) for detecting the presence of mycobacterial antigens in archived formalin-fixed paraffin-embedded (FFPE) tissues. Methods: FFPE tissues [surgical biopsies (n = 32) and post-mortem tissues (n = 8)] from clinically and histologically suggestive extrapulmonary tuberculosis (EPTB) cases at the Korle Bu Teaching Hospital, Accra, Ghana from 2015 to 2020 were stained with IHC (anti-Mycobacterium tuberculosis antibody) and Ziehl-Neelsen (ZN) stain. The staining outcomes of IHC and ZN were compared, and their sensitivity and specificity determined against histopathology as reference standard. Results: Lymph nodes were about 40% (16/40) of the samples analyzed. IHC stained positive in 43.8% (7/16) biopsies and 87.5% (4/5) post-mortem samples ranging from 43.8% (7/16) in lymph nodes to 80% (4/5) in gastrointestinal organs. The overall sensitivity for IHC was 52.50% (95% CI: 36.13%–68.49%) and 0% (95% CI: 0.00%–8.81%) for ZN. Specificity was 72.50% (95% CI: 56.11%–85.40%) and 75% (95% CI: 58.80–87.31%) for IHC and ZN respectively. Conclusions: IHC using anti-Mycobacterium tuberculosis antibody (ab905) can detect mycobacterial antigens in diverse range of paucibacillary extrapulmonary tissue sections. It is potentially a useful tool for the diagnosis of EPTB in FFPE tissues in a routine pathology laboratory.

The CelLock™ procedure kit is used to collect and prepare cellular specimens such as fine needle aspirates (FNA), cytology specimens, cultured cells, small tissue biopsies, and samples with scant tissue fragments or cells into a paraffin cell-block. This cell-block can be used for subsequent microtomy and staining using hematoxylin and eosin (H&E), special stains, immunohistochemistry (IHC), and applicable molecular techniques such as in situ hybridization (ISH). CelLock is a standardized method that provides optimal receipt, preservation, preparation, and processing of cell-blocks which, contain virtually all of the submitted specimens and are able to be embedded and sectioned in a reproducible fashion. The specimen contained within the cell-block is preserved such that all the cellular protein and genetic information is available for histological and ancillary testing. [ABSTRACT FROM AUTHOR]

Purpose: Diabetic kidney disease (DKD) is a serious complication of diabetes mellitus and a leading cause of chronic kidney disease and end-stage renal disease. One potential mechanism underlying cellular dysfunction contributing to kidney disease is aberrant protein post-translational modifications. Lysine acetylation is associated with cellular metabolic flux and is thought to be altered in patients with diabetes and dysfunctional renal metabolism., Experimental Design: A novel extraction and LC-MS/MS approach was adapted to quantify sites of lysine acetylation from formalin-fixed paraffin-embedded (FFPE) kidney tissue and from patients with DKD and non-diabetic donors (n = 5 and n = 7, respectively)., Results: Analysis of FFPE tissues identified 840 total proteins, with 225 of those significantly changing in patients with DKD. Acetylomic analysis quantified 289 acetylated peptides, with 69 of those significantly changing. Pathways impacted in DKD patients revealed numerous metabolic pathways, specifically mitochondrial function, oxidative phosphorylation, and sirtuin signaling. Differential protein acetylation in DKD patients impacted sirtuin signaling, valine, leucine, and isoleucine degradation, lactate metabolism, oxidative phosphorylation, and ketogenesis., Conclusions and Clinical Relevance: A quantitative acetylomics platform was developed for protein biomarker discovery in formalin-fixed and paraffin-embedded biopsies of kidney transplant patients suffering from DKD., (© 2024 Wiley‐VCH GmbH.)

