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This study evaluated two DNA-based next-generation sequencing approaches for detection of single-nucleotide variants (SNVs) and fusions in formalin-fixed, paraffin-embedded (FFPE) tissue specimens and liquid biopsies (AVENIO Targeted and Surveillance Panels). Reference standards (n = 7 with SNVs and structural variants) and real-world FFPE tissue specimens (n = 26 lung, colorectal, pancreas, ovary, breast, prostate, melanoma, and soft tissue cancer cases with n = 27 samples), liquid biopsies [n = 29 cases with n = 40 plasma/cell-free DNA (cfDNA) samples], and one pleural effusion (lung cancer) were analyzed by the AVENIO workflow for known SNVs (BRAF, BRCA1/2, CTNNB1, EGFR, KRAS, MET exon 14 skipping, NRAS, PIK3CA, and TP53), insertions and deletions (ERBB2 and KIT), and fusions (ALK and ROS1). Detection of SNVs, insertions and deletions, and fusions was reliable in 24 of 26 FFPE tissue specimen cases and at 1% allele frequency in 5 of 5 cfDNA reference standards and 37 of 40 plasma/cfDNA samples. Pitfalls were identified for the AVENIO workflow in calling and listing of clinically relevant variants, requiring additional manual inspection. Moreover, laboratory workflows are distinct for FFPE tissue specimens and liquid biopsies as well as time-consuming for sample quality control assays. In summary, the DNA-based next-generation sequencing approaches may be suitable for routine molecular pathology diagnostics on careful data interpretation and further optimization of the technical and laboratory workflows.

International audience; As opposed to surface marker staining, certain cell types can only be recognized by intracellular markers. Intracellular staining for use in cell sorting remains challenging. Fixation and permeabilization steps for intracellular staining and the presence of RNases notably affect preservation of high-quality mRNA. We report the work required for the optimization of a successful protocol for microarray analysis of intracellular target-sorted, formalin-fixed human bronchial club cells. Cells obtained from differentiated air-liquid interface cultures were stained with the most characteristic intracellular markers for club cell (SCGB1A1+) sorting. A benchmarked intracellular staining protocol was carried out before flow cytometry. The primary outcome was the extraction of RNA sufficient quality for microarray analysis as assessed by Bioanalyzer System. Fixation with 4% paraformaldehyde coupled with 0.1% Triton/0.1% saponin permeabilization obtained optimal results for SCGB1A1 staining. Addition of RNase inhibitors throughout the protocol and within the appropriate RNA extraction kit (Formalin-Fixed-Paraffin-Embedded) dramatically improved RNA quality, resulting in samples eligible for microarray analysis. The protocol resulted in successful cell sorting according to specific club cell intracellular marker without using cell surface marker. The protocol also preserved RNA of sufficient quality for subsequent microarray transcriptomic analysis, and we were able to generate transcriptomic signature of club cells.

Copy number alterations are genetic events that promote tumor initiation and progression and are used in clinical care as diagnostic, prognostic, and predictive biomarkers. Based on the length of the alteration, they are roughly classified as focal and arm-level alterations. Although genome-wide techniques to detect arm-level alterations are gaining momentum in hospital laboratories, the high precision and novelty of these techniques pose new challenges: there is no consensus on the definition of an arm-level alteration and there is a lack of tools to compute them for individual patients. Based on 376 clinical samples analyzed with the OncoScan formalin-fixed, paraffin-embedded assay, a bimodal distribution of the percentage of bases with copy number alterations within a chromosomal arm was observed, with the second peak starting at 90% of arm length. Two approaches were tested for the definition of arm-level alterations: sum of altered segments (SoS) >90%, or the longest segment (LS) >90%. These approaches were validated against expert annotation of 25 clinical cases. The SoS method outperformed the LS method as indicated by a higher concordance (SoS, 95.2%; LS, 79.9%). Some of the discordances ultimately were attributed to human error, highlighting the advantages of automation. The increase in reliability led to the development of publicly available software and its inclusion into routine clinical practice at Geneva University Hospitals.

Implementation of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) testing in the daily practice of pathology laboratories requires procedure adaptation to formalin-fixed, paraffin-embedded (FFPE) samples. So far, one study reported the feasibility of SARS-CoV-2 genome sequencing on FFPE tissues with only one contributory case of two. This study optimized SARS-CoV-2 genome sequencing using the Ion AmpliSeq SARS-CoV-2 Panel on 22 FFPE lung tissues from 16 deceased coronavirus disease 2019 (COVID-19) patients. SARS-CoV-2 was detected in all FFPE blocks using a real-time RT-qPCR targeting the E gene with crossing point (Cp) values ranging from 16.02 to 34.16. Sequencing was considered as contributory (i.e. with a uniformity >55%) for 17 FFPE blocks. Adapting the number of target amplification PCR cycles according to the RT-qPCR Cp values allowed optimization of the sequencing quality for the contributory blocks (i.e. 20 PCR cycles for blocks with a Cp value 30 were non-contributory. Comparison of matched frozen and FFPE tissues revealed discordance for only three FFPE blocks, all with a Cp value >28. Variant identification and clade classification was possible for 13 patients. This study validates SARS-CoV-2 genome sequencing on FFPE blocks and opens the possibility to explore correlation between virus genotype and histopathologic lesions., info:eu-repo/semantics/published

Gene mutation detection and the resulted precision-medicine therapy is transforming clinical practice. Here, we report the use of a custom-developed, medium-sized, pan-cancer probe panel for the detection of somatic and germline mutations. We used a hybridization capture-based NGS assay for targeted deep sequencing of all exons and selected introns of 181 key cancer driver genes, covering both inherited risks and somatic mutations. We performed paired-variant calling on tumor samples and their matched normal samples. We processed clinical patient samples of formalin-fixed, paraffin embedded tumors (FFPE samples) and cell-free peripheral blood (cfDNA samples). We found germline mutations of inherited cancer risk at 9%; and discovered a novel germline mutation in BRCA1. Somatic mutation rate in driver genes is at 73.1%, much higher than previously reported. On recommending precision-medicine therapeutics, we achieved 91.6% for patients with FFPE samples.

DNA junctions (DNAJs) frequently impact clinically relevant genes in tumors and are important for diagnostic and therapeutic purposes. Although routinely screened through fluorescence in situ hybridization assays, such testing only allows the interrogation of single-gene regions or known fusion partners. Comprehensive assessment of DNAJs present across the entire genome can only be determined from whole-genome sequencing. Structural variance analysis from whole-genome paired-end sequencing data is, however, frequently restricted to copy number changes without DNAJ detection. Through optimized whole-genome sequencing and specialized bioinformatics algorithms, complete structural variance analysis is reported, including DNAJs, from formalin-fixed DNA. Selective library assembly from larger fragments (500 bp) and economical sequencing depths (300 to 400 million reads) provide representative genomic coverage profiles and increased allelic coverage to levels compatible with DNAJ calling (40× to 60×). Although consistently fragmented, more recently formalin-fixed, specimens (2 years' storage) revealed consistent populations of larger DNA fragments. Optimized bioinformatics efficiently detected90% of DNAJs in two prostate tumors (approximately 60% tumor) previously analyzed by mate-pair sequencing on fresh frozen tissue, with evidence of at least one spanning-read in 99% of DNAJs. Rigorous masking with data from unrelated formalin-fixed tissue progressively eliminated many false-positive DNAJs, without loss of true positives, resulting in low numbers of false-positive passing current filters. This methodology enables more comprehensive clinical genomics testing on formalin-fixed clinical specimens.

