From Formalin-fixed And Paraffin-embedded Tissues Research Articles

Bovine Meat and Milk Factor-like Sequences Are Frequently Detected in Renal Cell Carcinoma Tissues.

Previous studies have indicated a potential role of diet in the pathogenesis of renal cell carcinoma (RCC). Recently, circular bovine meat and milk factor (BMMF) DNAs have been identified in peritumoral tissues of human colon and breast cancers. Here, we investigated the prevalence of the DNA of these novel human pathogenic infectious agents in RCC and adjacent peritumoral renal tissues. DNA was extracted from formalin-fixed and paraffin-embedded (FFPE) RCC and peritumoral kidney tissues, including a test (n = 11) and a validation (n = 152) collection. BMMF1 and BMMF2 consensus primers were designed to screen for the presence of BMMF1- and BMMF2-like DNA. In addition, BMMF-specific PCR was performed on selected cases to test for the presence of additional regions of BMMF1 and BMMF2 genomes. A reference collection of hepatocellular carcinomas (HCCs; n = 60) and adjacent peritumoral liver tissues (n = 50) was also included. Our results demonstrated that BMMF1 and BMMF2 DNAs are frequently found in human RCC tissues and are particularly more prevalent in peritumoral kidney tissues. Of note, BMMF1 and BMMF2 genotype heterogeneity was higher in peritumoral kidney tissues compared to RCC tissues. This is the first study to directly test human FFPE tissues for BMMF1- and BMMF2-like DNA using consensus PCR and demonstrate BMMF DNA in neoplastic and peritumoral kidney tissues. The findings are in line with the recently proposed indirect etiopathogenetic role of BMMFs in, e.g., colorectal carcinogenesis. Follow-up studies are needed to explore the potential role of BMMFs in the etiopathogenesis of RCC.

Atypical Mycosis in Psittacine Birds: A Retrospective Study.

A retrospective study was conducted on parrots submitted from necropsy to the Department of Veterinary Pathology, School of Biosciences and Veterinary, University of Camerino, Italy, from 2007 to 2018. From a total of 2,153 parrots examined at post-mortem, four cases were diagnosed with atypical mycosis and were considered for determination of the fungus species by PCR. A Fischer's lovebird (Agapornis fischeri), Peach-faced lovebirds (Agapornis roseicollis), and two Blue and Gold Macaws (Ara ararauna) from four different aviaries died after some days of lethargy and ruffled feathers. Records of gross necropsy and histopathological exams (H&E, PAS, and Grocott stain) were described and biomolecular analyses were carried out. No specific gross lesions were appreciated at necropsy, while histopathology evidenced a systemic mycosis in several organs, particularly in the lungs. In affected organs, broad and non-septate hyphae, suggestive of mycoses, were observed. Molecularly, Mucor racemosus (Fischer's lovebird) and M. circinelloides (Peach-faced lovebirds) were identified from formalin-fixed and paraffin-embedded (FFPE) lung and liver tissue. In addition, Alternaria alternata and Fusicladium spp. (respectively in male and female Blue and Gold macaws) were identified in FFPE tissue from several organs; whereas the role of Mucor spp. as true pathogens is well-demonstrated, and the behavior of A. alternata and Fusicladium spp. in macaws as opportunistic pathogens have been discussed. To our knowledge, this report is the first one reporting mucormycosis caused by M. racemosus and M. circinelloides in lovebirds, and A. alternata and Fusicladium spp. in macaws.

Nested PCR for the Diagnosis of Feline Sporotrichosis From Formalin-Fixed and Paraffin-Embedded Samples Using Different DNA Extraction Protocols.

