Embedded Tissue Sections Research Articles

Genetic Variations of Selected Genes Using Target Deep Sequencing in Colorectal Cancer Patients

Background: Colorectal carcinoma (CRC) is a burden problem in a developing country like Egypt since patients are usually admitted in late stage with bad prognosis and short overall survival. Because of genetic predisposition of CRC and introduction of advanced molecular techniques, efforts are directed to screen for potential pathogenic or disease-causing variants in CRC patients Methods: DNA was isolated from formalin fixed paraffin embedded tissue sections collected from 24 CRC confirmed diagnosed patients. TruSight CRC panel (Illumina) was used for detection of different variants in 15 genes. The generated reads were obtained from Illumina Miseq were clustered into single nucleotide polymorphism (SNPs) and small insertions/deletions (Indels). Further pathogenic variants with somatic and germline mutations were identified according to the recommended criteria. Some CRC patients were subjected to anti-EGFR target therapy. Results: Most of the variants were detected in TP53 gene 140 variants (65%); 105 short deletions none of them was pathogenic, 29 missense mutations and 6 SNPs at splicing sites. Next, ERBB2 has got 17 variants (8.8%) (missense and splicing), 8 of them were damaging disease causing variants. Besides, 16 pathogenic variants were identified in 12 patients (6 in TP53 and 7 in KRAS). Some pathogenic variants were not reported before in CRC e.g. TP53 C>A, rs121912654, Val157Phe. Additionally, patients carried different KRAS wild mutations showed variable response to anti-EGFR target therapy. Conclusion: The most affected pathway in CRC was TP53 pathway followed by ERBB2, NRAS, KRAS and PIK3CA genes. Variable response to target therapy suggested dependence on the type of pathogenic variant identified, also a possible role of ERBB2 which had a significant variant frequency.

Computer Assisted Immunohistochemical Score Prediction via Simplified Image Acquisition Technique

Background: techniques of image analysis have been used extensively to minimize interobserver variation of immunohistochemical scoring, yet; image acquisition procedures are often demanding, expensive and laborious. This study aims to assess the validity of image analysis to predict human observer’s score with a simplified image acquisition technique. Materials and methods: formalin fixed- paraffin embedded tissue sections for ameloblastomas and basal cell carcinomas were immunohistochemically stained with monoclonal antibodies to MMP-2 and MMP-9. The extent of antibody positivity was quantified using Imagej® based application on low power photomicrographs obtained with a conventional camera. Results of the software were employed to predict human visual scoring results with stepwise multiple regression analysis. Results: the overall prediction of epithelial score depicted as r square value was 0.26 (p<0.001) which was obviously higher than that of stromal score (0.10; p<0.01). Epithelial and stromal MMP-2 score prediction was generally higher than that of MMP-9. Collectively, ameloblastomas had a more efficient score prediction compared to basal cell carcinomas. Conclusion: there is a considerable variability in the prediction capacity of the technique with respect to different antibodies, different tumors and cellular versus stromal score.

Histomorphological spectrum of salivary gland tumors in a tertiary care hospital—A retrospective study

Background: Salivary gland tumors are rare, accounting for 3–10% of the total tumors of head and neck region. Approximately 80% of the salivary gland tumors are found in the parotid gland and 10–15% in the submandibular gland. Around 80% of parotid tumors and 50% of submandibular tumors are benign. Salivary gland tumors can show a striking range of morphological diversity between different tumor types and sometimes within an individual tumor mass. Hence, it is important to identify various histomorphological patterns and classify them accordingly. Objectives: The aim of this study was to recognize various histomorphological patterns of salivary gland tumors, their frequency, age, and site distribution. Materials and Methods: It was a retrospective study of 96 cases of salivary gland tumors, diagnosed on histopathological examination over a period of 2 years (July 2012 to June 2014) in the Department of Pathology of a tertiary care hospital. Histopathological examination was done on formalin fixed, paraffin embedded tissue sections stained with hematoxylin and eosin. Result: Out of 96 cases, 60 (62.5%) were benign and 36 (37.5%) were malignant. Benign tumors were frequent in the 3rd and 4th decade, whereas malignant tumors were common in 4th and 5th decades of life. Male:female ratio was 1.6:1. The most common site of occurrence for both benign and malignant tumors was the parotid gland. Pleomorphic adenoma was the most common benign tumor. Mucoepidermoid carcinoma (36.1%) was the most common malignant salivary gland tumor. Parotid was the most common site for mucoepidermoid carcinoma. Conclusion: Salivary gland tumors are relatively rare. Benign salivary gland tumors are more common than malignant ones. There is a male preponderance. The Parotid gland is the commonest site for occurrence of salivary gland tumors. Pleomorphic adenoma is the most frequent benign tumor whereas mucoepidermoid carcinoma is the most common malignant tumor of salivary glands. Histopathological examination is mandatory in the diagnosis of salivary gland tumors as they have a very wide spectrum of morphological patterns.

