IHC Findings Research Articles

The impact of N-acetylcysteine on hypoxia-induced testicular apoptosis in male rats: TUNEL and IHC findings

The present study aimed to evaluate the impact of N-acetylcysteine (NAC) on testicular hypoxia caused by varicocele, focusing specifically on the regulation of genes related to apoptosis and oxidative stress in the testes of mature Wistar rats.Thirty-two rats were divided into four groups: Control (Sham), hypoxia, testicular hypoxia treated with NAC (Hypoxia + NAC), and healthy animals treated with NAC. After the 8-week treatment period, testicular histopathology and the levels of oxidative stress markers—superoxide dismutase (SOD), glutathione peroxidase (GPx), and malondialdehyde (MDA)—in serum were examined. The expression of Bax and Bcl-2 mRNA was analyzed using immunocytochemistry and RT-qPCR assays, while the apoptosis rate was determined using the TUNEL method.Histopathological evaluations showed that parameters such as Johnsen's score, epithelium width, and seminiferous tubule diameter indicated significant improvement in the Hypoxia + NAC group compared to the Hypoxia group. NAC administration resulted in elevated serum levels of GPx and SOD, accompanied by a reduction in MDA levels (p < 0.003). Furthermore, the study revealed that NAC decreased Bax expression and enhanced Bcl-2 gene and protein expression compared to the varicocele group (p < 0.05). Additionally, NAC administration significantly decreased the rate of apoptosis in germ cells (p < 0.05).These findings suggest that NAC administration can mitigate testicular damage induced by hypoxia from varicocele in rats, primarily due to its antioxidant properties.

Lymphoid Aggregates in Canine Cutaneous and Subcutaneous Sarcomas: Immunohistochemical and Gene Expression Evidence for Tertiary Lymphoid Structures.

Canine cutaneous/subcutaneous soft-tissue sarcomas (STS) are diversely derived mesenchymal neoplasms with a risk of recurrence and/or metastasis depending on the extent of surgical excision and histologic grade. Lymphoid aggregates (LAs) are often described in these tumours but not characterised. In humans, LA characterised as tertiary lymphoid structures (TLSs) improve the prognosis of many tumours, including sarcomas. We sought to determine if LA meeting a size criterion (> 700 cells) in canine sarcomas met the criteria of TLS and the overall prevalence of LA of any size. RNA expression in large LAs versus aggregate-adjacent sarcoma tissue (AAS) was measured in laser capture microdissected tissue and compared to curl-derived RNA from aggregate-free sarcomas and lymph nodes. CD3, CD20, MUM-1 and PNAd expressions were measured using immunohistochemistry. CD20 and CD3 mRNA were more highly expressed in LA versus AAS (13.8 fold, p = 0.0003 and 2.3 fold, p = 0.043). This was supported by the IHC findings. The large LAs were also enriched in chemokine RNA expression characteristic of TLS (CXCR5 5.8 fold, p < 00001, CCL19 3.68 fold, p = 0.0209, CCL21 6.87 fold, p = 0.0209 and CXCL13 2.68 fold, p = 0.0924). Plasma cells and high endothelial venules were identified in LA containing tumours but not in control tissue. Large LAs were present in 12% of tumours, and LA of any size in 30%. We conclude that large LAs in canine STS are consistent with TLS.

Diagnostic Dilemma of Palatal Lesions: Polymorphous Adenocarcinoma (PAC) and its Insight into Updated Molecular Findings.

Polymorphous AdenoCarcinoma (PAC), a slow growing tumor primarily found in minor salivary glands, predominantly palate. Herein, we report a case of 52-year female with palatal swelling. This report highlights the recent updates on molecular and IHC findings of PAC and its sub variant CASG.

Efficacy of Topical Anticholinergic in Control of Symptoms in Patients with Vasomotor Rhinitis and non-Vasomotor Rhinitis Patients and Normal Individuals. A Controlled Comparative Study