Spatial proteomics profiling is an emerging set of technologies that has the potential to elucidate the cell types, interactions, and molecular signatures that make up complex tissue microenvironments, with applications in the study of cancer, immunity, and much more. An emerging technique in the field is CoDetection by indEXing, recently renamed as the PhenoCycler system. This is a highly multiplexed immunofluorescence imaging technology that relies on oligonucleotide-barcoded antibodies and cyclic immunofluorescence to visualize many antibody markers in a single specimen while preserving tissue architecture. Existing PhenoCycler panels are primarily designed for fresh frozen tissues. Formalin-fixed paraffin-embedded blocks offer several advantages in preclinical research, but few antibody clones have been identified in this setting for PhenoCycler imaging. Here, we present a novel PhenoCycler panel of 28 validated antibodies for murine formalin-fixed paraffin-embedded tissues. We describe our workflow for selecting and validating clones, barcoding antibodies, designing our panel, and performing multiplex imaging. We further detail our analysis pipeline for comparing marker expressions, clustering and phenotyping single-cell proteomics data, and quantifying spatial relationships. We then apply our panel and analysis protocol to profile the effects of 3 gene delivery nanoparticle formulations, in combination with systemic anti-PD1, on the murine melanoma tumor immune microenvironment. Intralesional delivery of genes expressing the costimulatory molecule 4-1BBL and the cytokine IL-12 led to a shift toward intratumoral M1 macrophage polarization and promoted closer associations between intratumoral CD8 T cells and macrophages. Delivery of interferon gamma, in addition to 4-1BBL and IL-12, not only further increased markers of antigen presentation on tumor cells and intratumoral antigen-presenting cells but also promoted greater expression of checkpoint marker PD-L1 and closer associations between intratumoral CD8 T cells and PD-L1-expressing tumor cells. These findings help explain the benefits of 4-1BBL and IL-12 delivery while offering additional mechanistic insights into the limitations of interferon gamma therapeutic efficacy., (Copyright © 2024. Published by Elsevier Inc.)

Fluorescence in situ hybridization (FISH) has revolutionized molecular cytogenetic analysis since the 1980s, enabling precise localization of DNA sequences in cells and tissues. Despite its relevance, applying FISH to formalin-fixed paraffin-embedded (FFPE) tissue samples encounters significant technical challenges. This review addresses the main issues encountered in this context, such as inadequate fixation, contamination, block and slide age, inadequate pretreatment, and FISH technique. Proposed solutions include optimized pretreatment protocols, monitoring of blockage, careful selection of probes, and thorough analysis of results. Implementing good laboratory practices and quality control strategies are essential to ensure reliable results. Additionally, the use of emerging technologies such as artificial intelligence and digital pathology offers new perspectives for improving the efficiency and accuracy of FISH in FFPE samples. This review highlights the importance of a careful and personalized approach to overcome the technical challenges associated with FISH in FFPE samples, strengthening its role in research and clinical diagnosis. RESEARCH HIGHLIGHTS: Few FISH studies on FFPE: The scarcity of studies specifically addressing FISH applications in FFPE tissues highlights a critical gap in the literature. Troubleshooting FISH in FFPE tissues: Identifying and addressing common challenges in FISH techniques when applied to FFPE samples, such as signal quality and hybridization efficiency. Critical aspects of FISH technique: Discuss the main technical considerations crucial for successful FISH in FFPE tissues, including sample preparation, probe selection, and protocol optimization., (© 2024 Wiley Periodicals LLC.)

Formalin-fixed paraffin-embedded (FFPE) tumour samples may provide crucial data regarding biomarkers for neoplasm progression. Analysis of gene expression is frequently used for this purpose. Therefore, mRNA expression needs to be normalized through comparison to reference genes. In this study, we establish which of the usually reported reference genes is the most reliable one in cutaneous malignant melanoma (MM) and cutaneous squamous cell carcinoma (CSCC). ACTB, TFRC, HPRT1 and TBP expression was quantified in 123 FFPE samples (74 MM and 49 CSCC biopsies) using qPCR. Expression stability was analysed by NormFinder and Bestkeeper softwares, and the direct comparison method between means and SD. The in-silico analysis with BestKeeper indicated that HPRT1 was more stable than ACTB and TFRC in MM (1.85 vs. 2.15) and CSCC tissues (2.09 vs. 2.33). The best option to NormFinder was ACTB gene (0.56) in MM and TFRC (0.26) in CSCC. The direct comparison method showed lower SD means of ACTB expression in MM (1.17) and TFRC expression in CSCC samples (1.00). When analysing the combination of two reference genes for improving stability, NormFinder indicated HPRT1 and ACTB to be the best for MM samples, and HPRT1 and TFRC genes for CSCC. In conclusion, HPRT1 and ACTB genes in combination are the most appropriate choice for normalization in gene expression studies in MM FFPE tissue, while the combination of HPRT1 and TFRC genes are the best option in analysing CSCC FFPE samples. These may be used consistently in forthcoming studies on gene expression in both tumours.

