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Pinto-Ribeiro, Ines, Rui M. Ferreira, Joana Pereira-Marques, Vanessa Pinto, Guilherme Macedo, Fátima Carneiro, and Ceu Figueiredo. "Evaluation of the Use of Formalin-Fixed and Paraffin-Embedded Archive Gastric Tissues for Microbiota Characterization Using Next-Generation Sequencing." International Journal of Molecular Sciences 21, no. 3 (February 7, 2020): 1096. http://dx.doi.org/10.3390/ijms21031096.
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Large numbers of well-characterized clinical samples are fundamental to establish relevant associations between the microbiota and disease. Formalin-fixed and paraffin-embedded (FFPE) tissues are routinely used and are widely available clinical materials. Since current approaches to study the microbiota are based on next-generation sequencing (NGS) targeting the bacterial 16S rRNA gene, our aim was to evaluate the feasibility of FFPE gastric tissues for NGS-based microbiota characterization. Analysis of sequencing data revealed the presence of bacteria in the paraffin control. After the subtraction of the operational taxonomic units (OTUs) present in the paraffin control to the FFPE tissue sample dataset, we evaluated the microbiota profiles between paired FFPE and frozen gastric tissues, and between different times of archiving. Compared with frozen gastric tissues, we detected a lower number of OTUs in the microbiota of paired FFPE tissues, regardless of the time of archiving. No major differences in microbial diversity were identified, but taxonomic variation in the relative abundance of phyla and orders was observed between the two preservation methods. This variation was also evident in each case and throughout the times of FFPE archiving. The use of FFPE tissues for NGS-based microbiota characterization should be considered carefully, as biases can be introduced by the embedding process and the time of tissue archiving.
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Michelsen, Nete V., Klaus Brusgaard, Qihua Tan, Mads Thomassen, Khalid Hussain, and Henrik T. Christesen. "Investigation of Archived Formalin-Fixed Paraffin-Embedded Pancreatic Tissue with Whole-Genome Gene Expression Microarray." ISRN Pathology 2011 (December 26, 2011): 1–12. http://dx.doi.org/10.5402/2011/275102.
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The use of formalin-fixed, paraffin-embedded (FFPE) tissue overcomes the most prominent issues related to research on relatively rare diseases: limited sample size, availability of control tissue, and time frame. The use of FFPE pancreatic tissue in GEM may be especially challenging due to its very high amounts of ribonucleases compared to other tissues/organs. In choosing pancreatic tissue, we therefore indirectly address the applicability of other FFPE tissues to gene expression microarray (GEM). GEM was performed on archived, routinely fixed, FFPE pancreatic tissue from patients with congenital hyperinsulinism (CHI), insulinoma, and deceased age-appropriate neonates, using whole-genome arrays. Although ribonuclease-rich, we obtained biologically relevant and disease-specific, significant genes; cancer-related genes; genes involved in (a) the regulation of insulin secretion and synthesis, (b) amino acid metabolism, and (c) calcium ion homeostasis. These results should encourage future research and GEM studies on FFPE tissue from the invaluable biobanks available at the departments of pathology worldwide.
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Asmann, Yan W., Vivekananda Sarangi, Bruce W. Eckloff, Julie M. Cunningham, Samantha J. McDonough, Yeon K. Lee, Eric D. Wieben, et al. "Comparison Of Single Nucleotide Mutations (SNVs) and Copy Number Variants (CNVs) Detection In Formalin Fixed Paraffin Embedded (FFPE) and Paired Frozen Tumor Tissues Using Target Capture and Sequencing Approach." Blood 122, no. 21 (November 15, 2013): 1784. http://dx.doi.org/10.1182/blood.v122.21.1784.1784.
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Abstract Background Next Generation sequencing (NGS) is a powerful tool to identify somatic mutations associated with tumor onset and drug response. While it is well suited for high quality fresh/frozen samples, NGS is not proven for FFPE tissue which is the most common type of clinical specimen. Since the nucleic acids can be readily extracted from FFPE samples for a variety of genomic analyses, a comparative mutational analysis of paired frozen and FFPE tissues is urgently needed. Our long term goal is to establish a lab protocol to detect mutations in FFPE tumors using a targeted capture and sequencing approach for genes of interest. This pilot study focuses on the comparison of FFPE and frozen samples to test the validity of using FFPE tissues in such application. Methods Gene Selection: 128 genes associated with known pathogenic mutations in lymphoma Sample Selection: 9 diffuse large B-cell lymphoma (DLBCL) cases with FFPE, frozen and germline samples, as well as 10 frozen normal lymphatic tissues as references for CNV detections Capture Probe Design: We targeted coding exons and UTR, as well as the evolutionarily conserved intronic regions. The capture probes were designed using the Agilent eArray tool. The titling density of the probes was set to 3 probes overlapping with every base in the target region to improve the capture efficiency in FFPE samples. The least stringent masking of the repeat regions was allowed to include regions with small repeats that are shorter than the length of the sequencing reads (100-bp). In addition, boosting parameters were picked to set various levels of probe replication in different regions in order to minimize the local coverage differences (e.g. between regions of different GC contents) Sequencing and Bioinformatics: The target capture and sequencing were performed by the Mayo Clinic Medical Genome Facility. The reads were mapped to Human Reference Genome Build 37 using Novalign, and SNVs were called using GATK. The CNVs were identified using an in-house developed algorithm, patternCNV. Results The designed probes covered 99.65937% of the target regions. We generated 2.2-6.7 Gbp of reads per sample, 57.4-71.5% of which were on target. This equalled an average coverage of 2100-6700 folds which is 10-30 times higher than the minimal coverage recommended by Agilent. Due to this high coverage, we observed duplicate reads that accounted for 7.7-73.5% of the total reads. When we analysed the data with and without the duplicated reads, the concordance of the called SNVs was between 84-93% out of 207-249 mutated positions per trio-sample. There were 7.8-8.9% and 1.1-2.2% unique SNVs per sample by excluding or including duplicate reads, respectively. The dis-concordances were mostly missed calls, where a SNV was observed in only 1 or 2 of the trio samples. The missed calls from frozen samples ranged from 0-10.4% compared to 1.4-10.4% from the FFPE tissues, with 0.88-2.4% more SNVs missed in FFPE. Further analyses showed that all of the missing calls came from the lack of or low coverage of the corresponding positions. There were also differences of the called SNVs between the trio samples. However, this was extremely rare. Only 2 out of the 9 trio samples at a total of 3 positions had disagreements in called SNVs between FFPE and frozen tissues, all due to the allelic imbalance where the percentage of reads supporting the alternative alleles were below 20%. Therefore, this dis-concordance can be removed by back-filling of the read-level information for each position. Unfortunately only 11.9-47.4% of the CNVs called in frozen tissues were identified in FFPE samples, due to the widely various coverage in FFPE samples. The consequent large noises of the log ratio values between the FFPEs and normal references significantly reduced the sensitivity for CNV calling. Conclusions This pilot study compared the performance of SNV and CNV detection in FFPE and paired frozen tissues using a target capture and sequencing approach. With a capture probe design strategized to benefit FFPE samples, we observed SNV detection rates in FFPE that were only slightly lower (0.88-2.4%) than those of frozen tissues due to poor coverage of some positions in FFPE samples. With a proper back-filling step, there was no dis-concordance of the called SNVs between FFPE and frozen samples. However, CNV detections in FFPE were more problematic due to the un-predictable regional coverage in FFPE samples. Disclosures: No relevant conflicts of interest to declare.
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Norgan, Andrew P., Lynne M. Sloan, and Bobbi S. Pritt. "Detection of Naegleria fowleri, Acanthamoeba spp, and Balamuthia mandrillaris in Formalin-Fixed, Paraffin-Embedded Tissues by Real-Time Multiplex Polymerase Chain Reaction." American Journal of Clinical Pathology 152, no. 6 (August 15, 2019): 799–807. http://dx.doi.org/10.1093/ajcp/aqz103.
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Abstract Objectives Pathogenic free-living amebae (FLAs) cause skin, ocular, and central nervous system (CNS) infections with significant morbidity and mortality. Diagnosis of FLA infections by pathologic examination of tissue sections can be aided using molecular assays. This study investigated the performance characteristics of a multiplex real-time polymerase chain reaction (PCR) assay (FLA-PCR) for detection and differentiation of FLAs in clinical specimens. Methods FLA-PCR was performed on 39 human specimens comprising one cutaneous, 14 corneal, and 24 CNS formalin-fixed, paraffin-embedded (FFPE) tissues with a histopathologic diagnosis of FLA infection and four CNS FFPE tissues with inflammation but no evidence of FLAs. In addition, clinical specificity and assay limit of detection were determined. Results FLA detection sensitivities ranged from 79% to 84% in FFPE tissues. No cross-reactivity was observed. Conclusions While sensitivity is limited, FLA-PCR assay may serve as a useful adjunct for detection or confirmation of FLA infections in FFPE tissues.
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Chung, Joon-Yong, Till Braunschweig, Reginald Williams, Natalie Guerrero, Karl M. Hoffmann, Mijung Kwon, Young K. Song, Steven K. Libutti, and Stephen M. Hewitt. "Factors in Tissue Handling and Processing That Impact RNA Obtained From Formalin-fixed, Paraffin-embedded Tissue." Journal of Histochemistry & Cytochemistry 56, no. 11 (July 21, 2008): 1033–42. http://dx.doi.org/10.1369/jhc.2008.951863.
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Formalin-fixed, paraffin-embedded (FFPE) tissue is the most common specimen available for molecular assays on tissue after diagnostic histopathological examination. RNA from FFPE tissue suffers from strand breakage and cross-linking. Despite excellent extraction methods, RNA quality from FFPE material remains variable. To address the RNA quality factors within FFPE tissues, we studied RNA quality, isolating individual elements of the tissue fixation and processing including length of fixation in formalin and the type of buffer incorporated in the fixative. We examined the impact of the length of the tissue processing cycle as well. The optimal fixation period of 12-24 hr in phosphate-buffered formalin resulted in better-quality RNA. Longer tissue processing times were associated with higher quality RNA. We determined that the middle region of gene suffers less damage by these processes as shown by real-time quantitative RT-PCR. These data provide key information for the development of methods of analysis of gene expression in archival FFPE tissues and contribute to the establishment of objective standards for the processing and handling of tissue in surgical pathology. This manuscript contains online supplemental material at www.jhc.org . Please visit this article online to view these materials.
