Paraffin-embedded Tissue Blocks Research Articles

Evaluation of biosafe alternative to eosin in hematoxylin and eosin staining procedure: A comparative study.

Hematoxylin and eosin staining is the globally accepted staining technique for histology and histopathology sections. Hematoxylin is a natural dye as compared to eosin which is a synthetic dye manufactured from chemicals. Eosin acts as a counterstain to hematoxylin giving a sharp contrast to its blue color. Eosin being a chemical, can cause health hazards and environmental pollution. Using eco-friendly materials and going organic is the demand of this era. Curcuma longa rhizomes are proven to contain curcuminoids that exhibit various medicinal properties and curcumin the bright yellow component of the plant, that is, being used as a colorant. This natural dye from C. longa provides a significant alternative to eosin as they are safer to use without health hazards and are biodegradable and have easy disposability. To evaluate and compare the staining qualities of C. longa (turmeric) with hematoxylin and eosin (H&E). A total of 100 paraffin-embedded tissue block specimens of various pathological tissues were stained with H and E and hematoxylin and curcumin (H&C). Evaluation and comparison of the staining efficacy were done for all the pathological tissues. The obtained data were subjected to statistical analysis. H&C staining gave comparable results with H and E staining with a statistically insignificant P value (p > 0.05). Curcumin has shown equivalent staining to that of eosin. Thus, it can be used as a better alternative.

Clinicopathological Significance of Cancer Stem Cell Markers (OCT-3/4 and SOX-2) in Oral Submucous Fibrosis and Oral Squamous Cell Carcinoma

Oral submucous fibrosis (OSMF) is highly prevalent in South East Asia with higher rates of malignant transformation in Indian subcontinent. Numerous biomarkers are now being studied to predict disease prognosis and detect malignant alterations at an early stage. Patients with clinically and biopsy-proven oral submucous fibrosis and oral squamous cell carcinoma were included in the study as the experimental group, while patients without a tobacco or betel nut habit who had their third molars surgically removed were included as the healthy control group. For the immunohistochemistry (IHC) investigation, 5-μm slices from formalin-fixed, paraffin-embedded tissue blocks (FFPE) were obtained. Fresh tissues (n = 45) from all three groups were collected and gene expression was studied using relative quantitation-based qPCR. The protein expression of octamer-binding transcription factor 3/4 (OCT 3/4) and sex-determining region Y-box 2 (SOX 2) was evaluated in the experimental group and compared with healthy controls. The IHC results showed a significant correlation with the expression of OCT 3/4 (p value = 0.000; χ2 = 20.244) and SOX 2 (p value = 0.006; χ2 = 10.101) among OSCC and OSMF patients in comparison to healthy controls. Both OCT 3/4 and SOX 2 showed overexpression of four-fold and three-fold in OSMF when compared to OSCC and healthy controls, respectively. This study shows the significant importance of cancer stem cell markers OCT 3/4 and SOX 2 to assess the disease prognosis in OSMF.

Perineural Invasion and Histological Grades of Prostatic Adenocarcinoma

Aim: Perineural invasion and Histological grades of Prostatic adenocarcinoma Study design: Analytical descriptive cross sectional Study setting: Department of Pathology, KEMU, Lahore in collaboration with other tertiary care hospitals. Duration of study: 6 months Methodology: 60 cases of prostatic carcinoma were enrolled in this study. Detailed demographics of included patients were recorded after taking informed written consent. Formalin fixed, processed and paraffin embedded tissue blocks of prostate adenocarcinoma collected. Results: Out of 60 biopsies, 57(95%) were TURP, 1(1.67%) was a TRUS guided biopsy, 1(1.67%) was a core needle biopsy and 1(1.67%) was a radical prostatectomy specimen. Perineural invasion was identified in 27 cases. There was insignificant association between perineural invasion and histological grades of prostatic carcinoma and p-value = 0.613. Among all high grade tumors with Gleason score (8-10), 23 cases were positive for perineural invasion and 25 were negative. Conclusion: Perineural invasion was identified in 27 cases i.e. 45%. There was insignificant association between perineural invasion and histological grades of prostatic carcinoma. Key words: Prostate, adenocarcinoma, perineural invasion, association, TURP,

Genomic Profiling of the Craniofacial Ossifying Fibroma by Next-Generation Sequencing.

