Formalin-fixed Tissue Samples Research Articles

Stromal expression of Fer suppresses tumor progression in renal cell carcinoma and is a predictor of survival.

Fps/Fes related (Fer) is a non-receptor tyrosine kinase that is expressed in fibroblasts, immune cells and endothelial cells. Fer serves an important pathological role in cell survival, angiogenesis and the immune system. However, the pathological role of Fer expression in the stromal cells surrounding renal cell carcinoma (RCC) has not been previously investigated. In the present study, immunohistochemical analysis of Fer was performed using the formalin-fixed tissue samples of 152 patients with RCC. The proliferative and apoptotic indices were used to represent the percentage of proliferation marker protein Ki-67- and cleaved caspase-3-positive cells, respectively. The microvessel density was defined as the number of cluster of differentiation (CD) 31-positively stained vessels/mm2. In addition, CD57+ and CD68+ cells were counted using semi-quantification of natural killer (NK) cells and macrophages. Fer expression in stromal cells was negatively associated with Fuhrman grade, pathological tumor stage and metastasis (P<0.001). Fer expression in stromal cells was negatively associated with CD68+ macrophage density, whereas it was positively associated with CD57+ NK cell density. Kaplan-Meier estimators indicated that decreased stromal Fer expression was a predictive marker of decreased cause-specific survival rate (P<0.001). Furthermore, low expression of Fer was identified as being an independent marker of decreased cause-specific survival using multivariate analysis (hazard ratio, 7.4; 95% confidence interval, 1.7-33.0; P<0.001). The results of the present study suggested that low Fer expression in stromal cells is associated with increased malignant aggressiveness and decreased survival in patients with RCC. CD57+ NK cell and CD68+ macrophage regulation in cancer-stromal tissue is considered to affect RCC pathology.

Pre-Analytical Considerations for Successful Next-Generation Sequencing (NGS): Challenges and Opportunities for Formalin-Fixed and Paraffin-Embedded Tumor Tissue (FFPE) Samples.

In cancer drug discovery, it is important to investigate the genetic determinants of response or resistance to cancer therapy as well as factors that contribute to adverse events in the course of clinical trials. Despite the emergence of new technologies and the ability to measure more diverse analytes (e.g., circulating tumor cell (CTC), circulating tumor DNA (ctDNA), etc.), tumor tissue is still the most common and reliable source for biomarker investigation. Because of its worldwide use and ability to preserve samples for many decades at ambient temperature, formalin-fixed, paraffin-embedded tumor tissue (FFPE) is likely to be the preferred choice for tissue preservation in clinical practice for the foreseeable future. Multiple analyses are routinely performed on the same FFPE samples (such as Immunohistochemistry (IHC), in situ hybridization, RNAseq, DNAseq, TILseq, Methyl-Seq, etc.). Thus, specimen prioritization and optimization of the isolation of analytes is critical to ensure successful completion of each assay. FFPE is notorious for producing suboptimal DNA quality and low DNA yield. However, commercial vendors tend to request higher DNA sample mass than what is actually required for downstream assays, which restricts the breadth of biomarker work that can be performed. We evaluated multiple genomics service laboratories to assess the current state of NGS pre-analytical processing of FFPE. Significant differences in pre-analytical capabilities were observed. Key aspects are highlighted and recommendations are made to improve the current practice in translational research.

Abstract 1465: Analysis of tumor-infiltrating lymphocytes (TILs) reveals biologically different subgroups of breast ductal carcinoma in situ