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Ghebeh, Hazem, Fatmah A. Mansour, Dilek Colak, Akram A. Alfuraydi, Amal A. Al-Thubiti, Dorota Monies, Monther Al-Alwan, Taher Al-Tweigeri, and Asma Tulbah. "Higher PD-L1 Immunohistochemical Detection Signal in Frozen Compared to Matched Paraffin-Embedded Formalin-Fixed Tissues." Antibodies 10, no. 3 (June 22, 2021): 24. http://dx.doi.org/10.3390/antib10030024.
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Purpose: Response to anti-PD-L1/PD-1 immunotherapy correlates with PD-L1 expression in breast cancer. However, the prevalence of PD-L1 positive breast cancer is variable, which could be due to differences in the population/cohort of patients tested or the preservation/detection technology used. To investigate this variability, we examined the effect of two tissue preservation methods on PD-L1 immunohistochemical detection in breast cancer. Methods: We compared PD-L1 expression in patient-matched frozen (FR) and formalin-fixed paraffin-embedded (FFPE) tissues of breast cancer patients. PD-L1 expression was assessed using tumor proportion score (TPS, simply PD-L1 score), and case positivity was determined with PD-L1 score ≥5. Results: In FFPE tissues, PD-L1 was positive in 7–10% of tested patients, depending on the antibody used. In patient-matched FR tissues, the same antibodies showed positive PD-L1 expression in 20–30% of cases. The impact of the antibody tested on the rate of PD-L1 positivity (% of PDL1 positive cases) was minor, as evident in the near perfect concordance between PD-L1 score obtained using the different antibodies whether tested in FR or FFPE tissues. However, there was a systematic drop by an average of 13–20% in the PD-L1 score obtained in FFPE tissues compared to their patient-matched FR tissues. Conclusions: In the tested patient-matched cohort, there was consistently a higher PD-L1 score in FR than FFPE tissues, regardless of the antibody used, demonstrating a significant effect on PD-L1 detection due to the preservation method. These findings should inspire further work to improve the sensitivity of PD-L1 detection and possibly search for more sensitive antibodies in FFPE tissues.
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Amemiya, Kei, Xiankun Zeng, Jeremy J. Bearss, Christopher K. Cote, Carl Soffler, Robert C. Bernhards, Jennifer L. Dankmeyer, et al. "Laser Scanning Confocal Microscopy Was Used to Validate the Presence of Burkholderia pseudomallei or B. mallei in Formalin-Fixed Paraffin Embedded Tissues." Tropical Medicine and Infectious Disease 5, no. 2 (April 29, 2020): 65. http://dx.doi.org/10.3390/tropicalmed5020065.
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Burkholderia pseudomallei and B. mallei are Gram-negative, facultative intracellular bacteria that cause melioidosis and glanders, respectively. Currently, there are no vaccines for these two diseases. Animal models have been developed to evaluate vaccines and therapeutics. Tissues from infected animals, however, must be fixed in formalin and embedded in paraffin (FFPE) before analysis. A brownish staining material in infected tissues that represents the exopolysaccharide of the pathogen was seen by bright field microscopy but not the actual microorganism. Because of these results, FFPE tissue was examined by laser scanning confocal microscopy (LSCM) in an attempt to see the microorganism. Archival FFPE tissues were examined from ten mice, and five nonhuman primates after exposure to B. pseudomallei or B. mallei by LSCM. Additionally, a historical spleen biopsy from a human suspected of exposure to B. mallei was examined. B. pseudomallei was seen in many of the infected tissues from mice. Four out of five nonhuman primates were positive for the pathogen. In the human sample, B. mallei was seen in pyogranulomas in the spleen biopsy. Thus, the presence of the pathogen was validated by LSCM in murine, nonhuman primate, and human FFPE tissues.
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Bao, Jian R., Richard B. Clark, Ronald N. Master, Kileen L. Shier, and Lynn L. Eklund. "Acid-fast bacterium detection and identification from paraffin-embedded tissues using a PCR-pyrosequencing method." Journal of Clinical Pathology 71, no. 2 (July 22, 2017): 148–53. http://dx.doi.org/10.1136/jclinpath-2016-204128.
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AimsAcid-fast bacterium (AFB) identification from formalin-fixed paraffin-embedded (FFPE) tissues is challenging and may not be readily available to the clinical laboratory. A method to detect and identify AFB from FFPE tissues using PCR and pyrosequencing (PCR-Seq) was developed and evaluated.MethodsThe method was validated using spiked cell-clotted paraffin blocks before use with patients’ specimens. DNA was extracted from tissue sections, and a 16S rRNA gene fragment was amplified and a signature sequence was produced on a PyroMark ID system. Sequences were aligned to established databases for AFB identification. Additional tissue sections were stained and examined for AFB.ResultsBoth sensitivity and specificity were 100% on spiked cell-clotted blocks without cross-reactivity with non-AFB. Of 302 FFPE tissues from patients, 116 (38%) were AFB-stain positive; 83 (72%) of these had AFB identified. The 21 AFB identified included Mycobacterium tuberculosis complex (14 cases), Mycobacterium leprae (3), Mycobacterium genavense (2), Mycobacterium marinum-ulcerans group (3) and 17 other AFB (61). Thirteen cases were AFB-stain indeterminate and 4 were positive by the PCR-Seq method. Of the AFB stain-negative cases, 167 were negative and 6 were positive by PCR-Seq.ConclusionsThe PCR-Seq method provided specific identification of various AFB species or complexes from FFPE tissues.
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Janecka-Widła, Anna, Agnieszka Adamczyk, Kaja Majchrzyk, Anna Cichocka, and Joanna Niemiec. "Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer." Diagnostyka Laboratoryjna 54, no. 3 (September 20, 2018): 145–50. http://dx.doi.org/10.5604/01.3001.0013.7699.
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Introduction: The chip-based protein separation using Agilent 2100 Bioanalyzer is a promising tool for proteomic analysis. However, it has not been defined if the above-mentioned device is suitable for analysis of formalin-fixed paraffin-embedded (FFPE) proteins extracts.<br>The aim of the study: Therefore we performed the analysis aimed at testing if extracts from FFPE tissues are suitable for proteins detection using Agilent 2100 Bioanalyzer.<br>Material and methods: FFPE tissues and cell cultures were used for experiments. Proteins were extracted from them using Qproteome FFPE Tissue Kit and RIPA buffer, respectively. For protein analysis Bioanalyzer Instrument, Western Blot and immunohistochemistry (IHC) were applied.<br>Results: In FFPE extracts, using Bioanalyzer we failed to detect β-actin. However, Western Blot analysis proved β-actin presence in them. One of possible explanations of this phenomenon might be lack of antibody-antigen interaction during immunoprecipitation preceding Bioanalyzer analysis. We suspected that, Extraction Buffer (from Qproteome FFPE Tissue Kit) or β-mercaptoethanol (both present in FFPE protein extracts) might be responsible for the above-mentioned blockade. Therefore, we applied IHC with detection of CD34 because, CD34 staining is robust against small methodological variations and this marker is always present in tumor tissue sections. Anti-CD34 antibody was diluted in Tris saline buffer with Extraction Buffer, β-mercaptoethanol or without. We did not observe CD34 immunopositivity only in the presence of Extraction Buffer.<br>Conclusion: Qproteome FFPE Tissue Kit is not suitable for protein analysis using Agilent 2100 Bioanalyzer, because the Extraction Buffer from the kit prevents an interaction between antibody and antigen during immunoprecipitation procedure.
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Hafy, Zen, Veny Larasati, Riana Sari Puspita, Novizar S, Haekal M, Rafdi A, and Sentani R. "Hubungan Lama Penyimpanan Sampel Arsip Jaringan Dalam Blok Parafin Terfiksasi Formalindengan Kualitas Hasil Ekstraksidna Mitokondria Jaringan." SRIWIJAYA JOURNAL OF MEDICINE 1, no. 3 (October 31, 2018): 157–62. http://dx.doi.org/10.32539/sjm.v1i3.31.
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Formalin-fixed paraffin-embedded (FFPE) archival tissue presents a readily available resource in molecular study nowadays. The quality of nucleid acid, especially mitoconhdrial DNA (mtDNA) extracted from FFPE tissue could be affected by the storage time. Thus, this study investigated if the FFPE tissue’s storage time had an effect on the quality of the extracted mtDNA at Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. DNA was extracted from 16 randomly selected archival FFPE tissues in Laboratory of Pathology Anatomy, RSUP dr. Mohammad Hoesin Palembang. The samples were grouped based on their storage time (less than 1 year and 1 to 5 yrs.’ old). The isolated DNA from each group was amplified using PCR with two primer pairs specifically designto amplify mtDNA of 320 bp and 142bp length, respectively. The PCR products were visualized by electrophoresismethod. None of the samples from both groups could be amplified witht the 320bp primers. However, the PCR result of the 149 bp primers showed positive for all of the samples from each study group. The study indicated that the storage time does not affect the quality of mtDNA isolated from the FFPE samples archived in Department of Pathology RSUP Dr. Mohammad Hoesin Palembang. Furthermore, the study showed that the mtDNA extracted from FFPE tissue has been degraded, therefore, the samples are not suitable for genetic studies require a long mtDNA amplicon.
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Maraschin, Bruna Jalfim, Viviane Palmeira da Silva, Leigha Rock, Huichen Sun, Fernanda Visioli, Pantelis Varvaki Rados, and Miriam P. Rosin. "Optimizing Fixation Protocols to Improve Molecular Analysis from FFPE Tissues." Brazilian Dental Journal 28, no. 1 (February 2017): 82–84. http://dx.doi.org/10.1590/0103-6440201701211.
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Abstract Most Departments of Pathology around the world have a considerable archive of formalin-fixed paraffin-embedded (FFPE) tissue suitable for molecular assessment. This article points out the potential DNA damage that may occur if basic steps are not followed during processing and storage of these samples. Furthermore, it hopes to establish parameters to optimize quality and quantity of DNA extracted from FFPE tissues.