Ossifying fibroma (OF) of the craniofacial skeleton is a fibro-osseous lesion characterized by various patterns of bone formation in a cellular fibroblastic stroma. The molecular landscape of OF remains mostly unknown. There are a few known pathogenic abnormalities in OF, including HRPT2 mutations in conventional OF and SATB2 translocations in juvenile psammomatoid OF. On the other hand, conflicting reports exist regarding MDM2 gene amplification and chromosomal copy number alterations (CNA) in OF. Surgically removed biopsies and curettage specimens from OF patients were obtained. Clinical, radiographic, and pathologic features of tumors were reviewed. Genomic DNA was extracted from formalin-fixed, paraffin-embedded blocks of tumor tissue. Capture-based DNA next-generation sequencing targeting the coding regions 529 cancer genes and select introns was performed. We identified 17 OF cases from 8 male and 8 female patients with mean age of 22years (range 1-58years). Nine case occurred in the gnathic bones and 8 in the extragnathic craniofacial bones. These cases included 3 juvenile psammomatoid OF, 6 conventional OF and 8 juvenile trabecular OF. Large-scale CNAs were present in 6 of 17 cases. Seven cases (41%) had focal amplifications including FOSB (n = 2, 11%), FOS (n = 4, 23%), COL1A1 (n = 4, 23%) and TBX3 (n = 5, 29%). Three cases (17%) had pathogenic CDC73 mutations. No cases showed focal MDM2 amplification. Here, we provided a comprehensive molecular characterization of OF that reveals a heterogeneous genetic profile with occasional large-scale CNAs (n = 6, 35%). FOS, FOSB, and TBX3 genes that regulate AP-1 transcriptional complex are frequently altered in OF (n = 7, 41%), chiefly in juvenile trabecular OF. These genes encode transcription factors that act as downstream effectors of the MAP kinase signaling pathway. MDM2 amplification is an exceedingly rare event in OF, if present at all, so identification of this event should continue to raise concern for low-grade gnathic osteosarcoma. In summary, our findings suggest that OF represents a heterogeneous group of tumors at the genetic level but dysregulation of the AP-1 pathway may play a role in pathogenesis of juvenile trabecular OF.

Comparative analysis of oral epithelial malignancies using routine stain and modified Cajal’s trichrome stain: An histopathological study

Diagnosis of initial epithelial pathology maybe difficult in Oral Squamous Cell Carcinoma (OSCC), early invasive squamous cell carcinoma and other atypical epithelial malignancies, under routine Haematoxylin and Eosin (H and E) stain. In this context, an appropriate differential stain capable of distinguishing epithelial cells in the connective tissue may well prove to be a valuable aid. In the present study, we endeavor to use Modified Cajal's trichrome stain for making diagnosis easier without resorting to expensive diagnostic aids. Aim of present study is to assess both the epithelial and connective tissue elements in early invasive carcinoma, verrucous carcinoma and oral squamous cell carcinoma in tissue sections stained with modified Cajal's trichrome stain (MCTS) and to compare it with H and E-stained sections. Formalin-fixed, paraffin-embedded tissue blocks of early invasive SCC (n = 10) Verrucous carcinoma (n = 10) OSCC (n = 10) were stained with MCTS and H&E. The sections were compared based on set histopathological criteria. In SCC cases stained with MCTS, invasion into connective tissue and keratin pearls were strikingly evident. Depth of invasion could be more accurately determined. Thus, CTS is a good differential stain, clearly delineating the epithelial elements from the connective tissue elements visually.

A Simple Molecular Tool for the Assessment of Kidney Transplant Biopsies.