Histochemical Staining of Collagen and Identification of Its Subtypes by Picrosirius Red Dye in Mouse Reproductive Tissues.

Collagen is one of the foremost components of tissue extracellular matrix (ECM). It provides strength, elasticity and architecture to the tissue enabling it to bear the wear and tear from external factors like physical stress as well as internal stress factors like inflammation or other pathological conditions. During normal pregnancy or pregnancy related pathological conditions like preterm premature rupture of membranes (PPROM), collagen of the fetal membrane undergoes dynamic remodeling defining biochemical properties of the fetal membrane. The protocol in this article describes the histochemical method to stain total collagen by Picrosirius red stain which is a simple, quick and reliable method. This protocol can be used on paraformaldehyde (PFA) and formaldehyde fixed paraffin embedded tissue sections. We further describe the staining and distribution of collagen in different mouse reproductive tissues and also demonstrate how this technique in combination with polarization microscopy is useful to detect the distribution of different subtypes of collagen.

Characterization of lymphocyte Infiltration in Molar pregnancies

Background: In normal pregnancy around 30% of stromal cells in decidualised endometrium are leucocytes, lymphocyte subtypes may play a role in the pathogenesis of molar pregnancies. The aim of the study is t o investigate the pattern of infiltrating lymphocytes in paraffin embedded tissue sections obtained from molar pregnancies. Material and Methods: This study included thirty-five archived paraffin-embedded samples of molar pregnancies; divided into 16 incomplete and 19 complete hydatid mole. Then dual immunofluorescence staining was used for phenotyping of lymphocytic infiltrate (CD3- CD19) and (CD4-CD8). Independent sample t-test was used to compare the mean cell counts between different study groups. Results: There is higher T cells (CD3) infiltrate in complete hydatid mole than normal placental tissue (p=0.003) and not significant with incomplete hydatid mole (p=0.089). Plasma cells (CD19) were higher in both complete hydatid mole CHM (p=0.012) and incomplete hydatid mole iCHM (p=0.013). Cytotoxic cells (CD8) were none significant in all groups while Helper cells (CD4) were significantly higher in CHM (p=0.017) and iCHM (p=0.025). Conclusion: The higher tissue infiltration with plasma cells and helper cells in both CHM and iCHM highlight the importance of these cells in molar pregnancy pathogenesis.

Extraskeletal Myxoid Chondrosarcomas are not Characterized by Loss of INI1/SMARCB1: Immunohistochemical Analysis of 16 Cases

Background: Extraskeletal myxoid chondrosarcoma (EMC) is a rare soft tissue sarcoma, characterized by t (9; 22) translocation, including EWSR1 and NR4A3 gene rearrangements, observed in most cases. On histopathologic examination, an EMC has certain diagnostic mimics, such as myoepithelial tumors, epithelioid malignant peripheral nerve sheath tumor and epithelioid sarcomas. All these tumors are included in the category of INI1/SMARCB1- deficient tumors. Lately, few studies have shown loss of INI1 in certain EMCs. Considering there is preclinical data regarding role of EZH2 inhibitor in “INI1 deficient” tumors, it is crucial to recognize such tumors. Objectives: To evaluate immunohistochemical features, including INI1/SMARCB1 immunostaining results in 16 prospectively diagnosed cases of EMC. Methods: Immunohistochemical staining was performed on formalin-fixed paraffin embedded tissue sections by immunoperoxidase method using a MACH 2 Universal HRP-Polymer detection kit. Two cases were tested for EWSR1 rearrangement by fluorescent in-situ hybridization (FISH) technique. Results: Sixteen EMCs occurred in 13 males and 3 females, most commonly in lower extremities; followed by chest wall, pelvis, including iliac fossa, shoulder and parasapinal region; in patients within age-range of 17-72 years (median=47.5). On histopathologic examination, most tumors displayed round to polygonal cells arranged in cords, trabeculae and pseudoglandular pattern in an abundant myxoid stroma. Three tumors revealed “rhabdoid” cells. By immunohistochemistry, tumor cells were positive for NSE (13/13) (100%), S100 protein (10/15) (66.6%), EMA (2/12) (16.6%), AE1/AE3 (0/9), P63 (0/2) and SMA (2/3), the latter in tumors containing rhabdoid-like cells. INI1/SMARCB1 was diffusely retained in all 16 tumors (100%). Two cases tested for EWSR1 rearrangement, were found to be positive for the same. Conclusion: An optimal immunohistochemical panel for differentiating an EMC from its diagnostic mimics may include antibody markers, such as NSE, S100 protein, AE1/AE3, EMA and SMA. EMCs, including those containing rhabdoid cells do not seem to be in the category of INI1-deficient tumors.