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He, Helen, Yu Yang, Zhongmin Xiang, Lunyin Yu, Jouhara Chouitar, Jie Yu, Natalie Roy D’Amore, et al. "A Sensitive IHC Method for Monitoring Autophagy-Specific Markers in Human Tumor Xenografts." Journal of Biomarkers 2016 (May 10, 2016): 1–11. http://dx.doi.org/10.1155/2016/1274603.
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Objective. Use of tyramide signal amplification (TSA) to detect autophagy biomarkers in formalin fixed and paraffin embedded (FFPE) xenograft tissue. Materials and Methods. Autophagy marker regulation was studied in xenograft tissues using Amp HQ IHC and standard IHC methods. Results. The data demonstrate the feasibility of using high sensitivity TSA IHC assays to measure low abundant autophagy markers in FFPE xenograft tissue.
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Chu, Wei-Sing, Qi Liang, Yao Tang, Randy King, Kondi Wong, Maokai Gong, Minqi Wei, et al. "Ultrasound-accelerated Tissue Fixation/Processing Achieves Superior Morphology and Macromolecule Integrity with Storage Stability." Journal of Histochemistry & Cytochemistry 54, no. 5 (January 6, 2006): 503–13. http://dx.doi.org/10.1369/jhc.5a6802.2005.
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We demonstrate that high-frequency and high-intensity ultrasound (US) can be applied to both tissue fixation and tissue processing to complete the conventional overnight formalin-fixation and paraffin-embedding (FFPE) procedures within 1 hr. US-facilitated FFPE retains superior tissue morphology and long-term room temperature storage stability than conventional FFPE. There is less alteration of protein antigenicity after US-FFPE preservation so that rapid immunohistochemical reactions occur with higher sensitivity and intensity, reducing the need for antigen retrieval pretreatment. US-FFPE tissues present storage stability so that room temperature storage up to 7 years does not significantly affect tissue morphology, protein antigenic properties, RNA distribution, localization, and quantitation. In addition, during fixation, tissue displays physical changes that can be monitored and reflected as changes in transmission US signals. As far as we know, this is the first effort to monitor tissue physical changes during fixation. Further study of this phenomenon may provide a method to control and to monitor the level of fixation for quality controls. The mechanism of less alteration of protein antigenicity by US-FFPE was discussed. (J Histochem Cytochem 54:503-513, 2006)
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Markou, Athina, George M. Yousef, Efstathios Stathopoulos, Vassilis Georgoulias, and Evi Lianidou. "Prognostic Significance of Metastasis-Related MicroRNAs in Early Breast Cancer Patients with a Long Follow-up." Clinical Chemistry 60, no. 1 (January 1, 2014): 197–205. http://dx.doi.org/10.1373/clinchem.2013.210542.
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Abstract BACKGROUND Stability of microRNAs (miRNAs) in formalin-fixed paraffin-embedded (FFPE) tissues enables their reliable analysis in archived FFPE tissue samples, which are an invaluable source for the evaluation of novel biomarkers. Especially in breast cancer, for which late relapses occur in many cases, analysis of miRNAs in FFPE tissues holds great potential, because it can lead to the discovery of novel biomarkers suitable for future routine clinical diagnostics for breast cancer. We investigated the prognostic significance of 6 metastasis-related miRNAs that can critically regulate various stages of migration and invasion and play critical roles in the multistep metastatic process. METHODS We quantified the expression of 6 mature miRNAs (namely miR-21, miR-205, miR-10b, miR-210, miR-335, and let-7a) by reverse-transcription quantitative PCR in FFPE tissues of 84 patients with early breast cancer and a long follow-up and 13 cancer-free breast tissue FFPE samples that were used as the control group. We further correlated individual miRNA over- or underexpression with the disease-free interval (DFI) and overall survival (OS). RESULTS Univariate analysis revealed that both miR-21 and miR-205 were significantly associated with DFI and only miR-205 with OS. Multivariate analysis demonstrated that miR-205 and miR-21 were independent factors associated with early disease relapse, whereas only miR-205 overexpression was associated with OS. CONCLUSIONS Our results clearly indicate that deregulation of metastasis-associated miRNAs in primary tumors is associated with clinical outcome in patients with early breast cancer and can differentiate patients with higher risk in well-characterized subgroups.
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Shiogama, Kazuya, Ken-ichi Inada, Michinori Kohara, Hidemi Teramoto, Yasuyoshi Mizutani, Takanori Onouchi, and Yutaka Tsutsumi. "Demonstration of Hepatitis C Virus RNA withIn SituHybridization Employing a Locked Nucleic Acid Probe in Humanized Liver of Infected Chimeric Mice and in Needle-Biopsied Human Liver." International Journal of Hepatology 2013 (2013): 1–7. http://dx.doi.org/10.1155/2013/249535.
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Background.In situhybridization (ISH) with high sensitivity has been requested to demonstrate hepatitis C virus (HCV) RNA in formalin-fixed, paraffin-embedded (FFPE) sections of the liver.Methods. ISH employing a locked-nucleic-acid- (LNA-)modified oligonucleotide probe and biotin-free catalyzed signal amplification system (CSAII) was applied to HCV-RNA detection in the liver tissue. Nested reverse-transcription polymerase chain reaction (RT-PCR) was performed for HCV genotyping using total RNA extracted from FFPE sections. The target tissues included FFPE tissue sections of humanized livers in HCV-infected chimeric mice (HCV genotypes 1a, 1b, and 2a and noninfected) and of needle-biopsied livers from HCV-infected patients.Results. HCV-RNA was demonstrated with the ISH technique in HCV-infected liver tissues from both chimeric mice and 9 (82%) of 11 patients with HCV infection. The HCV signals were sensitive to RNase. Nested RT-PCR confirmed the genotype in 8 (73%) of 11 livers (type 1b: 6 lesions and type 2a: 2 lesions). HCV-RNA was not identified in chronic hepatitis B lesions, fatty liver, autoimmune hepatitis, and hepatocellular carcinoma.Conclusion. ISH using the LNA-modified oligonucleotide probe and CSAII was applicable to detecting HCV-RNA in routinely prepared FFPE liver specimens.
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Wakamatsu, Nobuko, Daniel J. King, Bruce S. Seal, and Corrie C. Brown. "Detection of Newcastle Disease Virus RNA by Reverse Transcription-Polymerase Chain Reaction Using Formalin-Fixed, Paraffin-Embedded Tissue and Comparison with Immunohistochemistry and in situ Hybridization." Journal of Veterinary Diagnostic Investigation 19, no. 4 (July 2007): 396–400. http://dx.doi.org/10.1177/104063870701900410.
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The usefulness of reverse transcription-polymerase chain reaction (RT-PCR) from formalin-fixed, paraffin-embedded (FFPE) tissues was examined and compared to the immunohistochemistry (IHC) and in situ hybridization (ISH) assays for detection of Newcastle disease virus (NDV). Spleen and lung tissues were collected from chickens experimentally infected with either of 2 NDV isolates: a low virulent virus (LaSota) and a virulent virus (from the 2002–2003 California outbreak). The tissues were harvested immediately postmortem and fixed in 10% neutral buffered formalin for approximately 52 hours. Also, just before euthanasia, oral and cloacal swabs were collected for virus isolation. RNA was obtained from the FFPE tissues by digestion with proteinase K and subsequent extraction with phenol, chloroform, and isoamyl alcohol. By seminested RT-PCR with primers for the NDV matrix gene, a 232-base pair (bp) product was generated and visualized by electrophoresis. The results of PCR were compared to those of IHC for viral nucleoprotein and ISH for matrix gene (850 bp) on 3-μm sections and to those of virus isolation from swabs. All samples from infected chickens were positive by RT-PCR, including samples that were negative by both IHC and ISH. The RT-PCR positives included tissue from chickens that were no longer shedding virus detectable by virus isolation. The RT-PCR was an effective and sensitive method to detect NDV in FFPE tissues. To the authorsn' knowledge, this is the first report of NDV detection in FFPE tissues as a diagnostic approach possibly suitable for archival materials.
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Kissel, Heather D., Thomas G. Paulson, Karen Liu, Xiaohong Li, Elizabeth Swisher, Rochelle Garcia, Carissa A. Sanchez, Brian J. Reid, Susan D. Reed, and Jennifer Anne Doherty. "Feasibility of RNA and DNA Extraction from Fresh Pipelle and Archival Endometrial Tissues for Use in Gene Expression and SNP Arrays." Obstetrics and Gynecology International 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/576842.
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Identifying molecular markers of endometrial hyperplasia (neoplasia) progression is critical to cancer prevention. To assess RNA and DNA quantity and quality from routinely collected endometrial samples and evaluate the performance of RNA- and DNA-based arrays across endometrial tissue types, we collected fresh frozen (FF) Pipelle, FF curettage, and formalin-fixed paraffin-embedded (FFPE) hysterectomy specimens (benign indications) from eight women. Additionally, neoplastic and uninvolved tissues from 24 FFPE archival hysterectomy specimens with endometrial hyperplasias and carcinomas were assessed. RNA was extracted from 15 of 16 FF and 51 of 51 FFPE samples, with yields >1.2 μg for 13/15 (87%) FF and 50/51 (98%) FFPE samples. Extracted RNA was of high quality; all samples performed successfully on the Illumina whole-genome cDNA-mediated annealing, selection, extension, and ligation (WG-DASL) array and performance did not vary by tissue type. While DNA quantity from FFPE samples was excellent, quality was not sufficient for successful performance on the Affymetrix SNP Array 6.0. In conclusion, FF Pipelle samples, which are minimally invasive, yielded excellent quantity and quality of RNA for gene expression arrays (similar to FF curettage) and should be considered for use in genomic studies. FFPE-derived DNA should be evaluated on new rapidly evolving sequencing platforms.