Formalin-fixed, paraffin-embedded (FFPE) tissue is the most commonly used material for tumor molecular profiling, therapy selection, and prognostication. Tumor tissue enrichment by tissue dissection is highly recommended to generate quality data reproducibly for use in downstream assays, such as real-time PCR and next-generation sequencing. The aim of this study was to evaluate the performance of the automated tissue dissection tool AVENIO Millisect System compared with a manual dissection method using 18 FFPE tissue specimens. The study assessed performance of these two methods with paraffinized and deparaffinized sections at 5- and 10-μm thickness as well as at low (5% to 10%) and high (>50%) tumor content. In addition, compatibility with various nucleic acid and protein extraction methods was assessed. Overall, dissection by Millisect resulted in statistically significantly higher yields of nucleic acids and protein compared with manual dissection (P = 0.00524). In downstream analysis on a statistically nonpowered sample set, EGFR mutation testing by PCR led to highly concordant results, and next-generation sequencing testing yielded significantly higher allelic frequencies when tissue was dissected by Millisect compared with manual scraping, demonstrating noninferiority of the automated method. In summary, the AVENIO Millisect System may replace manual labor and support automation of FFPE tumor tissue workflows in clinical molecular laboratories with high testing volumes with adequate validation.

RNA sequencing (RNA-seq) is a well-validated tool for detecting gene fusions in fresh-frozen tumors; however, RNA-seq is much more challenging to use with formalin-fixed, paraffin-embedded (FFPE) tumor samples. We evaluated the performance of RNA-seq to detect gene fusions in clinical FFPE tumor samples. Our assay identified all 15 spiked-in NTRK fusions from RNA reference material and six known fusions from five cancer cell lines. Limit of detection for the assay was assessed with a series of dilutions of RNA from the cell line H2228. These fusions can be detected when the dilution is down to 10%. Good intra-assay and interassay reproducibility was observed in three specimens. For clinical validation, the assay detected 10 of 12 fusions initially identified by a DNA panel (covering 23 gene fusions) in clinical specimens (83.3% sensitivity), whereas one fusion (MET fusion) was identified in another 34 fusion-negative tumor specimens as determined by the DNA panel (negative prediction value of 94.3%). This MET fusion was confirmed by RT-PCR and Sanger sequencing, which found that this is a false-negative result for the DNA panel. The assay also identified 26 extra fusions not covered by the DNA panel, 20 (76.9%) of which were validated by RT-PCR and Sanger sequencing. Therefore, this RNA assay has reasonable performance and could complement DNA-based next-generation sequencing assays.

Coronavirus Disease-19 (COVID-19), caused by the coronavirus SARS-CoV-2, was initially recognized in Wuhan, China and subsequently spread to all continents. The disease primarily affects the lower respiratory system, but may involve other organs and systems. Histopathologic evaluation of tissue from affected patients is crucial for diagnostic purposes, but also for advancing our understanding of the disease. For that reason, we developed immunohistochemical (IHC) and in situ hybridization (ISH) assays for detection of the. virus. A total of eight autopsy lungs, one placenta, and ten kidney biopsies from COVID-19 patients were stained with a panel of commercially available antibodies for IHC and commercially available RNA probes for ISH. Similarly, autopsy lungs, placentas and renal biopsies from non-COVID-19 patients were stained with the same antibodies and probes. All eight lungs and the placenta from COVID-19 patients stained positive by IHC and ISH, while the kidney biopsies stained negative by both methodologies. As expected, all specimens from non-COVID-19 patients were IHC and ISH negative. These two assays represent a sensitive and specific method for detecting the virus in tissue samples. We provide the protocols and the list of commercially available antibodies and probes for these assays, so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes., Detailed protocols for immunohistochemical and in situ hybridization assays for the detection of SARS-CoV-2 are provide so they can be readily implemented in pathology laboratories and medical examiner offices for diagnostic and research purposes. These assays were found to represent a sensitive and specific method for detecting the virus in tissue samples.

This study aims to compare the potential of standard RNA-sequencing (RNA-Seq) and 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing for the analysis of fresh tissue and describes transcriptome profiling of formalin-fixed paraffin-embedded (FFPE) archival human samples by MACE. To compare MACE to standard RNA-Seq on fresh tissue, four healthy conjunctiva from four subjects were collected during vitreoretinal surgery, halved and immediately transferred to RNA lysis buffer without prior fixation and then processed for either standard RNA-Seq or MACE RNA-Seq analysis. To assess the impact of FFPE preparation on MACE, a third part was fixed in formalin and processed for paraffin embedding, and its transcriptional profile was compared with the unfixed specimens analyzed by MACE. To investigate the impact of FFPE storage time on MACE results, 24 FFPE-treated conjunctival samples from 24 patients were analyzed as well. Nineteen thousand six hundred fifty-nine transcribed genes were detected by both MACE and standard RNA-Seq on fresh tissue, while 3251 and 2213 transcripts were identified explicitly by MACE or RNA-Seq, respectively. Standard RNA-Seq tended to yield longer detected transcripts more often than MACE technology despite normalization, indicating that the MACE technology is less susceptible to a length bias. FFPE processing revealed negligible effects on MACE sequencing results. Several quality-control measurements showed that long-term storage in paraffin did not decrease the diversity of MACE libraries. We noted a nonlinear relation between storage time and the number of raw reads with an accelerated decrease within the first 1000 days in paraffin, while the numbers remained relatively stable in older samples. Interestingly, the number of transcribed genes detected was independent on FFPE storage time. RNA of sufficient quality and quantity can be extracted from FFPE samples to obtain comprehensive transcriptome profiling using MACE technology. We thus present MACE as a novel opportunity for utilizing FFPE samples stored in histological archives., RNA of sufficient quality and quantity can be extracted from formalin-fixed paraffin-embedded (FFPE) samples to obtain comprehensive transcriptome profiling using the 3′ massive analysis of c-DNA ends (MACE) RNA-sequencing technology. Thus, MACE provides an opportunity for utilizing FFPE samples stored in histological archives.