Abstract Objective In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). Results The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62–0.81), 0.59 (0.47–0.71) and 0.80 (0.73–0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39–0.68), 0.43 (0.31–0.56) and 0.70 (0.59–0.80), respectively. Bland–Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined.

Bone marrow biopsy depends on tissue morphology, immunohistochemical staining, and moleculardetection. Tissue pretreatment is required for bone marrow samples, from clinical specimen acquisition to pathological reporting, but during the process, proteins and nucleic acids are often altered because of the acid in fixation and decalcification solutions. In our study, we present an easy and effective pretreatment protocol and compared this novel pretreatment protocol (Set 2) with an existing traditional pretreatment process (Set 1) using tissue morphology, IHC staining, and molecular pathological analyses. Granulocytic IHC markers showed more intensive staining in samples of Set 2 than in those of Set 1. The Set 2 protocol provided a higher DNA yield and less fragmentation; moreover, samples processed with the Set 2 protocol could be subsequently used in FISH and DNA sequencing assays. Our optimized novel pretreatment protocol could better protect proteins and DNA molecules while maintaining good cell morphology compared to traditional pretreatment The novel pretreatment reagents could role as a reference by more laboratories for pretreating bone marrow biopsy samples and scientific research. [ABSTRACT FROM AUTHOR]

The purpose of the study is to assess the etiology detection ability of Metagenomic Next-Generation Sequencing (mNGS) on formalin fixation and paraffin embedding (FFPE) tissue from postoperative biopsy specimens. We prospectively enrolled specimens from patients undergone surgery biopsy due to undefinite diagnosis and pathologically indicated granulomatous lesions. FFPE tissues were tested by mNGS and histopathology. The etiology detection rate of mNGS was calculated and compared with histopathology.. Among the 69 cases eventually included, 41 (59.42%) were diagnosed with infectious granuloma. The overall fungi and mycobacteria etiology detection rates of mNGS in granuloma lesions was 87.80% (36/41). The mNGS increased the detection rate by 68.29% (28/41) compared with histopathology, the difference was statistically significant (χ2 = 28.97, P = 0.00). The detection rates of mNGS in fungal infections (12/12,100%) and in mycobacterium infections (22/27, 81.48%) were significant higher than those of histopathology (8/12, 66.67% and 0/27, 0.00%; both P = 0.00). Two (2/2.100%) cases of co-infection were detected at one time by mNGS. All mNGS-based clinical decisions were made within 2 days. The mNGS could accurately and quickly detect fungi and mycobacteria in FFPE specimens from postoperative granuloma specimens and identify the pathogens to the species level. China Clinical Trial Registry ChiCTR2000035464 [ABSTRACT FROM AUTHOR]

Routinely available clinical samples of all stages of pancreatic cancer are used in the present study to elucidate its molecular mechanisms and identify novel therapeutic targets. We evaluated the use of next‐generation sequencing (NGS) of endoscopically obtained pancreatic cancer tissues. We enrolled 147 patients who underwent endoscopic ultrasound‐guided fine‐needle aspiration or endoscopic biopsy. The quantity and quality of the extracted DNA was assessed. Tissue samples were used for NGS of 78 cancer‐related genes, from which gene alterations and microsatellite instability (MSI) were extracted. NGS was successful in 141 out of 147 (96%) cases. Gene alterations were detected in 134 out of 141 (91%) samples, among which eight out of 10 samples with a DNA concentration below the detection limit had some type of gene alteration. Targetable genes were detected in 28 (19.9%) cases. MSI and germline mutations in homologous recombination repair associated genes were detected in 5% and 3% of cases, respectively. Cox regression analysis revealed that metastasis (P <.005; hazard ratio [HR], 3.30) was associated with poor prognosis in all pancreatic cancer patients. In addition, fewer than three mutations (P =.03; HR, 2.48) and serum carcinoembryonic antigen levels >5 ng/mL (P <.005; HR, 3.94) were associated with worse prognosis in cases without and with metastasis, respectively. Targeted sequencing of all stages of pancreatic cancer using available samples from real clinical practice could be used to determine the relationship between gene alterations and prognosis to help determine treatment choices. [ABSTRACT FROM AUTHOR]

