Specific Staining Research Articles

Review: Aplikasi Histopatologi Untuk Praktik Forensik

Forensics plays an important role in investigating crimes because it can reveal everything related to a murder case, including the time and cause of the murder. Histopathology is the study of damage to body tissues and organs which can be used to help investigate crimes. The aim of this literature review is to study the role of histopathology applications for forensic practice. The method used is searching journals and books using Google Scholar, ScienceDirect, NCBI, and PubMed with the keywords histopathology, forensics, diagnosis, death, microscopy, disease, staining, and autopsy. The histology approach helps the pathologist make a clear determination on the cause of death. The aim of histopathology is to perfect the macroscopic diagnosis and act as an additional examination in forensic case management. One of the histopathology methods in the forensic field is the paraffin method with hematoxylin eosin staining and special stains, immunohistochemistry, and MITS. The parameters observed with this method are measurements of pulmonary fat embolism; determining the age of myocardial infarction; assessment of liver steatosis, fibrosis, and cirrhosis; determination of the age of thrombus or thromboembolism; and diagnosis of shock with various diseases. The prospect of forensic histopathology is to complement molecular diagnostic methods, so that the combination of molecular and histology methods can play a maximum role in the field of forensic pathology. The conclusion of this literature review is that histopathology can be used to confirm the diagnosis in forensic cases and has good future prospects if combined with other methods such as molecular.

Anabasis articulata (Forssk.) Moq. food aqueous extract triggers oxidative stress-induced senescence and reduces metastatic power in MDA-MB-231 cells

Ancient ethnobotanical practices handed down through traditional knowledge are still commonly employed to treat various pathologies, although the scientific reasons underlying their biological effects have not been clarified yet. In this contribution, the potential antitumoral activity of the aqueous extract from A. articulata (AAE) was investigated to validate the hypothesis of the Algerian folk medicine which would suggest this plant derivative as a functional food for treating breast cancer. A. articulata phytocomplex, isolated by maceration following exactly the African recipe, has been already characterized by our research group in previous works. Thus, the antiproliferative function of AAE against MDA-MB-231, a highly aggressive human breast adenocarcinoma cell line, was evaluated. Slowing down of cell growth, absence of cytotoxicity and DNA fragmentation, and cell cycle arrest at the G2/M phase were observed after treatment with AAE at different doses (0.3–6 mg of dried plant material equivalent per mL of culture medium) for 24 and 48 h. Wound and transwell assays proved that AAE possessed both antimigration and antiinvasive capacities, evidence also supported by molecular analyses focused on Metalloproteases (MMP-2 and MMP-9), Vimentin and ανβ3-Integrin. These results, together with the demonstration of the activation of p53/p21WAF1/Cip1/p27Kip1 pathway and the increase of oxygen reactive species levels, suggested that AAE triggered a senescence process. The final confirmation was obtained by a specific kit staining senescent cells. All our data would explain the efficacy of the Algerian medicinal remedy based on the intake of the investigated functional plant food and would highlight the basics for developing novel natural pharmacological products based on AAE and showing preventive and therapeutic antineoplastic potentialities against highly aggressive breast cancers.

Testicular Regression Syndrome: Histopathological Evaluation of 20 cases from Rural India with Review of Literature

ABSTRACT Context: Testicular regression syndrome (TRS), clinically termed “vanishing testes,” is a condition with atrophy of testes in late fetal or early neonatal life. The spermatic cord ends blindly or into a small fibrovascular nubbin. Histopathology reveals a fibrovascular nodule with associated hemosiderin-laden macrophages and dystrophic calcification. Few cases show the presence of seminiferous tubules and germ cells. The presence of germ cells is associated with the risk of developing a testicular germ cell tumor. Aims: To study the histopathological features of testicular nubbins and to determine the presence of germ cells and associated neoplasm. Settings and Design: It is a retro-prospective observational study carried out on patients referred to a pediatric surgical pathology center in Miraj, who were clinically diagnosed with undescended testis, over a period of 1 year. Data were collected after the study was approved by the IEC-MGCH on December 19, 2022. Materials and Methods: Twenty patients diagnosed with cryptorchidism, undescended testis, or atrophic testis were included. Findings of hematoxylin and eosin-stained slides of each case were noted, and Prussian Blue (special staining) was performed in four cases. Statistical analysis used: As this is a descriptive observational study, simple arithmetic calculations were performed. No unique statistical analysis was performed. Results: Among twenty cases of vanishing testes syndrome, 20 cases showed fibrovascular tissue only, and 1 case showed the presence of seminiferous tubules with Sertoli cells. Conclusions: Vanishing testes syndrome is a rare condition that is difficult to diagnose on clinical examination, and a definitive diagnosis may be missed if the pathologist is unaware of the condition. Also, there is a risk of germ cell tumor if there are residual germ cells in the testicular nubbin.