Sporotrichosis is a chronic, cosmopolitan granulomatous mycosis that affects humans and animals. The infection is caused by the dimorphic fungi Sporothrix sp. The aims of the present study were to evaluate, standardize and validate a nested PCR technique using two DNA purification kits for the extraction of DNA from formalin fixed and paraffin-embedded tissues (FFPE) for Sporothrix sp. detection. FFPE mycological culture pellet samples of different Sporothrix species (S. chilensis, S. mexicana, S. pallida, S. globosa, S. brasiliensis and S. schenckii) were used as positive controls and clinical FFPE tissue samples of animals positive for Cryptococcus sp., Leishmania infantum and Histoplasma sp. were used as negative controls. Ten clinical FFPE skin samples from cats with sporotrichosis were used to validate the nested PCR. These samples were cut into two distinct paraffin sectioning protocols (5 and 16 μm thick). The paraffin sections were subjected to two different DNA extraction kits (chemical and thermal extractions). A nested PCR was performed on the extracted DNA to identify the genus Sporothrix. The chemical extraction protocol with the 5 μm thick paraffin section was more effective in extracting DNA from Sporothrix sp. from FFPE samples and the nested PCR technique showed the highest sensitivities (100% in the positive controls and of 50% in the skin samples of cats) and specificity (100%). Therefore, the nested PCR using this protocol has great potential to be applied in Sporothrix sp. diagnosis in FFPE samples of cats.

Fatal avian malaria in captive Atlantic puffins (Fratercula arctica) in Switzerland.

Avian malaria is a vector-borne disease caused by Plasmodium species, which may affect a broad spectrum of bird families worldwide. In most endemic and migratory birds, Plasmodium infections seem not to cause severe harm; however, non-indigenous species kept in human care such as penguins may experience high morbidity and mortality rates. Fatal avian malaria may also occur in other non-native seabirds such as puffins (Fratercula spp.), but reported cases are scarce.The aim of this study was to analyze seven cases of sudden death in captive Atlantic puffins (Fratercula arctica) at Berne Animal Park in Switzerland between 2010 and 2020, and to determine the involvement of haemosporidian parasites in the fatal outcome. In all cases, lymphoplasmacytic inflammation, necrotic lesions in several organs and presence of protozoan stages within tissues/erythrocytes or accumulation of iron-based pigment were observed histologically. A one-step multiplex PCR designed to simultaneously detect and discriminate haemosporidia belonging to the genera Plasmodium, Haemoproteus and Leucocytozoon, and a nested PCR detecting Plasmodium and Haemoproteus infections were performed on DNA extracted from formalin-fixed and paraffin-embedded (FFPE) or fresh liver and spleen tissues from five and two birds, respectively. Plasmodium spp. DNA was detected in the tissues from six of seven birds by the one-step multiplex PCR and in five of seven individuals by the nested PCR protocol. Direct sequencing of the amplification products allowed the molecular identification of Plasmodium relictum SGS1 as the involved species in three individuals and Plasmodium matutinum LINN1 in two of these fatal cases. In one bird, no haemosporidian DNA could be amplified from FFPE tissues despite of suggestive histopathological findings. These results indicate that avian malaria represents an important cause of death in captive puffins and it should be considered as a differential diagnosis in unclear or fatal cases in this threatened bird species.

Molecular identification of pathogenic fungi in formalin-fixed and paraffin-embedded tissues.