The Banff Classification for Allograft Pathology recommendations for the diagnosis of kidney transplant rejection includes molecular assessment of the transplant biopsy. However, implementation of molecular tools in clinical practice is still limited, partly due to the required expertise and financial investment. The reverse transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) assay is a simple, rapid, and inexpensive assay that permits simultaneous evaluation of a restricted gene panel using paraffin-embedded tissue blocks. The aim of this study was to develop and validate a RT-MLPA assay for diagnosis and classification of rejection. A retrospective cohort of 220 kidney transplant biopsies from two centers, which included 52 antibody-mediated rejection, 51 T-cell-mediated rejection, and 117 no-rejection controls, was assessed. A 17-gene panel was identified on the basis of relevant pathophysiological pathways. A support vector machine classifier was developed. A subset of 109 biopsies was also assessed using the Nanostring Banff Human Organ Transplant panel to compare the two assays. The support vector machine classifier train and test accuracy scores were 0.84 and 0.83, respectively. In the test cohort, the F1 score for antibody-mediated rejection, T-cell-mediated rejection, and control were 0.88, 0.86, and 0.69, respectively. Using receiver-operating characteristic curves, the area under the curve for class predictions was 0.96, 0.89, and 0.91, respectively, with a weighted average at 0.94. Classifiers' performances were highest for antibody-mediated rejection diagnosis with 94% correct predictions, compared with 88% correct predictions for control biopsies and 60% for T-cell-mediated rejection biopsies. Gene expression levels assessed by RT-MLPA and Nanostring were correlated: r = 0.68, P < 0.001. Equivalent gene expression profiles were obtained with both assays in 81% of the samples. The 17-gene panel RT-MLPA assay, developed here for formalin-fixed paraffin-embedded kidney transplant biopsies, classified kidney transplant rejection with an overall accurate prediction ratio of 0.83. This article contains a podcast at https://dts.podtrac.com/redirect.mp3/www.asn-online.org/media/podcast/CJASN/2023_04_10_CJN10100822.mp3.

A deep learning-based histopathology classifier for Focal Cortical Dysplasia

A light microscopy-based histopathology diagnosis of human brain specimens obtained from epilepsy surgery remains the gold standard to confirm the underlying cause of a patient’s focal epilepsy and further inform postsurgical patient management. The differential diagnosis of neocortical specimens in the realm of epilepsy surgery remains, however, challenging. Herein, we developed an open access, deep learning-based classifier to histopathologically assess whole slide microscopy images (WSI) and to automatically recognize various subtypes of Focal Cortical Dysplasia (FCD), according to the ILAE consensus classification update of 2022. We trained a convolutional neuronal network (CNN) with fully digitalized WSI of hematoxylin–eosin stainings obtained from 125 patients covering the spectrum of mild malformation of cortical development (mMCD), mMCD with oligodendroglial hyperplasia in epilepsy (MOGHE), FCD ILAE Type 1a, 2a and 2b using 414 formalin-fixed and paraffin-embedded archival tissue blocks. An additional series of 198 postmortem tissue blocks from 59 patients without neurological disorders served as control to train the CNN for homotypic frontal, temporal and occipital areas and heterotypic Brodmann areas 4 and 17, entorhinal cortex and dentate gyrus. Special stains and immunohistochemical reactions were used to comprehensively annotate the region of interest. We then programmed a novel tile extraction pipeline and graphical dashboard to visualize all areas on the WSI recognized by the CNN. Our deep learning-based classifier is able to compute 1000 × 1000 µm large tiles and recognizes 25 anatomical regions and FCD categories with an accuracy of 98.8% (F1 score = 0.82). Microscopic review of regions predicted by the network confirmed these results. This deep learning-based classifier will be made available as online web application to support the differential histopathology diagnosis in neocortical human brain specimens obtained from epilepsy surgery. It will also serve as blueprint to build a digital histopathology slide suite addressing all major brain diseases encountered in patients with surgically amenable focal epilepsy.

Associations between Folate and Alcohol Consumption with Colorectal Tumor Ki67 Expression in the Southern Community Cohort Study

Folate is hypothesized to accelerate cell proliferation in colorectal cancer (CRC) by supporting DNA synthesis, while alcohol is also linked to gastrointestinal epithelial proliferation, despite biological antagonism of folate. We report associations between folate and alcohol consumption with the proliferation marker Ki67 in CRC tumors from the Southern Community Cohort Study. Tumor samples were obtained from formalin-fixed paraffin-embedded tissue blocks. The percentage of cells expressing Ki67 was measured immunohistochemically. Exposures were assessed via questionnaire pre-diagnosis. Associations were assessed via linear regression. In 248 cases (40–78 years), neither dietary folate, folic acid supplements, nor total folate intake were associated with Ki67. Folic acid supplement use was associated with Ki67 in distal/rectal tumors (β [95% confidence interval]: 7.5 [1.2-13.8], p = .02) but not proximal tumors (-1.4 [-7.1-4.3], p=.62). A positive trend for total folate was observed for distal/rectal tumors (1.6 [0.0-3.3] per 200 μcg, p-trend=.05). Heavy drinking (women: ≥1 drink/day, men: ≥2 drinks/day) was associated with higher Ki67 (6.4 [1.0-11.9], vs. nondrinkers, p=.02), especially for distal/rectal tumors (10.4 [1.6-19.1], p=.02). Negative interaction between alcohol, total folate was observed for distal/rectal tumors (p-interaction=.06). Modest associations between folate, alcohol consumption and distal/rectal tumor Ki67 expression suggest accelerated proliferation, consistent with folate’s role in DNA synthesis.

