scholarly journals A Pilot Study of Rare Renal Amyloidosis Based on FFPE Proteomics

Molecules ◽  
2021 ◽  
Vol 26 (23) ◽  
pp. 7234
Author(s):  
Shuang Meng ◽  
Wenwen Xia ◽  
Li Xia ◽  
Li Zhou ◽  
Jing Xu ◽  
...  
Keyword(s):  
Pilot Study ◽  
Bioinformatics Analysis ◽  
Great Promise ◽  
Individual Treatment ◽  
Renal Amyloidosis ◽  
Tissue Samples ◽  
Retrospective Diagnosis ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
End Stage

Renal amyloidosis typically manifests albuminuria, nephrotic-range proteinuria, and ultimately progresses to end-stage renal failure if diagnosed late. Different types of renal amyloidosis have completely different treatments and outcomes. Therefore, amyloidosis typing is essential for disease prognosis, genetic counseling and treatment. Thirty-six distinct proteins currently known to cause amyloidosis that have been described as amyloidogenic precursors, immunohistochemistry (IHC) or immunofluorescence (IF), can be challenging for amyloidosis typing especially in rare or hereditary amyloidosis in clinical practice. We made a pilot study that optimized the proteomics pre-processing procedures for trace renal amyloidosis formalin-fixed paraffin-embedded (FFPE) tissue samples, combined with statistical and bioinformatics analysis to screen out the amyloidosis-related proteins to accurately type or subtype renal amyloidosis in order to achieve individual treatment. A sensitive, specific and reliable FFPE-based proteomics analysis for trace sample manipulation was developed for amyloidosis typing. Our results not only underlined the great promise of traditional proteomics and bioinformatics analysis using FFPE tissues for amyloidosis typing, but also proved that retrospective diagnosis and analysis of previous cases laid a solid foundation for personalized treatment.

scholarly journals Multi-Omics and Informatics Analysis of FFPE Tissues Derived from Melanoma Patients with Long/Short Responses to Anti-PD1 Therapy Reveals Pathways of Response

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3515
Author(s):  
Saurabh K. Garg ◽  
Eric A. Welsh ◽  
Bin Fang ◽  
Yuliana I. Hernandez ◽  
Trevor Rose ◽  
...  
Keyword(s):  
Antigen Processing ◽  
Expression Profiles ◽  
Stage Iii ◽  
Poor Responders ◽  
Tissue Samples ◽  
Immune Therapies ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Melanoma Patients ◽  
Pathway Analyses

Anti-PD-1 based immune therapies are thought to be dependent on antigen processing and presentation mechanisms. To characterize the immune-dependent mechanisms that predispose stage III/IV melanoma patients to respond to anti-PD-1 therapies, we performed a multi-omics study consisting of expression proteomics and targeted immune-oncology-based mRNA sequencing. Formalin-fixed paraffin-embedded tissue samples were obtained from stage III/IV patients with melanoma prior to anti-PD-1 therapy. The patients were first stratified into poor and good responders based on whether their tumors had or had not progressed while on anti-PD-1 therapy for 1 year. We identified 263 protein/gene candidates that displayed differential expression, of which 223 were identified via proteomics and 40 via targeted-mRNA analyses. The downstream analyses of expression profiles using MetaCore software demonstrated an enrichment of immune system pathways involved in antigen processing/presentation and cytokine production/signaling. Pathway analyses showed interferon (IFN)-γ-mediated signaling via NF-κB and JAK/STAT pathways to affect immune processes in a cell-specific manner and to interact with the inducible nitric oxide synthase. We review these findings within the context of available literature on the efficacy of anti-PD-1 therapy. The comparison of good and poor responders, using efficacy of PD-1-based therapy at 1 year, elucidated the role of antigen presentation in mediating response or resistance to anti-PD-1 blockade.

scholarly journals Molecular Markers for Prostate Cancer in Formalin-Fixed Paraffin-Embedded Tissues