Abstract Background The use of a topical anticholinergic medication, ipratropium bromide, and its ability to inhibit methacholine and rhinitis-induced hypersecretion is emphasized. Ipratropium bromide appears to be both safe and effective in reducing this troublesome symptom. This study is designed to show the therapeutic effect of anticholinergic local treatment on nasal mucosa in patients with vasomotor rhinitis. Objective To assess the therapeutic effect of local anticholinergic treatment on nasal mucosa in patients with vasomotor rhinitis compared to non-vasomotor (allergic) patients and normal individuals, and if there any down regulation of the muscarinic receptors or not. Patients and Methods This prospective interventional study was conducted in Otorhinolaryngology Department, Faculty of Medicine, Ain Shams University. This study was conducted on 60 cases. All patients were divided into 3 groups: Study group (1): includes 20 patients diagnosed clinically non allergic rhinitis suggestive to be vasomotor rhinitis. Study group (2): includes 20 patients diagnosed as allergic rhinitis. Study group (3): includes 20 patients performing surgery for non-vasomotor rhinitis non allergic causes (as septoplasty or rhinoplasty). Results Comparison between the pre and post SNOT questionnaire of symptoms among vasomotor group showed that there was a significant difference between the pre and post treatment SNOT questionnaire symptoms; Nasal obstruction, runny nose, post nasal drip, thick nasal discharge and lack of good night sleep. Among allergic cases group, there was a significant difference between the pre and post treatment immunohistochemistry findings considering epithelium, glands, arteries and veins. 20% of cases had grade 3 epithelium before treatment while after treatment, this was dropped to 0%. Comparison between the pre and post SNOT questionnaire of symptoms among allergic group showed that there was a significant difference between the pre and post treatment SNOT questionnaire symptoms; runny nose, post nasal drip, and thick nasal discharge. Among non- vasomotor rhinitis non allergic causes (as septoplasty or rhinoplasty) group, there was a significant difference between the pre and post treatment IHC findings considering epithelium, glands, and arteries, however no significant difference between the pre and post IHC veins grade. Also, there was no significant difference between the pre and post treatment SNOT questionnaire symptoms. Conclusion Topical anticholinergic such as ipratropium bromide nasal spray are effective in treating rhinorrhea symptoms in allergic and non-allergic rhinitis. Further controlled studies with larger sample size and longer follow-up are needed to confirm our results and to identify risk factors of adverse events.

Network Pharmacology, Molecular Docking, and Experimental Verification to Reveal the Mitophagy-Associated Mechanism of Tangshen Formula in the Treatment of Diabetic Nephropathy.

This study investigated the mechanism of TSF in treating DN through network pharmacology, molecular docking, and experimental validation. To identify critical active ingredients, targets, and DN genes in TSF, multiple databases were utilized for screening purposes. The drug-compound-target network was constructed using Cytoscape 3.9.1 software for network topological analysis. The protein interaction relationship was analyzed using the String database platform. Metascape database conducted enrichment analysis on the key targets using Gene Ontology and the Kyoto Encyclopedia of Genes and Genomes. The renoprotective effect was evaluated using a mouse model of diabetic nephropathy (db/db mice) that occurred spontaneously. Validation of the associated targets and pathways was performed using Western Blot (WB), Polymerase Chain Reaction (PCR), and Immunohistochemical methods (IHC). The network analysis showed that the TSF pathway network targeted 24 important targets and 149 significant pathways. TSF might have an impact by focusing on essential objectives such as TP53, PTEN, AKT1, BCL2, BCL2L1, PINK-1, PARKIN, LC3B, and NFE2L2, along with various growth-inducing routes. Our findings demonstrated that TSF effectively repaired the structure of mitochondria in db/db mice. TSF greatly enhanced the mRNA levels of PINK-1. WB and IHC findings indicated that TSF had a notable impact on activating the PINK-1/PARKIN signaling pathway in db/db mice, significantly increasing LC3 and NRF2 expression. Our results indicate that TSF effectively addresses DN by activating the PINK-1/PARKIN signaling pathway and enhancing Mitochondrion structure in experimental diabetic nephropathy.

Immune strategies of silver pomfret (Pampus argenteus) infected with Nocardia seriolae at different infection stages

Nocardia seriolae, a widespread opportunistic pathogen, has the ability to infect various economically farmed fishes, leading to prolonged disease courses and high mortality rates. In our current study, we focused on the silver pomfret (Pampus argenteus) and examined the liver, spleen, and kidney tissues at three specific N. seriolae infection phases: control (0 day), L group (infection for 5 days), and H group (infection for 15 days). Utilizing HE staining, we observed typical granulomatous nodules displaying stratified concentric layers, including caseous necrotic cells, epithelioid cells and multinucleated giant cells, fibroblasts and fibrocyte envelopes. The TUNEL assay revealed a significant presence of apoptotic cells concentrated within the renal nodules. Following the infection, there was a noticeable increase in AKP, ACP, and LZM levels in all tissues, while LPS exhibited a consistent decline. Through combined analysis of WGCNA and KEGG pathways derived from the tissue transcriptome, we identified several highly enriched pathways, such as the PI3K-Akt signaling pathway and p53 signaling pathway, in all three tissues. Key DEGs from these pathways underwent further investigation through RT-qPCR. In comparison to the control group, the L group exhibited up-regulation of genes associated with biosynthesis catabolism, whereas genes related to immune response, apoptosis, and ion transport displayed down-regulation. Interestingly, the H group showcased a stark reversal of this trend. To validate these DEGs expressions in vitro, we established a Pampus argenteus spleen cell line (PaS). By evaluating the invasion metrics of NS into PaS, we determined an optimal invasion threshold of 105 CFU/ml. The TUNEL assay demonstrated a notable presence of apoptotic signals in the 5 h and 7 h treatment groups. JC-1 assays indicated a gradual decline in membrane potential starting from the 3-h mark. As the duration of treatment increased, RT-qPCR and IHC findings highlighted an initial up-regulation followed by a subsequent down-regulation of genes associated with biosynthesis catabolism. In contrast, genes related to innate immunity displayed up-regulation, whereas genes related to adaptive immunity and ion transport exhibited an initial surge followed by a gradual decrease. Additionally, genes inhibiting apoptosis showed down-regulation. In conclusion, these findings provide valuable insights into the apoptosis mediated by N. seriolae in piscine hosts. These effects are potentially attributed to the increased expression of ion transport-related genes. Moreover, our study sheds light on the intricate molecular mechanisms underlying piscine immunological responses throughout the entire infection timeline, offering promising opportunities for disease control.