Introduction. Histopathological examination (HPE) of tissue helps in the diagnosis of invasive fungal infections (IFIs) but cannot identify the fungus to the genus/species levelGap Statement Available protocols for the molecular identification of fungi from formalin-fixed and paraffin-embedded (FFPE) tissues have limitations in terms of extraction and target selection, and standardisation.Aim. Development of sequence-based fungal identification protocol after extraction of DNA from formalin-fixed and paraffin-embedded (FFPE) tissues.Methodology. A total of 63 FFPE tissues from histopathology proven IFI cases were used to standardize the DNA extraction (commercial QIAamp kit-based extraction and conventional phenol-chloroform-isoamyl alcohol [PCI] method) and sequence-based fungal identification protocols. The PCR targeted different ribosomal DNA (rDNA) regions including complete internal transcribed spacer (ITS1-5.8S-ITS2), separate ITS1 and ITS2, 18S and D1/D2 of 28S regions. Semi-nested PCR targeting Mucorales-specific 18S rDNA region was performed in tissues having aseptate hyphae. The optimized ITS1-PCR protocol was evaluated in 119 FFPE tissues containing septate hyphae or yeast, and Mucorales-specific semi-nested PCR in 126 FFPE tissues containing aseptate hyphae.Results. The DNA yield by conventional PCI method was significantly higher (P<0.0001) than commercial kit, though the quality of DNA was similar by both protocols. The test accuracy was best while using ITS1 (61.9 %) as the target compared to 7.9, 29.9 and 22.2 % on targeting ITS1-5.8S-ITS2, ITS2, the D1/D2 region of 28S, respectively. The test accuracies of ITS1-PCR in tissues containing septate hyphae, aseptate hyphae and yeasts were 75.5, 18.7 and 100 %, respectively. The amplification (targeting ITS1 region) improved by increasing the thickness of tissue section (up to 50 µm) used for DNA extraction. ITS1-PCR protocol could amplify fungal DNA in 76 (63.8 %) tissues and Mucorales-specific semi-nested PCR in 86 (68.3 %) tissues.Conclusion. Conventional PCI-based DNA extraction from thick tissue (50 µm) may be used until optimal commercial fungal DNA extraction kit is developed. Subsequent ITS1-PCR for septate fungi and yeast, and semi-nested PCR targeting 18S rDNA for Mucorales are recommended to identify the fungus in FFPE tissues.

Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin-embedded skin tissue.

The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. Over a 30-year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953-1.000). The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.

UPLC‑MS/MS‑based metabolomic characterization and comparison of pancreatic adenocarcinoma tissues using formalin‑fixed, paraffin‑embedded and optimal cutting temperature‑embedded materials

The purpose of the present study was to compare metabolites from formalin-fixed and paraffin-embedded (FFPE) pancreatic tissue blocks with those identified in optimal cutting temperature (OCT)-embedded pancreatic tissue blocks. Thus, ultra-performance liquid chromatograph-mass spectrometry/mass spectrometry-based metabolic profiling was performed in paired frozen (n=13) and FFPE (n=13) human pancreatic adenocarcinoma tissue samples, in addition to their benign counterparts. A total of 206 metabolites were identified in both OCT-embedded and FFPE tissue samples. The method feasibility was confirmed through reproducibility and a consistency assessment. Partial least-squares discriminant analysis and heatmap analysis reliably distinguished tumor and normal tissue phenotypes. The expression of 10 compounds, including N-acetylaspartate and creatinine, was significantly different in both OCT-embedded and FFPE tumor samples. These ten compounds may be viable candidate biomarkers of malignant pancreatic tissues. The super-categories to which they belonged exhibited no significant differences between FFPE and OCT-embedded samples. Furthermore, purine, arginine and proline, and pyrimidine metabolism used a shared pathway found in both OCT-embedded and FFPE tissue samples. These results supported the notion that metabolomic data acquired from FFPE pancreatic cancer specimens are reliable for use in retrospective and clinical studies.

On-Tissue Hydrogel-Mediated Nondestructive Proteomic Characterization: Application to fr/fr and FFPE Tissues and Insights for Quantitative Proteomics Using a Case of Cardiac Myxoma.

The application of a methodology for quantitative protein analysis from formalin-fixed and paraffin-embedded (FFPE) tissue by using hydrogels. Miniaturized polymeric gels are placed onto histologically defined tissue regions in order to perform localized digestion for bottom-up proteomics. Hydrogel-extracted peptides are then labeled with tandem mass tags (TMT) reagents for relative protein quantification. A cardiac myxoma biopsy is used. Multiple hydrogels, incorporating the proteolytic enzyme trypsin, are placed on serial tissue sections, and processed for digestion and TMT derivatization. SCX fractionation before LC-MS/MS analysis and bioinformatics analysis are carried out. Two histologically different areas on both FFPE and frozen sections of the same cardiac myxoma biopsy are compared. In total, 1949 (FFPE) and 2491 (frozen) proteins are identified, with a total overlap of 56%. The quantitative comparison highlighted 15 (FFPE) and 138 (frozen) differentially expressed proteins between myxoma regions. The methodology successfully detects numerous protein signals from FFPE and frozen specimens and is able to differentiate between tissue regions. A fast and reliable tissue preparation for quantitative protein analysis by minimum sample manipulation is developed. This offers an option for on-tissue proteomics analysis while preserving the inherent spatial information on the tissue.