The clinical and pathological features of Kimura disease in pediatric patients.

Kimura disease is characterized by inflammation, with its underlying causes remaining uncertain. There is a lack of comprehensive and systematic research on the pathology of this condition in pediatric patients. Our objective is to study the clinical and pathological attributes of Kimura disease in pediatric patients and investigate the potential diagnostic significance of immunoglobulin E (IgE) in this context. Clinical and laboratory information, pathological characteristics, and follow-up data were correlated to examine the distinctive features. Immunohistochemistry, acid-fast staining, and molecular assay were used to identify the presence of IgE and pathogens. We conducted an analysis of five cases of Kimura disease in pediatric patients at our hospital. The patients' ages ranged from 5 years and 7 months to 14 years and 2 months, with 4 (80%) being male. The most common site was the head and neck region, particularly the postauricular subcutaneous area. Eosinophilia was observed in four patients (80%), and two patients (40%) had elevated serum immunoglobulin E (IgE) levels. Histopathological changes included eosinophilic infiltrates, follicular hyperplasia, and the proliferation of postcapillary venules. Immunohistochemical results supported the reactive nature of the lymphoid process and IgE deposition in the follicle, while no specific pathogens were discovered by special staining. All patients underwent surgical excision, and none experienced recurrence in their original location. Children with Kimura disease show distinct eosinophilic and IgE alterations in both laboratory findings and pathological features. The application of immunohistochemical staining of IgE could serve as a promising marker for diagnosing Kimura disease.

Two-Photon-Responsive "TICT + AIE" Active Naphthyridine-BF2 Photoremovable Protecting Group: Application for Specific Staining and Killing of Cancer Cells.

The combined effects of twisted intramolecular charge transfer (TICT) and aggregation-induced emission (AIE) phenomena have demonstrated a significant influence on excited-state chemistry. These combined TICT and AIE features have been extensively utilized to enhance photodynamic and photothermal therapy. Herein, we demonstrated the synergistic capabilities of TICT and AIE phenomena in the design of the photoremovable protecting group (PRPG), namely, NMe2-Napy-BF2. This innovative PRPG incorporates TICT and AIE characteristics, resulting in four remarkable properties: (i) red-shifted absorption wavelength, (ii) strong near-infrared (NIR) emission, (iii) viscosity-sensitive emission property, and (iv) accelerated photorelease rate. Inspired by these intriguing attributes, we developed a nanodrug delivery system (nano-DDS) using our PRPG for cancer treatment. In vitro studies showed that our nano-DDS manifested effective cellular internalization, specific staining of cancer cells, high-resolution confocal imaging of cancerous cells in the NIR region, and controlled release of the anticancer drug chlorambucil upon exposure to light, leading to cancer cell eradication. Most notably, our nano-DDS exhibited a substantially increased two-photon (TP) absorption cross section (435 GM), exhibiting its potential for in vivo applications. This development holds promise for significant advancements in cancer treatment strategies.

Isolated bovine pancreatic islets as an alternate in vitro model for diabetes research.

Background & objectives Isolation of functional pancreatic islets for diabetes research and clinical islet transplantation stands as a big challenge despite the advancements in the field. In this context, the non-availability of human/animal tissues is one of the major impediments to islet-based research, which has tremendous scope for translation. The current study explores the feasibility of using the bovine pancreas as an alternative source to isolate pancreatic islets and assess its functionality for in vitro studies. Methods The bovine pancreas was collected from a registered slaughterhouse and transported in an ice-cold medium - Hank's Balanced Salt Solution (HBSS) to the laboratory. Islets were isolated by sequential collagenase digestion followed by a two-step filtration and purification by density gradient separation method. After isolation, islets were identified with dithizone staining and the islet function was assayed in vitro for assessing the dynamic insulin secretory function by monitoring the glucose-stimulated insulin secretion (GSIS), in response to low and high glucose. Staining techniques were also used to understand the cytoarchitecture of the bovine pancreas. Results The islet yield was 157±23 islets per gram of pancreas and was viable. The cold ischaemia time was reduced to 60-75 min. The islets released insulin with glucose stimulation. The insulin release was observed more with high glucose (28 mM) than with low glucose (2.8 mM). Dithizone staining confirmed the presence of islets after isolation and the size of islets ranged from 50 to 600 µm size. The mantled islets (islets with acinar tissue) were also noted with the pure islets in culture. Hematoxylin and eosin (H&E) and aldehyde- fuchsin showed islets interspersed in the acinar tissue of the bovine pancreas. Special stain defined the islets better than regular staining. Fluorescent and diaminobenzidine (DAB) staining with insulin, glucagon and somatostatin revealed the arrangement of the cells in each islet. The beta cells were majorly found in the islet core with alpha cells interspersed with the delta cells in the periphery. Interpretation & conclusions The isolation procedure described in this study yielded viable islets for in vitro studies which showed a differential response to glucose challenge, confirming their viability. We provide a simple and reproducible method for small-scale isolation of functional islets from the bovine pancreas. This model proffers the beginner a hands-on in islet experiments and helps to re-iterate the process that could be extrapolated to other pancreatic tissues as well as to expand on diabetes research.