Endoscopic resection of gastric calcifying fibrous tumor: An incidental finding

Calcifying fibrous tumor (CFT) is benign mesenchymal lesion. CFTs of the GI tract are quite rare with less than 60 cases of gastric CFTs reported in the English literature. In a healthy 49 old female, routine Endoscopic ultrasonography (EUS) revealed an internally isoechoic, homogeneous 12mm lesion mainly within the submucosa in the distal body of stomach with no significant past history. An endoscopic submucosal dissection (ESD) was done to rule out malignancy. The histopathological examination showed a hypocellular lesion with few spindle cells interspersed in a hyalinized, collagenous matrix with occasional lymphoplasmacytic cells. Foci of psammomatous, dystrophic calcification were noted. Immunohistochemical examination revealed tumor cells positive for vimentin and negative for CD34, CD117, DOG1, desmin, SMA and S100. Based on histopathological and IHC findings, a diagnosis of gastric calcifying fibrous tumour was made. Only a handful of CFT cases resected by endoscopic submucosal dissection has been reported in the literature. So herein we report a rare case of endoscopically resected gastric calcifying fibrous tumor.

Osteosarcoma of orbital bone-chondroblastic morphology mimics to chondrosarcoma-A rare case report &amp; review of literature

Conventional osteosarcoma, a subgroup of intramedullary osteosarcoma(OS) ,is the most common osteosarcoma that occurs in adolescents and early adulthood. We present a case of 22‑year‑old male patient with the chief complaint of proptosis, pain and swelling in the left orbital region &amp; diagnosed as chrondoblastic osteosarcoma(COS) of left orbital bone. COS is very rare at orbital location, this report aimed to discuss clinical, radiographic, histopathologic, IHC findings and diagnostic pitfalls &amp; manangement of COS in view of the literature.

Abstract 6350: Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy

Abstract BST2, a raft-associated type II transmembrane protein, functions as a restriction factor and inhibits HIV release by cross-linking virions onto infected cell surfaces. It also engages ILT7, a specific inhibitory receptor on plasmacytoid dendritic cells, which are associated with poor prognosis when infiltrate patients’ breast tumors. Two groups have identified BST2 isoforms of different lengths, generated by post-transcriptional modification. Based on these findings and our work on protein interaction of ILT7-BST2, we hypothesized that the expression of BST2 isoforms differed in normal and cancer tissues. To demonstrate that, we generated and characterized anti-long BST2 isoform monoclonal antibodies (mAbs). Results were compared with our previously developed mAb anti-BST2 full length molecule, 26F8, that recognizes a common epitope to all BST2 isoforms. By comparing the amino acid sequences of different isoforms of BST2, we identified a region of 24 residues on the extracellular domain of BST2, restricted to its long isoform. We then generated mAbs by hybridoma technology, immunizing BALB/c mice with the 24 mers peptide-KLH conjugated. Screening and positive selection of several clones were performed following our lab SOP. One of the mAbs, LA5, was selected based on positive ELISA against the immunogen and FACS staining of recombinant HEK293 cells expressing full-length BST2 protein. Pharmacokinetic determined using OCTET platform, revealed a KD of 4.61+/- 0.074 × 10−9 M for LA5. Epitope mapping of LA5 on BST2 overlapping peptides, confirmed specific recognition of full-length BST2 protein and the peptide used as immunogen. LA5 heavy and light chains were sequenced and analysis was performed by IgBlast and IMGTV. Binding of LA5 and 26F8 to the myeloma cell lines MM1 (B-lymphoblast) and U266 (B-lymphocyte) by flow cytometry, indicated 97-100% positive cells with both mAbs, with higher MFI values observed for clone 26F8: 5.8 and 1.8-fold-increase in MM1 and U266 cells respectively. IHC on formalin-fixed paraffin embedded tissues of human ductal breast carcinoma, revealed that LA5 staining was able to identify infiltrating carcinoma cells but not the normal surrounding acinar cells. In contrast, 26F8 staining did not discriminate between BST2+ malignant and surrounding normal acinar cells. Currently, we are analyzing a panel of &amp;gt;105 breast cancer tissue samples of different molecular classifications. Initial data revealed a high-level of expression of BST2 (by 26F8) on the epithelial area of the tumors, lower in the TME (tumor microenvironment-infiltrating immune cells) and almost negative expression in the surrounding fibroblasts rich stroma. Evaluation of IHC staining by LA5 is being processed and yet to be established. These IHC findings indicate a potential diagnostic value of LA5 to distinct between tumor and normal cells in patients’ specimens. Treatment prospects may arise during the process. Citation Format: Laura C. Bover, Ahmed Muhsin, Long T. Vien, Felipe Amaya Manzanares, Zhuang Wu, Janis D. Johnson, Diego Castro Reyes, Myrthala Moreno-Smith, Estefania Labanca, Irene Sorin, Silvia Saucedo, Silvina Gazzaniga, Alicia I. Bravo. Monoclonal antibody against BST2 isoform: potential tool for diagnostic and cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6350.