What Does This Mutation Mean? The Tools and Pitfalls of Variant Interpretation in Lymphoid Malignancies.

High throughput sequencing (HTS) is increasingly important in determining cancer diagnoses, with subsequent prognostic and therapeutic implications. The biology of cancer is becoming increasingly deciphered and it is clear that therapy needs to be individually tailored. Whilst translational research plays an important role in lymphoid malignancies, few guidelines exist to guide biologists and routine laboratories through this constantly evolving field. In this article, we review the challenges of interpreting HTS in lymphoid malignancies and provide a toolkit to interpret single nucleotide variants obtained from HTS. We define the pre-analytical issues such as sequencing DNA obtained from formalin-fixed and paraffin-embedded tissue (FFPE), the acquisition of germline DNA, or the bioinformatic pitfalls, the analytical issues encountered and how to manage them. We describe the main constitutional and cancer databases, their characteristics and limitations, with an emphasis on variant interpretation in lymphoid malignancies. Finally, we discuss the challenges of predictions that one can make using in silico or in vitro modelling, pharmacogenomic screening, and the limits of those prediction tools. This description of the current status in genomic interpretation highlights the need for new large databases and international collaboration in the lymphoma field.

A Metastatic Biology Gene Expression Assay to Predict the Risk of Distant Metastases in Patients With Localized Prostate Cancer Treated With Primary Radical Treatment

Approximately 20% of patients with organ confined prostate cancer (PCa) will develop disease recurrence following radical treatment (surgery or external beam radiotherapy (EBRT)). We hypothesized that a molecular subgroup of early PCa may have metastatic potential at presentation, resulting in disease recurrence. Using unsupervised hierarchical clustering of gene expression from a PCa dataset we identified a novel molecular subgroup with a transcriptional profile similar to metastatic disease. We developed a 70-gene expression assay to prospectively identify patients within the subgroup from formalin fixed and paraffin embedded tissue (FFPE). Initial assessment found the assay to be prognostic in three independent publicly available prostatectomy datasets. We therefore assessed the prognostic value of the assay in FFPE clinical samples collected from multiple international sites. FFPE tumor resections and tumor biopsy specimens were obtained from 322 surgical patients and 248 patients treated with EBRT. Regions of highest Gleason grade were identified for macrodissection, RNA extraction, and gene expression analysis. Samples were dichotomized as metastatic biology assay positive or negative using a pre-specified cut-off. The association of assay results with biochemical failure (BF) and distant metastases (DM) was tested on multivariate (MVA). The assay was significantly associated with BF on MVA (HR = 1.59 [1.11-2.29]; P = 0.0128), (HR = 1.86 [1.07-3.22]; P = 0.0277) and DM on MVA (HR = 3.09 [1.70-5.61]; P = 0.0002), (HR = 2.83 [1.13-7.11]; P = 0.0273) for surgery and EBRT cohorts respectively. Importantly, in a combined model, the assay demonstrated additional information to the commonly used CAPRA clinical tool for prediction of DM, HR = 2.72 [2.10-3.51]; P < 0.0001, and HR = 2.72 [1.42-5.20]; P = 0.0026 (Prostate Metastatic Assay combined with CAPRA-S and CAPRA, respectively). The metastatic biology assay predicts BF and DM in PCa patients treated with surgery or EBRT. The assay may help to select patients at risk of metastatic disease for additional treatment aimed at preventing disease recurrence.

Estimation of age-related DNA degradation from formalin-fixed and paraffin-embedded tissue according to the extraction methods.