Abstract Background: Treatment of breast ductal carcinoma in situ (DCIS) aims to prevent invasive recurrence. Recent studies have shown an important impact of TILs on the outcome of invasive breast cancer, however their influence on breast DCIS prognosis has not been fully explored. In this study we investigated whether the amount and/or phenotype of TILs can help recognizing DCIS subgroups of different biology and recurrence risk. Methods: The study included 134 patients, diagnosed and treated for a DCIS from 2001 to 2005 in our institution. Formalin-fixed and paraffin-embedded (FFPE) cancer tissue samples, taken before any treatment, were retrospectively collected. H&amp;E whole tissue sections served for assessment of the cancer size, grade, histotype, architecture, mitotic index and amount of stromal lymphocytic (Ly) infiltrate. The latter was semi-quantitatively graded into 4 grades (0 - absent, 1 - mild, 2 - moderate, 3 - intense). Ly-phenotype was assessed by immunohistochemistry (IHC) on tissue microarrays (TMAs), constructed by sampling each case at the area of the densest Ly-infiltration (3 cores of 0.6 mm diameter per case). The number/mm2 of the CD8+, CD4+, FOXP3+, CD20+ and CD38+ mononuclear cells was determined. TMAs served also for estrogen receptor (ER), progesterone receptor (PR), HER2 and Ki67 IHC staining, as well as for HER2 amplification status. Results: There were 97 DCIS and 37 microinvasive DCIS (micDCIS). The micDCIS displayed significantly more diffuse architecture, frequent HER2 amplification (HER2amp), higher grade, lower ER expression (0.029&amp;lt;p&amp;lt;0.044 for each) and more peritumoral Ly-infiltrate (grades 2+3, micDCIS vs DCIS, 51.5% vs. 39.0%, p = 0.036). All but CD20+ cells were more numerous in micDCIS than in DCIS (0.0016&amp;lt;p&amp;lt;0.05). Within the entire cohort, the cases having the (CD8+/CD4+):(CD20+/CD38+) ratio higher than 1 had a significantly greater risk of containing a microinvasive component (OR 3.47 (1.26-9.57), p = 0.029). Interestingly, that ratio was significantly higher (p = 0.012) in micDCIS than in the DCIS with grade 2 or 3 Ly-infiltrate (Ly-DCIS, n = 38). On the other side, there was no difference between micDCIS and Ly-DCIS in architecture, histograde, HER2amp rate and ER expression. Cluster analysis further confirmed significant similarities between micDCIS and Ly-DCIS, putting them both apart from non-Ly-DCIS (p = 0.0034). The overall 10-year recurrence rate was 13% (18/134 pts). No parameter significantly correlated with recurrence risk, however the micDCIS have received significantly more treatment than DCIS (axillary lymphadenectomy (p&amp;lt;10-7), chemotherapy (p = 0.031) or hormonal therapy (p = 5.4×10-6)). Conclusion: These results indicate that Ly-DCIS might be biologically and immunologically similar to micDCIS. TILs in DCIS are worth investigating in larger studies, as they could be a marker of microinvasion and help tailoring the initial treatment of the disease. Citation Format: Marie Beguinot-Cornillon, Marie-Melanie Dauplat, Fabrice Kwiatkowski, Guillaume Lebouedec, Lucie Tixier, Christophe Pomel, Frederique Penault-Llorca, Nina Radosevic-Robin. Analysis of tumor-infiltrating lymphocytes (TILs) reveals biologically different subgroups of breast ductal carcinoma in situ. [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 1465.

Microscopic Study of Testicular Tissue Structure and Spermatogenesis Following Long Term Dose Dependent Administration of Monosodium Glutamate in Adult Diabetic Rats

Abstract Background and aims: Diabetic hyperglycemia leads to structural and functional alterations in body organs including testis. Monosodium glutamate (MSG) is a food additive which has toxic effects on human and animal’s tissues. The aim of this study was to evaluate the effects of MSG on diabetic complications of testicular tissue. Material and Methods: 48 adult rats were divided into six groups. Diabetes was induced by Streptozotocin (45 mg/kg i.p.). Monosodium glutamate was administrated in two doses (6 and 60 mg/kg) to control and diabetic groups. After eight weeks, the body weight was measured and blood samples were collected for analysis of the pituitary-testicular axis hormones. Formalin fixed tissue samples were prepared through routine histologic methods. Results: MSG led dose dependently to weight gain of control groups and prevented weight loss of diabetic rats. The blood levels of luteinizing hormone and testosterone were reduced in diabetic rats following administration of MSG. Reduction of cellular population of germinal epithelium, seminiferous tubules diameter decrement and decrease of indices of spermatogenesis were observed with more intensity in MSG treated diabetic rats. Conclusions: The results of this study suggest that the long term administration of MSG may induce in a dose dependent manner structural and functional alterations of testicular tissue in diabetic rats.

How Suitable is Matrix-Assisted Laser Desorption/Ionization-Time-of-Flight for Metabolite Imaging from Clinical Formalin-Fixed and Paraffin-Embedded Tissue Samples in Comparison to Matrix-Assisted Laser Desorption/Ionization-Fourier Transform Ion Cyclotron Resonance Mass Spectrometry?