Primary intracranial malignant melanoma in an adolescent female: a case report

BackgroundPrimary central nervous system melanoma is an extremely rare entity and even rarer in children and adolescents as compared to adults. It is often difficult to consider a diagnosis of intracranial melanoma pre-operatively without any clinical evidence of neurocutaneous melanosis.Case presentationHerein, a case of primary melanoma of the brain is reported in a 17-year-old female who presented with headache, vomiting, and focal neurological deficit in the form of left-sided facial palsy and limb weakness. A contrast-enhanced computed tomography of head was performed which revealed a heterogeneously hyperattenuating mass lesion at left gangliocapsular region showing peripheral enhancement with internal non-enhancing cystic component. The patient underwent left frontotemporal craniotomy. The diagnosis was made on histopathological examination, which showed an invasive tumor comprising of epithelioid to spindled cells arranged in sheets, nests, and singly scattered. The special stains and immunohistochemical markers proved very helpful in establishing the diagnosis.ConclusionsThe case highlights the uncommon occurrence of primary intracranial melanoma in the pediatric age group, the perplexing histological features, and the rapid and fatal course.

Clinicopathological features of Sjogren's syndrome complicated with liver injury

Objective: To study the clinicopathological features of Sjogren's syndrome (SS) with liver injury and to improve the understanding of this disease. Methods: Forty-nine patients with SS complicated with liver injury were collected from Beijing Ditan Hospital, Capital Medical University from October 2008 to January 2022. All patients underwent ultrasound-guided liver biopsy, and all specimens were stained with HE. The histopathologic characteristics were observed and the pathologic indexes were graded. Immunohistochemical stains for CK7, CK19, CD38, MUM1 and CD10 were performed by EnVision method; and special histochemical stains for reticulin, Masson's trichrome, Rhodanine, Prussian blue, periodic acid Schiff (PAS) and D-PAS stains were conducted. Results: The age of patients ranged from 31 to 66 years, including 3 males and 46 females. SS combined with drug-induced liver injury was the most common (22 cases, 44.9%), followed by autoimmune liver disease (13 cases, 26.5%, including primary biliary cholangitis in eight cases, autoimmune hepatitis in 3 cases, and PBC-AIH overlap syndrome in 2 cases), non-alcoholic fatty liver disease (NAFLD, 9 cases, 18.4%) and other lesions (5 cases, 10.2%; including 3 cases of nonspecific liver inflammation, 1 case of liver amyloidosis, and 1 case of porto-sinusoidal vascular disease). Among them, 28 cases (57.1%) were associated with obvious interlobular bile duct injury, mainly in SS combined with PBC group and drug-induced liver injury group. Twenty-three cases (46.9%) were associated with hepatocyte steatosis of varying degrees. In SS with autoimmune liver disease group, ISHAK score, degree of fibrosis bile duct injury, bile duct remodeling, lymphocyte infiltration of portal area, and plasma cell infiltration, MUM1 and CD38 expression; serum ALP and GGT, IgM; elevated globulin; positive AMA, proportion of AMA-M2 positive and IgM positive were all significantly higher than those in other groups(all P<0.05). Serum ALT, direct bilirubin and SSA positive ratio in SS combined with drug liver group were significantly higher than those in other groups(all P<0.05). The serum total cholesterol level in SS combined with PBC group (P=0.006) and NALFD group (P=0.011) were significantly higher than those in other groups (P<0.05). Conclusions: The pathologic manifestations of SS patients with liver injury are varied. The inflammatory lesions of SS patients with autoimmune liver disease are the most serious, and the inflammatory lesions of SS patients with non-alcoholic fatty liver disease and non-specific inflammation are mild. Comprehensive analysis of liver histopathologic changes and laboratory findings is helpful for the diagnosis of SS complicated with different types of liver injury.

