IHC Results Research Articles

Experimental and proteomics evidence revealed the protective mechanisms of Shemazhichuan Liquid in attenuating neutrophilic asthma

BackgroundNeutrophilic asthma (NA) is one of the most important phenotypes of non-Th2 asthma and is often insensitive to glucocorticoid therapy, making current treatment difficult. Shemazhichuan Liquid (SMZCL), a Chinese medicine compound preparation, has unique advantages in the treatment of asthma. However, the underlying mechanisms of SMZCL in treating NA are not fully understood. PurposeThe efficacy and underlying mechanisms of SMZCL on NA were investigated by TMT-labeled quantitative proteomics analysis and in vivo and in vitro experiments. MethodsNA mouse model was constructed by OVA/CFA sensitization followed by a 10-day challenge with 5 % OVA. Lung histopathology, leukocyte counts and cell sorting counts, inflammatory cytokines levels, as well as expression of autophagy markers were then assessed. The specific pathways and proteins of SMZCL for treating NA were further illustrated through TMT-based quantitative proteomics. In addition, RAW264.7 cells were induced by LPS to further explore the mechanism of the main active ingredient of SMZCL on autophagy pathway. ResultsIn vivo, SMZCL contributed to attenuating airway inflammation and collagen disposition, markedly reduced the number of leukocytes, especially neutrophils in bronchoalveolar lavage fluid (BALF), as well as decreased IgE and inflammatory cytokine levels (TNF-α, IL-1β, IL-6 and IL-8) in BALF and serum. Besides, SMZCL elevated the levels of LC3 and ATG5 while inhibiting the expression of p62 and mTOR. Mzb1 and Rab3ip were identified as the critical overlapping DEPs whose expression was inhibited by SMZCL and rapamycin. KEGG enrichment analysis showed that necroptosis process was a key pathway for SMZCL to treat NA airway inflammation. IHC and WB results confirmed that SMZCL and rapamycin inhibited the phosphorylation of RIPK1, PIPK3 and MLKL. In vitro, ATG5 and LC3 proteins were obviously increased while p-mTOR expression was inhibited after amygdalin treatment. ConclusionSMZCL attenuated airway inflammation in NA mainly through inhibition of the mTOR pathway, along with inhibition of the necroptosis pathway regulated by the RIPK1/RIPK3/MLKL axis and inhibition of Mzb1 and Rab3ip expression.

DEK::AFF2 Fusion-Associated Squamous Cell Carcinoma: A Case Series with Literature Review on an Emerging and Challenging Entity.

DEK::AFF2 fusion-associated squamous cell carcinoma (DEK::AFF2 SCC), also reported in the literature as low-grade papillary sinonasal (Schneiderian) carcinoma (LGPSC), is a rare, primarily bland-appearing, but locally aggressive neoplasm. Morphologically, these tumors can closely resemble sinonasal papilloma (SP), especially on small or limited biopsy, often leading to misdiagnosis. DEK::AFF2 SCC is devoid of the underlying mutually exclusive EGFR or KRAS driver mutations of SP, suggesting it may represent a distinct unique entity. In this study, we conducted a retrospective search of "unusual" SP reported either as atypical, dysplastic, or suspicious for malignant transformation at our institution in the last 13 years (2010-2023), to identify potential cases of DEK::AFF2 SCC. Of the 201 SP cases during this time period, 30 "unusual" SP cases were identified. On morphologic review of these 30 cases, 6 were worrisome for DEK::AFF2 SCC and were selected for AFF2 immunohistochemical stain (IHC), of which 3 cases were positive. All 3 AFF2 IHC positive cases were also positive for DEK::AFF2 fusion by fluorescence in situ hybridization (FISH), thereby, confirming IHC results. This study highlights that AFF2 IHC can be an invaluable surrogate marker to FISH in identifying DEK::AFF2 SCC in challenging cases to avoid misdiagnosis. Detailed clinical and pathologic data were collected to gain a better understanding of this emerging challenging entity. A literature review was performed to enrich our knowledge of DEK::AFF2 SCC.

Diagnosis of autoimmune bullous dermatoses: Comparative analysis of immunohistochemical staining using C4d, C3d, IgG, and IgG4 in lesional tissues and perilesional frozen skin samples