Summary The diagnosis of microscopic colitis (MC) relies on specific histopathological findings in colon biopsies. The number of biopsies needed to diagnose MC remains disputed. The aim of the study was to determine the number and site of biopsies necessary for the diagnosis and the effect of perpendicular orientation when embedding the biopsies. This retrospective multicenter European study included 42 patients with a consensus diagnosis of collagenous colitis (CC), 51 patients with lymphocytic colitis (LC), and three patients with incomplete LC (LCi). The number of individual diagnostic biopsies from each patient was determined. The diagnostic rate of 744 individual biopsies from 96 patients with MC was 69.5% for the specific MC subgroup, 79.4% for MC and 93.4% for MC plus incomplete MC (MCi). The risk of missing a diagnosis of the specific subgroup of MC when analyzing four biopsies was 0.87%, decreasing to 0.18% for MC and 0.0019% for MC plus MCi. More biopsies from the right colon were diagnostic of the specific MC subgroup (76.3% vs. 64.0%, p = 0.0014). Perpendicular orientation of biopsies increased the diagnostic rate of the specific MC subgroup (73.1% vs. 65.0%, p = 0.0201). Histological changes diagnostic of MC were present in almost all biopsies from the right colon, with orientated biopsies more often being diagnostic of the specific MC subgroup. The results of this study indicate that four biopsies from the colon, rectum excluded, are sufficient to diagnose MC.

Laser-capture microdissection of formalin-fixed and paraffin-embedded (FFPE) tissue is a powerful tool for the analysis of cells present in small cell numbers. It allows retrospective studies of healthy and diseased tissue specimens considering the huge repositories for FFPE tissue. A protocol is described with detailed evaluation of each sample preparation step including protein extraction from unstained and immunohistochemical stained tissue, SP3 digestion and TMT labeling. Finally, the established method was applied to limited sample amounts of human substantia nigra., Graphical Abstract Highlights A detailed investigation of the protein extraction step from FFPE tissue is shown. Acidification during peptide wash increased peptide recovery of the SP3 method. LCM of substantia nigra enriched neuron-specific proteins including TH. >5,600 proteins were quantified using 3000 cells per sample from substantia nigra., Laser-capture microdissection (LCM) allows the visualization and isolation of morphologically distinct subpopulations of cells from heterogeneous tissue specimens. In combination with formalin-fixed and paraffin-embedded (FFPE) tissue it provides a powerful tool for retrospective and clinically relevant studies of tissue proteins in a healthy and diseased context. We first optimized the protocol for efficient LCM analysis of FFPE tissue specimens. The use of SDS containing extraction buffer in combination with the single-pot solid-phase-enhanced sample preparation (SP3) digest method gave the best results regarding protein yield and protein/peptide identifications. Microdissected FFPE human substantia nigra tissue samples (∼3,000 cells) were then analyzed, using tandem mass tag (TMT) labeling and LC-MS/MS, resulting in the quantification of >5,600 protein groups. Nigral proteins were classified and analyzed by abundance, showing an enrichment of extracellular exosome and neuron-specific gene ontology (GO) terms among the higher abundance proteins. Comparison of microdissected samples with intact tissue sections, using a label-free shotgun approach, revealed an enrichment of neuronal cell type markers, such as tyrosine hydroxylase and alpha-synuclein, as well as proteins annotated with neuron-specific GO terms. Overall, this study provides a detailed protocol for laser-capture proteomics using FFPE tissue and demonstrates the efficiency of LCM analysis of distinct cell subpopulations for proteomic analysis using low sample amounts.

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.

microRNAs play prominent regulatory roles by inducing target mRNA degradation or translational repression. Several studies have highlighted the power of microRNAs as biomarkers and potential mechanistic mediators of human kidney disease. Small RNA deep sequencing libraries can be generated from minute amounts of formalin-fixed paraffin-embedded tissues including specific tissue regions. Such analyses have unique advantages and may complement analyses of fresh kidney specimens to advance tissue functional genomics-based precision medicine for human kidney disease.

The limited accuracy of endoscopic biopsy in detecting high-grade dysplasia or adenocarcinoma within ampullary adenoma or dysplasia has been reported. The natural history of ampullary dysplasia is also unclear, and there are no established guidelines to determine which patients with ampullary dysplasia require resection versus surveillance endoscopy. DNA flow cytometry was performed on 47 ampullary biopsies with low-grade dysplasia, 18 high-grade dysplasia, and 23 negative for dysplasia, as well as 11 cases of ampullary adenocarcinoma. Abnormal DNA content (aneuploidy or elevated 4N fraction > 6%) was identified in 9 (82%) of adenocarcinoma, 13 (72%) of high-grade dysplasia, 7 (15%) of low-grade dysplasia, and none (0%) of non-dysplastic mucosa. One-, 2-, and 7-year detection rates of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with abnormal DNA content were 57%, 86%, and 88%, respectively, whereas low-grade dysplasia patients in the setting of normal DNA content had 1-, 2-, and 7-year detection rates of 10%, 10%, and 10%, respectively. The univariate and multivariate hazard ratios (HRs) for subsequent detection of high-grade dysplasia or adenocarcinoma in low-grade dysplasia patients with DNA content abnormality were 16.8 (p =

Summary Objectives Vocal fold nodules (VFNs) are benign disorders affecting the superficial lamina propria of the true vocal folds. The etiology of VFNs still remains unclear but laryngeal trauma caused by vocal abuse, tobacco, alcohol, and laryngopharyngeal reflux (LPR) plays a crucial role on the pathogenesis. The aim of this study was to assess the presence of pepsin in formalin-fixed, paraffin-embedded (FEPE) specimens of VFNs to evaluate the role of LPR as a risk factor for VFNs. Materials and Methods A total of 28 pathology specimens of patients suffering from VFNs who had undergone laser microsurgery under general anesthesia were evaluated. The specimens were maintained in paraffin blocks in the pathology department. Western blot (WB) and enzyme-linked immunosorbent assay (ELISA) analyses were used to measure pepsin enzyme levels in the VFNs tissue specimens. Signs of LPR were assessed according to the reflux finding score. Results The mean reflux finding score of the patients was 13.6 ± 2.89 (8–21). According to WB and ELISA analyses, pepsin was detected with both the WB the ELISA tests in positive controls, but there was no pepsin enzyme in any of the 28 laryngeal FEPE VFNs specimens. Conclusion The pepsin enzyme was not detected in any of the FEPE VFNs specimens, and it is concluded that further studies are needed to reveal the role of pepsin in the etiology of VFNs.

Accuracy in morphological diagnosis is the cornerstone of anatomic pathology. Proficiency with the microscope offers values to the health care system that cannot be overestimated. However, that skill is only possible if high-quality histological substrates are available for assessment, particularly focusing on hematoxylin and eosin (H&E)-stained slides. This brief review considers the several steps that are necessary to control in the preparation of high-quality H&E sections, including those dealing with fixation, embedding, microtomy, histochemical staining, and coverslipping. A table for the troubleshooting of problem slides is also included.