Verification of the expression trend and interaction prediction of innate immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis.

This study aimed to explore the expression trends of innate immune cells and immune-checkpoint molecules validated by data calculation in the process of oral mucosal carcinogenesis, as well as to explore methods of suppressing oral mucosal carcinogenesis based on immunotherapy by predicting their interactions. Me-thods 1) The cancer genome atlas (TCGA) database comprehensively scores immune cells and immune-checkpoint molecules in the process of oral mucosal carcinogenesis and screens out intrinsic immune cells and immune-checkpoint molecules that interfere with tumor immune escape. 2) Clinical patient blood routine data were collected for the statistical analysis of peripheral blood immune cells during the progression of oral mucosal carcinogenesis. Immune cells in peripheral blood that may affect the progression of oral mucosal carcinogenesis were screened. 3) Immunohistochemical staining was performed on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation in various stages of oral mucosal carcinogenesis. 4) Special staining was used to identify innate immune cells in various stages of oral mucosal carcinogenesis based on data-calculation verification. 5) Survival analysis was conducted on intrinsic immune cells and immune-checkpoint molecules validated based on data calculation during the process of oral mucosal carcinogenesis. The association of intrinsic immune cells and immune-checkpoint molecules with the prognosis of oral squamous cell carcinoma was verified. The expression of monocytes and neutrophils increased during the process of oral mucosal carcinogenesis. The expression of eosinophils showed a single peak trend of up and down. The expression of mast cells decreased. In the process of oral mucosal carcinogenesis, the expression of the immune-checkpoint molecules cytotoxic T-lymphocyte-associated protein 4 (CTLA4) and programmed cell death-ligand (PD-L1) increased. The expression trends of monocytes, neutrophils, and eosinophils were positively correlated with those of CTLA4 and PD-L1 immune-checkpoint molecules. The expression trend of mast cells was negatively correlated with the expression of CTLA4 and PD-L1. Monocytes, neutrophils, and eosinophils may promote tumor immune escape mediated by CTLA4 and/or PD-L1, thereby accelerating the progression of oral mucosal carcinogenesis. Mast cells may inhibit tumor immune escape mediated by CTLA4 and/or PD-L1, delaying the progression of oral mucosal carcinogenesis. Therefore, interference with specific immune cells in innate immunity can regulate the expression of CTLA4 and/or PD-L1 to a certain extent, inhibit tumor immune escape, and delay the progression of oral mucosal carcinogenesis.

Clinicopathological study of masses of nasal cavity and paranasal sinuses and its correlation with radiology

Abstract Background: Nasal and paranasal abnormalities frequently represent the primary observations in the otorhinolaryngology domain. Thorough clinical evaluation and radiological methodologies aid in establishing a preliminary diagnosis; however, histopathological examination (HPE) remains indispensable for confirming a definitive diagnosis. The objective is to determine the demographic breakdown of patients exhibiting sinonasal lesions and to evaluate and correlate clinical and radiological observations with histopathological diagnoses. Materials and Methods: This ambispective observational study involved 128 sinonasal lesions diagnosed via histopathology spanning 3 years (2020–2023). All pertinent clinical radiological observations were recorded, and tissue samples underwent staining with hematoxylin and eosin (H&amp;E), special stains, and immunohistochemistry (IHC) when necessary. The collected data were organized into tables, and statistical analyses were conducted. Results: Among the 128 cases examined, 98 (76.56%) were identified as non-neoplastic lesions, 14 (10.93%) as benign neoplasms, and 16 (12.50%) as malignant lesions through HPE. The male-to-female ratio stood at 1.2:1. The highest number of cases was observed within the age bracket of 21–40 years. Nasal obstruction emerged as the predominant symptom, accounting for 46 (35.93%) cases. Conclusion: A broad range of lesions can impact the sinonasal tract. While clinical and radiological characteristics may exhibit similarities, histopathology is the ultimate standard for confirming a definitive diagnosis.