Genetic heterogeneity and enrichment of variants in DNA-repair genes in ameloblastoma.

Ameloblastomas are a group of relativelycommon odontogenic tumors that frequentlyoriginate from the dental epithelium. These tumors are aggressive in nature and present as slow-growing painless cortical expansion of the jaw. Histologically, the follicular and plexiform subtypes constitute two-thirds of solid/multicystic ameloblastomas. The objective of this study was to understand the genetic architecture of follicular and plexiform ameloblastomas using deep whole-exome sequencing. Archived formalin-fixed paraffin-embedded tissue blocks of follicular (n= 4) and plexiform (n= 6) ameloblastomas were retrieved and genomic DNAs were isolated from the tumor tissue dissected from the formalin-fixed paraffin-embedded block. The exomes were enriched using the Integrated DNA TechnologiesExome Research Panel (IDT, Coralville, IA) and paired-end sequencing was completed on an Illumina NovaSeq 6000 with an average output of 20 GB of data resulting in a mean coverage of 400×. Variant analysis was completed using custom-developed software: Rapid Understanding of Nucleotide variant Effect Software and variant integration and knowledge interpretation in genomes. Our analyses focused on examining somatic variants (gnomAD minor allele frequency ≤1%) in genes found on an FoodandDrugAdministration-approved clinical cancer sequencing panel (FoundationOne®CDx). In follicular tumors, variants (>20% of the reads) were identified in BRAF, KMT2D, and ABL1 genes. In plexiform tumors, variants (>20% of the reads) were identified in ALK, BRAF, KRAS, KMT2D, SMO, KMT2A, and BRCA2 genes. Enrichment analysis showed a significant role of DNA repair genes in the development of these tumors. The variants identified in follicular and plexiform ameloblastomas were enriched in DNA-repair genes. The observed genetic heterogeneity in these ameloblastomas may contribute to the aggressive nature and recurrence risk of these tumors.

The Pattern of Expression of Human Placental Lactogen Across Normal, Lactational, and Malignant Mammary Epithelium.

The immunoexpression of human placental lactogen (hPL) in mammary epithelium is not well studied in the literature. Our overall objective was to delineate the distribution pattern of hPL across mammary epithelia of varying levels of differentiation. This is the first research to study the level of expression of hPL in human lactational change epithelium. Immunohistochemistry (IHC) for hPL was performed on archival formalin-fixed paraffin-embedded tissue blocks of 97 cases. These consisted of 53 invasive ductal carcinomas, 21 lactational change cases, and 23 cases of normal mammary tissue. The results of this study show underexpression of hPL in malignant epithelium compared to normal and lactational groups individually and combined as a non-malignant group. However, a higher expression of hPL was noted in mammary carcinoma of axillary lymph node (ALN)-positive patients compared to ALN-negative cases. There was no statistically significant difference between hPL expression and tumor grade, estrogen receptors (ER), progesterone receptors (PR), or human epidermal growth factor receptor 2 (HER2) status. The comparison of the immunoexpression of hPL in malignant epithelium versus lactational change epithelium may provide the basis for future studies on the possible role of hPL in the protective mechanism of lactation tissue from carcinogenesis. Our results could be explained by the proposed mechanism in the literature, which is that breast cancer cells have a potential inhibitory effect on the translation of human chorionic somatotropin hormone (CSH) mRNA into hPL protein. Our results support the literature findings of a poorer prognostic outcome for breast malignancies when hPL is expressed but require further studies using a more comprehensive range of clinical parameters.

Measurement of LAG-3 Expression Across Multiple Staining Platforms With the 17B4 Antibody Clone.