MRNA in situ Hybridization Analysis of Vascular Endothelial Growth Factor and Matrix Metalloproteinase-1 in Colorectal Cancer

Colorectal cancer is a major global health problem. The aim of the current study is to determine the expression of vascular endothelial growth factor (VEGF) and matrix metalloproteinase -1 (MMP-1), in colorectal cancer patients using in situ hybridization technique, formalin-fixed and paraffin embedded tissue sections.
 Thirty colorectal cancer patients were obtained from the archive of the pathology laboratory of Al-Yarmouk Teaching Hospital from January 2013 to July 2014. In addition to, formalin-fixed, paraffin embedded tissue sections of twenty normal colon tissues were collected and used as a control group. Each tissue block was subjected to cut as serial thin sections of (4μm) thickness and then fixed on positive charged slides, to be used for In situ hybridization for the evaluation of MMP-1 and VEGF expression.
 A highly significant difference (p<0.01) was noted in the mean percentage of MMP-1 and VEGF mRNA in situ expression respectively between the colorectal cancer group and control group. The study revealed a highly significant positive correlation (P<0.01) between MMP-1 and VEGF expression in colorectal cancer group. In conclusion, abnormal high expressions of VEGF and MMP-1 may represent the early molecular changes in the development of colorectal cancer

Effects of rosiglitazone on hyperlipidemic rats with severe acute pancreatitis secondary to lung injury

Objective To explore the effects of rosiglitazone (ROSI), a peroxisome proliferator-activated receptors-gamma (PPAR-γ) ligand, on hyperlipidemia in rats with severe acute pancreatitis (SAP) associated with lung injury. Methods A total of 120 male SD rats received intragastric administration of high fat diet for two weeks to induce experimental hyperlipemia. The hyperlipidemic rats were randomly(random number) divided into six groups: hyperlipidemia (HL) group (n=20), hyperlipidemia with SAP (HP) group (n=20), hyperlipidemia with rosiglitazone intervention (HRP) group (n=20), hyperlipidemia with rosiglitazone and antagonist to rosiglitazone (HRGP) group (n=20), rosiglitazone control (HR) group (n=20) and antagonist control (HG) group (n=20). The SAP was induced by a retrograde infusion of 5%sodium tauroholate into bile-pancreatic duct, and the SAP was established in HP group, HRP group and HRGP group. In HL group, HR group and HG group, equivalent volume of normal saline was used instead of sodium taurocholate. In HRP group and HR group, ROSI (6 mg/kg) was administered via the femoral vein 1 hour prior to the administration of sodium taurocholate. In HRGP group, GW9662 (0.3 mg/kg), an antagonist to PPRA-gamma, was given via the femoral vein 30 min prior to the administration of ROSI. In HG group, only GW9662 (0.3 mg/kg) was given via the left femoral vein 30 min prior to pretend SAP modeling. Rats from each group were sacrificed by exsanguination 12 h after SAP modeling. Blood samples were taken from all subjects to measure serum amylase (AMY), total cholesterol (TC), triglycerides (TG), Successive sections of the paraffin embedded tissue from pancreas and lung were taken for pathological examination with hematoxylin-eosin (HE) staining. Histopathological changes of pancreatic and pulmonary tissues observed under light microscope were evaluated. In pulmonary tissue, nuclear factor-kappa B(NF-κB)p65 expression was assayed by immuno- histochemistry. Intercellular adhesion molecule (ICAM-1) protein and tumor necrosis factor-α (TNF-α) protein levels were studied using Western blot analysis. Results The serum levels of TC and TG in HL group and HP group were significantly higher than those in HR group and HRP group (1.24±0.28, 1.14±0.08 vs. 0.41±0.17, 0.58±0.12; 14.86±1.47, 12.42±0.96 vs. 6.52±2.04, 7.36±0.95, all P 0.05). Conclusions Our study demonstrates that ROSI exerts anti-hyperlipidemic effect and anti-inflammatory effect on hyperlipidemia in rats with sodium taurocholate-induced severe acute pancreatitis associated with lung injury by inhibiting NF-κB and down-regulating the expression of TNF-α and ICAM-1. Key words: Rosiglitazone; Hyperlipidemia; Severe acute pancreatitis; Lung injury; NF-κB