Clinical biomarker studies are often hindered by the scarcity or suboptimal quality of biological specimens. EdgeSeq, a transcriptomics analysis platform, combines quantitative nuclease protection assay technology with next-generation sequencing, using small amounts of starting material and delivering reproducible gene expression profiles from challenging material, such as formalin-fixed, paraffin-embedded (FFPE) tissue. To evaluate EdgeSeq for analysis of archives of stained FFPE tissue, EdgeSeq was performed on unstained, hematoxylin and eosin (HE)-stained, and immunohistochemistry-stained slides from patients with small-cell and non-small-cell lung cancer. Pairwise comparisons of gene expression profiles from stained and unstained slides showed higher Pearson correlation coefficients with HE staining (0.86 to 0.97) than with immunohistochemistry staining (0.21 to 0.56). A 25-gene interferon-γ signature score from unstained slides showed a Pearson correlation coefficient of 0.92 with HE-stained slides and a significant Spearman correlation (P = 0.0025) with immune scores. To test gene expression profiling in small samples, FFPE sample equivalents were examined from 5.0 to 0.08 mm

Infectious haemolytic anaemia (IHA) in dogs share similar clinical signs including fever, lethargy, icterus, paleness of mucous membranes and splenomegaly. Postmortal findings are similar and, without additional diagnostic methods, an accurate aetiological diagnosis is difficult to achieve. In order to investigate causes of lethal IHA in Croatian dogs, we performed a retrospective study on archived formalin-fixed, paraffin-embedded tissue blocks (FFPEB) from dogs that died due to haemolytic crisis, using microscopic and molecular diagnostic tools to determine the aetiological cause of disease. Molecular analysis was performed on kidney, lung, myocardium and spleen on FFPEB from all dogs. The originally stated aetiological diagnosis of B. canis or leptospirosis was confirmed in only 53% of the dogs. PCR and sequencing revealed that, in addition to the expected pathogens, B. canis and Leptospira interrogans, the presence of previously undiagnosed "new" pathogens causing anaemia including Candidatus Neoehrlichia mikurensis and Anaplasma phagocytophilum. Furthermore, Theileria capreoli was detected for the first time in a dog with postmortal descriptions of lesions. Intensive extravascular hemolysis was noticeable as jaundice of the mucosa, subcutis and fat tissue, green or yellow discoloration of renal parenchyma caused by bilirubin excretion in the renal tubules and bile accumulation within the liver in 90% of the dogs. This work highlights the value of molecular diagnostics to complement traditional ante-mortem and post-mortem diagnostic protocols for the aetiological diagnosis of pathogens associated with IHA.

The vast majority of archived research and clinical pathological specimens are stored in the form of formalin fixed, paraffin-embedded (FFPE) tissues, but, unlike fresh frozen tissue samples, highly quantitative measures in FFPE tissues are limited to immunohistochemical and immunofluorescence thresholding image analysis studies, cell counting, and ordinal ranking systems. This poses a significant obstacle for clinical investigations that aim to correlate diagnostic markers of neurodegenerative diseases like Alzheimer's disease (AD) with parameters like age, gender, drug exposures, genotype, disease stage, co-morbidities, or environmental factors. To overcome this limitation, we have developed Luminex-based techniques and protocols for the quantification of amyloid β and hyperphosphorylated Tau in FFPE brain sections. We validated the Luminex assay in FFPE sections from prefrontal cortex, hippocampus, and neostriatum from 30 cases that underwent prior neuropathological diagnostic assessment of AD following the current NIA-AA recommendations for AD: 10 cases diagnosed as not or low, 10 cases as intermediate, and 10 cases as high AD neuropathologic change. Consistent with the neuropathologic assessment, Luminex assay detected high amounts of amyloid beta in the frontal cortex and striatum, and high amounts of hyperphosphorylated Tau in the frontal cortex and hippocampus, of cases with high AD neuropathologic change. This assay can be expanded to detect diverse antigenic targets of interest, as we show here with IBA1 and GFAP. This novel approach supports multiplexed highly quantitative, molecularly specific neuropathology measures to further explore mechanisms of neurodegeneration in AD.

The clinical management and therapy of many solid tumor malignancies depends on detection of medically actionable or diagnostically relevant genetic variation. However, a principal challenge for genetic assays from tumors is the fragmented and chemically damaged state of DNA in formalin-fixed, paraffin-embedded (FFPE) samples. From highly fragmented DNA and RNA there is no current technology for generating long-range DNA sequence data as is required to detect genomic structural variation or long-range genotype phasing. We have developed a high-throughput chromosome conformation capture approach for FFPE samples that we call Fix-C, which is similar in concept to Hi-C. Fix-C enables structural variation detection from archival FFPE samples. This method was applied to 15 clinical adenocarcinoma- and sarcoma-positive control specimens spanning a broad range of tumor purities. In this panel, Fix-C analysis achieves a 90% concordance rate with fluorescence in situ hybridization assays, the current clinical gold standard. In addition, novel structural variation undetected by other methods could be identified, and long-range chromatin configuration information recovered from these FFPE samples harboring highly degraded DNA. This powerful approach will enable detailed resolution of global genome rearrangement events during cancer progression from FFPE material and will inform the development of targeted molecular diagnostic assays for patient care.

Patient stratification, prognosis and disease monitoring are three important aspects of personalized cancer medicine. With traditional serum tumour protein biomarkers showing lack of specificity and sensitivity, and tumour heterogeneity affecting the response to targeted therapy based on tissue biomarkers, the focus has shifted to the use of molecular tumour signatures as specific biomarkers. Multiplex microsphere-based panels are robust and cost-effective, high throughput molecular assays, which can accurately characterize tumours even from small amounts of poor quality nucleic acids. Only few studies have reported the use of microspheres (beads) to quantify RNA expression of targets of interest simultaneously (multiplexing). This review is an overview of the various applications of bead-based RNA panels in molecular oncology, with focus on the Invitrogen™ QuantiGene™ Plex Assay (Thermo Fisher Scientific), and provides a comparison with PCR-based and other methodologies. The advantages of multiplex bead assays are exemplified by the quantification of RNA expression in formalin-fixed, paraffin embedded (FFPE) archival tissue and the simultaneous detection of biomarkers in low input samples, including quantification of markers in microdissected tissue material, to characterise heterogeneous tumour sites within a sample, and by the detection of markers in low numbers of circulating tumour cells.

Immunohistochemistry (IHC) specifically localizes proteins in cells and tissues, but methodologies vary widely. Therefore, we performed a methodological IHC optimization and validation study. First, we compared advantages and disadvantages of cryostat sections versus paraffin sections. Second, we compared and optimized antigen retrieval in paraffin sections using citrate buffer and Tris/EDTA buffer. Third, aminoethyl carbazole (AEC) and 3,3'-diaminobenzidine (DAB) were tested as horseradish peroxidase (HRP) substrates to obtain a water-insoluble coloured end product to visualize antigens. Fourth, secondary antibodies conjugated with either mono-HRP or poly-HRP were compared. The study was performed using serial sections of human tonsil. IHC was performed with primary antibodies against endothelial cell marker CD31, smooth muscle actin (SMA), chemokine stromal-derived factor-1α (SDF-1α) and its receptor C-X-C receptor type 4 (CXCR4), macrophage marker CD68 and proliferation marker Ki67. DAB rather than AEC, and cryostat sections rather than paraffin sections gave optimum staining at highest primary antibody dilutions, whereas tissue morphology in paraffin sections was superior. Loss of antigenicity in paraffin sections by formaldehyde fixation, heat and/or masking of epitopes was counteracted by antigen retrieval but not for all antigens. Two out of six antigens (CD31 and CD68) could not be retrieved irrespective time and type of retrieval. Tris-EDTA was superior to citrate buffer for antigen retrieval. The use of mono-HRP or poly-HRP depended on the affinity of the primary antibody for its antigen. We conclude that IHC methodology optimization and validation are crucial steps for each antibody and each research question.