Immunohistochemical staining with immunoglobulins and complements may aid the diagnosis of patients whose clinical and histological findings are consistent with autoimmune bullous dermatoses (AIBD). We aimed to investigate the diagnostic value of immunohistochemical markers in lesional biopsy and perilesional frozen samples in AIBD. We included 136 cases from whom lesional biopsies and perilesional samples for direct immunofluorescence (DIF) examination were collected with a preliminary diagnosis of AIBD between January 2019 and January 2023. All diagnoses were reconfirmed by evaluating the clinical, histopathological, and serological findings and DIF results (C3, IgG, IgA, or IgM positivity compatible with the clinical diagnosis) altogether, although DIF results were considered a priority. After confirming the diagnoses, the samples were categorized as AIBD or the others. The perilesional tissues obtained for DIF simultaneously with skin biopsy and stored at −80 °C were thawed, and FFPE tissues were prepared. We performed immunohistochemical staining (C4d, C3d, IgG, and IgG4) on FFPE tissues of both lesional and perilesional samples. Strong, linear, or granular staining patterns at the dermoepidermal junction or the intraepidermal blistering space were considered positive in line with the diagnosis of the case. Cases other than AIBD were used as negative control tissues to assess the specificity of immunohistochemical markers. Of the 136 cases, 52 were diagnosed with AIBD. In lesional samples, the sensitivity of C4d, C3d, IgG, and IgG4 was 80.6 %, 69.4 %, 75 %, and 5.7 % with corresponding specificity of 100 %, 98.7 %, 89.6 %, and 97.4 %, respectively in pemphigoid diseases compared to a sensitivity of 18.2 %, 9.1 %, 70 %, and 9.1 % and specificity of 98.7 %, 100 %, 89.6 %, and 97.4 %, respectively in pemphigus diseases. In frozen samples, we detected expression in a limited number of cases. The sensitivity of C4d, C3d, IgG, and IgG4 was 8.7 %, 2.2 %, 19.4 %, and 2.2 %, with corresponding specificity of 100 %, 100 %, 98.5 %, and 98.6, respectively. There was a none to slight concordance rate between the IHC results of lesional tissues and perilesional frozen samples. Kappa coefficients for C4d, C3d, IgG, and IgG4 were 0.120 (P = 0.029), 0.111 (P = 0.050), 0.203 (P = 0.003), and - 0.15 (P = 0.846), respectively. Immunohistochemical staining with C4d, C3d, IgG, and IgG4 on biopsy samples collected from lesions may guide the diagnosis of AIBD, thereby eliminating the need for an additional biopsy and accelerating the diagnostic process.

Kaempferia galanga L. alleviates the expression of COX-2 and NF-kappaB-p65 in the oral mucosa ulcer of Wistar rats and exhibits no irritation toward the vascular membrane of chicken eggs and the skin of albino rabbits

The global incidence of recurrent aphthous stomatitis in 2018 reached 5–66 % of the population, while in Indonesia 8 %. Moreover, the prevalence of oral mucosal fibrosis and recurrent aphthous stomatitis among male doctors and nurses in China was 21.24 % and 24.27 %, respectively. Our previous study has shown that the ethanol extract of Kaempferia galanga L. rhizome (EKGR) revealed an accelerated wound-healing effect in the oral mucosa ulcer of Wistar rats. This study aims to explore the effects of EKGR on the expression of NF-kappaB-p65 and COX-2 in the tongue tissue of male Wistar rats by Western blot analysis and immunohistochemistry technique, its safety towards the vascular membrane of the egg chorioallantoic membrane, and its single-dose application on the skin of male rabbits. The rats were randomly assigned into 7 groups: the normal control; the negative control; the positive control (treated with triamcinolone acetonide); and 4 treatment groups of EKGR (0.5 %; 1 %; 2 %; 4 %). Western blot and immunohistochemistry methods were used to measure the expression of NF-kappaB-p65 and COX-2. The hen's egg test-chorioallantoic membrane assay was employed to predict the safety of EKGR towards the vascular membrane. Moreover, the effect of 200 mg/kg BW EKGR application on the dorsal skin of male albino rabbits was also evaluated. EKGR inhibits the expression of NF-kappaB-p65 and COX-2 as proven by WB and IHC results. In the HET-CAM assay, all concentrations of EKGR do not induce irritation responses, which elicits the safety of EKGR. The administration of EKGR causes mild irritation to the dorsal skin of male rabbits but does not induce erythema and edema, no significant changes in BW, no toxic effects on organ macroscopic examination or histopathology, and does not induce abnormalities in the hematological profile of male albino rabbits. EKGR has confirmed its anti-inflammatory activity by suppressing the expression of COX-2 and NF-kappaB-p65 in the oral mucosa ulcer of Wistar rats. EKGR is safe as it does not exhibit irritating potential and harmful effects.

Timeliness and Completeness of Endometrial Cancer Pathology Reports Before and After the FIGO 2023 Endometrial Cancer Staging Guidelines Update

Introduction: Uterine cancer is the most common gynecologic malignancy among women in the United States with an estimated 66,200 new cases and 13,030 deaths in 2023.1 These cancers are divided into endometrial adenocarcinomas (EAC) and uterine sarcomas. In 2013, the Cancer Genome Atlas (TCGA) proposed four new molecular classifications based on genome and exome sequencing of EACs including POLE-mutant, mismatch repair deficiency (MMRd), p53 abnormal, and no specific molecular profile (NSMP).4 In June of 2023, the International Federation of Gynecology and Obstetrics (FIGO) published an updated staging system for uterine cancer to incorporate these molecular indices along with the traditional anatomical distribution of disease and histology used in older FIGO staging guidelines.5 The primary objective of this project was to analyze the timeliness and completeness of pathology reports and IHC staining of patients who underwent hysterectomies for uterine cancer at Detroit Medical Center from October 2022 – December 2023. Methods: Patients were identified from the list of patients presented at the Karmanos Cancer Insitute’s (KCI) Gynecologic Oncology Tumor Board. Given the June publication timing of the FIGO 2023 staging guidelines, this served as the intervention for the study. The pre-intervention group consists of surgical cases from October 2022 through June 2023, and the post-intervention group consists of cases from July 2023-December 2023. Differences in time from surgery to pathology report, surgery to IHC result, pathology report to IHC result as well as completeness of IHC results were compared pre- and post-intervention. Because neither group was normally distributed, a MannWhitney-U test was used to calculate P-value. IHC result completeness pre- and post-intervention was calculated using a Fisher exact test. Results: A total of 106 cases were included, of which 67 were preintervention (63%) and 39 were postintervention. The median number of days from surgery to final pathology report was statistically significant (p=0.003) between the groups. The median number of days from pathology report to IHC result was not a statistically significant difference (p=0.19). The median number of days from surgery to completed IHC also not statistically significant. In terms of IHC staining, there was a statistically significant difference in the number of specimens pre- and post-intervention for p53 (p=0.004) and HER2 (p=0.04). There was no change in the MMR and MSI testing between the two groups. Conclusions: Following the implementation of the FIGO 2023 staging guidelines in June of 2023, there was a significant decrease in the time from surgery to finalized pathology report and a significant change in the completeness of staining for p53 and HER2 on hysterectomy specimens. By finalizing the results of both pathology and IHC for endometrial cancer specimens in a timelier manner, patients can be informed of their diagnosis sooner. Additionally, with a complete pathologic and molecular diagnosis, our gynecologic oncologists can also more efficiently and effectively communicate within a multidisciplinary team of providers including radiation oncology and genetic counseling colleagues to formulate a treatment plan.