Techniques for the extraction and use of nucleic acids from formalin-fixed and paraffin-embedded (FFPE) tissues, preserved over long time periods in libraries, have been developed. However, DNA extracted from FFPE tissues is generally damaged, and long-term storage may affect DNA quality. Therefore, it is important to elucidate the effect of long-term storage on FFPE tissues and evaluate the techniques used to extract DNA from them. In the present study, the yield, purity, and integrity of DNA in FFPE tissue samples was evaluated. Two DNA extraction techniques were used: A silica-binding DNA collection method using QIAamp DNA FFPE Tissue kit (QIA) and a total tissue DNA collection method using a WaxFree DNA extraction kit (WAX). A total of 25 FFPE tissues from lung adenocarcinomas were studied, which had been surgically resected and fixed at Okayama University Hospital prior to examination and subsequent storage at room temperature for 0.5, 3, 6, 9 and 12 years. Extracted DNA was quantified using ultraviolet absorbance, fluorescent dye, and quantitative polymerase chain reaction (qPCR). The quality of the DNA was defined by the absorbance ratio of 260 to 280 nm (A260/280) and Q-score, which is the quantitative value of qPCR product size ratio. The results demonstrated that the yield of total DNA extracted using WAX was significantly greater than when QIA was used (P<0.01); however, DNA extracted using WAX included more contaminants and was significantly more fragmented compared with DNA extracted using QIA (P<0.01). Aging had no significant effect on absolute DNA yield or DNA purity, although it did significantly contribute to increased DNA degradation for both QIA and WAX extraction (QIA P=0.02, WAX P=0.03; 0.5 years vs. 3 years, QIA P<0.01, WAX P=0.03; 9 years vs. 12 years). Both extraction methods are viable depending on whether high yield or high quality of extracted DNA is required. However, due to the increased degradation with age, storage time limits the available DNA in FFPE tissues regardless of the extraction method.

A metastatic biology gene expression assay to predict the risk of distant metastases in patients with localized prostate cancer treated with primary radical treatment.

11 Background: Approximately 20% of patients with organ confined prostate cancer (PCa) will develop disease recurrence following radical treatment (surgery or external beam radiotherapy (EBRT)). We hypothesized that a molecular subgroup of early PCa may have metastatic potential at presentation, resulting in disease recurrence. Methods: Using unsupervised hierarchical clustering of gene expression from a PCa dataset we identified a novel molecular subgroup with a transcriptional profile similar to metastatic disease. We developed a 70 gene expression assay to prospectively identify patients within the subgroup from formalin fixed and paraffin embedded tissue (FFPE). Initial assessment found the assay to be prognostic in three independent publicly available prostatectomy datasets. We therefore assessed the prognostic value of the assay in FFPE clinical samples collected from multiple international sites. FFPE tumor resections and tumor biopsy specimens were obtained from 322 surgical patients and 248 patients treated with EBRT. Regions of highest Gleason grade were identified for macrodissection, RNA extraction and gene expression analysis. Samples were dichotomized as metastatic biology assay positive or negative using a pre-specified cut-off. The association of assay results with biochemical failure (BF) and distant metastases (DM) was tested on multivariate (MVA). Results: The assay was significantly associated with BF on MVA (HR 1.67 [1.16-2.38]; p = 0.0059), (HR 2.26 [1.26-4.04]; p = 0.0062) and DM on MVA (HR 3.39 [1.88-6.12]; p = 0.0001), (HR 3.26 [1.27-8.30]; p = 0.0137) for surgery and EBRT cohorts respectively. Importantly, in a combined model, the assay demonstrated additional information to the commonly used CAPRA clinical tool for prediction of DM, HR 2.72 [2.10-3.51]; p &lt; 0.0001, and HR 2.72 [1.42-5.20]; p = 0.0026 (Prostate Metastatic Assay combined with CAPRA-S and CAPRA respectively). Conclusions: The metastatic biology assay predicts BF and DM in PCa patients treated with surgery or EBRT. The assay may help to select patients at risk of metastatic disease for additional treatment aimed at preventing disease recurrence.