2013 ◽  
Vol 2013 ◽  
pp. 1-15 ◽  
Author(s):  
Tamara Sequeiros ◽  
Marta García ◽  
Melania Montes ◽  
Mireia Oliván ◽  
Marina Rigau ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Molecular Markers ◽  
Tumor Grade ◽  
Developed Countries ◽  
Response To Therapy ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Prostate cancer (PCa) is the most frequently diagnosed type of cancer in developed countries. The decisive method of diagnosis is based on the results of biopsies, morphologically evaluated to determine the presence or absence of cancer. Although this approach leads to a confident diagnosis in most cases, it can be improved by using the molecular markers present in the tissue. Both miRNAs and proteins are considered excellent candidates for biomarkers in formalin-fixed paraffin-embedded (FFPE) tissues, due to their stability over long periods of time. In the last few years, a concerted effort has been made to develop the necessary tools for their reliable measurement in these types of samples. Furthermore, the use of these kinds of markers may also help in establishing tumor grade and aggressiveness, as well as predicting the possible outcomes in each particular case for the different treatments available. This would aid clinicians in the decision-making process. In this review, we attempt to summarize and discuss the potential use of microRNA and protein profiles in FFPE tissue samples as markers to better predict PCa diagnosis, progression, and response to therapy.

scholarly journals Early upregulation of AR and steroidogenesis enzyme expression after 3 months of androgen-deprivation therapy.

2020 ◽  
Author(s):  
Agus Rizal A.H. Hamid ◽  
Harun Kusuma Putra ◽  
Ningrum Paramita Sari ◽  
Putri Diana ◽  
Saras Serani Sesari ◽  
...  
Keyword(s):  
Gene Expression ◽  
Protein Expression ◽  
Androgen Deprivation Therapy ◽  
Androgen Deprivation ◽  
Relative Gene Expression ◽  
Steroidogenic Enzymes ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Relative Gene

Abstract Background: Androgen deprivation therapy (ADT) is a standard treatment for advanced prostate cancer (PCa). However, PCa recurrence and progression rates during ADT are high. Until now, there has been no evidence regarding when progression begins. This study evaluated the gene expression of intraprostatic androgen receptor (AR) and steroidogenic enzymes in the early stages of ADT. Methods: Prostate tissue samples were taken from PCa patients with urinary retention who received ADT (ADT-PCa; n=10) and were further subgrouped into ADT ≤12 months (n=4) and ADT >12 months (n=6). The ADT-PCa tissues were then compared with BPH (n=12) and primary (no treatment) PCa tissues (n=16). mRNA for gene expression analysis of AR and steroidogenic enzymes was extracted from formalin-fixed paraffin embedded (FFPE) tissues and analyzed by real-time PCR. Protein expression was evaluated by immunohistochemistry with specific antibodies. Results: AR gene expression was higher in the ADT-PCa group than in the BPH or primary PCa group. Both the ADT ≤12 and > 12 months subgroups had significantly higher relative gene expression levels of AR (p<0.01 and 0.03, respectively) than the primary PCa group. In the ADT-PCa group, AR protein expression showed an increasing trend in the ADT ≤12 months subgroup and was significantly elevated in the ADT >12 months subgroup compared with the PCa group (100%; p <0.01). Half (50%) of the patients in the ADT ≤12 months subgroup were found to have upregulation of AR, and one showed upregulation beginning at 3 months of ADT. A trend toward elevated relative gene expression of SRD5A3 was also apparent in the ADT groups. Conclusion: AR and steroidogenic enzymes are upregulated in ADT-PCa patients as early as 3 months, without PSA elevation. Steroidogenic enzymes, particularly SRD5A3, were also upregulated before PSA rose.

scholarly journals Methods for restoration of ki67 antigenicity in aged paraffin tissue blocks