Gene Regulation of Neutrophils Mediated Liver and Lung Injury through NETosis in Acute Pancreatitis.

Acute pancreatitis (AP) is one of the most common gastrointestinal emergencies, often resulting in self-digestion, edema, hemorrhage, and even necrosis of pancreatic tissue. When AP progresses to severe acute pancreatitis (SAP), it often causes multi-organ damage, leading to a high mortality rate. However, the molecular mechanisms underlying SAP-mediated organ damage remain unclear. This study aims to systematically mine SAP data from public databases and combine experimental validation to identify key molecules involved in multi-organ damage caused by SAP. Retrieve transcriptomic data of mice pancreatic tissue for AP, lung and liver tissue for SAP, and corresponding normal tissue from the Gene Expression Omnibus (GEO) database. Conduct gene differential analysis using Limma and DEseq2 methods. Perform enrichment analysis using the clusterProfiler package in R software. Score immune cells and immune status in various organs using single-sample gene set enrichment analysis (ssGSEA). Evaluate mRNA expression levels of core genes using reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry. Validate serum amylase, TNF-α, IL-1β, and IL-6 levels in peripheral blood using enzyme-linked immunosorbent assay (ELISA), and detect the formation of neutrophil extracellular traps (NETs) in mice pancreatic, liver, and lung tissues using immunofluorescence. Differential analysis reveals that 46 genes exhibit expression dysregulation in mice pancreatic tissue for AP, liver and lung tissue for SAP, as well as peripheral blood in humans. Functional enrichment analysis indicates that these genes are primarily associated with neutrophil-related biological processes. ROC curve analysis indicates that 12 neutrophil-related genes have diagnostic potential for SAP. Immune infiltration analysis reveals high neutrophil infiltration in various organs affected by SAP. Single-cell sequencing analysis shows that these genes are predominantly expressed in neutrophils and macrophages. FPR1, ITGAM, and C5AR1 are identified as key genes involved in the formation of NETs and activation of neutrophils. qPCR and IHC results demonstrate upregulation of FPR1, ITGAM, and C5AR1 expression in pancreatic, liver, and lung tissues of mice with SAP. Immunofluorescence staining shows increased levels of neutrophils and NETs in SAP mice. Inhibition of NETs formation can alleviate the severity of SAP as well as the levels of inflammation in the liver and lung tissues. This study identified key genes involved in the formation of NETs, namely FPR1, ITGAM, and C5AR1, which are upregulated during multi-organ damage in SAP. Inhibition of NETs release effectively reduces the systemic inflammatory response and liver-lung damage in SAP. This research provides new therapeutic targets for the multi-organ damage associated with SAP.

Predicting locoregional recurrence in triple-negative breast cancer using novel molecular subtyping.

e12549 Background: Triple-negative breast cancer (TNBC) presents a poorer prognosis compared to other breast cancer subtypes, characterized by a higher likelihood of early recurrence and increased incidence of visceral metastases. Evidence from numerous randomized clinical trials indicates that postmastectomy radiotherapy (PMRT) reduces locoregional recurrence (LRR) and improves breast cancer survival in TNBC. Previous reports creatively divided TNBC into four molecular subtypes for personalized treatment. The present study aims to elucidate patterns of LRR in different subtypes of TNBC after PMRT. Methods: From 2019 to 2022, data from 349 AJCC 7th edition stage II-III breast cancer patients treated with mastectomy and PMRT were retrospectively analyzed. These patients were classified into the basal-like immune-suppressed (BLIS) and non-BLIS groups based on the immunohistochemistry biomarkers. The cumulative incidence function was applied to estimated LRR rates, with death as a competing risk, using Gray's test for comparisons. Results: According to the IHC results, a total of 255 patients belonged to the non-BLIS group and 94 to the BLIS group. The 3-year LRR for the entire cohort was 5.16%. There were 9 cases each in the non-BLIS subtype (3.5%) and BLIS subtype (9.6%). The cumulative incidence of LRR between the two groups was significantly different, with a rate of 4.0% in the non-BLIS group and 9.0% in the BLIS group ( P = 0.02). Among the 18 patients with LRR in the whole population, 11 had isolated LRR and 7 were diagnosed with LRR with concurrent distant metastases. The most common area of recurrence in both subtypes was the supraclavicular region. The majority of recurrences occurred within the RT field (in-field), and no isolated out-of-field relapse was observed. In multivariate analysis, independent factors predicting higher LRR were the BLIS subtype (HR = 3.06, P = 0.02). Conclusions: Study results suggest that TNBC of the BLIS subtype is at higher risk for LRR. Immunohistochemistry-based molecular typing may serve as a prognostic biomarker to guide postmastectomy radiotherapy in patients with TNBC. New strategies are needed to improve local control rates in patients with BLIS subtype.