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis (Adv. Healthcare Mater. 10/2024)

Amplified and Specific Staining of Protein Dimerization on Cell Membrane Catalyzed by Responsively Installed DNA Nanomachines for Cancer Diagnosis (Adv. Healthcare Mater. 10/2024)

Gross as well as microscopic anatomy and physiological functions of fetal placenta in Jaffrabadi buffaloes

The present study was conducted to know the gross morphology and histo-morphological structure of fetal placenta in Jaffrabadi buffaloes. Parameters like calf weight, placental weight, numbers, and size of cotyledons were observed in Jaffrabadi buffaloes and morphologically, fetal cotyledons were convex and non-pendunculated in Jaffrabadi buffaloes were found. Histological studies of small and large cotyledons showed extensive branching of secondary and tertiary villi that were longer, slender, and well developed in Jaffrabadi buffalo. A less developed basal lamina was seen in small cotyledon whereas developed basal lamina with numerous capillaries and connective tissue were observed in the large cotyledon. The diameter of trophoblast giant cells (TGC) in larger cotyledons were significantly (P&lt;0.05) than the small cotyledons in expelled placenta at full term in Jaffrabadi buffaloes. A distinct distribution of carbohydrate and lipids in cotyledons were observed between large and small cotyledons as evident by acid mucopolysaccharides, neutral polysaccharides, and sudanophilic staining. Specific staining for calcium with Alizarin red stain showed that calcium is not present in a noticeable amount in small and large cotyledons. Isolation and culture of Jaffrabadi placental cells in M-199 medium with antibiotics and 2% FBS results in the efficient production of progesterone, estrogen, and testosterone. This study has shown that trophoblast cells are the actual sites for steroid hormone production. These cultured placental cells (1x106 cells/ ml) produce Progesterone, Estradiol-17β and Testosterone in the range of 1.72 to 2.12, 16.03 to 19.51 and 0.51 to 0.58 ng/ml, respectively in Jaffrabadi buffalo.

In Vivo Fluorescent Labeling of Foam Cell-Derived Extracellular Vesicles as Circulating Biomarkers for In Vitro Detection of Atherosclerosis.

Real-time monitoring of the development of atherosclerosis (AS) is key to the management of cardiovascular disease (CVD). However, existing laboratory approaches lack sensitivity and specificity, mostly due to the dearth of reliable AS biomarkers. Herein, we developed an in vivo fluorescent labeling strategy that allows specific staining of the foam cell-derived extracellular vesicles (EVs) in atherosclerotic plaques, which are released into the blood as circulating biomarkers for in vitro detection of AS. This strategy relies on a self-assembled nanoprobe that could recognize foam cells specifically, where the probe is degraded by the intracellular HClO to produce a trifluoromethyl-bearing boron-dipyrromethene fluorophore (termed B-CF3), a lipophilic dye that can be transferred to the exosomal membranes. These circulating B-CF3-stained EVs can be detected directly on a fluorescence spectrometer or microplate reader without resorting to any sophisticated analytical method. This liquid-biopsy format enables early detection and real-time differentiation of lesion vulnerability during AS progression, facilitating effective CVD management.

Number of intraepithelial lymphocytes and presence of a subepithelial band in normal colonic mucosa differs according to stainings and evaluation method

Chronic watery diarrhea is a frequent symptom. In approximately 10% of the patients, a diagnosis of microscopic colitis (MC) is established. The diagnosis relies on specific, but sometimes subtle, histopathological findings. As the histology of normal intestinal mucosa vary, discriminating subtle features of MC from normal tissue can be challenging and therefore auxiliary stainings are increasingly used. The aim of this study was to determine the variance in number of intraepithelial lymphocytes (IELs) and presence of a subepithelial band in normal ileum and colonic mucosa, according to different stains and digital assessment. Sixty-one patients without diarrhea referred to screening colonoscopy due to a positive feacal blood test and presenting with endoscopically normal mucosa were included. Basic histological features, number of IELs, and thickness of a subepithelial band was manually evaluated and a deep learning-based algorithm was developed to digitally determine the number of IELs in each of the two compartments; surface epithelium and cryptal epithelium, and the density of lymphocytes in the lamina propria compartment. The number of IELs was significantly higher on CD3-stained slides compared with slides stained with hematoxylin-and-eosin (HE) (p<0.001), and even higher numbers were reached using digital analysis. No significant difference between right and left colon in IELs or density of CD3-positive lymphocytes in lamina propria was found. No subepithelial band was present in HE-stained slides while a thin band was visualized on special stains. Conclusively, in this cohort of prospectively collected ileum and colonic biopsies from asymptomatic patients, the range of IELs and detection of a subepithelial collagenous band varied depending on the stain and method used for assessment. As assessment of biopsies from patients with diarrhea constitute a considerable workload in the pathology departments digital image analysis is highly desired. Knowledge provided by the present study highlight important differences that should be considered before introducing this method in the clinic.