In research and clinical settings, formalin-fixed and paraffin-embedded (FFPE) tissue specimens are collected routinely and therefore this material constitutes a highly valuable source to gather insight in metabolic changes of diseases. Among mass spectrometry techniques to examine the molecular content of FFPE tissue, mass spectrometry imaging (MSI) is the most appropriate when morphological and histological features are to be related to metabolic information. Currently, high-resolution mass spectrometers are widely used for metabolomics studies. However, with regards to matrix-assisted laser desorption/ionization (MALDI) MSI, no study has so far addressed the necessity of instrumental mass resolving power in terms of clinical diagnosis and prognosis using archived FFPE tissue. For this matter we performed for the first time a comprehensive comparison between a high mass resolution Fourier-transform ion cyclotron resonance (FTICR) mass spectrometer and a time-of-flight (TOF) instrument with lower mass resolving power. Spectra analysis revealed that about one-third of the detected peaks remained unresolved by MALDI-TOF, which led to a 3-5 times lower number of m/z features compared to FTICR measurements. Overlaid peak information and background noise in TOF images made a precise assignment of molecular attributes to morphological features more difficult and limited classification approaches. This clearly demonstrates the need for high-mass resolution capabilities for metabolite imaging. Nevertheless, MALDI-TOF allowed reproducing and verifying individual markers identified previously by MALDI-FTICR MSI. The systematic comparison gives rise to a synergistic combination of the different MSI platforms for high-throughput discovery and validation of biomarkers.

Cytogenetic and immunohistochemical biomarker profiling of therapy-relevant factors in salivary gland carcinomas.

There is currently no established algorithm for the molecular profiling of therapy-relevant defects in salivary gland carcinomas (SGC). HER2 overexpression in a subfraction of SGC and low frequencies of EGFR mutations are known. Here, we established receptor and cell signalling profiles of 17 therapy-relevant factors and propose a molecular diagnostic algorithm for SGC. Formalin-fixed and paraffin-embedded tissue samples from SGC (n = 38) were analysed with immunohistochemistry and fluorescence in situ hybridisation (FISH). Two or more expressed receptors and/or receptor gene amplification were detectable in eight of 38 (21%) tumours: HER2 3+/AR 1+, HER3 gene amplification/AR 1+/EGFR 1+, ER 3+/AR 1+, EGFR 2+/PR 1+ and EGFR 2+/PR 1+/AR 1+. No FGFR1-3, MET, ALK1, ROS1, RET, BRAF nor VEGFA defects were detectable, and ERCC1 was not overexpressed. No PD1+ tumour-infiltrating T cells were detectable. Personalised therapy of patients with salivary gland carcinomas should include HER2 and EGFR signalling testing and, in negative cases, evaluation of rare potential target molecules. ERCC1 and PD1 do not appear to be reliable markers for the decision for or against chemotherapy or immunotherapy, respectively.

Aquaporin 1 protein expression is associated with BRAF V600 mutation and adverse prognosis in cutaneous melanoma.

Despite experimental findings suggesting the prognostic significance of Aquaporin 1 (AQP1) in human melanoma, no published clinical data are available. We studied the expression of AQP1 protein in cutaneous melanoma, correlated our findings with standard histological and genetic markers, and long-term clinical follow-up. Our study evaluated the AQP1 protein expression in 78 melanoma patients, representing two predefined risk cohorts using the immune labeling technique with commercially available anti-AQP1 antibodies on routinely formalin-fixed and paraffin-embedded tumor tissue samples. BRAF V600E mutation analyses were carried out successfully in 70 patients using PCR and restriction fragment length polymorphism analyses, followed by confirmatory analysis with the Sanger sequencing technique. AQP1-expressing melanoma cells were found in 52 cases (66.7%, median H-score=124.24). Significantly higher AQP1 H-scores (P=0.047) were found in the 'high-risk' patients. No correlations were found with the established histological markers, such as mitotic index (P=0.42), Clark level (P=0.95), and Breslow thickness (P=0.51). BRAF V600 mutation analyses were successful in 89%, and showed a two times higher mutation frequency in the 'high-risk' group. The BRAF V600 mutations were significantly associated with AQP1 expression (P=0.014). Long-term follow-up indicated a reduced progression-free survival (P=0.036) and overall survival (P=0.017) for the AQP1-positive cutaneous melanoma patients. AQP1 expression is likely to be associated with an adverse prognosis in cutaneous melanoma.