Expression and prognostic significance of KAP1 gene in malignant pleural mesothelioma

Objective: To explore the expression of KAP1 (KRAB-associated protein 1, KAP1) in Malignant pleural mesothelioma (MPM) based on the cancer genome atlas (TCGA) and clinical trials. And elucidate the correlation between the expression of KAP1 and the clinical pathological parameters of patients with MPM and its prognosis. Methods: In April 2022, Based on the second generation KAP1mRNA sequencing data and clinicopathological data of MPM patients downloaded from TCGA database, the correlation between KAP1mRNA expression and clinical parameters was analyzed, and the correlation between KAP1 protein expression and clinicopathological parameters and its prognostic value were analyzed based on Chuxiong data set cohort clinical samples. The expression of KAP1 mRNA in MPM samples and matched normal tumor adjacent tissues was detected by qRT-PCR, and the expression of KAP1 protein in MPM and normal pleural tissues was detected by immunohistochemistry and Westernblotting. To construct a Kaplan-Meier model to explore the effect of KAP1 expression on the prognosis of MPM patients, and to analyze the prognostic factors of MPM patients by Cox regression. Results: qRT-PCR and Western blotting detection showed that the expression levels of KAP1 gene in four different MPM cells (NCI-H28, NCI-H2052, NCI-H2452, and MTSO-211H) were significantly higher than those in normal pleural mesothelial cells Met-5A. qRT-PCR, Western blotting and IHC results demonstrated that the mRNA and protein expression levels of KAP1 in MPM tissues was significantly higher than that in matching normal mesothelial tissues, and the expression level of KAP1 protein was correlated with TP 53 protein expression levels and serum CEA levels (P<0.05) . The mRNA expression level was significantly correlated with the prognosis, The overall survival time of mesothelioma patients with high KAP1mRNA expression was significantly shorter (HR=3.7, Logrank P<0.001) . Tumor type, age and the mRNA expression were related to the prognosis of MPM patients (P<0.05) . Multivariate analysis showed that tumor type and KAP1 mRNA expression level were independent prognostic factors of MPM patients (P<0.05) . Conclusion: In this study, TCGA database and Chuxiong cohort experiment samples were used to collect the relevant information of KAP1 expression in malignant melanoma tissues. It was confirmed that KAP1 is highly expressed in MPM tissues. The mRNA expression level and pathological type are correlated with the prognosis of patients.

Abstract 1509: Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity

Abstract Introduction: It is well known that tumor-associated macrophages/microglia (TAMs) play a vital role in brain tumor progression. Our previous studies suggested that the activated microglia could reduce the cytotoxic effect of chemotherapeutic drugs on glioma cells in a 2D/3D model. This study delves into TAMs within diverse brain TME regions, emphasizing the distinction of resident microglia from infiltrating macrophages in invasion areas and their role in glioma migration and invasion. Material and Method: We employed the murine orthotopic astrocytoma model (ALTS1C1), dissecting brain tumor tissues into normal, invasion, edge, and core regions. Immunofluorescence staining, utilizing TMEM119 and CD68 markers to identify resident microglia and infiltrating macrophages, respectively. The IHC results were analyzed with QuPath software and validated via flow cytometry. Microglia (BV2) and macrophage (RAW264.7) cell lines were directly co-cultured with ALTS1C1 in 2D and 3D migration/invasion models. Bulk RNA sequencing analysis unraveled the differences between BV2 and RAW264.7. Bone marrow-derived macrophages (BMDM) were used for the TAMs activation test, and the cathepsin inhibitor, E64, was applied to elucidate cathepsin's role in microglia within co-culture models. Results: The IHC results reveal more TMEM119+ microglia than CD68+ infiltrating macrophages in the tumor invasion area. On the other hand, there are more CD68+ infiltrating macrophages than TMEM119+ microglia in the tumor core regions, signifying a specific role of microglia in tumor invasion. Flow cytometry further supported that more CD11b+CD45low microglia prevalence in tumor surrounding tissues than in tumor core. ALTS1C1 astrocytoma cells, in the presence of microglia, exhibited more increased invasion and migration ability than the presence of macrophages in 2D/3D models. RNA sequencing highlighted M2 activation and cathepsin gene upregulation in microglia over macrophages. M2-polarized TAMs enhanced 2D migration, while cathepsin inhibition significantly decreased glioma's 3D invasion potential. Conclusion: This study identifies microglia as primary TAMs in brain tumor invasion areas and their roles in glioma cell survival and dissemination. Notable differences in activation states and the critical impact of cathepsin in microglia for their roles on brain tumor invasion and survival offer promising therapeutic avenues. Targeting cathepsin in microglia emerges as a novel glioma prevention and treatment strategy. These findings contribute valuable insights to enhance clinical interventions for glioma patients. Citation Format: Sheng-Yan Wu, Chi-Shiun Chiang. Microglial dominance in glioma dissemination: Unraveling TAMs' impact and therapeutic implications through modulating cathepsin activity [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 1509.