Abstract 6330: A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors

Abstract Introduction: Chimeric Antigen Receptor (CAR)-T cell therapy is a highly innovative form of immunotherapy that has proven to be successful in the treatment of B cell malignancies and multiple myeloma. As this treatment modality continues to evolve toward the targeting of novel tumor antigens and engineering of CAR constructs that enhance cell persistence, there is a need for reagents that can be leveraged to selectively interrogate multiple aspects of CAR-T cell biology. Here, we report on a novel and highly versatile recombinant rabbit monoclonal antibody raised against the Gly4Ser peptide linker, which is commonly integrated into single-chain variable fragment (scFv)-based CARs. This antibody can be leveraged for flow cytometry-based CAR detection, bead-based CAR-T cell enrichment, and selective activation of CAR transduced cells. Methods: The recombinant monoclonal antibody, E7O2V, was generated by immunizing rabbits with a synthetic peptide containing multiple repeats of the Gly4Ser core pentapeptide. E7O2V was directly conjugated to a panel of fluorophores and validated for specificity in a live cell flow cytometry assay using non-transduced and CAR-transduced primary human T cells. To assess its utility in cell enrichment using bead-based sorting, a biotinylated conjugate of E7O2V was used in combination with streptavidin releasable magnetic meads to immunoaffinity purify CAR transduced cells from a mixed population. Finally, a plate-bound antibody stimulation assay was leveraged to assess the ability of E7O2V to selectively activate CAR transduced cells. Results: Live cell flow cytometric analysis of CAR-transduced primary human T cells revealed that E7O2V could detect surface expressed CARs, independent of scFv specificity. No specific staining was observed on non-transduced cells. Furthermore, flow cytometric analysis of bead-free, purified cells revealed that biotinylated E7O2V could enrich CAR transduced cells to a high degree of purity and viability. E7O2V was found to selectively activate CAR transduced cells, but not non-transduced cells, as demonstrated by upregulation of cell surface activation markers, including CD69 and CD25. Conclusions: Exploiting the commonly used Gly4Ser linker of scFv-based CARs for antibody discovery, we identified a novel and highly versatile monoclonal antibody, E7O2V. This antibody binds to CARs of varying antigen specificity and can be leveraged in multiple assay formats to interrogate various attributes of CAR transduced cells, including CAR expression, CAR signaling, and transcriptome profiling enabled by bead-based enrichment. Citation Format: Amrik Singh, Sarah L'Heureux, Jeremy Fisher. A novel monoclonal antibody for the detection, enrichment and activation of cells expressing scFv-based chimeric antigen receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6330.

Abstract 2589: Novel workflow to generate natural killer cell sensitivity prediction model based on the epithelial mesenchymal transition related reprogramming at a single cell resolution

Abstract Single cell technology allows us to examine cell heterogeneity and identify rare cell populations; however, the high cost of single cell RNAseq limits its adoption into clinical and research environments. As an inexpensive alternative, immunofluorescent assays analyzing cell morphologies have been used in high content screening to examine cell phenotype and response to external stresses. Natural Killer cells (NK) are the bodies first line of defense and gained traction as a potential target in treating cancer. A study showed that cancer cells undergoing the process epithelial mesenchymal transition (EMT) increase their vulnerability to NK mediated cytotoxicity. We hypothesize that the EM morphology change can be correlated with NK sensitivity. Our goal is to develop an image-based, cost-effective, and efficient workflow capable of extracting cancer cell morphological features to predict its NK sensitivity at the single-cell level. To optimize this workflow, we use A549 cell line cells treated with transforming growth factor- β (TGF-β) as a representative models of cells with various EM phenotypes as NK sensitivities. We utilized a Cell Painting inspired novel image based live cell cytotoxicity assay. To capture cell morphological features, we optimized an organelle specific and live cell compatible staining panel that targets the nucleus, nucleic acid, cell membrane, F-actin, and mitochondria. The cell viability is assessed by Propidium Iodide, which stains dead cell nuclei. Our image analysis pipeline can extract the morphological features and track the cells of interest during the experiment and assess their viability. Subsequently, the morphology and viability data will be processed by a classification algorithm that could discern morphological features of cancer cells that indicate high NK vulnerability. We started with treating A549 cells with 5ng/mL TGF-β for 0, 24, 48, and 72 hours, and successfully applied the organelle staining panel. We observed distinct cell morphologies at each timepoint of TGF-β treatment, with cells shifting from a cobblestone (no TGF-β) to a spindle-like (72 hours TGF-β) morphology. Notably, the F-actin signal was significantly overexpressed in the 72 hours TGF-β treated A549 cells. A CellProfiler based preliminary image analysis pipeline was established for cell segmentation, cell features extraction, and cell viability tracking with quality control filters. Further optimization for cell segmentation, viability tracking, and classification algorithms will be needed to enhance the robustness of the workflow. Our established workflow demonstrates the feasibility of live cell functional assay with organelle staining. This workflow could be adopted to correlate and predict single tumor cell susceptibility to cytotoxic immune cells, with the goal of developing a therapeutic index. Citation Format: Yin Zou, Harrison Ball, Cade Harris, Nithya Ramnath, Venkateshwar Keshamouni, Sunitha Nagrath. Novel workflow to generate natural killer cell sensitivity prediction model based on the epithelial mesenchymal transition related reprogramming at a single cell resolution [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2589.