Protocol for qRT-PCR analysis from formalin fixed paraffin embedded tissue sections from diffuse large b-cell lymphoma: Validation of the six-gene predictor score.

As a part of an international study on the molecular analysis of Diffuse Large B-cell Lymphoma (DLBCL), a robust protocol for gene expression analysis from RNA extraction to qRT-PCR using Formalin Fixed Paraffin Embedded tissues was developed. Here a study was conducted to define a strategy to validate the previously reported 6-gene (LMO2, BCL6, FN1, CCND2, SCYA3 and BCL2) model as predictor of prognosis in DLBCL. To avoid variation, all samples were tested in a single centre and single platform. This study comprised 8 countries (Brazil, Chile, Hungary, India, Philippines, S. Korea, Thailand and Turkey). Using the Kaplan-Meier and log rank test on patients (n=162) and two mortality risk groups (with those above and below the mean representing high and low risk groups) confirmed that the 6-gene predictor score correlates significantly with overall survival (OS, p<0.01) but not with event free survival (EFS, p=0.18). Adding the International Prognostic Index (IPI) shows that the 6-gene predictor score correlates significantly with high IPI scores for OS (p<0.05), whereas those with low IPI scores show a trend not reaching significance (p=0.08). This study defined an effective and economical qRT-PCR strategy and validated the 6-gene score as a predictor of OS in an international setting.

Genetic Analyses of Eosinophilic Esophagitis (EoE) Biopsies Does Not Explain the Rarity of EoE in Hispanics Compared to Caucasians Patients

Introduction: Eosinophilic esophagitis (EoE) is a rare chronic immune-mediated inflammatory disorder. EoE in population reviews have mainly focused on white or Caucasian decent compared with other races or ethnicities with specifically no population based study in the United States addressing the prevalence of EoE in Hispanics. Our recent study in a tertiary care center where 80% of the patients are Hispanics showed a prevalence of EoE of 0.96% compared to 7.26% in Caucasians. The purpose of this study is to compare genetic markers of EoE in Caucasians and Hispanics to help understand this disparity. Methods: Biopsy samples were randomly selected from patients who were diagnosed with EoE over the last 16 monthsfrom 07/2015to undergo molecular analysis to confirm the diagnosis of EoE as well as to compare genetic markers in Caucasians and Hispanics. We sent a total of 9 biopsy samples: 3 with positive EoE biopsies in Hispanic patients, 3 with positive EoE biopsies in Caucasian patients, and 3 random non-EoE patients as controls who had GERD. The EoE diagnostic panel (EDP) consists of,namely RNA extraction, EDP panel qPCR, and data analysis. RNA is extracted from a fresh patient esophageal biopsy or formalin-fixed paraffin embedded tissue sections. An aliquot of the RNA sample is subjected to reverse transcription reaction, and the resulting cDNA is loaded onto the 384-well fluidic card (4 patients) for qPCR amplification. The qPCR data are subjected to dual algorithms, namely signature analysis (heat map clustering) and dimensionality reduction (EoE score) to establish molecular EoE diagnosis. Results: A total of 9 samples were analyzed for molecular genetic testing. All six samples that were histologically diagnosed as positive for EoE had molecular results that confirmed the diagnosis. There were no significant genetic differences between the biopsies for the 3 Caucasian and the 3 Hispanic patients. The 3 biopsies that were histologically negative for EoE also underwent molecular analysis which confirmed that they did not have EoE. Conclusion: Although EoE is rare in the Hispanic populations, 7 fold less than Caucasians, our data indicate no significant differences between the two ethnicities regarding the genetic composition of the esophageal tissues. Thus the rarity of EoE in Hispanics vs Caucasians cannot be explained by genetics. We speculate that this difference in prevalence is related to environmental factors and recommend further population and epidemiologic research in this field.

Inverse SORS for detecting a low Raman-active turbid sample placed inside a highly Raman-active diffusely scattering matrix - A feasibility study.