Abstract 2611: Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 immunotherapy

Abstract Background: Antibody-drug conjugates (ADCs) play a key role in targeted therapy of solid tumors. However, few studies have examined the impact of ADCs on the tumor microenvironment (TME), particularly on innate immune cells. To elucidate this, this study focuses on investigating the comprehensive impacts of ICAM1-DXd on the tumor microenvironment, especially on tumor-associated neutrophils (TAN) and its synergistic effect with PD-1 immunotherapy. Methods: Firstly, the efficacy and prognosis of ICAM1-DXd, a potent ADC for cervical cancer, were determined in pre-clinical tumor models, providing crucial transcriptomic and biological function information by using RNA-seq. Then, pathological studies were performed to evaluate the abundance and location of TAN (Ly6G+/MPO+) in TME post-ADC treatment. The interaction between TAN and ICAM1-DXd and subsequent impacts on adaptive immune cells (e.g., CD8+ cells) was examined by flow cytometry (FACS), confocal fluorescent imaging, and western blot techniques (WB). Eventually, the synergistic effect of ICAM1-DXd and PD-1 monoclonal antibody were investigated in an immunocompetent animal model. Results: ICAM1-DXd potently inhibits cervical tumor growth in preclinical models, and RNA-seq analysis revealed that ICAM1-DXd treatment induces a strong down-regulation of neutrophil activation, especially for Ly6G+/MPO+ TANs, a subtype with pro-tumor function. IHC and FACS results confirmed that pro-tumor TANs were efficiently eliminated in TME by ICAM1-DXd treatment via non-specific phagocytosis in comparison with peripheral blood neutrophils. ICAM1-DXd-phagocytised TANs also elicit a strong bystander killing effect by cleaving the quadra peptide linker of ICAM1-DXD by cathepsin B. This ADC-mediated TAN elimination effectively ameliorates immunosuppressive TME and works in synergy with PD-1 immunotherapy in preclinical models. Conclusion: In preclinical models of cervical cancer, ICAM1-DXd not only directly ablates cancer cells via antigen recognition but also non-specifically kills tumor-promoting TANs, resulting in an improved tumor immune microenvironment, providing a shred of mechanistic evidence supporting ADC and PD-1 combination therapy. Citation Format: Shili Yao, Lu Sun, Xiu Zhu, Hanfei Xie, Tong Yang, Peng Guo, Huarong Tang, Tao Zhu. Non-specific elimination of tumor-associated neutrophils by antibody-drug conjugates boosts anti-tumor immunity for PD-1 immunotherapy [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 2611.

Abstract 7408: Robust profiling of cancer-related gene fusions: Analytical validation of PANCARNA for multiple cancer types

Abstract The impact of examining gene fusion presence in cancer patients is often overlooked, and as such, no well-established workflow currently exists for robust profiling of tumor samples in this area. We established a targeted RNA sequencing panel (PANCARNA) for investigating gene fusions across multiple cancer types and performed analytical validation to confirm performance. Furthermore, we report on the applicability of using targeted RNA sequencing for the purposes of gene expression profiling. This panel covers 117 genes associated with solid tumors. Thirty-nine FFPE samples with gene fusion status validated using an orthogonal method were used to evaluate analytical performance. Of the 39 standard samples, 33 samples were positive for gene translocation events and 6 were negative controls. Samples were collected from across 10 cancer types. To examine the robustness of the panel, 12 samples with known fusions were selected to undergo testing. Each sample was tested in triplicate across two experimental batches. Seraseq Fusion RNA Mix V4 (SeraCare, Cat. 0710-0497) standard samples were used to test the limit of detection for PANCARNA. Additionally, we explored a cohort of 22 samples with over expressed genes and 4 samples without overexpressed genes to test the expression profiling capabilities of PANCARNA and compared the results to immunohistochemistry stains. Of the 39 samples tested using the orthogonal method and targeted RNA-Seq, 38 were found to have concordant fusion calls in both methods (Positive percent agreement = 97.0%, 32/33; Negative percent agreement = 100%, 6/6; Overall percent agreement = 97.4%, 38/39). In reproducibility studies, the assay produced consistent intrarun and interrun results in all samples (Repeatability = 100%, 12/12; Reproducibility = 100%, 12/12). The limit of detection for multiple fusions of interest, such as EML4-ALK, CD74-ROS1, TPM3-NTRK1, and SLC34A2-BRAF, is found to be 0.5-1 copies per ng of RNA. ETV6-NTRK1 can be reliably detected at 2-4 copies per ng of RNA. MET ex14 skipping events can also be detected in a few as 1-2 copies per ng of RNA. To validate the capability of PANCARNA to quantify gene expression, we tested 26 samples with known gene expression levels for three genes of interest (ERBB2, EGFR, MET). ERBB2, EGFR and MET amplification detection through PANCARNA was found to have a high concordance to IHC results (Sensitivity = 96.2%, 100%, 94.7%, respectively). In conclusion, PANCARNA demonstrated highly accurate and sensitive detection of clinically relevant gene fusions. This assay produces reliable results when detecting fusions and offers high concordance to results obtained through IHC/FISH, indicating that targeted RNA seq analysis can assist with profiling gene fusion events in a clinical setting. Citation Format: Haimeng Tang, Hua Bao, Rui Liu, Shuyu Wu, Sisi Liu, Xue Wu, Yang Shao. Robust profiling of cancer-related gene fusions: Analytical validation of PANCARNA for multiple cancer types [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 7408.