Telomeres in most somatic cells shorten with each cell division, and critically short telomeres lead to cellular dysfunction, cell cycle arrest, and senescence. Thus, telomere shortening is an important hallmark of human cellular senescence. Quantitative fluorescence in situ hybridization (Q-FISH) using formalin-fixed paraffin-embedded (FFPE) tissue sections allows the estimation of telomere lengths in individual cells in histological sections. In our Q-FISH method, fluorescently labelled peptide nucleic acid (PNA) probes are hybridized to telomeric and centromeric sequences in FFPE human tissue sections, and relative telomere lengths (telomere signal intensities relative to centromere signal intensities) are measured. This chapter describes our Q-FISH protocols for assessing relative telomere lengths in FFPE human tissue sections., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Incidence of human papillomavirus (HPV)-associated oral cancers is on the rise. However, epidemiological data of this subset of cancers are limited. Dental hospital poses a unique advantage in detection of HPV-positive oral malignancies. We assessed the utility of formalin-fixed paraffin-embedded (FFPE) tissues, which are readily available, for evaluation of high-risk HPV infection in oral cancer. For protocol standardization, we used 20 prospectively collected paired FFPE and fresh tissues of histopathologically confirmed oral cancer cases reported in Oral Medicine department of a dental hospital for comparative study. Only short PCRs (~ 200 bp) of DNA isolated using a modified xylene-free method displayed a concordant HPV result. For HPV analysis, we used additional 30 retrospectively collected FFPE tissues. DNA isolated from these specimens showed an overall 23.4% (11/47) HPV positivity with detection of HPV18. Comparison of HPV positivity from dental hospital FFPE specimens with overall HPV positivity of freshly collected oral cancer specimens (n = 55) from three cancer care hospitals of the same region showed notable difference (12.7%; 7/55). Further, cancer hospital specimens showed HPV16 positivity and displayed a characteristic difference in reported sub-sites and patient spectrum. Overall, using a xylene-free FFPE DNA isolation method clubbed with short amplicon PCR, we showed detection of HPV-positive oral cancer in dental hospitals. [ABSTRACT FROM AUTHOR]

The presence of wild-type RAS alleles, as determined by genotyping codons 12, 13, 59, 61, 117, and 146, is a prerequisite for personalized anti-EGFR treatment of metastatic colorectal cancer (mCRC) patients. Here we describe analytical validation of in-house developed massively parallel sequencing technology (MPS) in comparison to the in vitro diagnostics (IVD) certified qPCR method. DNA extracted from FFPE samples from CRC patients (n=703) and reference standards (n=33) were tested for KRAS and NRAS mutations in 6 codons of exons 2, 3, and 4 using deep amplicon sequencing (DAS) on a MiSeq benchtop sequencer (Illumina). Two different amplicon lengths and two different library preparation methods (long-RAS and short-RAS) were tested in order to evaluate their impact on DAS performance. In parallel, identical tumor DNA was tested by the following IVD assays: therascreen KRAS RGQ PCR Kit (Qiagen), cobas® KRAS Mutation Test (Roche Diagnostics), and SNaPshot assay (Thermo Fisher Scientific). Both DAS assays detected all the mutations present in reference standards and external quality control samples, except for the artificially generated KRAS codon 146 mutation. The DAS assays performed sufficient analytical specificity and sensitivity (≥0.95). The use of shorter amplicons prolonged the preparation steps but significantly improved the sequencing success rate of FFPE-derived DNA. RAS mutation frequencies in the Czech CRC patients were similar to previous reports, although rare mutations were also detected. DAS with short amplicons is a good strategy for routine assessment of somatic mutations in low-quality FFPE-derived DNA. [ABSTRACT FROM AUTHOR]