Abstract 7391: Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&amp;E images

Abstract Background: In breast cancer diagnostics, the accurate assessment of HER2 status is essential for treatment stratification. Conventionally, HER2 immunohistochemistry (IHC) scoring necessitates specialized staining protocols, which are resource- and time-intensive. This study presents a virtual IHC (vIHC) digital assay to predict HER2 scores directly from hematoxylin and eosin (H&amp;E) stained images, which are routinely acquired for all specimens. This methodology aims to accelerate standard practices by reducing the need for additional IHC staining, thereby decreasing laboratory workload, costs, and turnaround time. It also presents an opportunity to screen a wider array of samples promptly, potentially identifying candidates for HER2-targeted therapy that might otherwise be delayed or overlooked. Methods: Serially sectioned breast core needle biopsy samples were stained with H&amp;E and HER2-IHC following Leica-Bond staining protocols. Stained slides were imaged using the Hologic GeniusTM Digital Diagnostics System with Research Use Only software for Whole Slide Imaging. From this initial image dataset, 240 regions of interest (ROIs) were manually selected in order to create a dataset that uniformly covers: (i) all HER2-IHC scores (0-3), (ii) a wide range of HER2 stain intensities, and (iii) expression patterns. The IHC scores were assigned to each selected ROI by a pathology expert, following the scoring recommendations of ASCO/CAP American Society of Clinical Oncology and College of American Pathologists, with the caveat that ductal carcinoma in situ was also given a score for the purpose of training the model. These ROIs were then extracted from the images of the serially sectioned H&amp;E-stained slides using Reveal Bio’s proprietary co-registration digital assay. A predictive digital assay was developed at Reveal Biosciences to classify the 240 H&amp;E-stained breast tissue ROIs into four HER2 IHC score categories: 0, 1, 2, and 3. For model evaluation, cross-validation was performed 10 times by randomly splitting the dataset of H&amp;E ROIs into 80% for training MIL and 20% for testing the model, followed by averaging the performance metrics across the 10 dataset splits. The performance metrics included classification accuracy, precision, recall, and F1 scores for each HER2 score category. Results: The vIHC digital assay for predicting the HER2 scores directly from H&amp;E stained breast tissue images achieved the average classification accuracy of 93% across the four HER2 score categories on the test set of ROIs. This performance demonstrates that the model is capable of successfully distinguishing between the HER2 score categories directly from H&amp;E images without the need for IHC staining. The results suggest that the vIHC digital assay could be a reliable and cost-effective tool for preliminary breast cancer screening, providing a valuable adjunct to traditional histopathological analysis. Citation Format: Misagh Naderi, Nikola Mitic, Nikola Spasic, Djordje Cikic, Kristen Ruf, Nikolaus Wagner, Igor Mihajlovic, Sinisa Todorovic, Ray Jenoski, Sid Mayer, Michael Quick, Claire Weston. Beyond traditional staining: A virtual-IHC digital assay for HER2 score prediction from routine H&amp;E images [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 7391.

Concomitant Tuberculous and Lepromatous Lymphadenitis: Clues and Pitfalls of Leprosy Concealed by Tuberculosis in Lymph Nodes.