2021 ◽  
Author(s):  
Federica Grillo ◽  
Michela Campora ◽  
Simona Pigozzi ◽  
Silvia Bonadio ◽  
Luca Valle ◽  
...  
Keyword(s):  
Central Area ◽  
Antigen Retrieval ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Prolonged Heat ◽  
Paraffin Tissue ◽  
Visual Colour ◽  
Formalin Fixed

AbstractPathology archives are a treasure trove of paraffin embedded tissue spanning many years and covering a wide variety of tissues and diseases. The possibility of using old archival formalin fixed paraffin embedded (FFPE) tissues for diagnostic updates and research projects is a widespread need and it requires archives of stable, well-preserved samples. Immunohistochemistry performed on old archival paraffin blocks may give unreliable results, in particular for some antigens, such as Ki67. In consideration of this phenomenon, our aim is to comprehensively test and identify methods which may be used to obtain Ki67 immunohistochemical reactions of good quality from old archival FFPE blocks. Various methods were tested in order to evaluate their possible efficacy in increasing Ki67 immunointensity in a collection of 40-year-old, archival blocks including re-embedding, with deeper sectioning of tissue from the block and increasing heat-based pretreatment times (20 cases) and re-processing (20 cases). All reactions were performed using an automated immunostainer and Ki67 stained immunosections compared using a visual colour-based scale (the first immunostained section was considered as baseline). The combination of deep sectioning (1000 µM) and prolonged heat-based pretreatment (64 min) markedly increased immunoreactivity for Ki67. Re-embedding and reprocessing did not have a significant effect. Large tissue samples showed heterogeneity of Ki67 immunoexpression between the periphery of the sample and the central area. In conclusion, the study defines a useful protocol to increase antigen retrieval applicable to dated archival tissues.

scholarly journals Z Probe, an Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues

2018 ◽  
Author(s):  
Manish Tripathi ◽  
Chidi Zacheaus ◽  
Kyle Doxtater ◽  
Fatemeh Keramatnia ◽  
Lani Gao ◽  
...  
Keyword(s):  
Tissue Samples ◽  
Protein Coding ◽  
Dna And Rna ◽  
Non Coding Rna ◽  
Formalin Fixed Paraffin ◽  
Lncrna Malat1 ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Single Transcript ◽  
Long Non Coding Rna

Formalin-Fixed Paraffin Embedded (FFPE) tissues are a valuable resource in studying different markers and mechanistic molecules (protein, DNA and RNA) in order to understand the etiology of different cancers as well as many other diseases. Degradation and modification of RNA is the major challenge in utilizing FFPE tissue samples in medical research. Recently, non-protein coding transcripts long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. There is no validated method except qRTPCR or RNAseq to evaluate and study lncRNA expression. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to identify, localize and quantitate lncRNA in FFPE tissues. This assay is sensitive to single transcript and localizes lncRNA in individual cells within tumor. We have characterized a tumor suppressor lncRNA-NRON (non coding repressor of NFAT), which is scarcely expressed, a moderately expressed oncogeneic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. MALAT1 expression increased with the progression of the stage in colorectal cancer and invasiveness in breast cancer.

scholarly journals Comparison between immunofluorescence and immunohistochemistry for Listeria monocytogenes detection in formalin-fixed paraffin-embedded tissues

2022 ◽  
Vol 52 (3) ◽  
Author(s):  
Kelen Regina Ascoli Baldi ◽  
Jéssica Line Farias de Lima ◽  
Isabela Gimenes da Silva ◽  
Fernanda Felicetti Perosa ◽  
Ricardo Evandro Mendes ◽  
...  
Keyword(s):  
Listeria Monocytogenes ◽  
Primary Antibody ◽  
Economic Losses ◽  
Secondary Antibody ◽  
Tissue Samples ◽  
Formalin Fixed Paraffin ◽  
Rabbit Igg ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