Objective: In previous studies using Illumina Infinium methylation arrays, we have identified DNA methylation marks associated with cancer predisposition and progression. In the present study, we have sought to find appropriate technology to both technically validate our data and expand our understanding of DNA methylation in these genomic regions. Here, we aimed to assess the repeatability of methylation measures made using QIAseq targeted methyl panel and to compare them with those obtained from the Illumina HumanMethylation450 (HM450K) assay. We included in the analysis high molecular weight DNA extracted from whole blood (WB) and DNA extracted from formalin-fixed paraffin-embedded tissues (FFPE). Results: The repeatability of QIAseq-methylation measures was assessed at 40 CpGs, using the Intraclass Correlation Coefficient (ICC). The mean ICCs and 95% confidence intervals (CI) were 0.72 (0.62–0.81), 0.59 (0.47–0.71) and 0.80 (0.73–0.88) for WB, FFPE and both sample types combined, respectively. For technical replicates measured using QIAseq and HM450K, the mean ICCs (95% CI) were 0.53 (0.39–0.68), 0.43 (0.31–0.56) and 0.70 (0.59–0.80), respectively. Bland–Altman plots indicated good agreement between QIAseq and HM450K measurements. These results demonstrate that the QIAseq targeted methyl panel produces reliable and reproducible methylation measurements across the 40 CpGs that were examined. [ABSTRACT FROM AUTHOR]

Abstract Background The utility of formalin-fixed paraffin-embedded (FFPE) corneal tissue specimens for retrospective diagnosis of microsporidial keratitis was evaluated by transmission electron microscopy (TEM) analysis and the possible second case of microsporidial keratitis after Descemet stripping automated endothelial keratoplasty (DSAEK) was described. Case presentation A 68-year-old man presented with multiple crystalline opacities in the corneal stroma that progressed extremely slowly after DSAEK. Fungiflora Y staining of corneal scrapings from the affected regions revealed an oval microorganism. Topical voriconazole administration was ineffective and penetrating keratoplasty was performed. Histological and molecular analyses were carried out on the excised cornea. Ziehl–Neelsen staining revealed an acid-fast, oval organism that was visible by ultraviolet illumination after Fungiflora Y and Uvitex 2B staining, whereas periodic acid-Schiff and Grocott’s staining did not yield any significant findings. Microsporidium was detected by TEM of FFPE tissue. Nosema or Vittaforma sp. was suspected as the causative microorganism by PCR of FFPE tissue and by the fact that those species are known to cause eye infection. The corneal graft has maintained transparency at 1 year and half postoperatively. Conclusions This is the first known case of microsporidial keratitis diagnosed retrospectively by molecular and ultrastructural study of FFPE tissue, and the possible second case of microsporidial keratitis after DSAEK. Microsporidial keratitis should be considered when corneal opacity refractory to conventionally known therapy would occur after DSAEK. Our findings suggest that more microsporidial keratitis cases than have been reported to date can be identified by TEM or PCR examination of FFPE corneal specimens.

Abstract Background The Fluorescence In Situ Hybridization (FISH) technique is a very useful tool for diagnostic and prognostic purposes in molecular pathology. However, clinical testing on patient tissue is challenging due to variables of tissue processing that can influence the quality of the results. This emphasizes the necessity of a standardized FISH protocol with a high hybridization efficiency. We present a pretreatment protocol that is easy, reproducible, cost-effective, and facilitates FISH on all types of patient material simultaneously with good quality results. During validation, FISH analysis was performed simultaneously on formalin-fixed paraffin-embedded, fresh frozen and cytological patient material in combination with commercial probes using our optimized one-fits-all pretreatment protocol. An optimally processed sample is characterized by strong specific signals, intact nuclear membranes, non-disturbing autofluorescence and a homogeneous DAPI staining. Results In our retrospective cohort of 3881 patient samples, overall 93% of the FISH samples displayed good quality results leading to a patient diagnosis. All FISH were assessed on quality aspects such as adequacy and consistency of signal strength (brightness), lack of background and / or cross-hybridization signals, and additionally the presence of appropriate control signals were evaluated to assure probe accuracy. In our analysis 38 different FISH probes from 3 commercial manufacturers were used (Cytocell, Vysis and ZytoLight). The majority of the patients in this cohort displayed good signal quality and barely non-specific background fluorescence on all tissue types independent of which commercial probe was used. Conclusion The optimized one-fits-all FISH method is robust, reliable and reproducible to deliver an accurate result for patient diagnostics in a lean workflow and cost-effective manner. This protocol can be used for widespread application in cancer and non-cancer diagnostics and research.