Mixed Epithelial Stromal Tumour Kidney: Report of Two Cases, Benign and with Malignant Transformation

Mixed Epithelial Stromal Tumor kidney (MESTK), is a rare renal tumor, with stromal as well as epithelial components. It is predominantly benign with rare malignant transformation as morphology resembling that of a stromal sarcoma, carcinosarcoma, rhabdomyosarcoma, chondrosarcoma, undifferentiated sarcoma, or malignant tumour with rhabdoid features. Here we present 2 cases of MEST of kidney both are middle aged females with history of episodes of hematuria. Radiology of both showed heterogenous lesion. Histopathological and IHC findings favoured a diagnosis of first case as MEST kidney benign entity and second case as MEST with malignant transformation.

Discordance between germline genetic findings and abnormal tumor immunohistochemistry staining of mismatch repair proteins in individuals with suspected Lynch syndrome.

Tumor immunohistochemical staining (IHC) of DNA mismatch repair (MMR) proteins is often used to guide germline genetic testing and variant classification for patients with suspected Lynch syndrome. This analysis examined the spectrum of germline findings in a cohort of individuals showing abnormal tumor IHC. We assessed individuals with reported abnormal IHC findings and referred for testing with a six-gene syndrome-specific panel (n=703). Pathogenic variants (PVs) and variants of uncertain significance (VUS) in MMR genes were designated expected/unexpected relative to IHC results. The PV positive rate was 23.2% (163/703; 95% confidence interval [CI], 20.1%-26.5%); 8.0% (13/163; 95% CI, 4.3%-13.3%) of PV carriers had a PV in an unexpected MMR gene. Overall, 121 individuals carried VUS in MMR genes expected to be mutated based on IHC results. Based on independent evidence, in 47.1% (57/121; 95% CI, 38.0%-56.4%) of these individuals the VUSs were later reclassified as benign and in 14.0% (17/121; 95% CI, 8.4%-21.5%) of these individuals the VUSs were reclassified as pathogenic. Among patients with abnormal IHC findings, IHC-guided single-gene genetic testing may miss 8% of individuals with Lynch syndrome. In addition, in patients with VUS identified in MMR genes predicted to be mutated by IHC, extreme caution must be taken when the IHC results are considered in variant classification.

High-Grade Sarcoma of the Hyoid Bone

Our case was a suprahyoid mass in the neck with the probable epicenter in the hyoid bone. Cytopathology of suprahyoid neck masses, without imaging guidance, may lead to misdiagnoses. Imaging was suggestive of the hyoid bone as the probable epicenter. Based on the morphology and IHC findings of the core biopsy, a diagnosis of high-grade sarcoma most likely of myofibroblastic origin was made, which was also confirmed in the main surgical specimen. En bloc surgical excision of the mass was performed preserving the rest of the laryngeal framework and the thyroid gland followed by adjuvant radiotherapy. However, the patient developed distant metastasis within a few months of treatment completion highlighting the high-grade nature of the sarcoma.

Evaluation of Progesterone Receptor Antagonist and Maxi-K Channel Agonist as Neuroprotective in Feeney's Weight Drop Model of TBI.