An immunohistochemistry (IHC) assay developed to detect lymphocyte-activation gene 3 (LAG-3), a novel immune checkpoint inhibitor target, has demonstrated high analytic precision and interlaboratory reproducibility using a Leica staining platform, but it has not been investigated on other IHC staining platforms. To evaluate the performance of LAG-3 IHC assays using the 17B4 antibody clone across widely used IHC staining platforms: Agilent/Dako Autostainer Link 48 and VENTANA BenchMark ULTRA compared to Leica BOND-RX (BOND-RX). Eighty formalin-fixed, paraffin-embedded melanoma tissue blocks were cut into consecutive sections and evaluated using staining platform-specific IHC assays with the 17B4 antibody clone. Duplicate testing was performed on the BOND-RX platform to assess intraplatform agreement. LAG-3 expression using a numeric score was evaluated by a pathologist and with a digital scoring algorithm. LAG-3 positivity was determined from manual scores using a 1% or greater cutoff. LAG-3 IHC staining patterns and intensities were visually similar across all 3 staining platforms. Spearman and Pearson correlations were 0.75 or greater for interplatform and BOND-RX intraplatform concordance when LAG-3 expression was evaluated with a numeric score determined by a pathologist. Correlation increased with a numeric score determined with a digital scoring algorithm (Spearman and Pearson correlations ≥0.88 for all comparisons). Overall percentage agreement was 77.5% or greater for interplatform and BOND-RX intraplatform comparisons when LAG-3 positivity was determined using a 1% or greater cutoff. Data presented here demonstrate that LAG-3 expression can be robustly and reproducibly assessed across 3 major commercial IHC staining platforms using the 17B4 antibody clone.

Cortactin expression in muscle-invasive and nonmuscle-invasive urinary bladder carcinoma in patients live in Sohag City, Egypt

Abstract Background Bladder carcinoma (BC) is the ninth most prevalent cancer worldwide. However, it represents the most frequently occurring cancer in Egypt, where infection by Schistosoma haematobium is an endemic health problem. BCs are clinically subdivided into different stages according to invasion of the muscle layer and distant metastasis. Invasion of the surrounding tissues and metastasis are characteristic issues in malignant tumors. Malignant cells rearrange the intracellular actin filaments in the form of an assembly of branched network in the direction of cell movement. The well-formed actin assembly must be anchored by an adhesive-like protein named cortactin (CTTN). CTTN overexpression has been detected in various human malignant neoplasms. The aim of this study was to evaluate the expression of CTTN in BC and to correlate such expression with different parameters such as depth of invasion, nodal metastasis, and bilharzial infestation. Patients and methods The current study enrolled 66 patients with BCs. Formalin-fixed and paraffin-embedded tissue blocks were prepared from each specimen. Hematoxylin and eosin tissue sections were prepared to confirm the diagnosis of BCs, determine tumor phenotype, assess degree of invasion, and detect any evidence of bilharzial infestation. Corresponding tissue sections were stained by antihuman CTTN antibody using the immunohistochemical approach. Results Overexpression of CTTN was associated with muscle invasion by neoplastic cells (P&lt;0.001), advanced pathological T category (P=0.002), and presence of metastatic deposits in the regional lymph nodes (P&lt;0.001). Conclusion Overexpression of CTTN in BC is associated with increased tumor invasion and nodal metastasis.

Over-expression of Anterior Gradient 3 Is Associated With Tumor Progression and Poor Survival in Gastric Cancer.

Anterior gradient (AGR) proteins, including AGR1, AGR2, and AGR3, which are members of the protein disulfide isomerase family, have been reported as biomarkers for various carcinogenesis processes. Although AGR2 and AGR1 have been demonstrated to be associated with gastric cancer (GC) progression and poor survival, the effect of AGR3 on the progression and prognosis of GC remains unknown. Therefore, our study aimed to examine the expression and prognostic significance of AGR3 in patients with GC. We investigated 271 GC patients receiving curative surgery. Formalin-fixed and paraffin-embedded tissue blocks were obtained, and long-term survival analysis was performed. The expression of AGR3 in GC tissues was investigated by quantitative reverse transcription-polymerase chain reaction, western blotting, and immunohistochemistry. AGR3 was over-expressed in GC tissue compared with paired normal tissue at the mRNA and protein levels. AGR3 over-expression was significantly associated with larger tumor size, deeper tumor invasion, lymph node metastasis, and advanced tumor stage. The overall survival of patients with positive AGR3 expression was significantly lower than that of patients without positive AGR3 expression. Multivariate analysis demonstrated that AGR3 and age were independent prognostic factors associated with overall survival. Over-expression of AGR3 was significantly associated with tumor progression and poor survival of GC patients. Therefore, AGR3 may be a novel biomarker and prognostic factor for GC.