Abstract Background Necrotising enterocolitis (NEC) is a common cause of death in preterm infants and is closely linked to the gut microbiota. Spontaneous intestinal perforation (SIP) also occurs in preterm neonates, but results in lower mortality and less adverse neonatal outcomes than NEC. Existing studies are largely limited to non-invasive stool samples, which may not be reflective of the anatomical site of disease. Therefore, we analysed historical formalin-fixed paraffin-embedded (FFPE) tissue from NEC and SIP preterm infants. A total of 13 NEC and 16 SIP infants were included. Extracted DNA from FFPE tissue blocks underwent 16S rRNA gene sequencing. For a subset of infants, diseased tissue and marginal healthy tissue from the same infant were compared. Results Xylene provided a cost and time effective means of deparaffinization. Tissue from the site of disease was highly comparable to adjacent healthier tissue. Comparing only diseased tissue from all infants showed significantly lower Shannon diversity in NEC (P = 0.026). The overall bacterial communities were also significantly different in NEC samples compared to SIP (P = 0.038), and large variability within NEC infants was observed. While no single OTU or genus was significantly associated with NEC or SIP, at the phylum level Proteobacteria (P = 0.045) and Bacteroidetes (P = 0.024) were significantly higher in NEC and SIP infants, respectively. Conclusions Existing banks of intestinal FFPE blocks provide a robust and specific sample for profiling the microbiota at the site of disease. We showed preterm infants with NEC have lower diversity and different bacterial communities when compared to SIP controls.

Most tissue biopsies from patients in hospital environments are formalin-fixed and paraffin-embedded (FFPE) for long-term storage. This fixation process produces a modification in the proteins called “crosslinks”, which improves protein stability necessary for their conservation. Currently, these samples are mainly used in clinical practice for performing immunohistochemical analysis, since these modifications do not suppose a drawback for this technique; however, crosslinks difficult the protein extraction process. Accordingly, these modifications make the development of a good protein extraction protocol necessary. Due to the specific characteristics of each tissue, the same extraction buffers or deparaffinization protocols are not equally effective in all cases. Therefore, it is necessary to obtain a specific protocol for each tissue. The present work aims to establish a deparaffinization and protein extraction protocol from FFPE kidney samples to obtain protein enough of high quality for the subsequent proteomic analysis. Different deparaffination, protocols and protein extraction buffers will be tested in FFPE kidney samples. The optimized conditions will be applied in the identification by LC-MS/MS analysis of proteins extracted from 5, 10, and 15 glomeruli obtained through the microdissection of FFPE renal samples.

Analysis of formalin-fixed paraffin-embedded (FFPE) tissue by immunohistochemistry (IHC) is commonplace in clinical and research laboratories. However, reports suggest that IHC results can be compromised by biospecimen preanalytical factors. The National Cancer Institute's Biospecimen Preanalytical Variables Program conducted a systematic study to examine the potential effects of delay to fixation (DTF) and time in fixative (TIF) on IHC using 24 cancer biomarkers. Differences in IHC staining, relative to controls with a DTF of 1 hr, were observed in FFPE kidney tumor specimens after a DTF of ≥2 hr. Reductions in H-score and/or staining intensity were observed for c-MET, p53, PAX2, PAX8, pAKT, and survivin, whereas increases were observed for RCC1, EGFR, and CD10. Prolonged TIF of 72 hr resulted in significantly reduced H-scores of CD44 and c-Met in kidney tumor specimens, compared with controls with 12-hr TIF. An elevated probability of altered staining intensity due to DTF was observed for nine antigens, whereas for prolonged TIF an elevated probability was observed for one antigen. Results reported here and elsewhere across tumor types and antigens support limiting DTF to ≤1 hr when possible and fixing tissues in formalin for 12–24 hr to avoid confounding effects of these preanalytical factors on IHC. [ABSTRACT FROM AUTHOR]