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Johnston, P. G., K. Mulligan, E. Kay, J. Black, S. Moore, U. Mc Dermott, R. Wilson, and D. Harkin. "A genetic signature of relapse in stage II colorectal cancer derived from formalin fixed paraffin embedded tissue (FFPE) tissue using a unique disease specific colorectal array." Journal of Clinical Oncology 24, no. 18_suppl (June 20, 2006): 3519. http://dx.doi.org/10.1200/jco.2006.24.18_suppl.3519.
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3519 Background: We have developed the first disease specific microarray for colorectal cancer using a transcriptome-based approach. This unique tool has been specially designed and optimised for analysis of gene expression profiles from formalin fixed paraffin embedded tissues (FFPE). To evaluate this tool we are conducting a study in patients with Dukes B (stage II) colorectal cancer (CRC) using FFPE tissues to determine a prognostic genetic signature predicting disease recurrence. Methods: The colorectal array was developed using a high-throughput transcriptome-based approach and encodes over 52,500 transcripts expressed in normal and diseased colorectal tissue. To date, tumours from 32 patients have been selected (mean FFPE tissue block age - 10.4 years) from the stage II CRC cohort of a phase III randomized trial comparing 5-FU/Leucovorin versus no adjuvant treatment (NI240). The patients were from the observation alone arm;19 of whom were disease-free for 5 years post-randomisation, while 13 suffered relapse prior to 3 years. Results: RNA was extracted from all 32 FFPE tissue samples, amplified, labelled and hybridised to the colorectal cancer disease specific array. Raw data was normalised using scalar, median-polish and z-score strategies and scalar was selected as the best strategy in relation to predictive error. Within this data set genes selected by correlation using ANOVA performed best in both KNN and support vector machine algorithms (supervised). Using this approach a gene signature containing 48 genes demonstrated 100% accuracy in the prediction of relapse in stage II CRC (p<0.001). This signature was also able to separate samples at the meta-node level using unsupervised hierarchical clustering. Conclusion: We have developed the first colorectal cancer disease specific microarray and demonstrated its use using FFPE tissues. Using this approach we have derived a gene signature that predicts for a high likelihood of early relapse in stage II CRC. [Table: see text]
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Khan, Muhammad Nouman, Qianqian Wang, Bushra Sana Idrees, Geer Teng, Xutai Cui, and Kai Wei. "Discrimination of Melanoma Using Laser-Induced Breakdown Spectroscopy Conducted on Human Tissue Samples." Journal of Spectroscopy 2020 (December 30, 2020): 1–11. http://dx.doi.org/10.1155/2020/8826243.
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Discrimination and identification of melanoma (a kind of skin cancer) by using laser-induced breakdown spectroscopy (LIBS) combined with chemometrics methods are reported. The human melanoma and normal tissues are used in the form of formalin-fixed paraffin-embedded (FFPE) blocks as samples. The results demonstrated higher LIBS signal intensities of phosphorus (P), potassium (K), sodium (Na), magnesium (Mg), and calcium (Ca) in melanoma FFPE samples while lower signal intensities in normal FFPE tissue samples. Chemometric methods, artificial neural network (ANN), linear discriminant analysis (LDA), quadratic discriminant analysis (QDA), and partial least square discriminant analysis (PLS-DA) are used to build the classification models. Different preprocessing methods, standard normal variate (SNV), mean-centering, normalization by total area, and autoscaling, were compared. A good performance of the model (sensitivity, specificity, and accuracy) for melanoma and normal FFPE tissues has been achieved by the ANN and PLS-DA models (all were 100%). The results revealed that LIBS combined with chemometric methods for detection and discrimination of human malignancies is a reliable, accurate, and precise technique.
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Lee, Taebum, Hee Young Na, Sun-ju Byeon, Kyoung-Mee Kim, Hey Seung Lee, Sung-Hye Park, Ji-Young Choe, and Kyoung Chan Choi. "Mycobiome analysis in fungal infected formalin-fixed and paraffin-embedded tissues for identification of pathogenic fungi: a pilot study." F1000Research 9 (July 22, 2020): 758. http://dx.doi.org/10.12688/f1000research.25126.1.
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Background: Fungal organisms are frequently observed in surgical pathological diagnosis. In order to more accurately identify fungi in formalin-fixed and paraffin-embedded (FFPE) tissues, it is necessary to use genomic information. The purpose of our pilot study is to identify the factors to be considered for the identification of pathogenic fungi using mycobiome analysis in FFPE tissues. Methods: We selected 49 cases in five hospitals. In each case, FFPE tissue was cut into 50 µm and DNA was extracted. Multiplex PCR with four primers (ITS1, ITS2, ITS3 and ITS4) was performed. Multiplex sequencing was performed using a MinION device according to the manufacturer’s protocol. Sequences of each case were searched using BLASTN with an ITS database from NCBI RefSeq Targeted Loci Project with default parameters. Results: A total of 2,526 DNA sequences were sequenced. We were able to identify 342 fungal sequences in 24 (49.0%, 24/49) cases. The median number of detected fungal sequences per case was 3 (1Q: 1 and 3Q: 14.25). Of the fungal DNA sequences, 215 (62.87%) contained the entire region of ITS1 or ITS2. The remaining 127 fungal DNA sequences were identified as fungi using a partial sequence of ITS1, ITS2, 5.8S, LSU or SSU. Conclusion: In conclusion, we have identified the possibility of finding pathogenic fungi through mycobiome analysis in fungal infected FFPE tissues using nanopore sequencing. However, we have also found several limitations to be solved for further studies. If we develop a method to characterize pathogenic fungi in FFPE tissues in a follow-up study, we think it will help patients to use appropriate antifungal agents.
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Razvi, M. A. N., Ahmed Bakry, A. Saeed, S. M. Afzal, Y. F. AL-Hadeethi, Jaudah AL-Maghrabi, and Saad AL-Muhayawi. "Diagnosis of Oral Squamous Cell Carcinoma (OSCC) Using Laser Induced Fluorescence." Science of Advanced Materials 12, no. 6 (June 1, 2020): 853–60. http://dx.doi.org/10.1166/sam.2020.3759.
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Cancer is a dreaded disease; a large number of deaths occur every year due to this disease. Oral squamous cell carcinoma (OSCC) is the most common cancer of the head and neck, which is approximately 16% to 40% of all malignancies. In this study, Laser induced fluorescence (LIF) spectroscopy has been utilized to discriminate OSCC against healthy (normal) tissues and to investigate whether the LIF could provide information from formalin-fixed paraffin-embedded (FFPE) tissue samples similar to that reported using fresh tissues. Samples were prepared after biopsy from ten patients using standard FFPE tissues methods. LIF system consists of a continuous wave (CW) He–Cd laser at 325 nm, a seven-core optical fiber cable coupled to the laser, a spectrometer with cooled charge coupled device (CCD) detector, and a computer for acquisition of the LIF spectra. Spectra were decomposed using second derivative and curve fitting analysis to reveal the changes in molecular composition of the samples. Moreover, samples spectra were discriminated by hierarchical cluster analysis (HCA) and principal components analysis (PCA). Spectral results showed differences in peak areas and positions between normal and OSCC tissues. LIF spectroscopy revealed significant decrease in the peak area of collagen and decrease in peak area of coenzymes of OSCC tissues. In addition, significant shift in the peak position of coenzymes was recorded. HCA and PCA of LIF indicated a very clear discrimination of the normal and FFPE-OSCC tissues. The achieved discrimination between elliptic polygons of normal and OSCC tissues was 96.3% by PCA. This study confirms that the LIF spectroscopy is a good diagnostic tool for OSCC and it could be used with samples that are prepared using FFPE tissues methods.
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Almeida, Paula R., Caroline P. Andrade, Laura L. Almeida, Luiz G. S. Oliveira, Luiza A. Castro, Priscila Zlotowski, Sérgio C. da Silva, and David Driemeier. "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." Pesquisa Veterinária Brasileira 32, no. 8 (August 2012): 715–20. http://dx.doi.org/10.1590/s0100-736x2012000800006.
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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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Bhatnagar, Julu, Marlene DeLeon-Carnes, Kathryn L. Kellar, Kakali Bandyopadhyay, Zoi-Anna Antoniadou, Wun-Ju Shieh, Christopher D. Paddock, and Sherif R. Zaki. "Rapid, Simultaneous Detection ofClostridium sordelliiandClostridium perfringensin Archived Tissues by a Novel PCR-Based Microsphere Assay: Diagnostic Implications for Pregnancy-Associated Toxic Shock Syndrome Cases." Infectious Diseases in Obstetrics and Gynecology 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/972845.
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Clostridium sordelliiandClostridium perfringensare infrequent human pathogens; however, the case-fatality rates for the infections are very high, particularly in obstetricC. sordelliiinfections (>90%). Deaths fromClostridium sordelliiandClostridium perfringenstoxic shock (CTS) are sudden, and diagnosis is often challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues usually are the only specimens available for sudden fatal cases, and immunohistochemistry (IHC) for Clostridia is generally performed but it cannot identify species. A clear need exists for a rapid, species-specific diagnostic assay for FFPE tissues. We developed a duplex PCR-based microsphere assay for simultaneous detection ofC. sordelliiandC. perfringensand evaluated DNA extracted from 42Clostridiumisolates and FFPE tissues of 28 patients with toxic shock/endometritis (20 CTS, 8 non-CTS, as confirmed by PCR and sequencing). The microsphere assay correctly identifiedC. sordelliiandC. perfringensin all known isolates and in all CTS patients (10C. sordellii, 8C. perfringens, 2 both) and showed 100% concordance with PCR and sequencing results. The microsphere assay is a rapid, specific, and cost-effective method for the diagnosis of CTS and offers the advantage of simultaneous testing forC. sordelliiandC. perfringensin FFPE tissues using a limited amount of DNA.
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Surat, Güzin, William A. Wallace, Ian F. Laurenson, and Amie-Louise Seagar. "Rapid real-time PCR for detection of Mycobacterium tuberculosis complex DNA in formalin-fixed paraffin embedded tissues: 16% of histological ‘sarcoid’ may contain such DNA." Journal of Clinical Pathology 67, no. 12 (August 28, 2014): 1084–87. http://dx.doi.org/10.1136/jclinpath-2014-202307.