Background Objective of our work was to develop a sequential double nonfluorescent immunostaining method which allows the selective identification of myelinated motor fibers in paraffin-embedded samples of peripheral nerves. Motor recovery after a nerve gap-lesion repaired by artificial nerve-guides ("conduits") is often less complete and slower than sensory recovery. The mechanism for this is not fully understood. New method Incubation in sheep polyclonal choline acetyltransferase antibody (Abcam 18,736) at dilution of 1:150 was followed by incubation in mouse monoclonal anti-myelin basic protein antibody (Abcam 62,631) at a dilution of 1:5000. Counterstaining was performed with hematoxylin QS (Vector Labs H-3404). Results Immunostaining of choline acetyltransferase and myelin basic protein can be combined together and results show a good contrast between the light brown of the choline acetyltransferase reaction product and the green of myelin basic protein reaction product. Cell nuclei are stained blue. This new protocol retains the advantages of paraffin embedded sections such as (i) having a relatively simple methodology, (ii) years-long storage life, and (iii) easy sharing among laboratories. Comparison with existing method. This specific combinatorial protocol has never been used before on paraffin embedded sections. It has been named "reciprocal nerve staining" (RNS). Conclusions Routine combination of choline acetyltransferase and myelin basic protein immunostaining provides a highly specific, highly contrasted paraffin-embedded sections where optical differentiation of myelinated motor fibers is easy and straightforward. This method will likely simplify and speed-up the routine histological study of nerve regeneration and will contribute a better identification of the nerve motor component.

To implement Hyperspectral Imaging (HSI) as a tool for quantifying inflammatory cells in tissue specimens by the example of myocarditis in a collective of forensic patients.44 consecutive patients with suspected myocardial inflammation at autopsy, diagnosed between 2013 and 2018 at the Institute of ForensicMedicine, Medical University of Innsbruck, were selected for this study. Using the IMEC SNAPSCAN camera, visible and near infrared hyperspectral images were collected from slides stained with CD3 and CD45 to assess quantity and spatial distribution of positive cells. Results were compared with visual assessment (VA) and conventional digital image analysis (DIA).Finally, specimens of 40 patients were evaluated, of whom 36 patients (90%) suffered from myocarditis, two patients (5%) had suspected healing/healed myocarditis, and two did no have myocarditis (5%). The amount of CD3 and CD45 positive cells did not differ significantly between VA, HSI, and DIA (pHSI is a reliable and objective method to count inflammatory cells in tissue slides of suspected myocarditis. Implementation of HSI in digital pathology might further expand the possibility of a sophisticated method.

Recent advances in high-throughput screening of human tumors have enabled the identification of numerous tumor markers. However, the clinical utility of these newly identified molecules remains enigmatic until they are well validated in patient samples. Although many long non-coding RNA molecules of clinical importance have been identified in the differential diagnosis of prostate cancer, the consistency of many of them in practice is still controversial. Therefore, short non-coding RNA molecules such as miRNAs are coming to the forefront in the differential diagnosis of the disease because of their stability. Accordingly, in the present study, we aimed to reveal the clinical significance of miR-130a, miR-301a, miR-454 expression levels in formalin fixed paraffin embedded (FFPE) tissue samples of prostate cancer patients. miRNA expression signatures were determined by RT-qPCR method. Notably, we found that miR-301a and miR-454 were significantly upregulated whereas miR-130a were downregulated in cancerous tissues of prostate cancer patients compared to adjacent healthy tissue samples. Moreover, differential expression of these miRNAs was significantly associated with patients' clinicopathological findings, such as Gleason score, lymphovascular invasion, perineural invasion, and extra-prostatic extension. Collectively, our observations indicate that these miRNAs may be of clinical importance in the differential diagnosis of prostate cancer.

Formalin-fixed paraffin-embedded (FFPE) tissues stored in thousands of human and animal pathology laboratories around the globe represent mines of stored genetic information. In recent years, the use of FFPE tissues as a viable source of DNA for diverse genetic studies has attracted attention for interrogating microbiomes from this sample type. These studies have proven that 16S rRNA amplicon sequencing-based microbiome studies are possible from FFPE samples but present some particular challenges. In this review, we summarize all aspects of microbiome studies from FFPE tissues including the challenges associated with working highly degraded DNA, best practices for reducing environmental contamination, and we propose solutions to address these issues. Finally, we discuss how the combination of FFPE microbiome studies and Laser Capture Microdissection and/or Laser Microdissection could enable to determine the spatial heterogeneity underlying complex bacterial communities.

The tissue processing technique is used to preserve the biological structure of tissue samples harvested from biopsy as closely as possible to their in vivo state for the diagnoses and study of disease on the cellular level. This process includes fixation, dehydration, clearing, paraffin infiltration. The protocol follows paraffin block embedding, microtome slicing, staining, and microscope slide studying. Tissue processing is a time-sensitive task as histopathologists must rapidly prevent the decomposition of tissue samples and promptly provide diagnoses on time. However, there are different tissue processing protocols for processing tissues with different types and sizes. Fatty tissues and tissues thicker than 1.5 mm are more susceptible to human errors when choosing a proper tissue processing protocol when preparing separate batches of tissues. In this research, a novel automated variable power cold microwave tissue processor was developed using a universal tissue processing protocol (processing time of 97 min) to simultaneously process all tissue types up to 4 mm in thickness. The tissue processor operated with a relatively smaller number of reagents without formaldehyde or xylene. These materials cause severe health and safety issues for humans and the environment. The quality of healthy and diseased processed tissues (sizes 1×1×1 to 24×15×4 mm) of fatty, thyroid, breast, placenta, skin, prostate, stomach, and bladder was examined under a light microscope by defining MAS (morphology, artifacts, and staining) criteria for evaluating cellular details, tissue arrangement, tissue integrity, stain uptake, and visual distinction of a tissue structure in light microscopy. It was found that the new tissue processor has successfully processed both healthy and diseased fatty and nonfatty tissue samples, while all tissue samples also met MAS criteria. Light microscopy showed outstanding integrity and arrangement in the tissue structures with an excellent visual distinction.