Neuroprotection in traumatic brain injury (TBI) is an unmet medical need. We evaluated two agents, aglepristone (progesterone receptor antagonist) and N-salicyloyltryptamine (STP) (activator of Maxi-K channel in GH3 cells), for neuroprotection in Feeney's weight drop model of TBI. Forty-eight male Wistar rats were divided into six groups (n = 8 per group). A battery of six neurobehavioral tests was evaluated at the end of the first week (EO1W), second week (EO2W), and third week (EO3W). In addition, histopathological and immunohistochemistry (BAX, Bcl-2, and M30 Cytodeath) tests were performed at EO3W. Aglepristone at 10 mg/kg showed significant neuroprotection compared to control as assessed by Rota-rod test at EO1W, VEFP right paw and 28-point neurobehavioral test at EO2W, MWM test at EO3W, and positive histopathological and IHC findings. Aglepristone at 20 mg/kg showed negative results as assessed by BAX expression, downregulation of Bcl-2, and positive M30 Cytodeath, thereby suggesting toxicity at higher doses. STP 100 mg/kg showed modest neuroprotective activity but failed to show a dose-response relationship at a dose of 50 mg/kg. The study shows that progesterone receptor antagonists have neuroprotection at lower doses and toxicity at higher doses.

Abstract P1-04-07: Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer

Abstract BACKGROUND: Inflammatory breast cancer (IBC) is a rare, but aggressive type of locally advanced breast cancer for which a specific immune response seems to be an important driver (Van Laere et al., CCR 2013). The tumor immune micro-environment (TIME) of IBC is characterized by an influx of tumor-associated macrophages (TAMs) and patients with spatial colocalization of TAMs with tumor cells seem to have a worse prognosis (Van Berckelaer et al., SABCS 2019). In contrast with TAMs, the presence of CD8+ T cells was associated with a better prognosis in IBC. Cytotoxic T cells mediate killing of the tumor by inducing apoptosis after activation of effector caspases-3/-7, regulated by the X-Linked Inhibitor of Apoptosis protein (XIAP). Recently, we reported that XIAP in breast cancer is associated with shorter survival and resistance to chemotherapy (Devi et al, Cancers 2021). In IBC, XIAP is translationally upregulated during cellular stress and induces, via NFkB, an immunosuppressive signaling (Evan et al, Cancer Res 2018). Therefore, we investigated the interplay between tumor cell driven XIAP expression and the TIME. METHODOLOGY: IBC patient clinicopathological variables (n=75) along with Affymetrix data (n=30/75) were collected. Immunostainings for XIAP, CD8 and CD163 (Hematoxylin-DAB) were done according to validated protocols. All slides were digitized and evaluated in Visiopharm to quantify the number of DAB+ immune cells and the relative marker area (RMA: the DAB+ area/total area of interest). Spatial interactions were examined using an effector index (EI) that counts the number of immune cells in a circle with a radius of 30 mcm around a CD8+ T cell or a tumor cell. We evaluated correlations between XIAP protein and mRNA expression, immune cell signatures (using CIBERSORT) &amp; gene-expression pathways as a validation of our IHC findings. RESULTS: High XIAP expression was detected in 45.3% of the IBC samples (n= 34/75). Although, XIAP expression was not associated with any clinicopathological parameters like grade, molecular subtype, or disease stage. High XIAP expressing tumors had more sTIL infiltration (P= 0.008), PDL1 expression (P= 0.012) and infiltration with TAMs (RMA: 1.03% (0.03% – 5.97%) vs. 2.73% (0.01% – 11.3%), P= 0.003). Furthermore, in these XIAP high samples TAMs were also located closer to the tumor cells (EI: 0.95 cells (0.06 – 3.48) vs. 2.04 cells (0.28 – 5.20), P&amp;lt; 0.001) and the CD8+ T cells (EI: 2.85 cells (0.26 – 8.85) vs. 5.13 cells (0.87 – 10.16), P&amp;lt; 0.001), while there was no correlation between XIAP and the total number of CD8+ cells. Although gene expression data analysis did not reveal any correlation between protein and mRNA expression of XIAP, there was a significant correlation between XIAP activation signature and protein expression (P&amp;lt;0.001). Furthermore, we discovered a strong association between XIAP protein and cellular stress response- &amp; inflammation-related gene sets. Finally, in the CIBERSORT data, we observed the strongest correlation between XIAP expression and M2 macrophages &amp; activated dendritic cells. CONCLUSION: This is the first study showing high XIAP expressing IBC tumors have increased infiltration of immunosuppressive TAMs, which co-localized near tumor and cytotoxic T cells. We also demonstrated an association between XIAP protein expression and inflammation related &amp; cellular stress response gene signatures. This is relevant as XIAP function depends on translational upregulation during cellular stress which leads to immunosuppressive signaling and an aggressive breast cancer phenotype. XIAP antagonists are in clinical trials and these data suggest potential benefit in targeting XIAP to enhance immunotherapeutic efficacy in IBC therapy. FUNDING: DoDs-W81XWH-17-1-0297,W81XWH-20-1-0153, ACS Mission Boost Grants(GRD) Citation Format: Christophe Van Berckelaer, Steven Van Laere, Joseph Gerardts, Luc Dirix, Michael Morse, Mark Kockx, François Bertucci, Peter Van Dam, Gayathri R Devi. Xiap expression is associated with infiltration of cd163+ tumor-associated macrophages in the tumor micro-environment of inflammatory breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P1-04-07.