Comorbidities between tuberculosis and leprosy are expected in endemic regions. Pulmonary tuberculosis and cutaneous leprosy are the most prevalent coinfections. One of the common manifestations of tuberculosis is generalized lymphadenopathy. In contrast, leprosy is clinically less suspected to manifest as a generalized lymphadenopathy, and it is pathologically unusual to diagnose leprosy primarily in lymph nodes. Concomitant tuberculous and lepromatous lymphadenitis are unprecedented and clinically unexpected, particularly in nonendemic countries. This imposes diagnostic challenges. We report concurrent tuberculosis and leprosy that were diagnosed in a lymph node in 45-year-old man with generalized lymphadenopathy. The effaced lymph node was predominantly replaced by caseating epithelioid granulomas alternating with foamy histiocytes. Ziehl-Neelsen stain showed positive acid-fast bacilli in the necrotizing granulomas only. The initial differential diagnosis of the nodal foamy macrophages included fungal infections, leishmaniasis, and Whipple disease, for which the special stains were negative. The vacuolated macrophages were disregarded as nonspecific lipogranuloma. A modified acid-fast stain was not considered. The histopathologic clues to nodal lepromatous leprosy included the presence of intracytoplasmic globi, intermixed microabscesses, and lymphoplasmacytic infiltrate and involved pericapsular nerves. Wade-Fite stain was subsequently performed. It revealed numerous lepra bacilli within the foamy histiocytes. The final diagnosis was concurrent disseminated tuberculosis and leprosy. Nodal lepromatous leprosy could be missed when compounded by concurrent nodal tuberculosis, particularly in developed countries. The clinicians and pathologists should have a high index of suspicion, particularly in patients from or with history of travel from endemic regions. Certain histopathologic features are helpful clues to avoid pitfalls.

Cytological, Histopathological and Immunohistochemical Studies on Naturally Occurring Cutaneous Melanomas in Cattle in and Around Namakkal, Tamil Nadu, India

Background: Melanomas are tumours that originate from melanoblasts. The studies on bovine tumours are of clinical concern since some melanocytic tumours are congenital and occur in young animals. The present work was carried out to study the pathomorphological characteristics of melanomas in cattle for early diagnosis and intervention. Methods: Tumour samples were collected from suspected cases of neoplasia from four animals during the study period. Data pertaining to the animals were collected. Fine needle aspiration cytology and impression smear were stained with Giemsa stain. Tissue samples were collected in 10 per cent neutral buffered formalin for histopathological examination. Special staining with Fontana silver stain and immunohistochemistry with Melan- A and S- 100 markers for confirmatory diagnosis of melanin pigment was done. Result: Cytology revealed neoplastic cells with vacuolation and pleomorphism. Cytoplasm contained abundant melanin pigments. Microscopically, the cytoplasm contained abundant brown, black coloured melanin pigment granules. Tissue sections stained with Fontana silver impregnation method revealed the presence of black coloured granules. Immunohistochemistry with Melan-A and S-100 revealed strong expression of brown coloured reaction in the cytoplasm of the neoplastic cells.

Impact of Alcian blue and periodic acid Schiff expression on the prognosis of gastric signet ring cell carcinoma.

The Alcian blue (AB) and periodic acid Schiff (PAS) stains are representative mucus markers in gastric signet ring cell carcinoma (SRCC). They are low-cost special staining methods used to detect acidic mucus and neutral mucus, respectively. However, the clinical importance of the special combined AB and PAS stain is unclear. To investigate AB expression, PAS expression and the AB-to-PAS (A/P) ratio in gastric SRCC patients and to assess patient prognosis. Paraffin-embedded sections from 83 patients with gastric SRCC were stained with AB and PAS, and signet ring cell positivity was assessed quantitatively. Immunohistochemical staining for Ki67, protein 53 (P53) and human epidermal growth factor receptor 2 (HER2) was performed simultaneously. The cancer-specific survival (CSS) rate was estimated via Kaplan-Meier analysis. Cox proportional hazards models were used for univariate and multivariate survival analyses. Kaplan-Meier survival analysis revealed that the 3-year CSS rate was significantly greater in the high-PAS-expression subgroup than in the low-PAS-expression subgroup (P < 0.001). The 3-year CSS rate in the A/P ≤ 0.5 group was significantly greater than that in the A/P > 0.5 group (P = 0.042). Univariate Cox regression analysis revealed that the factors affecting prognosis included tumor diameter, lymph node metastasis, vessel carcinoma embolus, tumor stage, the A/P ratio and the expression of Ki67, P53 and the PAS. Cox multivariate regression analysis confirmed that low PAS expression [hazard ratio (HR) = 3.809, 95% confidence interval (CI): 1.563-9.283, P = 0.003] and large tumor diameter (HR = 2.761, 95%CI: 1.086-7.020, P = 0.033) were independent risk factors for poor prognosis. A/P > 0.5 is potentially a risk factor for prognosis, and low PAS expression is an independent risk factor in the prognosis of gastric SRCC. PAS expression and the A/P ratio could help in predicting the clinical prognosis of patients with SRCC.