Developments in quantitative proteomics and data-independent acquisition (DIA) methodology is enabling quantification of proteins in biological samples. Currently, there are a few reports on DIA mass spectrometry (MS) approaches for proteome analysis of formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, to facilitate detection and quantification of immune- and glioblastoma (GBM)-relevant proteins from FFPE patient materials, we established a simple and precise DIA-MS workflow. We first evaluated different lysis buffers for their efficiency in protein extractions from FFPE GBM tissues. Our results showed that more than 1700 proteins were detected and over 1400 proteins were quantified from GBM FFPE tissue microdissections. GBM-relevant proteins (e.g., GFAP, FN1, VIM, and MBP) were quantified with high precision (median coefficient of variation

In mice, the labyrinth zone of the placenta exchanges nutrients and gases between mother and fetus. This placental zone is complex in structure and defects in its morphogenesis can compromise substrate exchange and thus, fetal growth and viability. Numerous mouse models involving genetic and environmental manipulation show abnormalities in labyrinth zone size. However, further structural analysis, normally undertaken using ultrathin resin sections, can pose practical constraints. Here, we validate the use of stereology on paraffin-embedded sections double-labelled for lectin and cytokeratin as a cheap, fast and robust alternative for analysing the structure of the mouse placental labyrinth. ispartof: Placenta vol:94 pages:44-47 ispartof: location:Netherlands status: published

Highlights • A comprehensive high-throughput viral sequencing strategy (ViroCap) was adapted for use with feline tumors. • Papillomavirus was not commonly associated with feline oral squamous cell carcinoma. • Feline oral squamous cell carcinoma is a good model for HPV-negative head and neck squamous cell carcinoma in people. • The virome of FOSCC and normal feline oral mucosa included feline foamy virus, torque teno virus, herpes and papillomavirus, FIV and EBV. • Co-occurrence of Epstein Barr Virus and feline papillomavirus-3 was found found in a feline oral squamous cell carcinoma sample., Feline oral squamous cell carcinoma (FOSCC) may be the best naturally-occurring model of human head and neck squamous cell carcinoma (HNSCC). HNSCC can be broadly divided into human papillomavirus (HPV)-negative cancers and HPV-positive cancers where HPV is the causative agent. Previous studies in FOSCC have used both species-specific and species-nonspecific PCR primers that may be insensitive to the detection of PVs and other viruses that may be divergent from known sequences. ViroCap is a targeted capture and next generation sequencing tool that was designed to identify all known vertebrate DNA and RNA viruses. In this study we used a metagenomic approach using ViroCap for DNA viruses in 20 FOSCC, 9 normal feline oral mucosal, and 8 suspected PV positive control samples. We tested the hypothesis that viruses would be enriched in FOSCC compared to normal oral mucosa. The virome of the FOSCC and normal feline oral mucosa consisted of feline foamy virus in 7/20 and 2/9 (35% and 22%), feline torque teno virus in 2/20 and 0/9 (10% and 0%), alphaherpesvirus in 2/10 and 0/9 (10% and 0%), FIV (0% and 22%), Epstein-Barr virus in 1/20 and 0/9 (5% and 0%) and feline papillomavirus in 1/20 and 0/9 samples (5% and 0% respectively). Felis catus papillomavirus-3 was found in 1 of 20 FOSCC samples. A virus was not associated consistently with FOSCC. If PVs have a role in FOSCC it is at most a supplementary or uncommon role. FOSCC appears most closely related to HPV-negative HNSCC. Future research on FOSCC should focus on identifying genetic and environmental causes.

The gut microbiomes of rainbow trout (Oncorhynchus mykiss) reared at 16° and 22 °C were determined using formalin-fixed paraffin-embedded tissues (FFPE) and compared to fresh frozen tissue. The data revealed microbiomes could be successfully determined using FFPE tissue opening a new horizon in studying intestinal microbiota using archived histological samples.

Analysis of somatic mutations in solid tumors and hematologic malignancies using targeted next generation sequencing (NGS)-based assays has become part of routine oncology practice as well as clinical trials. The use of paired tumor-normal DNA samples increases confidence of somatic calls. NGS assays that utilize unique patient identifiers (SNP IDs) allow further comparison of samples within a run or paired tumor/normal samples. The sources of germline DNA include peripheral blood (PB) and formalin-fixed paraffin-embedded tissue (FFPE). However, the source of normal can be problematic, especially in transplant setting. Herein, we report two cases of NGS-based molecular testing in a patient with mycosis fungoides treated with stem cell transplant [SCT] (Pt1) and a patient with lung adenocarcinoma who previously had acute leukemia cured by SCT. These cases highlight the importance of selecting an appropriate normal sample for excluding germline polymorphisms during somatic mutation testing. Initial analyses that included concurrent PB sample failed to filter known germline polymorphisms. Repeat analyses using pre-transplant PB/bone marrow allowed for the successful subtraction of germline variants. Somatic mutations in PTEN and ERBB4 (Pt1) and CDKN2A, KRAS, KDR, and TP53 (Pt2) were reported with confidence. Selection of an appropriate source of germline DNA for NGS-based somatic mutation testing for patients with SCT transplant can be challenging. Particular attention to the clinical history is crucial for accurate interpretation and reporting.

Cytological examination of urine is the most widely used noninvasive pathologic screen for bladder urothelial carcinoma (BLCA); however, inadequate diagnostic accuracy remains a major challenge. We performed mass spectrometry-based proteomic analysis of urine samples of ten patients with BLCA and ten paired patients with benign urothelial lesion (BUL) to identify ancillary proteomic markers for use in liquid-based cytology (LBC). A total of 4,839 proteins were identified and 112 proteins were confirmed as expressed at significantly different levels between the two groups. We also performed an independent proteomic profiling of tumor tissue samples where we identified 7,916 proteins of which 758 were differentially expressed. Cross-platform comparisons of these data with comparative mRNA expression profiles from The Cancer Genome Atlas identified four putative candidate proteins, AHNAK, EPPK1, MYH14 and OLFM4. To determine their immunocytochemical expression levels in LBC, we examined protein expression data from The Human Protein Atlas and in-house FFPE samples. We further investigated the expression of the four candidate proteins in urine cytology samples from two independent validation cohorts. These analyses revealed AHNAK as a unique intracellular protein differing in immunohistochemical expression and subcellular localization between tumor and non-tumor cells. In conclusion, this study identified a new biomarker, AHNAK, applicable to discrimination between BLCA and BUL by LBC. To our knowledge, the present study provides the first identification of a clinical biomarker for LBC based on in-depth proteomics.

Adenovirus E1B 55-kilodalton (E1B-55K) mediated DAXX degradation represents a potential mechanism by which E1B-55K sensitizes cancer cells to chemotherapy. Here we report the effects of E1B-55K-mediated DAXX degradation in chemoresistant ovarian cancer cells on response to chemotherapy. Cells with E1B-55K expression were more sensitive to cisplatin than cells without E1B-55K expression. In vivo C13* xenograft studies showed that the combination of cisplatin and E1B-55K was markedly more effective to slow tumor growth and to confer prolonged survival of tumor-bearing mice than either cisplatin or E1B-55K alone. Our studies show that DAXX plays an important role in cisplatin resistance in ovarian cancer, and strategies that promote DAXX degradation such as E1B-55K expression in combination with cisplatin can overcome drug resistance and improve responses to standard chemotherapy. These results also indicate that E1B-55K might be a novel agent for enhancing treatment responses for cisplatin-resistant ovarian cancer.