Immunohistochemical expression of CXCL5 as a valuable prognostic marker in urinary bladder urothelial carcinoma

Abstract Background and aim Urothelial carcinoma (UC) is the most common histologic subtype of bladder carcinoma. Detection of novel biomarkers that could predict UC progression and prognosis represents an active focus of research in the oncology field. CXCL5 is a member of the CXC chemokine family. It has been implicated in progression of many cancers, but its prognostic value in UC remains unclear. This study aimed to evaluate the immunohistochemical expression of CXCL5 in noninvasive and invasive UC of the urinary bladder and to correlate its expression with different studied clinicopathological characteristics to evaluate its potential prognostic value. Patients and methods A total of 60 specimens of primary invasive and noninvasive urothelial bladder carcinomas were included in this study. Paraffin-embedded tissue blocks were prepared from the specimens. Two serial tissue sections were obtained from each specimen. One tissue section was stained by hematoxylin and eosin stain and examined to confirm diagnosis, histologic subtype, tumor grade, muscle invasion, and presence or absence of bilharzial infestation, and the second tissue section was subjected to immunohistochemical using anti-human CXCL5 antibody. Results Studying the association between CXCL5 expression and different clinicopathologic parameters in urothelial bladder carcinoma revealed a statistically significant correlation between its expression and poor prognostic indicators. CXCL5 expression is significantly correlated with poorly differentiated (P=0.025), advanced stage (P=0.039), and invasive tumors (P=0.002). Conclusion The current study revealed that CXCL5 overexpression in urothelial bladder carcinoma is strongly associated with adverse prognostic factors. CXCL5 might be considered as a reliable biomarker in predicting tumor progression and poor prognosis.

Evaluation of Immunohistochemical Expression of Heparanase in Helicobacter pylori-Associated Chronic Gastritis.

Chronic gastritis (CG) is a very common disease. More than half of the worldwide population suffers from symptoms of CG. This disease has received great attention since the discovery of H. pylori as the most important cause of CG. Symptoms experienced by patients with CG are attributed to H. pylori-induced inflammatory reactions. Heparanase (HPSE) is a mammalian β-endoglucoronidase. In inflammation; HPSE degrades and remodels the extracellular matrix's heparan sulfate polysaccharide chains liberating heparan sulfate-bound cytokines and chemokines, HPSE also facilitates movement of inflammatory cells. This study aimed to detect the function of HPSE in CG by correlating levels of HPSE expression with histopathological features of CG, including H. pylori infection, acute and chronic inflammatory cells, mucosal atrophic and/or metaplastic features. Ninety-five upper endoscopic-guided gastric punch biopsies were enrolled in this study. From each specimen, formalin-fixed and paraffin-embedded tissue blocks were prepared. Tissue sections were stained by Hematoxylin and eosin, Giemsa, and anti-heparanase antibody. HPSE expression was statistically associated with H. pylori infection (P-value < .000), and intensity of chronic lymphocytic inflammatory infiltrate in the gastric mucosal tissues (P = .004). High levels of HPSE expression were also related to the presence of neutrophils in the gastric surface epithelium and lamina propria (P-value < .009). HPSE expression was upregulated in H. pylori-associated chronic gastritis. Thus, future therapeutic agents that could specifically inhibit HPSE enzyme activity, may aid in the reduction of sequelae of H. pylori infection.

EpCAM expression negatively regulates E-cadherin function in colorectal carcinomas.