A nomogram based on TFE3 IHC results and clinical factors as a preliminary screening scheme for TFE3-rearranged renal cell carcinoma.

TFE3 immunohistochemistry (TFE3-IHC) is controversial in the diagnosis of TFE3-rearranged renal cell carcinoma (TFE3-rearranged RCC). This study is to investigate the accuracy and sensitivity of IHC and establish a predictive model to diagnose TFE3-rearranged RCC. Retrospective analysis was performed by collecting IHC and fluorescence insitu hybridization (FISH) results from 228 patients. IHC results were evaluated using three scoring systems. Scoring system 1 is graded based on nuclear staining intensity, scoring system 2 is graded based on the percentage of stained tumor cell nuclei, and scoring system 3 is graded based on both the nuclear staining intensity and the percentage. We collected patients' IHC results and clinical information. Important variables were screened based on univariate logistic regression analysis. Then, independent risk factors were established through multivariate logistic regression, and a nomogram model was constructed. The model was validated in internal test set and external validation set. The receiver operating characteristic curve (ROC curve), calibration curve, and decision curve analysis (DCA) were generated to assess discriminative ability of the model. The accuracy of IHC based on three scoring systems were 0.829, 0.772, and 0.807, respectively. The model included four factors including age, gender, lymph node metastasis and IHC results. Area under the curve (AUC) values were 0.935 for the training set, 0.934 for the internal test set, 0.933 for all 228 patients, and 0.916 for the external validation set. TFE3 IHC has high accuracy in the diagnosis of TFE3-rearranged RCC. Clinical information such as age and lymph node metastasis are independent risk factors, which can be used as a supplement to the results of TFE3 IHC. This study confirms the value of IHC in the diagnosis of TFE3-rearranged RCC. The accuracy of the diagnosis can be improved by incorporating IHC with other clinical risk factors.

Abstract WMP114: EZH2 Inhibitor Improves Cognitive Behavior in CAA

Introduction: Cerebral amyloid angiopathy (CAA) is characterized by the deposition of amyloid-β (Aβ) in the leptomeningeal and cortical vessels. CAA may lead to dementia and stroke; however effective options currently are very limited to treat or halt CAA progression. EZH2 (enhancer of zeste 2 polycomb repressive complex 2) is a histone methyltransferase, a key regulator of inflammation and microglia phagocytosis. The present study investigates the therapeutic potential of an EZH2 inhibitor (GSK343) in CAA mice. Methods: Brain samples were collected from a cohort of 4 WT and 4 TgSwDI (CAA model) male mice and lysed for immunoblot analysis to quantify EZH2 and H3K27me3 expression levels. A second cohort of 10 WT and 20 TgSwDI male mice (15 months old) were randomly assigned into DMSO (vehicle) and GSK343 groups. After performing baseline behavioral testing (Barnes maze and NORT), WT and TgSwDI-DMSO groups were injected with 0.1% DMSO while TgSwDI-GSK343 groups were injected with GSK343 (5 mg/kg) every other day intraperitoneally. After 6 weeks of treatment, behavioral testing was performed, and brain tissues were collected for IHC. Data are presented as Mean±SEM. Results: Immunoblot assays showed elevated expression of EZH2 and H3K27me3 in TgSwDI in comparison to WT mice. TgSwDI mice injected with GSK343 had improvements in the ability to recognize a novel object (58.36%±1.84) as compared to TgSwDI controls (45.04%±3.41) in the NORT ( p = 0.0006). The latency to escape (63.08±2.86) was significantly faster in GSK343-treated mice compared to TgSwDI control mice (77.34±3.43) in the Barnes maze ( p = 0.003). IHC results were in line with behavioral testing as GSK343 reduced the expression of EZH2 and Aβ in the hippocampus and frontal cortex. Conclusions: Our study revealed that EZH2 expression is upregulated in the brain of CAA-model mice. Pharmacological inhibition of EZH2 improved cognitive outcomes in CAA mice, suggesting a possible therapeutic target.

The role of immunohistochemical examination in the differential diagnosis of atypical tumors and carcinomas of parathyroid glands

Studying the role of IHC analysis in the differential diagnosis of APT and PC. The study included 44 patients with morphologic diagnosis of the APT established after surgical treatment for primary hyperparathyroidism on the basis of Endocrinology Research Centre during 2018-2023. All cases underwent IHC examination with evaluation of CD31/CD34 and parathormone (PTH) expression for identification of vascular invasion, Ki-67, parafibromin. According to the results of IHC analysis in 8/44 patients (18.2%) the diagnosis of APT was revised in favor of the PC: in 7 of them vascular invasion was detected; in 1 patient the additional series of slices in the surrounding fatty tissue revealed foci of tumor growth, confirmed by positive reaction with antibodies to PTH. According to IHC results, the material was divided into 2 groups: APT and PC. There were no differences in clinical and morphological characteristics, Ki-67% level and parafibromin expression between the groups. Assessment of clinical and laboratory-instrumental data at the preoperative stage does not allow differentiating APT from PC. In case of APT diagnosis and detection of suspicious morphological features, it is necessary to perform IHC examination to exclude PC.