Purpose Droplet digital PCR (ddPCR) is a highly accurate method to determine DNA concentration and detect copy number variations. We developed an approach to assess HER2 gene amplification status using ddPCR with two sequences of TFF3 as reference probes. Experimental design. 76 templates of carcinoma DNA were prepared from formalin-fixed paraffin-embedded (FFPE) tissues. Digital PCR assay of the copy number of HER2 and TFF3 DNA was performed on the samples. The results were compared to prior fluorescent in-situ hybridation (FISH) assays performed on the same samples. Results The ddPCR assay had high concordance with the conventionally used immunohistochemistry (IHC) and FISH methods. The ddPCR method returned fewer indeterminate results than IHC. Concordance between a ddPCR plus FISH method and IHC plus FISH can rise to 98.7% (75/76) after validation is carried out. Conclusion It's potentially possible to improve the sensitivity and specifity of HER2 ddPCR assays using reference sequences not co-localized with HER2 on chromosome 17, and combining results from multiple sequences. Adopting an approach based on ddPCR HER2 assays plus FISH could lead to reduced costs, labour, and time consumption compared to current IHC plus FISH standard, while not losing precision.

Immunohistochemistry (IHC) provides clinically useful information on protein expression in cancer cells. However, quantification of colocalizing signals using conventional IHC and visual scores is challenging. Here we describe the application of quantitative immunofluorescence in angioimmunoblastic T-cell lymphoma (AITL), a peripheral T-cell lymphoma characterized by cellular heterogeneity that impedes IHC interpretation and quantification. A multiplexed immunofluorescence (IF) panel comprising T- and B-lymphocyte markers along with T-follicular helper (TFH) markers was validated for appropriate cellular localization in sections of benign tonsillar tissue and tested in two samples of AITL, using a Vectra microscope for spectral imaging and InForm software for analysis. We measured the percentage positivity of the TFH markers, BCL6 and PD1, in AITL CD4-positive cells to be approximately 26% and 45%, with 12% coexpressing both markers. The pattern is similar to CD4 cells within the germinal center of normal tonsils and clearly distinct from extragerminal CD4 cells. This study demonstrates the feasibility of automated and quantitative imaging of a multiplexed panel of cellular markers in formalin-fixed, paraffin-embedded tissue sections of a cellularly heterogenous lymphoma. Multiplexed IF allows the simultaneous scoring of markers in malignant and immune cell populations and could potentially increase accuracy for establishment of diagnostic thresholds.

More than a century ago in 1893, a revolutionary idea about fixing biological tissue specimens was introduced by Ferdinand Blum, a German physician. Since then, a plethora of fixation methods have been investigated and used. Formalin fixation with paraffin embedment became the most widely used types of fixation and preservation method, due to its proper architectural conservation of tissue structures and cellular shape. The huge collection of formalin-fixed, paraffin-embedded (FFPE) sample archives worldwide holds a large amount of unearthed information about diseases that could be the Holy Grail in contemporary biomarker research utilizing analytical omics based molecular diagnostics. The aim of this review is to critically evaluate the omics options for FFPE tissue sample analysis in the molecular diagnostics field.

The introduction of next generation sequencing (NGS) in the routine diagnostic setting is still in the development phase and has been limited by its complexity. Targeted NGS offers an attractive alternative to performing multiple single target assays and is very useful in meeting the increasing clinical demand for testing of multiple genetic aberrations in cancer specimens. To this end, we carried out a blinded validation study on 113 tumours in a diagnostic laboratory and compared mutation results from targeted NGS with those from Sanger sequencing, pyrosequencing, competitive allele specific TaqMan polymerase chain reaction (CAST PCR) and Cobas assays. DNA was extracted from formalin fixed, paraffin embedded (FFPE) tissue samples that included core biopsies, resections and cytology samples from three common and one rare cancer types [non-small cell lung cancer (NSCLC), colorectal cancer (CRC), malignant melanoma (MM) and gastrointestinal stromal tumour (GIST)]. Libraries were prepared using the TruSight Tumour 26 gene panel and NGS was carried out on the MiSeq instrument. Results from NGS were concordant with the mutational status determined by other platforms in 107 of the 113 cases tested (94.7%). The sequencing quality for NGS failed in four of the six false negative cases, while a further two samples gave false negative results because the c-KIT mutations were located outside the range of the NGS panel. One NSCLC sample contained an EGFR mutation previously detected by the Cobas assay. Reanalysis of the NGS data for this sample using a cut-off allele frequency of 1% revealed the mutation had an allele frequency of 2%, which was below the recommended software-determined threshold of 3%. NGS detected 113 additional mutations that were not previously known from analysis by the conventional methods. Twenty-six of these have known clinical importance, 37 have potential clinical significance, while 50 were novel mutations with unknown clinical significance. NGS detected variants using inputs of 10-20 ng of FFPE extracted DNA and from specimens with a tumour cell content less than 50%, for which when possible we recommend microdissection. We conclude that results from targeted NGS are highly concordant with those from other mutation testing platforms. Targeted NGS is suitable for a range of sample types received in the diagnostic pathology laboratory, including those with limited material or with low tumour cell content (TCC). This work has allowed us to determine the quality parameter settings required in order to obtain robust mutation data by NGS.

Objective Next Generation Sequencing (NGS) has become a useful tool for gene mutation testing which is required for targeted therapies. The aim of this study was to validate the GeneRead QIAact Actionable Insights Tumor Panel (Qiagen) on the GeneReader System in a diagnostic laboratory setting. Methods The GeneRead QIAact Actionable Insights Tumor Panel allows the analysis of 773 variant positions in 12 genes (ALK, BRAF, EGFR, ERBB2, ERBB3, ESR1, KIT, KRAS, NRAS, PDGFRA, PIK3CA and RAF1). For the validation of the panel we used a commercial available multiplex reference standard carrying 11 mutations in defined positions, samples from interlaboratory tests, and FFPE tumor samples from patients which were tested previously for mutations in KRAS, NRAS, BRAF, EGFR, KIT, and/or PDGFRA with pyrosequencing. Results Among the 122 tested samples, 121 samples (99.2%) were successfully sequenced. The sensitivity and specificity for detecting variants was 100% and results proved to be reproducible and precise. 119 (98.3%) results were concordant to the expected results. The differences between NGS and pyrosequencing observed in two samples were due to a wrong analysis by the pyrosequencing software which did not cover the present mutations. Conclusion Overall, the GeneRead QIAact Actionable Insights Tumor Panel was specific and sensitive for mutation analysis for targeted therapies and can be incorporated into laboratory diagnostics' daily practice.