This study aimed to characterise epithelial cell adhesion molecule (EpCAM) expression patterns in colorectal carcinomas (CRC) from Nigerian patients, its association with E-cadherin and tumour characteristics, to forecast patient selection for anti-EpCAM therapy among whom no data existed previously. Tissue microarray blocks of formalin-fixed and paraffin-embedded CRC tissues, with their non-cancer margins of resection, were sectioned and stained with EpCAM and E-cadherin primary antibodies. Scoring for antibody staining was done semiquantitatively by combining staining proportion and intensity. The outcome was correlated with patient age, gender and tumour histological parameters with p ≤ 0.05 regarded as statistically significant. Sixty-three carcinoma tissues had staining status for the two markers and were included in this study. Of these, 36 (57.1%) showed positive EpCAM expression (immunoscore ≥3) out of which 83% (30/36 positive cases) were overexpressed (combined immunoscore ≥4) while 12 (19%) tissues were positive for E-cadherin. Non-tumour margins of resection tissues showed less EpCAM positivity in 24% (6/25) of histospots. The difference in staining between tumour and non-tumour margin tissues with EpCAM was significant (p < 0.001). Also, EpCAM overexpression was significantly associated with reduced E-cadherin (p < 0.035) expression in tumour cells. Tumour extent within the gut wall was equal (50% each) for early and late pT stages among EpCAM overexpressing tumours but two-thirds (8/12) of cases expressing E-cadherin had later pT stage paradoxically, while distant metastasis was negligible among tumours bearing both markers. Also, tumours overexpressing EpCAM had significant association with tumour-associated lymphocytes (p < 0.02 each). CRC in this study preferentially overexpress EpCAM over E-cadherin whose strong cell-cell contact inhibitory role is weakened even when expressed, resulting in further local tumour spread. This, and the observed immune response, supports targeted therapy among eligible patients.

Identifying Active Progeny Virus Particles in Formalin-Fixed, Paraffin-Embedded Sections Using Correlative Light and Scanning Electron Microscopy

Immunohistochemical analysis of formalin-fixed paraffin-embedded (FFPE) tissue blocks is routinely used to identify virus-infected cells. However, detecting virus particles in FFPE sections using light microscopy is difficult because of the light diffraction resolution limitations of an optical microscope. In this study, light microscopy and field emission scanning electron microscopy were performed to observe 3-dimensional virus particles in FFPE sections in a nondestructive manner using NanoSuit or osmium conductive treatment methods. The virus particles in FFPE sections were immunostained with specific antibodies against the surface antigens of the viral particles and stained with 3,3′-diaminobenzidine. A metal solution (0.2% gold chloride or 2% osmium tetroxide) was applied to enhance the 3,3′-diaminobenzidine–stained area. This procedure is nondestructive for FFPE sections and is a simpler method than transmission electron microscopy. To validate the applicability of this technique, we performed 3-dimensional imaging of the virus particles of different sizes, such as human papillomavirus, cytomegalovirus, and varicella-zoster virus. Furthermore, ultrathin sections from the FFPE sections that were observed to harbor viral particles using field emission scanning electron microscopy were prepared and assessed using transmission electron microscopy. In the correlative areas, transmission electron microscopy confirmed the presence of large numbers of virus particles. These results indicated that the combination of marking viral particles with 3,3′-diaminobenzidine/metal staining and conductive treatment can identify active progeny virus particles in FFPE sections using scanning electron microscopy. This easy correlative imaging of field emission scanning electron microscopy of the identical area of FFPE in light microscopy may help elucidate new pathological mechanisms of virus-related diseases.

Correlation of amyloid and ameloblast-associated proteins to odontogenic cysts and tumors: A cross-sectional study.

Odontogenic cysts and tumors often form hard and soft structures that resemble odontogenesis. It is well known that amyloid is produced in Pindborgtumors; however, it is still debatable whether it is also formed in other odontogenic tumors and cysts. This study aimed to detect the presence of amyloid in different odontogenic cysts and tumors in correlation to matrix proteins secreted during enamel formation; namely amelogenin and odontogenic ameloblast-associated protein. This study included formalin fixed paraffin embedded tissue blocks of 106 different types of odontogenic cysts and tumors. Congo red and thioflavin T were performed to confirm the presence of amyloid; immunohistochemistry was used to detect amelogenin and odontogenic ameloblast-associated protein. Amyloid was detected in pindborg tumors (conventional), adenomatoid odontogenic tumors, odontogenic fibroma (Amyloid variant), follicular solid and unicystic ameloblastomas, radicular cysts, dentigerous cysts, dentinogenic ghost cell odontogenic tumor, ameloblastic fibroma, calcifying odontogenic cyst, and primordial Odontogenic tumor. Amelogenin was detected in 95.3% of the cases, while odontogenic ameloblast-associated protein was detected in 93.4% of the cases. The association between odontogenic ameloblast-associated protein and amyloid was highly significant at p < 0.01. However, there was no significant relationship between amelogenin and amyloid p > 0.05. Although pindborg tumor is the bonafide example of amyloid deposition in odontogenic tumors, this study concluded that amyloid may be deposited in traces to massive amounts in various odontogenic cysts and tumors, and it is significantly linked to odontogenic ameloblast-associated protein but not amelogenin matrix protein, since all amyloid cases were odontogenic ameloblast associated protein positive.