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AimsTo investigate the diagnostic accuracy of IS6110 real-time PCR for detection of Mycobacterium tuberculosis complex (MTBC) in DNA extracted from formalin-fixed paraffin embedded (FFPE) tissues using two different methods. In the absence of material submitted for tuberculosis (TB) culture, MTBC detection in FFPE tissue can be an important aid to diagnosis.MethodsWe collected 144 FFPE tissue blocks (lung and lymph node) for IS6110 real-time PCR. Two DNA extraction methods (QIAamp FFPE tissue kit and NucliSENS easyMAG) were assessed within a general laboratory setting. PCR results were compared with histology and culture.ResultsIn the histological MTBC and culture MTBC (TB-positive) groups, 72.4% were IS6110-positive and 27.6% negative. IS6110-negative results were obtained from 98%, 61.5% and 84% of the histologically MTBC-negative (TB-negative) group, histologically TB/no culture group and sarcoidosis group, respectively. Review of 19 IS6110-positive patients in the latter three groups showed that 15 had clinical TB. Thirteen of 15 (86.7%) IS6110-positive patients in the histological TB/no culture group and 2 of 4 (50%) IS6110-positive patients in the sarcoidosis group were clinically diagnosed with TB which highlights the difficulty of a pathological diagnosis.ConclusionsIS6110 real-time PCR using easyMAG extracted DNA is a moderately sensitive, specific and rapid method for MTBC detection in FFPE material, but must be interpreted in the overall clinical context. PCR results can be available in around 5 h from FFPE specimen receipt, with minimal hands-on time.
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Isabelle, Maxim, Michael Schirm, Gwenaël Pottiez, Rudolf Guilbaud, and Lorella Di Donato. "54 Patient stratification using clinical proteomics – validated multiplexed MRM assays to quantify HER2 and other biomarkers in clinical FFPE tissues." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A59. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0054.
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BackgroundThe advent of precision oncology has led a shift towards biomarker-driven clinical trial designs and molecular profiling of individual patients. Identification of patients with the target biomarker profile may be useful in guiding patient selection as an enrichment strategy for clinical trials. Targeted multiple reaction monitoring mass spectrometry (MRM-MS) analysis for multiplexed quantitation of biomarker proteins in FFPE tissue provides direct, more accurate and precise quantification over current ‘gold standard’ immunohistochemistry (IHC) methods. However, MRM-MS has not yet been broadly applied to clinical trials. In this study, we demonstrate the systematic development, optimization and analytical validation of quantitative, multiplexed MRM-MS assays for robust biomarker quantification in clinical FFPE tissues, including sample analysis under GCLP. Results from an MRM panel targeting 8 clinically relevant biomarker proteins will also be shown, including the measured HER2 levels in FFPE breast tumors classified by IHC as 0, 1+, 2+ or 3+.MethodsMRM-MS biomarker panels were developed and optimized for multiplexed quantitation of ≤12 proteins, in which unique peptides derived from each target protein were monitored as a surrogate measure of protein levels. Tumor regions from FFPE tissue sections were dissected using laser capture or macrodissection, solubilized, digested with trypsin to generate peptides for analysis, spiked with fixed levels of stable isotope labeled (SIL) peptide standards, and analyzed by MRM-MS. Analytical validation was performed per NCI CPTAC guidelines, including response curves, assay repeatability, selectivity, stability, and reproducibility of endogenous detection. Clinical performance was assessed using commercially sourced FFPE-tumor tissues, including a cohort of breast tumor tissues with a wide range of HER2 expression.ResultsAssay performance results were protein/peptide dependent, with sensitivity in the low pg/µg total protein range. For HER2, assay linearity was demonstrated over 2.5 to 3 orders of magnitude, with a precision and accuracy of <15% over 3 independent runs. In sample analysis, the MRM-MS was sufficiently sensitive to detect HER2 in 1 µg total protein from FFPE breast tumor classified by IHC as negative (0).ConclusionsGCLP-compliant quantitative multiplexed large-scale clinical analysis of protein biomarkers by MRM-MS in FFPE tissue is feasible and enables precise and accurate quantitation of proteins when IHC methods are unsuitable or unavailable. Data can be used for patient stratification, optimization of treatment outcomes, drug resistance prediction, and to support clinical development of novel therapies.
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Kaneko, Syuzo, Toutai Mitsuyama, Kouya Shiraishi, Noriko Ikawa, Kanto Shozu, Ai Dozen, Hidenori Machino, et al. "Genome-Wide Chromatin Analysis of FFPE Tissues Using a Dual-Arm Robot with Clinical Potential." Cancers 13, no. 9 (April 28, 2021): 2126. http://dx.doi.org/10.3390/cancers13092126.
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Although chromatin immunoprecipitation and next-generation sequencing (ChIP-seq) using formalin-fixed paraffin-embedded tissue (FFPE) has been reported, it remained elusive whether they retained accurate transcription factor binding. Here, we developed a method to identify the binding sites of the insulator transcription factor CTCF and the genome-wide distribution of histone modifications involved in transcriptional activation. Importantly, we provide evidence that the ChIP-seq datasets obtained from FFPE samples are similar to or even better than the data for corresponding fresh-frozen samples, indicating that FFPE samples are compatible with ChIP-seq analysis. H3K27ac ChIP-seq analyses of 69 FFPE samples using a dual-arm robot revealed that driver mutations in EGFR were distinguishable from pan-negative cases and were relatively homogeneous as a group in lung adenocarcinomas. Thus, our results demonstrate that FFPE samples are an important source for epigenomic research, enabling the study of histone modifications, nuclear chromatin structure, and clinical data.
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Iraola-Guzmán, Susana, Anna Brunet-Vega, Cinta Pegueroles, Ester Saus, Hrant Hovhannisyan, Alex Casalots, Carles Pericay, and Toni Gabaldón. "Target Enrichment Enables the Discovery of lncRNAs with Somatic Mutations or Altered Expression in Paraffin-Embedded Colorectal Cancer Samples." Cancers 12, no. 10 (October 1, 2020): 2844. http://dx.doi.org/10.3390/cancers12102844.
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Long non-coding RNAs (lncRNAs) play important roles in cancer and are potential new biomarkers or targets for therapy. However, given the low and tissue-specific expression of lncRNAs, linking these molecules to particular cancer types and processes through transcriptional profiling is challenging. Formalin-fixed, paraffin-embedded (FFPE) tissues are abundant resources for research but are prone to nucleic acid degradation, thereby complicating the study of lncRNAs. Here, we designed and validated a probe-based enrichment strategy to efficiently profile lncRNA expression in FFPE samples, and we applied it for the detection of lncRNAs associated with colorectal cancer (CRC). Our approach efficiently enriched targeted lncRNAs from FFPE samples, while preserving their relative abundance, and enabled the detection of tumor-specific mutations. We identified 379 lncRNAs differentially expressed between CRC tumors and matched healthy tissues and found tumor-specific lncRNA variants. Our results show that numerous lncRNAs are differentially expressed and/or accumulate variants in CRC tumors, thereby suggesting a role in CRC progression. More generally, our approach unlocks the study of lncRNAs in FFPE samples, thus enabling the retrospective use of abundant, well documented material available in hospital biobanks.
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Dear, Jonathan D., Jane E. Sykes, and Danika L. Bannasch. "Quality of DNA extracted from formalin-fixed, paraffin-embedded canine tissues." Journal of Veterinary Diagnostic Investigation 32, no. 4 (June 9, 2020): 556–59. http://dx.doi.org/10.1177/1040638720929637.
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Veterinary pathology tissue banks are valuable resources for genetic studies. However, limited data exist as to whether quality DNA can be extracted from these tissues for use in canine genotyping studies. We extracted DNA from 44 formalin-fixed, paraffin-embedded (FFPE) tissue blocks from dogs; 9 of these dogs had DNA available from whole blood samples that had been banked. We genotyped DNA from 30 of 44 tissue blocks and 9 whole blood samples on the Illumina CanineHD BeadChip; DNA quality was insufficient in 14 of 44 samples from tissue blocks. There was significant correlation between the 260/280 ratio and single-nucleotide variation (SNV) call rate ( p = 0.0276; r2 = 0.162); 23 of 30 samples from FFPE were genotyped with > 65% call rates. Median pairwise identical-by-state (IBS) analysis was 0.99 in 8 pairs of dogs with call rates > 65%. Neither age of tissue block nor specific tissue types were associated with significant differences in DNA concentration, 260/280 ratio, or SNV call rate. DNA extracted from tissue blocks can have variable quality, although comparable levels of homozygosity suggest that extracts from FFPE with call rates > 65% might provide similar results to samples from whole blood when analyzed on the Illumina CanineHD BeadChip.
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Do, Hongdo, and Alexander Dobrovic. "Sequence Artifacts in DNA from Formalin-Fixed Tissues: Causes and Strategies for Minimization." Clinical Chemistry 61, no. 1 (January 1, 2015): 64–71. http://dx.doi.org/10.1373/clinchem.2014.223040.
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Abstract BACKGROUND Precision medicine is dependent on identifying actionable mutations in tumors. Accurate detection of mutations is often problematic in formalin-fixed paraffin-embedded (FFPE) tissues. DNA extracted from formalin-fixed tissues is fragmented and also contains DNA lesions that are the sources of sequence artifacts. Sequence artifacts can be difficult to distinguish from true mutations, especially in the context of tumor heterogeneity, and are an increasing interpretive problem in this era of massively parallel sequencing. Understanding of the sources of sequence artifacts in FFPE tissues and implementation of preventative strategies are critical to improve the accurate detection of actionable mutations. CONTENT This mini-review focuses on DNA template lesions in FFPE tissues as the source of sequence artifacts in molecular analysis. In particular, fragmentation, base modification (including uracil and thymine deriving from cytosine deamination), and abasic sites are discussed as indirect or direct sources of sequence artifacts. We discuss strategies that can be implemented to minimize sequence artifacts and to distinguish true mutations from sequence artifacts. These strategies are applicable for the detection of actionable mutations in both single amplicon and massively parallel amplicon sequencing approaches. SUMMARY Because FFPE tissues are usually the only available material for DNA analysis, it is important to maximize the accurate informational content from FFPE DNA. Careful consideration of each step in the work flow is needed to minimize sequence artifacts. In addition, validation of actionable mutations either by appropriate experimental design or by orthogonal methods should be considered.