Matrix-assisted laser desorption/ionisation mass spectrometry imaging (MALDI-MSI) has been successfully used to elucidate the relative abundance and spatial mapping of analytes in situ. Currently, sample preparation workflows for soft formalin-fixed paraffin-embedded (FFPE) tissues, such as brain, liver, kidney, and heart, have been successfully developed. However, hard tissues, such as cartilage-bone, tooth, and whole mouse body, have resulted in the loss of morphology or tissue during the heat-induced epitope retrieval (HIER) step on commercially available conductive indium tin oxide (ITO) slides. Therefore, we have successfully developed a novel and cost-effective sample preparation workflow in which commercial conductive ITO slides are pre-coated with gelatin and chromium potassium sulfate dodecahydrate to improve the adherence of FFPE human osteoarthritic cartilage-bone tissue sections. Gelatin-coated ITO slides also resulted in overall higher N-glycan signal intensity for not only FFPE osteoarthritic cartilage-bone tissue but also for FFPE hard-boiled egg white used as a quality control to assess the quality of sample preparation and MALDI-MSI acquisition. In summary, we present a novel straightforward workflow to improve slide adherence and morphological preservation of FFPE cartilage-bone tissue sections during HIER while improving the signal intensity of N-glycans spatially mapped from the same tissue sections by MALDI-MSI. [ABSTRACT FROM AUTHOR]

The applicability of mass spectrometry imaging (MSI) has exponentially increased with the improvement of sample preparation, instrumentation (spatial resolution) and data analysis. The number of MSI publications listed in PubMed continues to grow with 378 published articles in 2020‐2021. Initially, MSI was just sensitive enough to identify molecular features correlating with distinct tissue regions, similar to the resolution achieved by visual inspection after standard immunohistochemical staining. Although the spatial resolution was limited compared with other imaging modalities, the molecular intensity mapping added a new exciting capability. Over the past decade, significant improvements in every step of the workflow and most importantly in instrumentation were made, which now enables the molecular analysis at a cellular and even subcellular level. Here, we summarize the latest developments in MSI, with a focus on the latest approaches for tissue‐based imaging described in 2020. [ABSTRACT FROM AUTHOR]

Immunofluorescence microscopy is routinely used in the diagnosis of and research on renal impairments. However, this highly specific technique is restricted in its maximum resolution to about 250 nm in the lateral and 700 nm in the axial directions and thus not sufficient to investigate the fine subcellular structure of the kidney's glomerular filtration barrier. In contrast, electron microscopy offers high resolution, but this comes at the cost of poor preservation of immunogenic epitopes and antibody penetration alongside a low throughput. Many of these drawbacks were overcome with the advent of super-resolution microscopy methods. So far, four different super-resolution approaches have been used to study the kidney: single-molecule localization microscopy (SMLM), stimulated emission depletion (STED) microscopy, structured illumination microscopy (SIM), and expansion microscopy (ExM), however, using different preservation methods and widely varying labelling strategies. In this work, all four methods were applied and critically compared on kidney slices obtained from samples treated with the most commonly used preservation technique: fixation by formalin and embedding in paraffin (FFPE). Strengths and weaknesses, as well as the practicalities of each method, are discussed to enable users of super-resolution microscopy in renal research make an informed decision on the best choice of technique. The methods discussed enable the efficient investigation of biopsies stored in kidney banks around the world. [ABSTRACT FROM AUTHOR]

The 1918 influenza pandemic was the most devastating respiratory pandemic in modern human history, with 50-100 million deaths worldwide. Here, we characterized the complete genomes of influenza A virus (IAV) from two fatal cases during the fall wave of 1918 influenza A (H1N1) pandemic in the United States, one from Walter Reed Army Hospital in Washington, DC, and the other from Camp Jackson, SC. The two complete IAV genomes were obtained by combining Illumina deep sequencing data from both total RNA and influenza viral genome-enriched libraries along with Sanger sequencing data from PCR across the sequencing gaps. This study confirms the previously reported 1918 IAV genomes and increases the total number of available complete or near-complete influenza viral genomes of the 1918 pandemic from four to six. Sequence comparisons among them confirm that the genomes of the 1918 pandemic virus were highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases. Interestingly, in the Washington, DC, case, evidence is presented of the first reported Rhodococcus -influenza virus co-infection., Importance: This study applied modern molecular biotechnology and high-throughput sequencing to formalin-fixed, paraffin-embedded autopsy lung samples from two fatal cases during the fall wave of the 1918 influenza A (H1N1) pandemic in the United States. Complete influenza genomes were obtained from both cases, which increases the total number of available complete or near-complete influenza genomes of the 1918 pandemic virus from four to six. Sequence analysis confirms that the 1918 pandemic virus was highly conserved during the main wave of the pandemic with geographic separation in North America and Europe. Metagenomic analyses revealed bacterial co-infections in both cases, including the first reported evidence of Rhodococcus -influenza co-infection. Overall, this study offers a detailed view at the molecular level of the very limited samples from the most devastating influenza pandemic in modern human history., Competing Interests: The authors declare no conflict of interest.