CD44 Expression in Oral Lichen Planus and Related Lesions-An Immunohistochemical Study.

Cluster of differentiation 44 (CD44) is a cell surface adhesion protein involved in the progression and metastasis of oral squamous cell carcinoma. The current study aims to evaluate the expression of CD44 in oral lichen planus and related lesions and thereby assess the relative risk of malignant transformation of these lesions. Formalin-fixed paraffin-embedded tissue blocks of 10 oral lichen planus (Group 1), 10 oral lichenoid lesions (Group 2), 8 with oral lichen planus with dysplasia (Group 3), and 5 with lichenoid dysplasia (Group 4) were included in the study. Immunostaining was done for the tissue sections using CD44 mouse monoclonal antibody. Staining density, staining intensity, and immunoreactive scores of CD44 were evaluated in all four groups. Statistical analysis was done by Statistical Package for the Social Sciences® software and the Kruskal-Wallis test was used. CD44 staining pattern of lichenoid dysplasia and lichen planus with dysplasia changed from membranous to cytoplasmic. The membranous CD44 immunoreactivity was mild with a score of 2.25 for Group 3 and 1.6 for Group 4 whereas moderate for other groups with a P-value of 0.009. The cytoplasmic immunoreactivity was significantly high in Group 3 (5.3 ± 2.6) followed by Group 4 (3.2 ± 1.2), Group 2 (1 ± 1.8), and Group 1 (0.7 ± 1.3) with a P-value of 0.001. The CD44 membranous immunoreactivity scores were low while the cytoplasmic immunoreactivity was high in oral lichen planus with dysplasia and oral lichenoid dysplasia when compared to oral lichen planus and oral lichenoid lesions. CD44 immunostaining pattern can help in assessing the malignant transformation of oral lichen planus or lichenoid lesions.

Evaluation of Her-2 protein in histologically tumor-free margins of oral squamous cell carcinoma: An immunohistochemical study.

The status of the 5-year survival rate and loco-regional recurrence in oral squamous cell carcinoma (OSCC) has remained unchanged over the decades. Recent advances in oral cancer research have revealed that the presence of molecular alterations in histologically tumor-free margins of OSCC has a prognostic significance and can aid in designing therapeutic strategies. However, the literature on molecular studies on histologically tumor-free margins is scant, especially in the Indian population. Considering the prognostic implications of Her-2 in malignancies of the breast, ovary, and OSCC, we aimed to assess the expression of Her-2 protein in histologically tumor-free margins of OSCC and to establish correlation with clinico-pathological parameters. 4 μ m thick sections from formalin-fixed paraffin-embedded tissue blocks of 40 histologically tumor-free margins of OSCC affecting the buccal mucosa and/or lower gingiva-buccal sulcus and 40 normal oral mucosa samples were subjected to immunohistochemical analysis for the Her-2 antibody. The obtained data were analyzed statistically. The mean ages in study and control groups were 49.83 years (SD ± 10.43) and 37.28 years (SD ± 8.61), respectively, with male predominance. Local recurrence was seen in 52.5% of patients. Follow-up data revealed that a total of 71.4% of patients succumbed to mortality and all of them had reported local recurrence. Overall, the presence of local recurrence and the status of survival showed a statistically significant association (p = 0.0001). All the samples in the study and control groups were negative for Her-2 immuno-expression. The study indicated the lack of Her-2 immuno-expression in histologically tumor-free margins of OSCC with several speculated explanations. As it is a preliminary study, further studies employing both immunohistochemistry (IHC) and gene amplification in histologically tumor-free margins of OSCC affecting different anatomical sites are warranted. This will aid in identifying the subset of patients that may benefit from targeted therapy.