ABSTRACT: Listeria monocytogenes is a bacterium that infect humans and animals and causes a zoonotic disease characterized by encephalitis, septicemia or abortion. In addition, listeriosis leads to significant economic losses due to animal death and sacrifice. This research compared the technique of immunofluorescence (IF) and immunohistochemistry (IHC) for the diagnosis of L. monocytogenes in formalin-fixed and paraffin-embedded (FFPE) tissues. A total of 30 tissue blocks from 15 animals with history and/or lesions compatible with listeriosis were selected. For both IHC and IF, the same diluted (1:200) polyclonal primary antibody was used against L. monocytogenes serotypes 1 and 4. For IHC, a polymer secondary antibody conjugated to peroxidase (HRP) was used. For IF, samples were incubated with a fluorescein-labeled anti-rabbit IgG secondary antibody. Each sample was classified according to the presence and percentage of immunolabeling area. From 30 samples, 10 were positive at least for one technique, whereas eight samples were positive for both IHC and IF with similar score. There was strong immunolabeling in tissue samples from bovines experimentally infected with L. monocytogenes ATCC 7644, as well as in nervous tissues from naturally infected ruminants. Additionally, IF did not show any difference in sensitivity when compared to IHC. Using processed biological materials for IF, instead of fresh tissues, is a quite unique technique, since there are few protocols described. Therefore, this study demonstrated that both techniques are efficient to detect L. monocytogenes in FFPE tissues.

scholarly journals Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues

2018 ◽  
Vol 4 (3) ◽  
pp. 20 ◽  
Author(s):  
Manish Tripathi ◽  
Chidi Zacheaus ◽  
Kyle Doxtater ◽  
Fatemeh Keramatnia ◽  
Cuilan Gao ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Cell Types ◽  
Rna Degradation ◽  
Activated T Cells ◽  
Tissue Samples ◽  
Protein Coding ◽  
Non Coding Rna ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

Formalin-fixed paraffin embedded (FFPE) tissues are a valuable resource for biomarker discovery in order to understand the etiology of different cancers and many other diseases. Proteins are the biomarkers of interest with respect to FFPE tissues as RNA degradation is the major challenge in these tissue samples. Recently, non-protein coding transcripts, long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. RNA sequencing (RNA-seq) or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) are the only validated methods to evaluate and study lncRNA expression and neither of them provides visual representation as immunohistochemistry (IHC) provides for proteins. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to visually identify and quantify lncRNA in FFPE tissues. This assay is highly sensitive and identifies transcripts visible within different cell types and tumors. We have detected a scarcely expressed tumor suppressor lncRNA NRON (non-coding repressor of nuclear factor of activated T-cells (NFAT)), a moderately expressed oncogenic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript 1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. Additionally, we have observed an increase in MALAT1 expression in different stages of colorectal cancer.

scholarly journals Expression of miR-127, miR-154, and miR-183 in Medullary Thyroid Carcinoma Tumors

2021 ◽  
Author(s):  
Mahsa RAHMANI SAMANI ◽  
Marjan ZARIF-YEGANEH ◽  
Atefeh MEHRABI ◽  
Amir Nader EMAMI RAZAVI ◽  
Sara SHEIKHOLESLAMI ◽  
...  
Keyword(s):  
Thyroid Cancer ◽  
Rna Extraction ◽  
Control Group ◽  
Tissue Samples ◽  
Altered Expression ◽  
Medullary Thyroid ◽  
Formalin Fixed Paraffin ◽  
Tumor Tissues ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues

Background: Medullary thyroid cancer (MTC) accounts for 5%–10% of all thyroid cancers, but causes 13% of all thyroid cancer related deaths. MicroRNAs (miRs) have key functions in the development and progression of MTC. Altered expression of some miRs has been reported in many human cancers, including Thyroid cancer. Therefore, we aimed to analyze the expression of miR-154, miR-183 and miR-127 in MTC tumor tissues. Methods: In this case-control study, 15 MTC Formalin-fixed, paraffin-embedded (FFPE) tissue samples and 15 adjacent normal thyroid FFPE tissues, as a control group, were collected from Taleghani, and Loghman Hakim Hospitals, Tehran, Iran since 2005 till 2015. After RNA extraction and cDNA synthesis, the expression of miR127, miR-154 and miR-183 was measured by quantitative Real-Time Polymerase Chain Reaction (qRT-PCR). Results: Our data showed a significant increase in the expression of miR-127 in MTC samples in comparison with the control group (P<0.05). Although miR-154 and miR-183 expression levels had increase expression in MTC tumors, this change was not statistically significant. Conclusion: The miR-127 could be considered as a prognostic, diagnostic and therapeutic marker for the management of MTC, and it is proposed for further investigation to fully establish the role of this miRNA in MTC.