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Lee, Tae Hee, Young Jun Kim, Woo Sun Rou, and Hyuk Soo Eun. "Fabrication of Formalin-Fixed, Paraffin-Embedded (FFPE) Circulating Tumor Cell (CTC) Block Using a Hydrogel Core-Mediated Method." Micromachines 12, no. 9 (September 20, 2021): 1128. http://dx.doi.org/10.3390/mi12091128.
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Circulating tumor cells (CTCs) are extremely low-frequency cells in the bloodstream. As those cells have detached from the primary tumor tissues and it circulates throughout the whole body, they are considered as promising diagnostic biomarkers for clinical application. However, the analysis of CTC is often restricted due to their rarity and heterogeneity, as well as their short-term presence. Here we proposed formalin-fixed, paraffin-embedded (FFPE) CTC block method, in combination manner with the hydrogel core-mediated CTC accumulation and conventional paraffin tissue block preparation. The hydrogel core specifically captures and releases cancer cells with high efficiency with an immunoaffinity manner. An additional shell structure protects the isolated cancer cells during the FFPE CTC block preparation process. The fabricated FFPE CTC block was sectioned and cytopathologically investigated just the same way as the conventional tissue block. Our results demonstrate that rare cells such as CTCs can also be prepared for FFPE cell blocks and shows great promise for cytopathological CTC studies.
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Klopfleisch, R., M. von Deetzen, A. Th Weiss, J. Weigner, F. Weigner, J. Plendl, and A. D. Gruber. "Weigners Fixative–An Alternative to Formalin Fixation for Histology With Improved Preservation of Nucleic Acids." Veterinary Pathology 50, no. 1 (April 26, 2012): 191–99. http://dx.doi.org/10.1177/0300985812441031.
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Formalin fixation and paraffin embedding (FFPE) is the standard method for tissue storage in histopathology. However, FFPE has disadvantages in terms of user health, environment, and nucleic acid integrity. Weigners fixative has been suggested as an alternative for embalming cadavers in human and veterinary anatomy. The present study tested the applicability of Weigners for histology and immunohistochemistry and the preservation of nucleic acids. To this end, a set of organs was fixed for 2 days and up to 6 months in Weigners (WFPE) or formalin. WFPE tissues from the skin, brain, lymphatic tissues, liver, and muscle had good morphologic preservation, comparable to formalin fixation. The quality of kidney and lung samples was inferior to FFPE material due to less accentuated nuclear staining and retention of proteinaceous interstitial fluids. Azan, Turnbull blue, toluidin, and immunohistochemical stainings for CD79a, cytokeratin, vimentin, and von Willebrand factor led to comparable results with both fixates. Of note, immunohistochemical detection of CD3 was possible after 6 months in WFPE but not in FFPE tissues. mRNA, miRNA, and DNA from WFPE tissues had superior quality and allowed for amplification of miRNA, 400-bp-long mRNA, and 1000-bp-long DNA fragments after 6 months of fixation in WFPE. In summary, Weigners fixative is a nonhazardous alternative to formalin, which provides a good morphologic preservation of most organs, a similar sensitivity for protein detection, and a superior preservation of nucleic acids. Weigners may therefore be a promising alternative to cryopreservation and may be embraced by people affected by formalin allergies.
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Abed, Firas M., and Michael J. Dark. "Determining the utility of veterinary tissue archives for retrospective DNA analysis." PeerJ 4 (May 3, 2016): e1996. http://dx.doi.org/10.7717/peerj.1996.
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Histopathology tissue archives can be an important source of specimens for retrospective studies, as these include samples covering a large number of diseases. In veterinary medicine, archives also contain samples from a large variety of species and may represent naturally-occurring models of human disease. The formalin-fixed, paraffin-embedded (FFPE) tissues comprising these archives are rich resources for retrospective molecular biology studies and pilot studies for biomarkers, as evidenced by a number of recent publications highlighting FFPE tissues as a resource for analysis of specific diseases. However, DNA extracted from FFPE specimens are modified and fragmented, making utilization challenging. The current study examines the utility of FFPE tissue samples from a veterinary diagnostic laboratory archive in five year intervals from 1977 to 2013, with 2015 as a control year, to determine how standard processing and storage conditions has affected their utility for future studies. There was a significant difference in our ability to obtain large amplicons from samples from 2015 than from the remaining years, as well as an inverse correlation between the age of the samples and product size obtainable. However, usable DNA samples were obtained in at least some of the samples from all years tested, despite variable storage, fixation, and processing conditions. This study will help make veterinary diagnostic laboratory archives more useful in future studies of human and veterinary disease.
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Mullins, Michael, Laurent Perreard, John F. Quackenbush, Nicholas Gauthier, Steven Bayer, Matthew Ellis, Joel Parker, Charles M. Perou, Aniko Szabo, and Philip S. Bernard. "Agreement in Breast Cancer Classification between Microarray and Quantitative Reverse Transcription PCR from Fresh-Frozen and Formalin-Fixed, Paraffin-Embedded Tissues." Clinical Chemistry 53, no. 7 (July 1, 2007): 1273–79. http://dx.doi.org/10.1373/clinchem.2006.083725.
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Abstract Background: Microarray studies have identified different molecular subtypes of breast cancer with prognostic significance. To transition these classifications into the clinical laboratory, we have developed a real-time quantitative reverse transcription (qRT)-PCR assay to diagnose the biological subtypes of breast cancer from fresh-frozen (FF) and formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: We used microarray data from 124 breast samples as a training set for classifying tumors into 4 previously defined molecular subtypes: Luminal, HER2+/ER−, basal-like, and normal-like. We used the training set data in 2 different centroid-based algorithms to predict sample class on 35 breast tumors (test set) procured as FF and FFPE tissues (70 samples). We classified samples on the basis of large and minimized gene sets. We used the minimized gene set in a real-time qRT-PCR assay to predict sample subtype from the FF and FFPE tissues. We evaluated primer set performance between procurement methods by use of several measures of agreement. Results: The centroid-based algorithms were in complete agreement in classification from FFPE tissues by use of qRT-PCR and the minimized “intrinsic” gene set (40 classifiers). There was 94% (33 of 35) concordance between the diagnostic algorithms when comparing subtype classification from FF tissue by use of microarray (large and minimized gene set) and qRT-PCR data. We found that the ratio of the diagonal SD to the dynamic range was the best method for assessing agreement on a gene-by-gene basis. Conclusions: Centroid-based algorithms are robust classifiers for breast cancer subtype assignment across platforms and procurement conditions.
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Farooq, Umar, Prashant Singh, Michael Gooch, Linda Burian, M. Melinda Sanders, and Richard Bernard Everson. "Comparisons of RNA-seq results for cryopreserved (CRYO) and formalin fixed, paraffin-embedded (FFPE) samples." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): e22211-e22211. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.e22211.
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e22211 Background: FFPE is the worldwide clinical standard for preservation of tissue samples. Although CRYO tissues are widely used for research, most gene expression assays in clinical use analyze FFPE by RT-PCR. Use of RT-PCR limits numbers of transcripts that can be studied with material available. Next Generation Sequencing promises to overcome those limitations, but for FFPE, there are concerns related to its accuracy including bias associated with RNA truncated at the 3’ end. Little data is available illustrating the extent of truncation or considering implications for bioinformatics analysis of sequencing results. Methods: Total RNA was extracted from patient consented specimens of tumor and normal bladder tissue split with matching halves processed by CRYO and FFPE. Paired end sequencing libraries 100 bp in length were generated using Illumina TruSeq RNA sample kits with minor modifications for FFPE samples. Sequencing was performed on the Illumina HiSeq 2000, aligned with TopHat using the hg19 reference sequence, and further analyzed using open source bioinformatics tools. Results: Comparing Cryo and FFPE reads for a representative specimen, the proportion of reads that mapped to the reference genome were 69% and 63% for normal, and 66% and 63% for tumor specimens. Mapped reads yielded over 25,000 significantly expressed transcripts, isoforms, and splice variants. The extent of 3’ bias was investigated by viewing data in the Integrated Genome Viewer and using Picard tools. Preliminary estimates of 3’ bias in FFPE showed 2.22 for normal and 1.48 for tumor. After accounting for bias, transcripts in CRYO vs FFPE were significantly correlated for both normal tissue and tumor (R2 = 0.76 and 0.95, respectively). Mutations previously identified in these samples by SureSelect hybrid capture system (Agilent) were also observed in the RNA-seq data. Conclusions: FFPE samples can be successfully analyzed by RNA-Seq, allowing the analysis of standard clinical samples and vast quantities of richly annotated archival specimens. Results from CRYO and FFPE analyses are similar when performed using minor modifications to sequencing library preparation and careful attention to appropriate analysis.
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Shi, Zonggao, Jeffrey J. Johnson, and M. Sharon Stack. "FluorescenceIn SituHybridization for MicroRNA Detection in Archived Oral Cancer Tissues." Journal of Oncology 2012 (2012): 1–8. http://dx.doi.org/10.1155/2012/903581.
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The noncoding RNA designated as microRNA (miRNA) is a large group of small single-stranded regulatory RNA and has generated wide-spread interest in human disease studies. To facilitate delineating the role of microRNAs in cancer pathology, we sought to explore the feasibility of detecting microRNA expression in formalin-fixed paraffin-embedded (FFPE) tissues. Using FFPE materials, we have compared fluorescentin situhybridization (FISH) procedures to detect miR-146a with (a) different synthetic probes: regular custom DNA oligonucleotides versus locked nucleic acid (LNA) incorporated DNA oligonucleotides; (b) different reporters for the probes: biotin versus digoxigenin (DIG); (c) different visualization: traditional versus tyramide signal amplification (TSA) system; (d) different blocking reagents for endogenous peroxidase. Finally, we performed miR-146a FISH on a commercially available oral cancer tissue microarray, which contains 40 cases of oral squamous cell carcinoma (OSCC) and 10 cases of normal epithelia from the human oral cavity. A sample FISH protocol for detecting miR-146a is provided. In summary, we have established reliablein situhybridization procedures for detecting the expression of microRNA in FFPE oral cancer tissues. This method is an important tool for studies on the involvement of microRNA in oral cancer pathology and may have potential prognostic or diagnostic value.