Comparison of Analytical Characteristics of Commercial and in-House Methods for DNA Isolation from Paraffin Histology Blocks

One of the hotspots in clinical research today is molecular genetic analysis of structural DNA alterations. Working with DNA obtained from formalin-fixed and paraffin-embedded (FFPE) tissue specimens is particularly challenging, due to cross-linking and fragmentation of DNA. We performed a comparative analysis of DNA extraction methods from FFPE tissue using two in-house protocols and Qiagen (QIAamp DNA FFPE Tissue Kit) and Roche (High Pure FFPET DNA Isolation Kit) commercial kits presented in the Russian market and used in clinical practice. To assess the quantity and quality of the isolated DNA, we used the real-time PCR to rate DNA yield, the inhibited impurity content and the degree of fragmentation. Our findings may be useful for a medical laboratory that performs testing of somatic mutations for the targeted therapy selection, and researchers who specialized in tumor genome structure studies in respect to patient prognosis and prediction of the sensitivity or resistance of tumor cells to therapy.

Diagnostic value of a semi-nested PCR for the diagnosis of mucormycosis and aspergillosis from paraffin-embedded tissue: A single center experience

ObjectiveThe main aim of our study was to investigate the diagnostic value of a molecular method for the diagnosis of mucormycosis and aspergillosis from formalin-fixed and paraffin-embedded (FFPE) tissues. MethodsA retrospective chart review identified all cases with histology reports mentioning the presence of fungi with morphological characteristics of either Aspergillus or mucormycetes, for the period 2005–2012. Paraffin blocks were retrieved from the archives of the Department of Pathology. A semi-nested PCR specific for the detection of mucormycetes and Aspergillus species was applied in FFPE tissue from the above blocks. Results were compared with those of histological (gold standard) and microbiological methods. ResultsTwenty cases with fungal hyphae in tissue were revealed. Mucormycetes were detected in 9 cases (45%) by PCR, in only 4 of which culture was available. Species of Aspergillus were detected in 8 cases (40%) by PCR, two of which were co-infection with mucormycetes. Five patients had other fungi, non-detectable with this specific PCR. At least one sample per patient was positive by PCR. Seven out of 30 samples tested overall were false negative. The calculated sensitivity of this method in our setting was 79.3% (95% CI: 60.3–91.9%); specificity was 100%. ConclusionsThe specific PCR used appears to be an easy and useful tool for the prompt and accurate diagnosis of mucormycosis and aspergillosis, in combination with histology and direct examination. Mucormycosis was more frequent than aspergillosis during the study period, highlighting the importance of continuous epidemiological surveillance of these serious infections.

PAT-ChIP coupled with laser microdissection allows the study of chromatin in selected cell populations from paraffin-embedded patient samples.

BackgroundThe recent introduction of pathology tissue-chromatin immunoprecipitation (PAT-ChIP), a technique allowing chromatin immunoprecipitation from formalin-fixed and paraffin-embedded (FFPE) tissues, has expanded the application potential of epigenetic studies in tissue samples. However, FFPE tissue section analysis is strongly limited by tissue heterogeneity, which hinders linking the observed epigenetic events to the corresponding cellular population. Thus, ideally, to take full advantage of PAT-ChIP approaches, procedures able to increase the purity and homogeneity of cell populations from FFPE tissues are required.ResultsIn this study, we tested the use of both core needle biopsies (CNBs) and laser microdissection (LMD), evaluating the compatibility of these methods with the PAT-ChIP procedure. Modifications of the original protocols were introduced in order to increase reproducibility and reduce experimental time. We first demonstrated that chromatin can be prepared and effectively immunoprecipitated starting from 0.6-mm-diameter CNBs. Subsequently, in order to assess the applicability of PAT-ChIP to LMD samples, we tested the effects of hematoxylin or eosin staining on chromatin extraction and immunoprecipitation, as well as the reproducibility of our technique when using particularly low quantities of starting material. Finally, we carried out the PAT-ChIP using chromatin extracted from either normal tissue or neoplastic lesions, the latter obtained by LMD from FFPE lung sections derived from mutant K-rasv12 transgenic mice or from human adeno- or squamous lung carcinoma samples. Well characterized histone post-translational modifications (HPTMs), such as H3K4me3, H3K27me3, H3K27Ac, and H3K9me3, were specifically immunoselected, as well as the CTCF transcription factor and RNA polymerase II (Pol II).ConclusionsEpigenetic profiling can be performed on enriched cell populations obtained from FFPE tissue sections. The improved PAT-ChIP protocol will be used for the discovery and/or validation of novel epigenetic biomarkers in FFPE human samples.