scholarly journals Application of the 3′ mRNA-Seq using unique molecular identifiers in highly degraded RNA derived from formalin-fixed, paraffin-embedded tissue

BMC Genomics ◽  
2021 ◽  
Vol 22 (1) ◽  
Author(s):  
Jin Sung Jang ◽  
Eileen Holicky ◽  
Julie Lau ◽  
Samantha McDonough ◽  
Mark Mutawe ◽  
...  
Keyword(s):  
Bias Correction ◽  
Exome Capture ◽  
Sequencing Data ◽  
Tissue Samples ◽  
Paired Samples ◽  
Pcr Bias ◽  
Formalin Fixed Paraffin ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

Abstract Background Archival formalin-fixed, paraffin-embedded (FFPE) tissue samples with clinical and histological data are a singularly valuable resource for developing new molecular biomarkers. However, transcriptome analysis remains challenging with standard mRNA-seq methods as FFPE derived-RNA samples are often highly modified and fragmented. The recently developed 3′ mRNA-seq method sequences the 3′ region of mRNA using unique molecular identifiers (UMI), thus generating gene expression data with minimal PCR bias. In this study, we evaluated the performance of 3′ mRNA-Seq using Lexogen QuantSeq 3′ mRNA-Seq Library Prep Kit FWD with UMI, comparing with TruSeq Stranded mRNA-Seq and RNA Exome Capture kit. The fresh-frozen (FF) and FFPE tissues yielded nucleotide sizes range from 13 to > 70% of DV200 values; input amounts ranged from 1 ng to 100 ng for validation. Results The total mapped reads of QuantSeq 3′ mRNA-Seq to the reference genome ranged from 99 to 74% across all samples. After PCR bias correction, 3 to 56% of total sequenced reads were retained. QuantSeq 3′ mRNA-Seq data showed highly reproducible data across replicates in Universal Human Reference RNA (UHR, R > 0.94) at input amounts from 1 ng to 100 ng, and FF and FFPE paired samples (R = 0.92) at 10 ng. Severely degraded FFPE RNA with ≤30% of DV200 value showed good concordance (R > 0.87) with 100 ng input. A moderate correlation was observed when directly comparing QuantSeq 3′ mRNA-Seq data with TruSeq Stranded mRNA-Seq (R = 0.78) and RNA Exome Capture data (R > 0.67). Conclusion In this study, QuantSeq 3′ mRNA-Seq with PCR bias correction using UMI is shown to be a suitable method for gene quantification in both FF and FFPE RNAs. 3′ mRNA-Seq with UMI may be applied to severely degraded RNA from FFPE tissues generating high-quality sequencing data.

scholarly journals Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

2012 ◽  
Vol 32 (8) ◽  
pp. 715-720 ◽  
Author(s):  
Paula R. Almeida ◽  
Caroline P. Andrade ◽  
Laura L. Almeida ◽  
Luiz G.S. Oliveira ◽  
Luiza A. Castro ◽  
...  
Keyword(s):  
Dna Amplification ◽  
Mycoplasma Hyopneumoniae ◽  
Ffpe Tissue ◽  
Tissue Samples ◽  
Histological Features ◽  
Frozen Tissue ◽  
Ffpe Samples ◽  
Formalin Fixed Paraffin Embedded ◽  
Ffpe Tissues ◽  
Formalin Fixed

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

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