Evaluation of iNOS Expression in Esophageal Cancer Patients

Background: Esophageal cancer is a public health concern around the world; this cancer is the sixth leading cause of death of cancer in the world with about 386,000 deaths per year. Its risk factors include environmental factors such as tobacco smoke, gastroesophageal reflux and genetic changes. iNOS is stated by the effect of various inflammatory factors and is thus called inducible NOS. Investigating iNOS expression is a powerful tool for understanding effective molecular parameters at tissue and cellular responses to external factors. In this research work, iNOS expression in patients with esophageal cancer was studied in Iran. Materials and Methods: 15 formalin-fixed and paraffin-embedded (FFPE) esophageal cancer tissue samples and 15 normal FFPE samples were collected from various medical centers (Zabol, Zahedan, Kashan) to measure iNOS expression by real-time reverse transcriptase polymerase chain reaction (real-time RT-PCR). All PCR reactions were conducted by three replicates for iNOS and internal control (β-actin) by 2^-ΔΔCT (Livak) method. Differences were measured in target gene expression in patients and control group using the t test. All statistical analyses were done using the SPSS software. Results: The results showed that there was no significant difference between iNOS expression in the case and control groups (p > 0.05); however, there was an increase in iNOS expression in the case group. On the other hand, there was a significant difference between iNOS expression in males and females in the two groups of healthy subjects and patients, and it was higher in women than in men. Conclusion: Further studies need to be conducted with larger sample sizes and in other populations to validate these findings.

Loop-mediated isothermal amplification combined with PCR and immunohistochemistry for detecting Porphyromonas gingivalis in periapical periodontitis.

Porphyromonas gingivalis is important in the development of marginal periodontitis. However, the precise role and localization of P. gingivalis in chronic periapical periodontitis remain unclear. Thus, methods that can detect P. gingivalis in formalin-fixed and paraffin-embedded (FFPE) tissue samples are needed. We assessed a technique combining loop-mediated isothermal amplification (LAMP) with PCR (PCR-LAMP) for detection of P. gingivalis, using 110 FFPE tissue samples of chronic apical periodontitis. PCR-LAMP specifically detected P. gingivalis with high sensitivity in FFPE tissue samples, and the sensitivity of the technique was higher than that of PCR or LAMP alone. The results of immunohistochemistry (IHC) confirmed the specificity of PCR-LAMP. IHC showed that P. gingivalis was localized in a granular layer of chronic apical periodontitis, a region that correlated with the localization of macrophages. This is the first study to describe the localization of P. gingivalis in human periapical periodontitis. In conclusion, PCR-LAMP was an effective tool for detecting P. gingivalis in periapical periodontitis. In addition, IHC results improve our understanding of the role of P. gingivalis in the progression of periapical periodontitis. (J Oral Sci 58, 163-169, 2016).

Retrospective analysis of TGF-β 1 expression in patients with thyroidectomy.

To investigate the relationship between the expression of transforming growth factor β1 (TGF-β1) and thyroid lesions by retrospective analysis in patients with thyroidectomy. Immunohistochemical analyses of TGF-β1 were performed with 120 archived formalin-fixed and paraffin-embedded thyroid resection tissue samples that included 60 cases of thyroid cancers (papillary thyroid carcinoma, follicular thyroid carcinoma), 14 cases of nodular goiter (NG), 16 cases of thyroid adenoma, 15 cases of Graves disease, 15 cases of Hashimoto's thyroiditis. The pathological analysis of resection thyroid showed a high coincidence with that diagnosed by preoperative fine needle aspiration biopsy (FNAB), with coincidence rate of 84.48%. The results showed that the expressions of TGF-β1 in the thyroid cancers were significantly higher than in other benign thyroid lesions and normal thyroid tissues (P < 0.05). Moreover, high expression of TGFβ1 was found to closely related with the occurrence of thyroid cancers, while there was no significant difference of TGFβ 1 expression between different thyroid cancers. The overexpression of TGF-β1 is associated with the occurrence of thyroid cancer, which can be used as a candidate for the diagnosis and prognosis of thyroid cancer.

Targeted or whole genome sequencing of formalin fixed tissue samples: potential applications in cancer genomics.