Targeted application of next-generation sequencing (NGS) technology allows detection of specific mutations that can provide treatment opportunities for cancer patients. We evaluated the applicability of the Ion AmpliSeq Cancer Hotspot Panel V2 (CHV2) using formalin-fixed, paraffin-embedded (FFPE) tissue of clinical specimens. Thirty-five FFPE tumour samples with known mutational status were collected from four different hospitals and sequenced with CHV2 using an Ion Chef System and Ion S5 XL system. Out of 35 cases, seven were sequenced with Oncomine focus Assay Panel for comparison. For the limit of detection test, we used an FFPE reference standard, a cell line that included an engineered 50% EGFR T790 M in an RKO cell line background. Coverage analysis results including number of mapped reads, on target percent, mean depth, and uniformity were not different according to hospitals. Sensitivity for mutation detection down to 3% was demonstrated. NGS results showed 100% concordance with the results from single molecular pathology tests Assay in 30 cases with 24 known positive mutations and 14 known negative mutations, and another NGS panel of the Oncomine focus in seven cases. The CHV2 NGS test for solid tumours using Ion chef system and S5 XL system in clinical molecular pathology laboratories for analysis of solid tumours could be routinely used and could replace some single molecular pathology tests after a stringent and thorough validation process.

Fourier-Transform Infrared micro-spectroscopy is an excellent method for biological analyses. In this paper, series metal coating films on ITO glass were prepared by the electrochemical method and the different thicknesses of paraffin embedding rat's brain tissue on the substrates were studied by IR micro-spetroscopy in attenuated total reflection (ATR) mode and transflection mode respectively. The Co-Ni-Cu alloy coating film with low cost is good reflection substrates for the IR analysis. The infrared microscopic transflection mode needs not to touch the sample at all and can get the IR spectra with higher signal to noise ratios. The Paraffin-embedding method allows tissues to be stored for a long time for re-analysis to ensure the traceability of the sample. Also it isolates the sample from the metal and avoids the interaction of biological tissue with the metals. The best thickness of the tissues is 4 μm.

Discussion of liver antibody-mediated rejection during the 2011, 2013, and 2015 Banff liver sessions raised concerns over reliability of complement fragment 4d (C4d) staining, precipitating a global survey followed by a tissue microarray staining quality assessment study among centers on formalin-fixed, paraffin-embedded tissue. Tissue microarray sections containing tissue plugs of resected native and allograft (with acute antibody-mediated rejection) liver, heart, and kidney (n = 33 total cores) were sent to 31 centers for C4d staining using local method(s) and pathologist scoring. Digital whole-slide images (n = 40) were then semiquantitatively scored by 7 experts for background, distribution, and intensity of portal vein and capillary, hepatic artery, sinusoidal, and central vein endothelia and portal and central stromal staining. Results showed that strong and diffuse portal vein and capillary C4d staining, as determined by both local and central pathologists, clearly distinguished allografts showing acute antibody-mediated rejection from native livers and from those with evidence of weaker donor-specific antibody. Downstream vascular endothelial cell C4d staining and assessment were more variable and difficult to identify. C4d staining in the majority of laboratories reliably detects acute liver allograft antibody-mediated rejection in formalin-fixed, paraffin-embedded tissues. Assessment should focus on portal veins and capillaries, sinusoids, and central veins present in peripheral core needle biopsies. C4d staining in one organ does not always translate to staining in another.

Targeted next-generation sequencing panels are increasingly used to assess the value of gene mutations for clinical diagnostic purposes. For assay development, amplicon-based methods have been preferentially used on the basis of short preparation time and small DNA input amounts. However, capture sequencing has emerged as an alternative approach because of high testing accuracy. We compared capture hybridization and amplicon sequencing approaches using fresh-frozen and formalin-fixed, paraffin-embedded tumor samples from eight lymphoma patients. Next, we developed a targeted sequencing pipeline using a 32-gene panel for accurate detection of actionable mutations in formalin-fixed, paraffin-embedded tumor samples of the most common lymphocytic malignancies: chronic lymphocytic leukemia, diffuse large B-cell lymphoma, and follicular lymphoma. We show that hybrid capture is superior to amplicon sequencing by providing deep more uniform coverage and yielding higher sensitivity for variant calling. Sanger sequencing of 588 variants identified specificity limits of thresholds for mutation calling, and orthogonal validation on 66 cases indicated 93% concordance with whole-genome sequencing. The developed pipeline and assay identified at least one actionable mutation in 91% of tumors from 219 lymphoma patients and revealed subtype-specific mutation patterns and frequencies consistent with the literature. This pipeline is an accurate and sensitive method for identifying actionable gene mutations in routinely acquired biopsy materials, suggesting further assessment of capture-based assays in the context of personalized lymphoma management.

Summary Melanoma patients with BRAF V600E and V600K mutations show complete or partial response to vemurafenib. Detection assays often scan for the common V600E mutation rather than the rare V600K variant, although this mutation can be found in a high proportion of melanoma patients in the South Pacific. Herein, we describe a BRAF high resolution melting (HRM) assay that can differentiate low level of V600E and V600K mutations using formalin fixed, paraffin embedded (FFPE) reference standards for assay validation. The assay is based on the competitive amplification of differentially melting amplicons (CADMA principle) and has a limit of detection of 0.8% mutant allele for V600K and 1.4% mutant allele for V600E. A differentiation between the two mutations based on the melting profile is possible even at low mutation level. Sixty FFPE specimens were scanned and mutations could be scored correctly as confirmed by castPCR. In summary, the developed HRM assay is suitable for detection of V600K and V600E mutations and proved to be reliable and cost effective in a diagnostic environment.

Objective Endometrioid (ENOC) and clear cell ovarian carcinoma (CCOC) share a common precursor lesion, endometriosis, hence the designation endometriosis associated ovarian cancers (EAOC). Long interspersed nuclear element 1 (LINE-1 or L1), is a family of mobile genetic elements activated in many cancers capable of moving neighboring DNA through 3′ transductions. Here we investigated the involvement of specific L1-mediated transductions in EAOCs. Methods Through whole genome sequencing, we identified active L1-mediated transductions originating within the TTC28 gene in 34% (10/29) of ENOC and 31% (11/35) of CCOC cases. We used PCR and capillary sequencing to assess the presence of specific TTC28 -L1 transductions in formalin-fixed paraffin-embedded (FFPE) blocks from six different anatomical sites (five tumors and one normal control) for four ENOC and three CCOC cases, and compared the results to the presence of single nucleotide variations (SNVs)/frame shift (fs) mutations detected using multiplex PCR and next generation sequencing. Results TTC28 -L1 mediated transductions were identified in at least three tumor samplings in all cases, and were present in all five tumor samplings in 5/7 (71%) cases. In these cases, KRAS , PIK3CA , CTNNB1 , ARID1A , and PTEN mutations were found across all tumor sites while other selected SNV/fs mutations of unknown significance were present at varying allelic frequencies. Conclusion The TTC28- L1 transductions along with classical driver mutations were near ubiquitous across the tumors, suggesting that L1 activation likely occurred early in the development of EAOCs. TTC28- L1 transductions could potentially be used to determine clonal relationships and to track ovarian cancer progression.