The broad range of applications of spatially-offset Raman spectroscopy (SORS) were found to involve samples having only marginal differences in Raman cross-sections between the surface and subsurface targets. We report the results of a feasibility study to evaluate the potential of the approach to identify the presence of a very low Raman-active turbid sample placed inside a highly Raman-active diffusely scattering matrix. Paraffin sandwiched tissue blocks prepared by embedding slices of chicken muscle tissue into solid paraffin blocks were employed as representative samples for the study. It was found that in contrast to the several millimetres of probing depth reported in the earlier applications, the Raman signatures of tissue were best recovered when it was located beneath the surface of the paraffin block at a depth of around a millimetre, beyond which the quality of recovery was increasingly poorer. However, the probing depth could be further increased by increasing the thickness of the embedded tissue sections. The results clearly suggest that though the probing depth achievable under the current condition is less than that found in previous applications, nevertheless it is sufficient for various other applications that would not require probing as deep as was required earlier.

Abstract 4451: Different patterns of SIRT1, KLF4, DAPK1 and SPG20 methylation in B lymphocytes correlate with the clinical parameters of non-Hodgkin lymphoma subtypes

Abstract Background. DNA methylation is one of the best studied epigenetic modifications and one major constituent of the epigenome of a cell. It contributes to normal development as well to carcinogenesis. Nowadays, many efforts are being made in order to use DNA methylation as a biomarker. The aim of our work is to characterize the expression and methylation of SIRT1, HIC1, BCL6, KLF4 and other genes relevant for Non-Hodgkin lymphomas (NHL) pathogenesis. Methods. Immunohistochemistry (IHC) on 72 formalin-fixed paraffin embedded tissue sections (FFPE). B-lymphocytes were purified from 36 biopsies of follicular hyperplasias (non-malignant B-lymphocytes), follicular lymphomas (FL) and diffuse large B-cell lymphomas (DLBCL). Gene expression were analysed by quantitative retrotranscribedPCR (qRTPCR). Quantitative CpG promoter methylation analysis was performed by pyrosequencing after bisulfite conversion or by Methyl II array qPCR on genomic DNA. Results. In a total of 72 FFPE samples of follicular hyperplasias (n = 17), FL (n = 36) and DLBCL (n = 19), SIRT1 staining is localized in the germinal center of the majority of follicular hyperplasias and FL samples. SIRT1 localizes preferentially in the centroblasts of the GC of the follicles where it correlates with Ki67. BCL6 is uniformly positive in follicular hyperplasias and FL, but heterogeneously distributed in DLBCL. Interestingly, SIRT1 and BCL6 expression correlate in FL. By quantitative pyrosequencing we analyzed 3 CpG sites for the SIRT1 promoter (corresponding to the binding sites for CREB, ARID and PPARG transcription factors). Follicular hyperplasias display higher methylation levels (52.88%) than FL (38.36%) and DLBCL (32.65%) on SIRT1 promoter suggesting a possible inverse correlation between tumor aggressiveness and SIRT1 methylation. Next, we selected a panel of genes whose expression is linked to lymphoma pathogenesis. By Methyl II array qPCR, we show that BCL6 methylation does not vary among samples. KLF4, DAPK1 and SPG20, show statistically significant methylation increases in FL and DLBCL compared to follicular hyperplasias, indicating a possible role of these proteins in lymphoma pathogenesis. On the contrary, no significant differences are observed for the other markers MZB1, MGMT, LMO2 and ASXL1. Notably, KLF4, DAPK1 and SPG20 mRNA expression levels anti-correlate with their promoter methylation in FL. Conclusions. Epigenetic changes in SIRT1 methylation inversely correlate with NHL aggressiveness (decreasing in the order: follicular hyperplasias - FL - DLBCL), while KLF4, DAPK1 and SPG20 show a methylation increase that correlates with tumor aggressiveness. These data suggest that different patterns of methylation correlate with the clinical and prognostic parameters of these NHL subtypes. Citation Format: Raffaele Frazzi, Eleonora Zanetti, Mariaelena Pistoni, Ione Tamagnini, Riccardo Valli, Francesco Merli. Different patterns of SIRT1, KLF4, DAPK1 and SPG20 methylation in B lymphocytes correlate with the clinical parameters of non-Hodgkin lymphoma subtypes. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4451.

An optimized xylene-free protein extraction method adapted to formalin-fixed paraffin embedded tissue sections for western blot analysis.