Current genomic studies are limited by the poor availability of fresh-frozen tissue samples. Although formalin-fixed diagnostic samples are in abundance, they are seldom used in current genomic studies because of the concern of formalin-fixation artifacts. Better characterization of these artifacts will allow the use of archived clinical specimens in translational and clinical research studies. To provide a systematic analysis of formalin-fixation artifacts on Illumina sequencing, we generated 26 DNA sequencing data sets from 13 pairs of matched formalin-fixed paraffin-embedded (FFPE) and fresh-frozen (FF) tissue samples. The results indicate high rate of concordant calls between matched FF/FFPE pairs at reference and variant positions in three commonly used sequencing approaches (whole genome, whole exome, and targeted exon sequencing). Global mismatch rates and C · G > T · A substitutions were comparable between matched FF/FFPE samples, and discordant rates were low (<0.26%) in all samples. Finally, low-pass whole genome sequencing produces similar pattern of copy number alterations between FF/FFPE pairs. The results from our studies suggest the potential use of diagnostic FFPE samples for cancer genomic studies to characterize and catalog variations in cancer genomes.

Application of bioinformatics in probe design enables detection of enteroviruses on different taxonomic levels by advanced in situ hybridization technology

BackgroundEnteroviral infections are common, affecting humans across all age groups. RT-PCR is widely used to detect these viruses in clinical samples. However, there is a need for sensitive and specific in situ detection methods for formalin-fixed tissues, allowing for the anatomical localization of the virus and identification of its serotype. ObjectivesThe aim was to design novel enterovirus probes, assess the impact of probe design for the detection and optimize the new single molecule in situ hybridization technology for the detection of enteroviruses in formalin-fixed paraffin-embedded samples. Study designFour enterovirus RNA-targeted oligonucleotide RNA probes – two probes for wide range enterovirus detection and two for serotype-targeted detection of Coxsackievirus B1 (CVB1) – were designed and validated for the commercially available QuantiGene ViewRNA in situ hybridization method. The probe specificities were tested using a panel of cell lines infected with different enterovirus serotypes and CVB infected mouse pancreata. ResultsThe two widely reactive probe sets recognized 19 and 20 of the 20 enterovirus serotypes tested, as well as 27 and 31 of the 31 CVB1 strains tested. The two CVB1 specific probe sets detected 30 and 14 of the 31 CVB1 strains, with only minor cross-reactivity to other serotypes. Similar results were observed in stained tissues from CVB –infected mice. ConclusionsThese novel in-house designed probe sets enable the detection of enteroviruses from formalin-fixed tissue samples. Optimization of probe sequences makes it possible to tailor the assay for the detection of enteroviruses on the serotype or species level.

CSF1R Protein Expression in Reactive Lymphoid Tissues and Lymphoma: Its Relevance in Classical Hodgkin Lymphoma.

Tumour-associated macrophages (TAMs) have been associated with survival in classic Hodgkin lymphoma (cHL) and other lymphoma types. The maturation and differentiation of tissue macrophages depends upon interactions between colony-stimulating factor 1 receptor (CSF1R) and its ligands. There remains, however, a lack of consistent information on CSF1R expression in TAMs. A new monoclonal antibody, FER216, was generated to investigate CSF1R protein distribution in formalin fixed tissue samples from 24 reactive lymphoid tissues and 187 different lymphoma types. We also analysed the distribution of CSF1R+, CD68+ and CD163+ macrophages by double immunostaining, and studied the relationship between CSF1R expression and survival in an independent series of 249 cHL patients. CSF1R+ TAMs were less frequent in B-cell lymphocytic leukaemia and lymphoblastic B-cell lymphoma than in diffuse large B-cell lymphoma, peripheral T-cell lymphoma, angioimmunoblastic T-cell lymphoma and cHL. HRS cells in cHL and, with the exception of three cases of anaplastic large cell lymphoma, the neoplastic cells in NHLs, lacked detectable CSF1R protein. A CSF1R+ enriched microenvironment in cHL was associated with shorter survival in an independent series of 249 cHL patients. CSF1R pathway activation was evident in the cHL and inactivation of this pathway could be a potential therapeutic target in cHL cases.

Bisulfite-Based DNA Methylation Analysis from Recent and Archived Formalin-Fixed, Paraffin Embedded Colorectal Tissue Samples.