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Lattanzio, Laura, Marzia Borgognone, Cristina Mocellini, Fabrizio Giordano, Ermanno Favata, Gaetano Fasano, Daniela Vivenza, et al. "MGMT Promoter Methylation and Glioblastoma: A Comparison of Analytical Methods and of Tumor Specimens." International Journal of Biological Markers 30, no. 2 (April 2015): 208–16. http://dx.doi.org/10.5301/jbm.5000126.
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It is already well known that hypermethylation of the O6-methylguanine DNA methyltransferase (MGMT) gene promoter is a predictive biomarker of response to temozolomide treatment and of favorable outcomes in terms of overall survival (OS) and progression-free survival (PFS) in glioblastoma (GBM) patients. Nevertheless, MGMT methylation status has not currently been introduced into routine clinical practice, as the choice of the ideal technique and tissue sample specimen is still controversial. The aim of this study was to compare 2 analytical methods, methylation-specific polymerase chain reaction (MSP) and pyrosequencing (PSQ), and their use on 2 different tissue type samples, snap-frozen and formalin-fixed paraffin-embedded (FFPE), obtained from a single-center and uniformly treated cohort of 46 GBM patients. We obtained methylation data from all frozen tissues, while no results were obtained for 5 FFPE samples. The highest concordance for methylation was found on frozen tissues (88.5%, 23/26 samples), using PSQ (76.7%, 23/30 samples). Moreover, we confirmed that OS and PFS for patients carrying methylation of the MGMT promoter were longer than for patients with an unmethylated promoter. In conclusion, we considered MSP a limited technique for FFPE tissues due to the high risk of false-positive results; in contrast, our data indicated PSQ as the most powerful method to stratify methylated/unmethylated patients as it allows reaching quantitative results with high sensitivity and specificity. Furthermore, frozen tumor tissues were shown to be the best specimens for MGMT methylation analysis, due to the low DNA degradation and homogeneity in methylation throughout the tumor.
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Belloni, Benedetta, Chiara Lambertini, Paolo Nuciforo, Jay Phillips, Eric Bruening, Stephane Wong, and Reinhard Dummer. "Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system." Journal of Clinical Pathology 66, no. 2 (November 3, 2012): 124–35. http://dx.doi.org/10.1136/jclinpath-2012-200983.
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Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device.AimsIn this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples.Methods12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry, DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used.ResultsMorphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues.ConclusionsThe switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.
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Landers, Mark, Rhonda Meredith, Jerry Lee, Yipeng Wang, Byung-In Lee, and Joseph Monforte. "Next-generation sequencing mutational analysis of triple-negative breast cancer patients from matched FFPE and fresh frozen samples." Journal of Clinical Oncology 30, no. 15_suppl (May 20, 2012): 1058. http://dx.doi.org/10.1200/jco.2012.30.15_suppl.1058.
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1058 Background: TNBC is an aggressive subtype of breast cancer accounting for 10-15% of all cases. TNBC tumors (ER-/PR-/HER-) are more common in patients with BRCA mutations. BRCA mutations leading to homologous DNA repair deficient tumors enhance the efficacy of chemotherapy and PARP inhibitors. BRCA mutations have been identified in 20% of patients without family history. Identification of germline and somatic BRCA mutations in unselected patients could increase the number of patients who benefit from these therapies. Determining BRCA mutational status from FFPE and fresh frozen specimens may enable clinical studies in these patient populations.We describe the development of an NGS BRCA mutational assay compatible with FFPE and fresh frozen samples using tumor/adjacent normal matched tissues. Methods: Matched samples were purchased from Cureline. gDNA was isolated by lysis/column purification (Qiagen) and enriched for BRCA exons/flanking regions (Halogenomics Selector). Fragment libraries were constructed (Ion Torrent frag express) and prepared for sequencing by emPCR (Ion Torrent Template Xpress). Libraries were sequenced for 65 cycles (Ion Torrent PGM) yielding 2-4M reads/sample. Variants were called from tMAP aligned reads by GATK and VarScan. Overlapped exonic variants were filtered by p-value (<0.0001) from VarScan. Results: In the first patient set normalized average depth of BRCA exon coverage was 64X and 72X per 150K reads in FFPE and fresh frozen tissues respectively covering 95-100% of target. hg19 alignment rates varied between 97-99% across all samples. Similar numbers of variants were called in both FFPE (12) vs. fresh frozen (13) with a corresponding mean duplicate removed depth of coverage of 23.3X and 42.4X at the called positions. 10/13 calls in fresh frozen overlapped with those in FFPE. A tumor specific somatic frameshift insertion in BRCA2 was detected in both FFPE and fresh frozen tissues. Conclusions: Results indicate that NGS mediated BRCA mutational analysis demonstrates equivalent utility in both FFPE and fresh frozen tumor samples although more sequencing reads are required to produce equivalent depth of coverage starting from FFPE samples.
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Fu, Rong, Xiaoshuang Chen, and Zonghong Shao. "Plasma DNA Methylation of p16 and shp1 in Patients with B-Cell Non-Hodgkin Lymphoma." Blood 124, no. 21 (December 6, 2014): 5369. http://dx.doi.org/10.1182/blood.v124.21.5369.5369.
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Abstract To discuss the clinical implications of cell free DNA, methylation status of the p16 and shp1 genes in plasma DNA from patients with B-NHL were analyzed. Methylation specific polymerase chain reaction (MSP) was used to detect the methylation status of these genes in plasma, peripheral blood leukocytes (PBLs), and formaldehyde-fixed, paraffin-embedded (FFPE) tumor tissues of 73 B-NHL patients. Results showed methylation frequencies of p16 in plasma, PBLs, and FFPE tumor tissues of B-NHL patients were 37%(27/73), 16% (12/73), 39% (16/41); Whereas those of shp1 were 47% (34/73), 25% (18/73), 63% (26/41). High methylation consistencies of p16/shp1 were observed between plasma and FFPE tumor tissues. In plasma and FFPE tumor tissues, there was a higher frequency of methylated p16 in B-NHL patients who had later disease stage, while no similar association was observed in PBLs samples; Higher frequency of methylated p16 was observed in B-NHL patients who had B symptoms and lower platelet count (<100×109/L) in all three samples. Methylated shp1 was frequently detected in patients with high serum LDH level. In summary, DNA methylation in plasma may be promising biomarkers for B-NHL diagnosis, prognosis evaluation, and targeted therapy. Disclosures No relevant conflicts of interest to declare.
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Hembrough, Todd A., Les Henderson, Brittany Rambo, Wei-Li Liao, Sheeno Thyparambil, Kathleen Bangali, Jamar Uzzell, et al. "Quantification of HER2 from gastroesophageal cancer (GEC) FFPE tissue by mass spectrometry (MS)." Journal of Clinical Oncology 32, no. 3_suppl (January 20, 2014): 17. http://dx.doi.org/10.1200/jco.2014.32.3_suppl.17.
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17 Background: Trastuzumab had a survival benefit in ‘HER2 positive’ GEC, determined by IHC/FISH. These companion diagnostics have limitations. IHC is semi-quantitative, subjective, and sensitive to antigen instability in FFPE; FISH is laborious, expensive, and subjective. Gene amplification may not correlate to protein expression. Moreover these are low throughput assays. There is increased recognition of profound interpatient molecular heterogeneity with several putative biomarkers, and only scarce tissue to assess for each one. We sought to evaluate our MS platform on GEC FFPE tissues for HER2 status compared to IHC/FISH. We also applied the “GEC-plex” of 11 other potentially predictive/prognostic markers for GEC. Methods: We utilized trypsin digestion mapping of rHER2 to identify unique peptides for SRM development. Stable isotope-labeled peptides were synthesized as internal standards, and standard curves were generated in a complex eukaryotic matrix (PC3 cells) to determine LOD, LLOQ, accuracy, precision and linearity of the assays. The assay was run on 17 GEC cell lines, in parallel with FISH/IHC, and expression thresholds were established for HER2+/HER2-; the sensitivity/specificity of the established cutoffs were then tested prospectively in FFPE GEC tissues on 10uM FFPE LCM slides (n=121). HER2 stability from FFPE sections was assessed by assaying 33 freshly cut FFPE samples; the adjacent sections were processed one year later. Results: The HER2 peptide chosen (ELVSEFSR) had a LOD of 100 amol and CV<20%. HER2 MS on GEC cell lines revealed concordance with FISH (HER2:CEP17) ratio (R2=0.96). The analysis suggested HER2 expression > 750 amol/ug was indicative of HER2 amplification. The assay was stable in archival FFPE sections (R2=0.76). For GEC FFPE cases, ‘any’ HER2 expression was seen in 69.4% of cases; 8.2% showed HER2 > 750 amol (10/121) - all were HER2 amplified. No cases <550 amol/ug were HER2 amplified. IHC/FISH results for cases with 550-750 amol/ug demonstrated a heterogeneous ‘equivocal’ zone, not unlike ‘IHC 2+’, which may require FISH confirmatory testing. Conclusions: ‘GEC-plex’ has a quantitative, sensitive, and specific HER2 assay that can be multiplexed along with other GEC biomarkers.
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Li, Jialu, Chunxiao Fu, Terence P. Speed, Wenyi Wang, and W. Fraser Symmans. "Accurate RNA Sequencing From Formalin-Fixed Cancer Tissue to Represent High-Quality Transcriptome From Frozen Tissue." JCO Precision Oncology, no. 2 (November 2018): 1–9. http://dx.doi.org/10.1200/po.17.00091.