Performance Evaluation of Kits for Bisulfite-Conversion of DNA from Tissues, Cell Lines, FFPE Tissues, Aspirates, Lavages, Effusions, Plasma, Serum, and Urine

DNA methylation analyses usually require a preceding bisulfite conversion of the DNA. The choice of an appropriate kit for a specific application should be based on the specific performance requirements with regard to the respective sample material. In this study, the performance of nine kits was evaluated: EpiTect Fast FFPE Bisulfite Kit, EpiTect Bisulfite Kit, EpiTect Fast DNA Bisulfite Kit (Qiagen), EZ DNA Methylation-Gold Kit, EZ DNA Methylation-Direct Kit, EZ DNA Methylation-Lightning Kit (Zymo Research), innuCONVERT Bisulfite All-In-One Kit, innuCONVERT Bisulfite Basic Kit, innuCONVERT Bisulfite Body Fluids Kit (Analytik Jena). The kit performance was compared with regard to DNA yield, DNA degradation, DNA purity, conversion efficiency, stability and handling using qPCR, UV, clone sequencing, HPLC, and agarose gel electrophoresis. All kits yielded highly pure DNA suitable for PCR analyses without PCR inhibition. Significantly higher yields were obtained when using the EZ DNA Methylation-Gold Kit and the innuCONVERT Bisulfite kits. Conversion efficiency ranged from 98.7% (EpiTect Bisulfite Kit) to 99.9% (EZ DNA Methylation-Direct Kit). The inappropriate conversion of methylated cytosines to thymines varied between 0.9% (innuCONVERT Bisulfite kits) and 2.7% (EZ DNA Methylation-Direct Kit). Time-to-result ranged from 131 min (innuCONVERT kits) to 402 min (EpiTect Bisulfite Kit). Hands-on-time was between 66 min (EZ DNA Methylation-Lightning Kit) and 104 min (EpiTect Fast FFPE and Fast DNA Bisulfite kits). Highest yields from formalin-fixed and paraffin-embedded (FFPE) tissue sections without prior extraction were obtained using the innuCONVERT Bisulfite All-In-One Kit while the EZ DNA Methylation-Direct Kit yielded DNA with only low PCR-amplifiability. The innuCONVERT Bisulfite All-In-One Kit exhibited the highest versatility regarding different input sample materials (extracted DNA, tissue, FFPE tissue, cell lines, urine sediment, and cellular fractions of bronchial aspirates, pleural effusions, ascites). The innuCONVERT Bisulfite Body Fluids Kit allowed for the analysis of 3 ml plasma, serum, ascites, pleural effusions and urine.

Comparison between the Effectiveness of Polymerase Chain Reaction and In Situ Hybridization in Detecting the Presence of Pathogenic Fungi by Using the Preserved DNA in Formalin-Fixed and Paraffin-Embedded Tissues

In situ hybridization (ISH) has been recognized as an important technique for identifying the causative fungi in the foci of infection observed in histopathological specimens which was processed from formalin-fixed and paraffin-embedded (FFPE) tissues. However, few basic studies have conducted an evaluation of the DNA preservation for use in ISH in comparison to polymerase chain reaction (PCR). The latter is a DNA amplification-based modality. In the present study, we analyzed 65 FFPE lung tissue specimens collected from autopsy cases for comparing the usefulness of ISH and PCR analysis. As a result, the positive identification rates for PCR were strikingly low; a majority of these results can be assumed to be false negative because the presence of fungi had been confirmed by histopathological analysis. In contrast, panfungal ISH targeting of the 28S rRNA showed a higher sensitivity than the 230-bp panfungal PCR primers did (80.0% versus 4.6%, respectively). Furthermore, over 60% of the samples we examined showed a favorable intensity of the ISH signal. Therefore, in conventional postmortem FFPE tissues, the state of DNA preservation may be more favorable for ISH than PCR analysis.