Dedifferentiated endometrial carcinoma: molecular characteristics and clinical outcomes

<h3>Objectives:</h3> We sought to describe the molecular characteristics of each histological component of undifferentiated and dedifferentiated endometrial carcinomas, as well as the clinical outcomes of cases from a large single institution cohort. <h3>Methods:</h3> Endometrial carcinoma cases with diagnosis of undifferentiated or dedifferentiated carcinoma from 2014-2017 were included. Demographic and clinical data were abstracted from the medical record. Central pathologic review was performed by expert gynecologic pathologist, and cases were excluded if final diagnoses was not dedifferentiated or undifferentiated endometrial carcinoma. Slides from hysterectomy specimen were evaluated via IHC for MMR (mismatch repair)proteins (MLH1, PMS2, MSH2, MSH6), p53, and PD-L1. Sanger sequencing for TP53 and POLE and MSI genotyping were performed on tumor DNA for each case. <h3>Results:</h3> A total of 12 of 772 cases (1.6%) with a clinical diagnosis of dedifferentiated or undifferentiated endometrial carcinoma were evaluated. A total of 2 were excluded after central pathologic review (carcinosarcoma, FIGO grade 3 endometrioid). All remaining cases were classified as dedifferentiated carcinoma. Seventy percent of patients presented with advanced stage disease. The majority of patients were treated with multimodal adjuvant therapy, however 30% progressed rapidly prior to completing treatment. Of those who completed primary treatment, no recurrences were noted. Ninety percent of cases showed MMR protein loss and 8/9 had concordant MMR IHC findings in the histologic subtypes. No <i>TP53</i> or <i>POLE</i> mutations were identified on tumor sequencing. <h3>Conclusions:</h3> Concordant MMR protein loss between histologic subtypes of dedifferentiated endometrial carcinoma lends credence to the theory that the undifferentiated component arises from the low-grade endometriod component. The high rate of MMR protein observed in dedifferentiated tumors points to immunotherapy checkpoint inhibitors an attractive treatment option. The current study does not offer molecular characteristics that can adequately explain the dichotomies in outcomes seen in our patients. Further research to identify prognostic and predictive biomarkers will greatly aid in expanding our ability to make informed decisions regarding optimal management strategies.

Abstract 1818: ZEB1 expression is associated with the PTEN loss and TMPRSS2 ERG fusion immune evasion phenotype in prostate cancer

Abstract Prostate cancer (PCa) is considered immunologically cold with a tumor microenvironment (TME) that evades effective immune responses. PTEN loss and TMPRSS2-ERG fusion are common somatic mutations in PCa and both alterations have recently been suggested to impact TME immune infiltration. Interestingly, both mutations are considered to play a role in epithelial to mesenchymal transition (EMT). ZEB1, a stemness and EMT driver has also been associated with immune evasion in different types of tumors. In this study, we investigated the role of ZEB1 in the immune TME of prostate cancer and the putative cooperation between ZEB1 expression, PTEN loss and TMPRSS2-ERG fusion. Immunohistochemistry for PTEN, ERG and ZEB1 were analyzed in 43 PCa cases collected after radical prostatectomy, correlation analysis of expression and immune cell abundance prediction were accessed using PCa expression data from TCGA (n=494). Correlation analysis between ZEB1 expression and 395 genes involved in immune response showed a positive correlation with ERG (r=0.1941, P&amp;lt;0.0001), PTEN (r=0.3514, P&amp;lt;0.0001), STAT3 (r=0.3129, P&amp;lt;0.0001), BRCA2 (r=0.2381, P&amp;lt;0.0001), CSF1R (r=0.6041, P&amp;lt;0.0001), PDCD1 (r=0.2536, P&amp;lt;0.0001), CD274 (r=0.4044, P&amp;lt;0.0001), IL10 (r=0.3655, P&amp;lt;0.0001), IL2 (r=0.1973, P&amp;lt;0.0001), CCR7 (r=0.3325, P&amp;lt;0.0001) and CXCL8 (r=0.424, P&amp;lt;0.0001). When the cohort was dichotomized to compare fusion-positive to fusion-negative samples, ZEB1 correlation with ERG remained positive in fusion-negative samples (r=0.6422, P&amp;lt;0.0001) but was negative (r=-0.2147, P=0.0022) in fusion-positive samples. Immune cell abundance prediction showed a positive correlation between ZEB1 and Tregs (r=0.35, P&amp;lt;0.0001), monocytes (r=0.3874, P&amp;lt;0.0001), dendritic cells (r=0.3934, P&amp;lt;0.0001) and macrophages M1 (r=0.2176, P&amp;lt;0.0001) and a negative correlation with plasma cells (r=-0.2003, P&amp;lt;0.0001), CD8+ naïve T cells (r=-0.3565, P&amp;lt;0.0001), th1 cells (r=-0.4562, P&amp;lt;0.0001) and pro B-cells (r=-0.3539, P&amp;lt;0.0001). Immunohistochemistry analysis showed a high expression of ZEB1 in non-neoplastic basal epithelium plus variable intensitiy of expression in adenocarcinomas and corroborated the correlation between ZEB1, PTEN and ERG expression levels. ZEB1 correlation with immunosuppressive cytokines and receptors such as CCR7, IL2, IL10, IL8 and PD-L1 provides evidence that ZEB1 and EMT may play an immune evasive role in PCa. The positive correlation with Tregs also supports this putative role. ZEB1 expression may indicate that stemness is associated with activation of downstream pathways modulated by PTEN and ERG. The correlation with BRCA2 may indicate an increased stemness-related role for this DNA damage response pathway. These observations, and our IHC findings, are in agreement with recently published data showing that ZEB1 is critical to a subset of basal stem cells in murine prostate samples. Citation Format: Luiz P. Chaves, Andre L. Caliari, Camila M. Melo, Fabiano P. Saggioro, Jeremy A. Squire. ZEB1 expression is associated with the PTEN loss and TMPRSS2 ERG fusion immune evasion phenotype in prostate cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1818.