Deparaffinization of formalin-fixed paraffin embedded (FFPE) tissues with xylene currently remains a major challenge to the biomedical community. We developed an efficient xylene-free protocol to isolate proteins from archived FFPE human tissue sections. A total of 79 different types of FFPE tissue sections of 8 µm thickness were obtained from various archived FFPE specimens. Deparaffinization was conducted by gently washing each section with around 1 ml of hot distilled water (≈80°C). The deparaffinized tissues were homogenized in lysis buffer, and the isolated proteins were quantified and efficiently resolved using western blot analysis for the presence of Protein kinase B (PKB/AKT) and β-actin. Moreover, a significant amount of proteins was successfully isolated with an average of 2.31 µg/µl. The migration pattern of AKT and β-actin obtained from the specimens was similar to the positive control obtained from protein lysates prepared from in vitro cultured MDA231 cancer cell lines. AKT was successfully identified in all specimens, and β-actin protein was resolved with an efficiency higher than 80%. The entire extraction procedure requires only 20 minutes. This newly developed technique is an efficient, safe, cost-effective, and rapid method to isolate proteins from FFPE tissue sections adequate for molecular analysis.

Identification of high risk basal like TNBC by an immunohistochemical approach.

e12571Background: Triple negative breast cancer (TNBC) is a heterogeneous disease and constitutes two molecular subtypes basal like and non-basal like. Immunohistochemical criterion is most suitable to identify these molecular subtypes as non-basal like carry a more favorable prognosis and increased chemotherapy sensitivity. Methods: In forty eight (Stage II-A & B) TNBC, basal markers CK5/6, CK14, CK17, EGFR and Vimentin along with C-Kit were immunolocalised on formalin fixed paraffin embedded tissue sections. On tumor cells, marker negativity and focal positivity scored as negative, and 1+ ( 50% cells positive) positivity scored as positive. The data was analysed by SPSS statistical software. Results: The combination of basal markers identified basal like phenotype in 39 (81.25%) and non-basal like phenotylpe in 9 (18.75% ) of TNBCs. The incidence of CK17 expression was higher (60%) followed by Vimentin (46%), EGFR (42%), CK5/6 (19%) and CK1...

Distribution pattern of bovine viral diarrhoea virus type 1 genome in lymphoid tissues of experimentally infected sheep.

In this study, cellular localization and the distribution pattern of BVDV genome in lymphoid tissues during the course of experimental acute BVDV-1 infection of sheep was investigated. Tonsils, mesenteric lymph nodes (MLN) and spleen were collected on 3, 6, 9, 12 and 15days post infection (dpi) from twenty 4-month-old lambs, experimentally inoculated intra-nasally with 5 × 10(5) TCID50 of a non-cytopathic (ncp) BVDV-1 isolate, Ind-17555. Tissues collected from ten mock-infected lambs served as controls. In situ hybridization (ISH) was carried out in paraformaldehyde fixed paraffin embedded tissue sections using digoxigenin labelled riboprobe targeting 5'-UTR of BVDV-1. BVDV genome was detected at all the intervals from 3 dpi to 15 dpi in the lymphoid tissues with variations between the intervals and also amongst the infected sheep. During the early phase of acute infection, presence of viral genome was more in tonsils than MLN and spleen, whereas the distribution was higher in MLN during later stages. BVDV-1 genome positive cells included lymphocytes, macrophages, plasma cells, reticular cells and sometimes crypt epithelial cells. Genome distribution was frequently observed in the lymphoid follicles of tonsils, MLN and spleen, besides the crypt epithelium in tonsils, paracortex and medullary sinus and cords of MLN. Most abundant and widespread distribution of BVDV-1 genome was observed on 6 dpi while there was a reduction in number and intensity of positive signals by 15 dpi in most of the infected animals. This is the first attempt made to study the localisation of BVDV-1 in lymphoid tissues of acutely infected sheep by in situ hybridization. The results show that the kinetics of BVDV-1 distribution in lymphoid tissues of experimentally infected non-pregnant sheep follows almost a similar pattern to that demonstrated in BVDV infected cattle.