MiRNAs Expression Analysis in Paired Fresh/Frozen and Dissected Formalin Fixed and Paraffin Embedded Glioblastoma Using Real-Time PCR

miRNAs are small molecules involved in gene regulation. Each tissue shows a characteristic miRNAs epression profile that could be altered during neoplastic transformation. Glioblastoma is the most aggressive brain tumour of the adult with a high rate of mortality. Recognizing a specific pattern of miRNAs for GBM could provide further boost for target therapy. The availability of fresh tissue for brain specimens is often limited and for this reason the possibility of starting from formalin fixed and paraffin embedded tissue (FFPE) could very helpful even in miRNAs expression analysis. We analysed a panel of 19 miRNAs in 30 paired samples starting both from FFPE and Fresh/Frozen material. Our data revealed that there is a good correlation in results obtained from FFPE in comparison with those obtained analysing miRNAs extracted from Fresh/Frozen specimen. In the few cases with a not good correlation value we noticed that the discrepancy could be due to dissection performed in FFPE samples. To the best of our knowledge this is the first paper demonstrating that the results obtained in miRNAs analysis using Real-Time PCR starting from FFPE specimens of glioblastoma are comparable with those obtained in Fresh/Frozen samples.

Abstract 2098: Reducing sequence artefacts from FFPE DNA for cancer diagnostics

Abstract Non-reproducible sequence artefacts have been frequently detected in DNA extracted from formalin-fixed and paraffin-embedded (FFPE) tissues. Studies of ancient DNA reveal that deamination of cytosine to uracil is a major cause of DNA damage. We hypothesised that similar lesions may occur in FFPE DNA and examined the role of uracil lesions on generation of transitional G:C to A:T artefacts in FFPE DNA by treatment with uracil DNA glycosylase (UDG). High resolution melting (HRM)-based assays were used for rapid and cost-effective screening of artefacts. The artefact rate was assessed by limited copy number-HRM (LCN-HRM) in FFPE DNA samples in which multiple G:C to A:T artefacts were found. The effect of UDG treatment on detection of true sequence changes was examined by testing mutational hotspots at the EGFR, KRAS and AKT1 genes. A high HRM positive rate as judged by the formation of heteroduplexes was found in FFPE DNA by LCN-HRM. This was markedly reduced after treatment with UDG. UDG treatment specifically suppressed the formation of artefacts in FFPE DNA and did not affect the detection of true KRAS codon 12 and EGFR exon 19 and 20 mutations. In conclusion, this study reports for the first time that uracil lesions due to cytosine deamination is one of the major causes of artefacts in FFPE DNA and the use of uracil-DNA-glycosylase that removes uracil lesions on DNA can lead to a significant reduction of artefacts. This finding will thus greatly improve biomarker studies using DNA samples derived from FFPE tissues, which has immediate implications especially to diagnostic settings. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2098. doi:1538-7445.AM2012-2098

Successful Protein Extraction from Over-Fixed and Long-Term Stored Formalin-Fixed Tissues

One of the major breakthroughs in molecular pathology during the last decade was the successful extraction of full-length proteins from formalin-fixed and paraffin-embedded (FFPE) clinical tissues. However, only limited data are available for the protein extraction efficiency of over-fixed tissues and FFPE blocks that had been stored for more than 15 years in pathology archives. In this study we evaluated the protein extraction efficiency of FFPE tissues which had been formalin-fixed for up to 144 hours and tissue blocks that were stored for 20 years, comparing an established and a new commercial buffer system. Although there is a decrease in protein yield with increasing fixation time, the new buffer system allows a protein recovery of 66% from 144 hours fixed tissues compared to tissues that were fixed for 6 hours. Using the established extraction procedure, less than 50% protein recovery was seen. Similarly, the protein extraction efficiency decreases with longer storage times of the paraffin blocks. Comparing the two buffer systems, we found that 50% more proteins can be extracted from FFPE blocks that were stored for 20 years when the new buffer system is used. Taken together, our data show that the new buffer system is superior compared to the established one. Because tissue fixation times vary in the routine clinical setting and pathology archives contain billions of FFPE tissues blocks, our data are highly relevant for research, diagnosis, and treatment of disease.