We aimed to test the applicability of formalin-fixed and paraffin-embedded (FFPE) tissue samples for gene specific DNA methylation analysis after using two commercially available DNA isolation kits. Genomic DNA was isolated from 5 colorectal adenocarcinomas and 5 normal adjacent tissues from "recent", collected within 6 months, and "archived", collected more than 5 years ago, FFPE tissues using either High Pure FFPET DNA Isolation kit or QIAamp DNA FFPE Tissue kit. DNA methylation analysis of MAL, SFRP1 and SFRP2 genes, known to be hypermethylated in CRC, was performed using methylation-sensitive high resolution melting (MS-HRM) analysis and sequencing. QIAamp (Q) method resulted in slightly higher recovery in archived (HP: 1.22 ± 3.18 μg DNA; Q: 3.00 ± 4.04 μg DNA) and significantly (p < 0.05) higher recovery in recent samples compared to High Pure method (HP) (HP: 4.10 ± 2.91 μg DNA; Q: 11.51 ± 7.50 μg DNA). Both OD260/280 and OD260/230 ratios were lower, but still high in the High Pure isolated archived and recent samples compared to those isolated with QIAamp. Identical DNA methylation patterns were detected for all 3 genes tested by MS-HRM with both isolation kits in the recent group. However, despite of higher DNA recovery in QIAamp slightly more reproducible methylation results were obtained from High Pure isolated archived samples. Sequencing confirmed DNA hypermethylation in CRCs. In conclusion, reproducible DNA methylation patterns were obtained from recent samples using both isolation kits. However, long term storage may affect the reliability of the results leading to moderate differences between the efficiency of isolation kits.

Using tissue samples for proteomic studies—Critical considerations

From my experience of 22 years working in a pathology research laboratory and overseeing dozens of collaborations with research groups from basic sciences and industry, I have the impression that researchers are rarely aware of the special issues related to acquisition and processing of frozen or formalin-fixed tissue samples for proteomic analysis. While challenges are expected for formalin-fixed tissues because of the cross-linking activities of formaldehyde, researchers believe when using frozen tissue samples they are safe and always have excellent material to analyze-but this is not always the case. It is alarming that many researchers do not question the quality of the tissue samples they are analyzing and focus only on their analytical technique. Standardization of the entire workflow from test ordering to the report of the proteomic assay, with special emphasis on the preanalytical phase, is crucial for successful integration of proteomic studies in the clinic as protein profiles may change due to sample processing before the proteomic analysis is performed. The aim of this review is to discuss the progress of proteomic studies with human tissues and to highlight the challenges that must be understood and addressed for successful translation of proteomic methods to clinical practice.

In-situ hybridization-based quantification of hTR: a possible biomarker in malignant melanoma.

Telomerase is reactivated in most cancers and there is accumulating evidence that this is a driver event in malignant melanoma (MM). Thus, our aim was to evaluate if in-situ hybridization (ISH)-based quantification of telomerase RNA (hTR) could be used to distinguish MM from naevi, and if there was a correlation with the Breslow thickness. We created a tissue microarray (TMA) from formalin-fixed and paraffin-embedded tissue samples from 17 MM and 23 naevi, performed ISH targeting hTR, and quantified the signals. We found a more than eightfold greater number of hTR signals per nucleus in the MM samples compared to the naevi, and a positive correlation (P=0.0381) between the number of hTR signals per nucleus and the Breslow thickness. Quantification of hTR ISH signals clearly distinguish MM from naevi (P<0.0001) and the number of signals per nucleus correlates with the Breslow thickness, suggesting that hTR might be a valuable biomarker in MM. Furthermore, as ISH-based detection requires the presence of both hTR and telomerase reverse transcriptase (hTERT), it might be an indicator of active telomerase and thus have future relevance as a predictive biomarker for anti-telomerase treatment.

Clinical significance of CD44s, CD44v3 and CD44v6 in breast cancer.

To evaluate levels of CD44 standard variant (CD44s), CD44 variant exon 3 (CD44v3) and CD44 variant exon 6 (CD44v6) protein in breast cancer tissue, and investigate their relationships with clinicopathological characteristics of the disease. Immunohistochemistry for CD44s, CD44v3 and CD44v6 was retrospectively performed on formalin-fixed paraffin wax-embedded breast cancer tissue samples. Tumour tissue samples from 60 patients with breast cancer were included. There was a significant relationship between CD44s positivity and tumour diameter and lymph node involvement. CD44v6 positivity was significantly associated with tumour-node-metastasis (TNM) stage and lymph node involvement. There were significant negative correlations between CD44s immunopositivity, tumour diameter and TNM stage, and significant positive correlations between CD44v6 immunopositivity, tumour diameter and TNM stage. CD44s and CD44v6 appear to play opposing roles in the development of breast cancer, but their precise functions and mechanisms of action remain unclear.