Expression of stem cell biomarker aldehyde dehydrogenase 1 (ALDH1) in canine mammary tumor

The study was conducted to investigate the immunohistochemical expression of a stem cell biomarker, ALDH1 in CMTs. ALDH1 is a cytosolic enzyme responsible for oxidizing intracellular aldehydes leading to the oxidation of retinol to retinoic acid, a key step involved in early stem cell differentiation. It was noticed that the cells expressing this marker possess ability to self renew. In present investigation, we assessed the expression of ALDH1 by immunohistochemistry on paraffin embedded tissue sections from 31 confirmed clinical cases of canine mammary tumors (CMT). The results of immunohistochemistry were analyzed semiquantitatively by calculating percentage score and staining intensity using light microscope and comparing it with the histological types. ALDH1 expression was detected in 26 (83%) of CMTs and it was observed in benign lesions, invasive cells and cells undergoing epithelial to mesenchymal transition (EMT). The high expression patterns of ALDH1 in various histological types of CMT indicated poor prognosis.

Abstract B05: Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing.

Abstract Mutations in TP53 have been shown to occur in around 96% of high-grade serous ovarian cancer (HGSOC). Different TP53 mutations can lead to accumulation of mutant p53 in the cell that support gain of function effects, or loss or abrogation of both wild-type and mutant p53. Success of new therapeutic strategies for HGSOC that target mutant p53 will likely depend on detailed knowledge of the type of TP53 mutation present and its influence on mutant p53 levels in the cell. The aim of this study was to identify the spectrum of TP53 mutations in a cohort of primary HGSOCs and compare this with p53 immunostaining patterns. In addition, we sought to identify TP53 mutations in the previously uncharacterised HGSOC cell line models OV167 and OV202, as well as the clear cell adenocarcinoma line OV207. DNA from fresh frozen tumors and cell line samples were processed for next generation sequencing using The Fluidigm Access TP53 Array System run on the Fluidigm Biomark HD™ Real-Time PCR fluidics system (Fluidigm Corporation). Amplicon libraries were pooled and analyses performed in a single massively parallel sequencing run using the MiSeq Sequencing System (Illumina Inc.). Unaligned BAM files were analyzed within the Broad Institute's Genome Analysis Toolkit (GATK) best practice pipeline. The ANNOVAR software tool was used to generate an annotated variants list. Variants were further filtered based on their frequency in the 1000 Genome Database and predicted SIFT scores. Mutations were visualized using the Broad Institute's Integrative Genomics Viewer. All samples in which a mutation was identified underwent Sanger sequencing using primers recommended in the International Agency for Research on Cancer TP53 database. Immunohistochemistry for p53 was performed on formalin fixed paraffin embedded tissue sections using a commercially available mouse monoclonal anti-human antibody (Protein Clone DO-7, cat. #M7001, Dako) on an automated staining platform (Leica BOND-III™ autostainer). The percentage of cells showing positive nuclear staining for p53 was reported. A mutation in TP53 was identified in 94% (68 of 72) of HGSOC. The majority of mutations identified (62%, 42 of 68) were missense, with all except one of these mutations located in the p53 DNA binding domain. Overall, 82% (56 of 68) of mutations occurred within the DNA binding domain. Frameshift, stop or splice mutations (38%, 26 of 68) were identified in exons or flanking intronic sequence between exons 4 – 10. The presence or location of a TP53 mutation did not influence overall survival. Sanger sequencing was unable to reliably detect variants at an allele frequency less than 25%. TP53 mutations were identified in all cell lines at a mutant allele frequency ≥99%. Based on percent of nuclei positive for p53, tumors were immunohistochemically graded as High, Intermediate or Low for p53. All missense mutations scored High for expression of the p53 protein. Considerable variability was observed in p53 staining levels in samples containing stop, frameshift or splice mutations, with samples in all of High, Intermediate and Low categories. Furthermore, a single sample shown to be wild-type for TP53 was graded as High for p53, raising the possibility that p53 had been activated in this tumor via an alternative mechanism. The level of p53 did not affect overall survival. Taken together, these results suggest that caution needs to be exercised when using p53 immunohistochemistry as a surrogate marker for either presence or type of TP53 mutation. A combination of next generation sequencing and p53 immunohistochemistry is required to robustly define both mutation type and effect on mutant p53 levels in the cell. Citation Format: Alexander J. Cole, Trisha Dwight, Ying Zhu, Anthony J. Gill, Kristie-Ann Dickson, Adele Clarkson, Gregory B. Gard, Jayne Maidens, Susan Valmadre, Roderick J. Clifton-Bligh, Deborah J. Marsh. Assessment of TP53 mutation status in primary high-grade serous ovarian cancer and cell line models: Comparison between immunohistochemistry and next-generation sequencing. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B05.