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Purpose Accurate transcriptional sequencing (RNA-seq) from formalin-fixed paraffin-embedded (FFPE) tumor samples presents an important challenge for translational research and diagnostic development. In addition, there are now several different protocols to prepare a sequencing library from total RNA. We evaluated the accuracy of RNA-seq data generated from FFPE samples in terms of expression profiling. Methods We designed a biospecimen study to directly compare gene expression results from different protocols to prepare libraries for RNA-seq from human breast cancer tissues, with randomization to fresh frozen (FF) or FFPE conditions. The protocols were compared using multiple computational methods to assess alignment of reads to a reference genome, the uniformity and continuity of coverage, the variance and correlation of overall gene expression, patterns of measuring coding sequence, phenotypic patterns of gene expression, and measurements from representative multigene signatures. Results The principal determinant of variance in gene expression was use of exon capture probes, followed by the conditions of preservation (FF v FFPE) and phenotypic differences between breast cancers. One protocol, with RNase H-based ribosomal RNA depletion, exhibited the least variability of gene expression measurements and strongest correlation between FF and FFPE samples and was generally representative of the transcriptome from standard FF RNA-seq protocols. Conclusion Method of RNA-seq library preparation from FFPE samples had a marked effect on the accuracy of gene expression measurement compared with matched FF samples. Nevertheless, some protocols produced highly concordant expression data from FFPE RNA-seq data, compared with RNA-seq results from matched frozen samples.
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Cooper, Melissa, Shu-Qiu Li, Tajinder Bhardwaj, Thomas Rohan, and Rita A. Kandel. "Evaluation of Oligonucleotide Arrays for Sequencing of the p53 Gene in DNA from Formalin-Fixed, Paraffin-Embedded Breast Cancer Specimens." Clinical Chemistry 50, no. 3 (March 1, 2004): 500–508. http://dx.doi.org/10.1373/clinchem.2003.025221.
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Abstract Background: Routine tissue processing has generated banks of paraffin-embedded tissue that could be used in retrospective cohort studies to study the molecular changes that occur during cancer development. The purpose of this study was to determine whether a p53 microarray could be used to sequence the p53 gene in DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissues. Methods: DNA was extracted from 70 FFPE breast cancer tissue specimens. p53 was sequenced with an oligonucleotide microarray (p53 GeneChip®; Affymetrix), and the results were compared with the results obtained from direct sequencing. Results: DNA was extracted from 62 of 70 cases. We identified 26 mutations in 24 of the 62 cases by the p53 GeneChip. No polymorphisms were detected, and exon 4 could not be evaluated in 20 cases. There were 43 genetic alterations detected by direct sequencing in 35 of the 62 cases. These consisted of 26 polymorphisms and 17 mutations in exons or splice sites. Fifteen mutations were identified by both methods. Direct sequencing detected significantly more gene alterations (43 of 54) in DNA extracted from FFPE tissue than the p53 GeneChip (26 of 54; P = 0.018). However, if the changes in exon 4 were eliminated from this comparison, the p53 GeneChip detected 26 of 27 mutations compared with direct sequencing, which identified 16 of 27 mutations. (P = 0.016). Conclusions: A combination of oligonucleotide microarray and direct sequencing may be necessary to accurately identify p53 gene alterations in FFPE breast cancer. The p53 GeneChip cannot be used to detect exon 4 polymorphisms (codon 72) in FFPE breast cancer tissue.
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Tripathi, Manish, Chidi Zacheaus, Kyle Doxtater, Fatemeh Keramatnia, Cuilan Gao, Murali Yallapu, Meena Jaggi, and Subhash Chauhan. "Z Probe, An Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues." Non-Coding RNA 4, no. 3 (September 5, 2018): 20. http://dx.doi.org/10.3390/ncrna4030020.
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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.
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Schmitt, C., A. Walijew, P. Hewitt, and J. H. Harleman. "P06: Gene expression profiling in toxicological studies using FFPE tissues." Experimental and Toxicologic Pathology 61, no. 4 (July 2009): 404–5. http://dx.doi.org/10.1016/j.etp.2009.02.092.
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Magdeldin, Sameh, and Tadashi Yamamoto. "Toward deciphering proteomes of formalin-fixed paraffin-embedded (FFPE) tissues." PROTEOMICS 12, no. 7 (April 2012): 1045–58. http://dx.doi.org/10.1002/pmic.201100550.
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Simmons, Alan J., and Ken S. Lau. "Deciphering tumor heterogeneity from FFPE tissues: Its promise and challenges." Molecular & Cellular Oncology 4, no. 1 (November 18, 2016): e1260191. http://dx.doi.org/10.1080/23723556.2016.1260191.
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Bonin, Serena, Falk Hlubek, Jean Benhattar, Carsten Denkert, Manfred Dietel, Pedro L. Fernandez, Gerald Höfler, et al. "Multicentre validation study of nucleic acids extraction from FFPE tissues." Virchows Archiv 457, no. 3 (July 28, 2010): 309–17. http://dx.doi.org/10.1007/s00428-010-0917-5.
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Yi, Qing-qing, Rong Yang, Jun-feng Shi, Nai-yan Zeng, Dong-yu Liang, Shuang Sha, and Qing Chang. "Effect of preservation time of formalin-fixed paraffin-embedded tissues on extractable DNA and RNA quantity." Journal of International Medical Research 48, no. 6 (June 2020): 030006052093125. http://dx.doi.org/10.1177/0300060520931259.
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Objectives This study aimed to investigate the factors affecting the quantity of DNA and RNA extractable from human formalin-fixed paraffin-embedded (FFPE) tissues stored for different lengths of time. Methods We randomly selected 20 FFPE specimens harvested from hysteromyoma patients with uterine fibroids during 2010, 2015, and 2017 at the Department of Pathology, Jiading District Central Hospital Affiliated Shanghai University of Medicine and Health Sciences. DNA and RNA extractions were performed using a DNA/RNA FFPE kit. DNA and RNA concentrations and their OD260/OD280 ratios were determined by a NanoDrop 2000 spectrophotometer. The human β-globin gene and aldehyde dehydrogenase-2 (ALDH2) gene were amplified from nucleic acids using a LightCycler 480 Real-Time PCR System, and PCR amplification products were electrophoresed on 1% agarose gels. Results Specimens that were stored for longer showed more degradation and a reduced concentration of DNA and RNA after nucleic acid extraction. However, there was no significant difference in DNA or RNA purity. β-globin and ALDH2 genes could be amplified from more than 99% of specimens. Conclusion We found that FFPE tissues stored for longer had a reduced quantity of extractable DNA and RNA. However, these tissues could be used for the analysis of some small target genes.
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Harbeck, Nadia, Inko Nimmrich, Arndt Hartmann, Jeffrey S. Ross, Tanja Cufer, Robert Grützmann, Glen Kristiansen, et al. "Multicenter Study Using Paraffin-Embedded Tumor Tissue Testing PITX2 DNA Methylation As a Marker for Outcome Prediction in Tamoxifen-Treated, Node-Negative Breast Cancer Patients." Journal of Clinical Oncology 26, no. 31 (November 1, 2008): 5036–42. http://dx.doi.org/10.1200/jco.2007.14.1697.
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Purpose We recently reported DNA methylation of the paired-like homeodomain transcription factor 2 (PITX2) gene to be strongly correlated with increased risk of recurrence in node-negative, hormone receptor–positive, tamoxifen-treated breast cancer patients using fresh frozen specimens. Aims of the present study were to establish determination of PITX2 methylation for routine analysis in formalin-fixed paraffin-embedded (FFPE) breast cancer tissue and to test PITX2 DNA methylation as a biomarker for outcome prediction in an independent patient cohort. Patients and Methods Real-time polymerase chain reaction (PCR) technology was validated for FFPE tissue by comparing methylation measurements in FFPE specimens with those in fresh frozen specimens from the same tumor. The impact of PITX2 methylation on time to distant metastasis was then evaluated in FFPE specimens from hormone receptor–positive, node-negative breast cancer patients (n = 399, adjuvant tamoxifen monotherapy). Results Reproducibility of the PCR assay in replicate measurements (rs ≥ 0.95; n = 150) and concordant measurements between fresh frozen and FFPE tissues (rs = 0.81; n = 89) were demonstrated. In a multivariate model, PITX2 methylation added significant information (hazard ratio = 2.35; 95% CI, 1.20 to 4.60) to established prognostic factors (tumor size, grade, and age). Conclusion PITX2 methylation can be reliably assessed by real-time PCR technology in FFPE tissue. Together with our earlier studies, we have accumulated substantial evidence that PITX2 methylation analysis holds promise as a practical assay for routine clinical use to predict outcome in node-negative, tamoxifen-treated breast cancer, which might allow, based on future validation studies, the identification of low-risk patients who may be treated by tamoxifen alone.
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Pełka, Kacper, Klaudia Klicka, Tomasz M. Grzywa, Agata Gondek, Janina M. Marczewska, Filip Garbicz, Kinga Szczepaniak, Wiktor Paskal, and Paweł K. Włodarski. "miR-96-5p, miR-134-5p, miR-181b-5p and miR-200b-3p heterogenous expression in sites of prostate cancer versus benign prostate hyperplasia—archival samples study." Histochemistry and Cell Biology 155, no. 3 (December 17, 2020): 423–33. http://dx.doi.org/10.1007/s00418-020-01941-2.
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AbstractMicroRNAs are involved in various pathologies including cancer. The aim of the study was to assess the level of expression of miR-96-5p, -134-5p, -181b-5p, -200b-3p in FFPE samples of prostate cancer, adjacent cancer-free tissue, and benign prostatic hyperplasia. Samples of 23 FFPE prostate cancer and 22 benign prostatic hyperplasias were dissected and HE stained. Compartments of tumor tissue and adjacent healthy glandular tissue were isolated from each sample using Laser Capture Microdissection. Total RNA was isolated from dissected tissues. Expression of miR-96-5p, miR-134-5p, 181b-5p, and miR-200b-3p was determined by real-time RT-qPCR method. The expression of miR-200b-3p was significantly higher in cancerous prostate: both in adenocarcinomatous glands and in the adjacent, apparently unaffected glands compared to BPH samples. The expression of miR-181b-5p was lower in in both prostate cancer tissues and adjacent tissue compared to BPH samples. Expression of miR-96-5p and miR-134-5p was lower in prostate cancer tissues compared to BPH. Levels of miR-96-5p, miR-134-5p, and 181b-5p negatively correlated with the Gleason score. Given further studies, miR-96-5p, miR-134-5p and especially miR-200b-3p and miR-181b-5p may differentiate BPH and PC.