Correlation of Immunohistochemistry, FISH and Clinical Outcomes in Primary Mediastinal (Thymic) Large B-Cell Lymphoma

IntroductionPrimary Mediastinal (thymic) B-cell lymphoma (PMBCL) is a distinct B-cell lymphoma which accounts for 2-4% of Non-Hodgkin's lymphoma. PMBCL is unique as although B-cell markers are readily expressed, morphologically and on gene expression profiling, PMBCL resembles classical Hodgkin's lymphoma. The poor prognostic impact of BCL2/BCL6 and MYC immunohistochemistry and FISH have been well published in Diffuse large B-cell lymphoma patients from other sites, however less is known about their significance in PMBCL. We retrospectively analysed 36 patients with PMBCL and correlated immunohistochemistry and FISH with clinical outcome.MethodsPatient selectionWe performed a search for all PMBCL cases in our pathology database from 2008-2016. 36 cases who had sufficient tissue and fulfilled the clinicopathological criteria were included.Histologic and Immunohistochemical MethodsImmunohistochemical studies were performed using formalin-fixed, paraffin-embedded tissue sections. Antibodies against CD3, CD20, CD79A, CD10, BCL6, MUM1, BCL2, C-MYC , Ki-67 and PDL1 were used for immunohistochemical studies. These studies were performed using an automated immunostainer (Benchmark XT) and a streptavidin-biotin peroxidase detection system. Tissue specimens were reviewed for percent tumour positivity and nuclear intensity. The cutoff scores for positive antibody staining were: 30% for CD10, 30% for BCL6, 60% for MUM1, 40% for CMYC and 70% for BCL2. PDL1 was graded positive if 3+/2+ (complete membranous staining in at least 10-30% of cells) and negative if 1+/0.In-situ hybridizationFor detection of Epstein-Barr virus encoded RNA (EBER), in situ hybridization was performed on formalin-fixed, paraffin-embedded tissue sections using the Ventana ready-to-use kit as prescribed by the manufacturer.Molecular fluorescence in situ hybridization (FISH) studiesFor detection of MYC/BCL2/BCL6 gene rearrangements, the Vysis LSI Dual Color, Break Apart Rearrangement Probes performed on formalin-fixed, paraffin-embedded tissue using the Vysis paraffin pre-treatment kit II system.ResultsOut of the 36 cases, 15 (42%) were of the germinal center phenotype while 21 (58%) were non germinal center phenotype by the Hans criteria. MYC was expressed in 26 cases (72%). 44% of cases were double expressors. In addition, there was also increased BCL6 (N=35, 97%), Ki67>60% (N=31, 86%), MUM1 (N=25, 69%) and PDL1 expression (N=24, 66%) noted. EBERISH was not present in any of the cases in this series. Table 1 summarizes the IHC findings. Of the 36 cases, only 1 case had unsatisfactory BCL2 FISH analysis. Out of 35 evaluable cases, there was 1 positive MYC FISH and 2 positive BCL6 FISH. There were no cases with positive BCL2 FISH.We compared our results with published results from DLBCL cases1,2 and found higher proportion of MYC/BCL6 positivity and Ki67% by IHC. BCL2 IHC expression was similar. FISH positivity for myc/bcl2/bcl6 translocations was lower in PMBCL compared to DLBCL. By Han's criteria, there was no significant difference in GC and non-GC, however a higher proportion of double expressors was noted. PDL1 expression was also higher than those published in DLBCL, similar to PDL1 expression in Hodgkin's lymphoma3. Table 2 summarizes the comparison between our data and results from other studies.In our cohort of 36 patients, the median age was 29 years (range 15-61) with 19 female patients. The majority had early stage (n= 24 ,66.7%) and bulky disease (n=24, 66.7%). All 36 patients were treated with immunochemotherapy (R-CHOP n=11, R-EPOCH n=21). With a median follow up of 41 months, PFS and OS was 95% and 81% respectively. There was no correlation between IHC/FISH markers, clinical prognostic markers (bulk, stage, age, IPI) with outcomes on univariate and multivariate analysis.ConclusionOur results validate that PMBCL is a unique lymphoma which has similarities with DLBCL yet bears resemblance to Hodgkin's lymphoma on IHC. The usual prognostic markers for DLBCL have not correlated with clinical outcome in statistical analysis suggesting that due to the uniqueness of this lymphoma, novel markers should be explored to improve prognostic accuracy and thus improve patient outcomes.