The Expression of DNA Methyltransferase DNMT3a in Classical and Fibrolamellar Hepatocellular Carcinoma

The epigenetic regulation of DNA-templated processes has been studied extensively during the last 15 years. As was revealed, DNA modification such as methylation possess great impact on cell fate and can result in abnormal protein expression patterns what can lead to the induction of carcinogenesis. DNA (cytosine-5)-methyltransferases are enzymes that catalyses the transfer of methyl groups to specific CpG structures in DNA. The methylation of these sequences can lead to inappropriate gene expression such as the silencing of tumor suppressor genes in cancer cells. DNA (cytosine-5)-methyltransferase 3A (DNMT3a) gene encodes a DNA methyltransferase that is thought to function mainly in de novo methylation. In the normal liver DNMT3a is usually expressed on the medium level, as was described in literature. Hepatocellular carcinoma (HCC) still remains one of the most common cause of death among patients with cancer. Fibrolamellar hepatocellular carcinoma (FL) represents rare subtype, which affects usually young people (an onset between 20-30 years) and its etiology is poorly understood. In our study we compared the presence of DNMT3a protein between two different types of HCC-common type and fibrolamellar one. We performed immunohistochemical staining of formalin fixed paraffin embedded tissue sections obtained from 30 patients (22 HCC and 8 FL). We found that DNMT3a immunoreactivity is significantly more pronounced in the non-fibrolamellar variant of HCC than in the fibrolamellar one. The DNMT3a immunoreactivity was predominantly localized in cancer cell nuclei in a form of separate large granules spotted in proximity to heterochromatin region. The reduced presence of DNMT3a in the fibrolamellar variant of HCC may suggest that different epigenetic mechanisms are involved in development of this particular type of liver cancer. Improving our understanding of the roles of DNMT proteins in hepatocarcinogenesis can benefit in the development of epigenetic –based therapy designed for specific HCC subtype.

Immunofluorescence on paraffin embedded renal biopsies: Experience of a tertiary care center with review of literature.

To describe the technique of immunofluorescence on paraffin embedded tissue sections and discuss the potential pitfalls with an in depth review of literature. Immunofluorescence is integral to diagnostic renal pathology. Immunofluorescence on paraffin embedded renal biopsies (IF-P) after enzyme treatment has been described in literature, however has not found widespread use in renal pathology laboratories. In our laboratory proteinase K digestion of paraffin embedded renal biopsy material was standardized and applied prospectively in cases where immunofluorescence on fresh frozen tissue was non contributory or not possible. Diagnostic utility was assessed and in a cohort of cases comparison of intensity of staining with routine immunofluorescence was performed. Over the 5-year study period, of the 3141 renal biopsies received IF-P was performed on 246 cases (7.7%) and was interpretable with optimal digestion in 214 cases (6.8%). It was of diagnostic utility in the majority of cases, which predominantly included glomerular disease. Non-diagnostic IF-P was found in membranous nephropathy (2 of 11 cases), membranoproliferative glomerulonephritis (2 of 32 cases), lupus nephritis (1 of 25 cases), post infectious glomerulonephritis (1 of 11 cases) and chronic glomerulonephritis (3 of 8 cases). Comparing cases with both routine IF and IF-P, 35 of 37 showed either equal intensity or a minor difference in intensity of staining (1+) for the diagnostic immunoglobulin/complement. Technically assessment of immunofluorescence on the paraffin embedded tissue was found to be easier with clearly observed morphology, however a false positive staining pattern was observed in under-digested tissue. As a "salvage" technique, immunofluorescence on paraffin embedded renal biopsies is of great diagnostic utility, however not without pitfalls.

A simple and effective heat induced antigen retrieval method

In this paper, we describe an additional step to the standard method of heat induced antigen retrieval to improve the detection of antibody staining of formalin fixed paraffin embedded tissue sections. Direct heating of tissues in buffer is an efficient epitope retrieval method but often results in the damage or loss of tissues. In this modified method, before keeping in buffer for heating, we overlapped the tissue on the slide with a plain slide by clipping one end using a normal paperclip, keeping a minimum gap between the slides. Tissues heated in this way in buffer had following advantages over normal heat treatment for epitope retrieval.•Tissues were intact even at high temperatures which improved the quality of staining by preventing fold, damage or detachment of tissues from the slides.•The method is very safe and economical compared to the methods using microwave or pressure cooker.•This simple method also appears to be very effective and less time consuming compared to the existing methods.