Downregulation of NEBL promotes migration and invasion of clear cell renal cell carcinoma by inducing epithelial-mesenchymal transition

As a member of the nebulin protein family and a structural protein of cytoskeleton, NEBL plays an important role in cardiac diseases. Recently, literature have reported the involvement of NEBL in the occurrence and development of various cancers except clear cell renal cell carcinoma (ccRCC). In this study, we found that mRNA and protein of NEBL are downregulated remarkably in ccRCC tissues based on both the TCGA database and clinical samples we collected. The areas under curve values of NEBL analyzed based on the TCGA database, qRT-PCR and IHC results were 0.9376, 0.9733 and 0.9807, respectively. The lower mRNA level of NEBL was associated with worse outcomes in ccRCC patients. When overexpressing NEBL in ccRCC cell lines, the proliferation, migration and invasion of ccRCC cells were suppressed significantly, suggesting a tumor suppressor role of NEBL. In addition, we identified that NEBL is closely related to epithelial-mesenchymal transition (EMT), thereby reducing the motility of ccRCC cells. Furthermore, the lower expression of NEBL was correlated with ccRCC patients with distant organ metastasis. In summary, we firstly described the aberrant expression of NEBL and revealed its tumor suppressor role in ccRCC. Our data support that NEBL could serve as a valuable diagnostic and prognostic biomarker in ccRCC, as well as a promising therapeutic target.

HER2 amplification level by in situ hybridization predicts survival outcome in advanced HER2-positive breast cancer treated with pertuzumab, trastuzumab, and docetaxel regardless of HER2 IHC results

BackgroundThe role of HER2 amplification level in predicting the effectiveness of HER2-directed therapies has been established. However, its association with survival outcomes in advanced HER2-positive breast cancer treated with dual HER2-blockade remains unexplored.MethodsThis is a single-center retrospective study of patients with advanced HER2-positive breast cancer treated with first-line pertuzumab, trastuzumab, and docetaxel. The primary objective was to ascertain the relationship between treatment outcomes and the level of HER2 amplification by in situ hybridization (ISH).ResultsA total of 152 patients were included with a median follow-up duration of 50.0 months. Among the 78 patients who received ISH, a higher HER2/CEP17 ratio correlated significantly with longer PFS (HR 0.50, p = 0.022) and OS (HR 0.28, p = 0.014) when dichotomized by the median. A higher HER2 copy number also correlated significantly with better PFS (HR 0.35, p < 0.001) and OS (HR 0.27, p = 0.009). In multivariate analysis, the HER2/CEP17 ratio was an independent predictive factor for PFS (HR 0.66, p = 0.004) and potentially for OS (HR 0.64, p = 0.054), along with HER2 copy number (PFS HR 0.85, p = 0.004; OS HR 0.84, p = 0.049). Furthermore, the correlation between HER2 amplification level by ISH with PFS and OS was consistent across the HER2 IHC 1+/2+ and 3+ categories.ConclusionsThis is the first study to report that a higher level of HER2 amplification by ISH is associated with improved PFS and OS in advanced HER2-positive breast cancer treated with dual HER2-blockade. Notably, HER2 amplification level had a predictive role regardless of IHC results. Even in patients with HER2 protein expression of 3+, treatment outcome to HER2-directed therapy was dependent on the level of HER2 gene amplification.

Pan-TRK immunohistochemistry as screening tool for NTRK fusions: A diagnostic workflow for the identification of positive patients in clinical practice.

Pan-TRK inhibitors Entrectinib and Larotrectinib have been recently approved as tumor-agnostic therapies in NTRK1-2-3 rearranged patients and there is therefore an urgent need to identify reliable and accessible biomarkers for capturing NTRK fusions in the real-world practice. We aim to assess the analytical validity of the recently released pan-TRK assay (Ventana), running a head-to-head comparison between immunohistochemistry and Archer FusionPlex Lung Panel (ArcherDX) that is designed to detect key fusions in 13 genes, also including NTRK1-3. Pan-TRK IHC and NGS analysis were conducted on a retrospective/prospective cohort of 124 cancer patients (carcinomas, 93 cases; soft tissue sarcomas, 19; primary central nervous system tumours, 10; and neuroblastomas, 2). FISH data were available in most of the IHC/NGS discordant cases. A comparison between IHC and NGS results was carried out in 117 cases: among 30 pan-TRK positive cases, NTRK rearrangement by NGS was found in 11 (37%), while one of the 87 (1.1%) pan-TRK negative cases (a case of NSCLC) showed a TPM3-NRTK1 rearrangement by NGS. Accordingly, sensitivity and specificity of IHC in predicting NTRK status were 91.7% and 81.9%, respectively, while negative (NPV) and positive predictive value (PPV) were 98.8% and 36.7%, respectively. These data lead to suggest that IHC with VENTANA pan-TRK antibody can be a reliable screening tool for the identification of patients potentially bearing NTRK rearranged tumours.