Clinicopathological Study of Locally Recurrent Breast Carcinoma with Special Reference to Resectability

The isolated local recurrence in a patient previously treated for early-stage invasive breast cancer presents a unique challenge to the oncologist. The main objectives of this study were to identify breast carcinoma patients who are prone to develop early recurrence, to study the feasibility of resection in case of locally recurrent breast carcinoma and determine the incidence of distant metastasis associated with locally recurrent breast carcinoma. The parameters namely age, previous clinical TNM staging, previous Histological grade, lymphovascular invasion, previous types of surgery, previous immunohistochemistry and previous chemoradiotherapy had shown very strong positive significance. There was significant association between previous findings of IHC and present findings of IHC (p=0.0087) but there was no significant association between clinical stage and resectability (p=0.95), between status of LVI and resectability (p=0.71) and between Histopathological Grade and resectability (p=0.43). Local recurrences after primary resection are seen to be mostly resectable with negative margin; however, whether it results in survival benefit can be studied in long term study.

Cutaneous leiomyosarcoma with visceral metastases in a White Carneau pigeon and literature review.

Cutaneous leiomyosarcomas are malignant mesenchymal tumors of smooth muscle origin and are reported occasionally in avian species. A 14-y-old male laboratory White Carneau pigeon (Columba livia) was presented for surgical excision of a cervical soft tissue mass. Ultrasonography with color flow Doppler imaging revealed multiple cavitations of mixed echogenicity within the mass and vascularization. Histologically, the dermis and subcutis were expanded by a densely cellular multinodular mass comprised of fusiform cells forming haphazardly arranged broad streams and short interwoven bundles, often surrounding blood vessels and variably sized cavitations. Neoplastic cells were strongly immunopositive for desmin and α-smooth muscle actin, and negative for pancytokeratin, S100, and von Willebrand factor. Based on histopathology and IHC findings, the cutaneous mass was diagnosed as leiomyosarcoma (LMS). The pigeon died 312 d post-operatively. Postmortem examination revealed masses infiltrating the left and right pulmonary airways and one hepatic nodule, but no regrowth at the surgical site. Histologic and IHC evaluation of the pulmonary and hepatic masses were consistent with LMS, representing metastatic foci from the primary cutaneous LMS. Our case highlights the malignant behavior and histomorphologic features of cutaneous LMS in an avian species.

An increase of CD8+\u2009T cell infiltration following recurrence is a good prognosticator in HNSCC

Programmed death-ligand 1 (PD-L1) expression and CD8-positive tumor-infiltrating lymphocyte (CD8+ TIL) infiltration are essential biomarkers for immune checkpoint inhibitor therapy. The objective of this study was to compare the expression of those biomarkers between initial and recurrent HNSCCs using paired analysis. Prognostic significance of those immunological changes was also investigated. Forty-two consecutive patients with locally recurrent HNSCCs were included. Immunohistochemical staining of CD8 and PD-L1 was done for both initial and recurrent tumor specimens. The IHC findings were verified with mRNA expression profiling. Also, the prognostic impact was analyzed based on overall survival (OS). Recurrent-to-initial (R/I) ratios of CD8+ TILs and PD-L1 were widely variable. CD8+ TIL density and PD-L1 expression decreased in 59.5% and 69% of patients, respectively (R/I ratio < 1). The R/I ratio of CD8A mRNA was significantly higher in patients with a CD8 R/I ratio > 1 (1.7 ± 1.5 vs. 0.6 ± 0.6, p = 0.042). CD8 R/I ratio (> 1) was a good prognosticator for OS (HR 0.293, 95% CI 0.091–0.945, p = 0.040). CD8+ TIL infiltration and PD-L1 expression changed variably following local recurrence of HNSCC. The increase of CD8+ TILs at recurrence was an excellent independent prognosticator.