Bio-Friendly Alternatives for Xylene - Carrot oil, Olive oil, Pine oil, Rose oil.

Xylene is a flammable liquid with characteristic petroleum or aromatic odours, it is miscible with most of the organic solvents and paraffin wax. Xylene clears tissues rapidly and renders transparency, facilitating clearing endpoint determination, this made it to be used as a clearing agent in routine histopathological techniques. Even though it is a good clearing agent, it causes damage to the tissues by its hardening effect particularly those fixed in non-protein coagulant fixatives. Apart from these tissue effects, it has severe, long lasting ill effects on health of technicians and pathologists when exposed to longer duration. Hence in order to overcome these effects and replace xylene with a safe alternative agent, the present study was carried out to assess the clearing ability and bio-friendly nature of four different natural oils i.e., Carrot oil, Olive oil, Pine oil and Rose oil in comparison with that of Xylene. According to Bernoulli's principle of fluid dynamics, to decrease viscosity of these oils and increase penetration into tissues for rapid clearing hot-air oven technique was used. To assess:1) Clearing ability and bio-friendly nature of four different oils i.e., Carrot oil, Olive oil, Pine oil, Rose oil in comparison with that of xylene, 2) Application of Bernoulli's principle of fluid dynamics in rapid clearing of tissues by using hot-air oven. Forty different formalin fixed tissue samples were taken. Each sample of tissue was cut into 5 bits (40x5=200 total bits) which were subjected for dehydration in differential alcohol gradients. Later, each bit is kept in 4 different oils such as Carrot oil, Olive oil, Pine oil, Rose oil and xylene and transferred into hot-air oven. Further routine steps of processing, sectioning and staining were done. Individual sections cleared in four different oils were assessed for cellular architecture, staining quality and a comparison was done between them. Results showed that all the four oils had ability to clear the tissues similar to that of xylene. Pine oil was superior in its physical and clearing properties, which also maintained good cellular architecture and distinct staining quality, when compared with other oils and xylene. Carrot oil, Olive oil, Pine oil, Rose oil, are not only bio friendly and economical but can also be used as clearing agent instead of xylene. In this study stability and longetivity of haematoxyllin and eosin staining was evaluated for over a period of one year and no significant difference in staining quality were observed.

PTEN sequence analysis in endometrial hyperplasia and endometrial carcinoma in Slovak women.

Phosphatase and tensin homolog (PTEN) is a protein that acts as a tumor suppressor by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate. Loss of PTEN function has been implicated in the pathogenesis of a number of different tumors, particularly endometrial carcinoma (ECa). ECa is the most common neoplasia of the female genital tract. Our study evaluates an association between the morphological appearance of endometrial hyperplasia and endometrial carcinoma and the degree of PTEN alterations. A total of 45 endometrial biopsies from Slovak women were included in present study. Formalin-fixed and paraffin-embedded tissue samples with simple hyperplasia (3), complex hyperplasia (5), atypical complex hyperplasia (7), endometrioid carcinomas G1 (20) and G3 (5), and serous carcinoma (5) were evaluated for the presence of mutations in coding regions of PTEN gene, the most frequently mutated tumor suppressor gene in endometrial carcinoma. 75% of the detected mutations were clustered in exons 5 and 8. Out of the 39 mutations detected in 24 cases, 20 were frameshifts and 19 were nonsense, missense, or silent mutations. Some specimens harboured more than one mutation. The results of current study on Slovak women were compared to a previous study performed on Polish population. The two sets of results were similar.

Next Generation Sequencing Analysis of Biofilms from Three Dogs with Postoperative Surgical Site Infection.

The composition of biofilms in chronic wound infections of dogs is unclear. In the present study, histologically identified biofilms attached to sutures in chronically infected wounds of three dogs were examined by next generation sequencing of total DNA extracted from formalin-fixed and paraffin-embedded tissue samples. The analysis identified an inhomogeneous bacterial composition in three tissues containing biofilms. Some of the identified bacterial families such as Staphylococci and Streptococci have been found before in biofilms associated with human and canine wounds but in this study were quantitatively in the minority. The majority of the reads classified as bacterial sequences had the highest identity with sequences belonging to the Porphyromonadaceae, Deinococcaceae, Methylococcaceae, Nocardiaceae, Alteromonadaceae, and Propionibacteriaceae and thus taxons of so far minor relevance in veterinary medicine.