Canine Gallbladder Erosion/Ulcer and Hemocholecyst: Clinicopathological Characteristics of 14 Cases.

(1) Background: Gallbladder mucosal erosion and/or ulceration are illnesses associated with unexpected gallbladder intra-cystic bleeding (hemocholecyst), an under-reported problem in dogs. (2) Methods: Clinicopathological characteristics of 14 dogs with gallbladder erosion/ulcer were investigated in this single-center retrospective study using clinical data and archived gallbladder tissues of client-owned dogs. (3) Results: Canine gallbladder erosion/ulcer tends to occur in older, neutered dogs of various breeds. Vomiting, lethargy, and anorexia are common. Concurrent gallbladder rupture occurred in 5/14 cases (35.7%), while rupture was absent in 6/14 cases (42.8%) and undetermined in 3/14 (21.4%) cases. Histologically, the gallbladder wall was markedly thickened due to mucosal hyperplasia, inflammatory infiltrates, fibrosis, edema, hemorrhage, and smooth muscle hyperplasia/hypertrophy. Twelve out of fourteen cases (85.7%) had concurrent cholecystitis of varying severity. Bacteria were detected by Giemsa or Warthin-Starry stain in 8/14 (57.1%) cases. Bacterial rods immunoreactive to the anti-Helicobacter antibody were present in one case. Mucosal epithelial cells of the gallbladder erosion/ulcer cohort were immunopositive for the cyclooxygenases COX-1 or COX-2 in only 5/14 (35.7%) cases. In contrast, COX-1 and COX-2 were more frequently expressed in a reference pool of cases of gallbladder mucocele (n = 5) and chronic cholecystitis (n = 5). COX-1 was expressed in 9/10 cases (90.0%) of gallbladder mucocele and chronic cholecystitis and in 10/10 cases (100%) for COX-2. (4) Conclusions: Canine gallbladder erosion/ulcer is an under-reported condition which requires active clinical intervention. Based on the clinicopathological information reported in this study in addition to the COX-1 and COX-2 IHC results, we suggest that canine gallbladder erosion/ulcer may be related to decreased cytoprotection physiologically provided by arachidonic acid, but which is decreased or absent due to reduced COX expression because of yet undetermined etiologies.

Cold Stress Induces Apoptosis in Silver Pomfret via DUSP-JNK Pathway.

We cultured silver pomfret for 20 days, decreasing water temperature from 18 to 8 ℃, and sampled muscle every 5 days. Muscle fiber degeneration and apoptosis began to increase at 13 ℃ detected by HE and TUNEL staining. Further analysis of transcriptome revealed that several apoptosis-related pathways were highly enriched by differentially expressed genes (DEGs). We analyzed 10 DEGs from these pathways by RT-qPCR during the temperature-decreasing process. JNK1, PIDD, CytC, Casp 3, and GADD45 were up-regulated after 15 and 20 days, while DUSP3, JNK2, and PARP genes were down-regulated after 15 and 20 days. DUSP5 was up-regulated from 10 to 20 days, and C-JUN was up-regulated after 20 days. We analyzed apoptosis in PaM cells under different temperatures (26 ℃, 23 ℃, 20 ℃, 17 ℃, and 14 ℃). The cell viability significantly declined from 14 to 20 ℃; the TUNEL and IHC results showed that the apoptosis signal increased with the temperature dropping, especially in 17 ℃ and 14 ℃; DUSP5, JNK1, CytC, C-JUN, Casp 3, and GADD45 were up-regulated at 17 ℃ and 14 ℃, and PIDD was up-regulated at 20 ℃, 17 ℃, and 14 ℃. DUSP3 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃, and PARP was down-regulated at 17 ℃ and 14 ℃. JNK2 was up-regulated at 20 ℃ but down-regulated at 17 ℃ and 14 ℃. Our results suggest that DUSP could help inhibit apoptosis in the initial stage of cold stress, but low temperature could down-regulate it and up-regulate JNK-C-JUN, inducing apoptosis in a later stage. These data provide a basis for the study of the response mechanism of fish to cold.

Placental Syphilis: A Comprehensive Review of Routine Histomorphology, HIV Co-infection, Penicillin Treatment, Immunohistochemistry, and Polymerase Chain Reaction

Introduction: Placental examination is valuable for diagnosing congenital syphilis, but the classic histological triad is not always observed. This study aimed to identify additional morphological clues, evaluate the sensitivity of IHC and qPCR, and investigate the impact of HIV co-infection and penicillin treatment on placental morphology. Materials and methods: Two hundred and fifteen placental specimens with treponemal infection were reviewed. Morphological findings, IHC, and qPCR results were analyzed. Results: Chronic villitis (94%), acute chorioamnionitis (91.6%), and villous immaturity (65.6%) were the most common abnormalities. HIV co-infection and penicillin treatment were associated with reduced frequencies of inflammatory lesions. IHC and qPCR exhibited sensitivities of 74.4 and 25.8%, respectively, confirming the diagnosis in 42 cases with negative or unknown serology. Conclusion: Villitis, chorioamnionitis, and villous immaturity were identified as the predominant placental abnormalities. HIV co-infection and penicillin treatment can impact morphology and hamper the diagnosis. IHC and q-PCR are valuable adjuncts when serology is negative.