The science of CellBlockistry highlights requirement for proper approach to process different types of cytopathology specimens with critical considerations during preparation of cell-blocks. Common cytopathology specimens which may be subjected for cell-blocking include FNA aspirates in addition to anterior fat pad aspirate, bone marrow aspirate, effusion fluids, and other fluids such as various washings and urine. In addition veterinary sciences and research fields including animal experiments and tissue/cell cultures may also be cell-blocked for improved diagnostic yield and research outcome. [ABSTRACT FROM AUTHOR]

The potential problems of DNA extraction from formalin‐fixed paraffin‐embedded (FFPE) tissue samples and amplification efficiency of Human papilloma virus (HPV) may occur in the molecular studies of head and neck squamous carcinoma (HNSCC). The aim of this study was to compare HPV detection rate in FFPE tissues of oral, oropharyngeal, hypopharyngeal, and laryngeal cancers using two silica‐based extraction kits and three amplification methods. A total of 50 FFPE specimens from HNSCC tissues were analyzed. The quality and quantity of the extracted DNA were tested by spectrophotometry. HPV DNA was detected using a single polymerase chain reaction (PCR), a nested PCR, and a Real‐time PCR kit. Statistically significantly higher DNA quality and quantity was observed using the QIAamp DNA FFPE Tissue Kit than when using the QIAamp DNA Mini Kit. There was not HPV amplification in any of the 50 FFPE samples using the single PCR and Real‐time PCR kits, whereas HPV DNA was detected in 22% of samples using nested PCR. Comparing results of the three different methods showed that HPV DNA was detected only with nested PCR. The results presented imply that nested PCR is the most appropriate method for the detection of HPV DNA in FFPE samples, along with adequate DNA extraction methods. [ABSTRACT FROM AUTHOR]

In people living with HIV (PLWH), chronic inflammation can lead to cancer initiation and progression, besides driving a dysregulated and diminished immune responsiveness. HIV infection also leads to increased transcription of Human Endogenous Retroviruses (HERVs), which could increase an inflammatory environment and create a tumor growth suppressive environment with high expression of pro-inflammatory cytokines. In order to determine the impact of HIV infection to HERV expression on the breast cancer microenvironment, we sequenced total RNA from formalin-fixed paraffin-embedded (FFPE) breast cancer samples of women HIV-negative and HIV-positive for transcriptome and retrotranscriptome analyses. We performed RNA extraction from FFPE samples, library preparation and total RNA sequencing (RNA-seq). The RNA-seq analysis shows 185 differentially expressed genes: 181 host genes (178 upregulated and three downregulated) and four upregulated HERV transcripts in HIV-positive samples. We also explored the impact of HERV expression in its neighboring breast cancer development genes (BRCA1, CCND1, NBS1/NBN, RAD50, KRAS, PI3K/PIK3CA) and in long non-coding RNA expression (AC060780.1, also known as RP11-242D8.1). We found a significant positive association of HERV expression with RAD50 and with AC060780.1, which suggest a possible role of HERV in regulating breast cancer genes from PLWH with breast cancer. In addition, we found immune system, extracellular matrix organization and metabolic signaling genes upregulated in HIV-positive breast cancer. In conclusion, our findings provide evidence of transcriptional and retrotranscriptional changes in breast cancer from PLWH compared to non-HIV breast cancer, including dysregulation of HERVs, suggesting an indirect effect of the virus on the breast cancer microenvironment.