Ki-67 Expression Research Articles

Abstract PO3-02-01: Educational Opportunities to Improve Community Healthcare Professionals’ Management of High-Risk HR-Positive/HER2-Negative Early Breast Cancer

Abstract Background: The treatment landscape for patients with hormone receptor–positive/HER2-negative early breast cancer (HR+/HER2- EBC) has evolved with the emergence of adjuvant CDK4/6 inhibitor therapy as a new option for patients with a high risk of disease recurrence. Adjuvant abemaciclib plus aromatase inhibitors (AIs) is guideline recommended for high clinical risk HR+ disease and reduces risk of recurrence regardless of Ki-67 expression level. Here, we examine healthcare professionals’ (HCPs’) awareness and practice regarding the use of adjuvant abemaciclib. Methods: Between November 2022 and June 2023, we conducted a series of regional educational activities for HCPs focused on management of high-risk HR+/HER2- EBC. Each activity included an interactive lecture led by a clinical investigator with expertise in managing patients with high-risk HR+/HER2- EBC and polling questions designed to assess key aspects of HCP knowledge and practice in the optimal management of high-risk HR+/HER2- EBC. A wider HCP audience was able to view an interactive on-demand webcast with the same content and embedded polling questions. Results: In total, 701 HCPs participated in one of 20 live (n = 373) activities or the one associated on-demand (n = 328) webcast. The distribution of HCP participation differed between the live and online activities with more physicians participating in the online activity vs the live activity (59% vs 34%, respectively); the converse was true for nurse participation (9% online vs 34% live). Polling result trends were similar between the live and online activities. At baseline, 38% (n/N = 182/405) of HCPs chose adjuvant therapy with an AI plus abemaciclib for a patient with high-risk, node-positive HR+/HER2- EBC consistent with expert and guideline recommendations. Following the education, 92% (n/N = 405/439) of HCPs selected AI plus abemaciclib for appropriate patients. Before education, 32% of HCPs (n/N = 147/454) were aware that adjuvant abemaciclib plus endocrine therapy improved invasive disease-free survival compared with endocrine therapy alone in patients with high-risk, node-positive HR+/HER2- EBC regardless of Ki-67 expression level, which increased to 87% (n/N = 370/425) following the education. Regarding the management of grade 2 neutropenia associated with adjuvant abemaciclib therapy, HCP knowledge was low at baseline (32%, n/N = 141/446) and improved to 80% (n/N = 332/415) after education. Conclusions: HCPs’ knowledge of and practice concerning adjuvant abemaciclib as a newer approved therapy for patients with high-risk HR+/HER2- EBC remain suboptimal. These results suggest an ongoing need for expert guidance and educational activities on the use of adjuvant abemaciclib therapy for appropriate patients with high-risk HR+/HER2- EBC. As the treatment landscape for HR+/HER2- EBC continues to evolve with the potential approval of additional new therapies, this need will only increase with a clear impact on optimizing and individualizing adjuvant therapy strategies for patients with HR+/HER2- EBC. Citation Format: Marie Becker, Timothy Quill, Erika Hamilton, Joyce O'Shaughnessy. Educational Opportunities to Improve Community Healthcare Professionals’ Management of High-Risk HR-Positive/HER2-Negative Early Breast Cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO3-02-01.

Abstract PO4-13-05: Spotlight on PSMA as a new theranostic biomarker for breast cancer

Abstract Breast cancer patients are risk-stratified on the basis of the subtype classification. However, this parameter is not completely accurate in discriminating between high- and low-risk disease, creating a need for a reliable marker to determine aggressiveness. Prostate-specific membrane antigen (PSMA) could fulfill this need given that it has been reported to be expressed by breast cancer tumor cells and the endothelial cells of tumor vessels. We analyzed 68 breast cancers (BC) of whom 14 luminal A, 28 Luminal B, 15 HER2 positive and 11 triple negative BC to assess whether PSMA expression by immunohistochemistry was different in the several tumor subtypes and was related to ki67 expression and stromal TILs. PSMA positivity was calculated as the number of positive vessels on 10 fields at 40X magnifications. Kruskal Wallis’s test was used to assess difference in PSMA positivity among BC subgroups and Dunn’s test was performed for post-hoc comparisons; Spearman’s rho coefficient was calculated to analyze the correlation among PSMA, KI67 and TILs. Our results show that median PSMA was higher both considering the number of positive vessels and the staining intensity in TNBC compared to Luminal A and B tumors (p< 0.001). Ki67 was higher in TNBC compared to Luminal A tumors (p< 0.001). We saw a correlation between PSMA and ki67, especially in HER2+ tumors (p=0.002), while a correlation between PSMA and TILs was observed in TNBC (p=0.014). The analysis of PSMA expression on the lymph nodes showed the same trend observed for the primary tumors given that it was higher in TNBC compared to HER2 positive and luminal cancers. Our results suggest that PSMA could be used as theranostic biomarker in BC considering that it is highly expressed in more aggressive tumors and the possibility to treat PSMA-expressing patients for anti-angiogenesis and/or radionuclide treatment. Citation Format: Sara Bravaccini, Stefania Cortecchia, Flavia Foca, Giovanni Martinelli, Sara Ravaioli, Maria Maddalena Tumedei, Federica Matteucci, Roberta Maltoni, Giovanni Paganelli, Francesca Poli, Maurizio Puccetti. Spotlight on PSMA as a new theranostic biomarker for breast cancer [abstract]. In: Proceedings of the 2023 San Antonio Breast Cancer Symposium; 2023 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2024;84(9 Suppl):Abstract nr PO4-13-05.

Clinicopathological characteristics and prognosis of Epstein-Barr virus-associated gastric cancer.

Epstein-Barr virus (EBV)-associated gastric cancer (EBVaGC) is a distinct molecular subtype of gastric cancer (GC). At present, the clinical characteristics and prognostic implications of EBV infection and the potential clinical benefits of immune checkpoint blockade in GC remain to be clarified. Hence, this study was designed to analyze the clinical and pathological characteristics of GC patients with varying EBV infection states and compare their overall survival (OS). A retrospective study was performed on 1031 consecutive GC patients who underwent gastrectomy at the Affiliated Hospital of Xuzhou Medical University from February 2018 to November 2022. EBV-encoded RNA (EBER) in situ hybridization (ISH) was used for EBV assessment, and immunohistochemical staining was used for evaluation of human epidermal growth factor receptor 2 (HER2), programmed death ligand 1 (PD-L1), and Ki67 expression. EBVaGC was defined as tumors with EBV positivity. In addition, EBV-negative GC (EBVnGC) patients were matched with EBVaGC patients based on seven clinicopathological parameters (age, gender, anatomic subsite, tumor size, Lauren classification, degree of differentiation, and tumor-node-metastasis [TNM] stage). The correlations of clinical features with HER2, PD-L1, and Ki67 expression were evaluated statistically. The survival of patients was assessed through medical records, telephone, or WeChat communication, and prognostic analysis was performed using the logrank test as well as univariable and multivariable regression analysis. Out of 1031 GC patients tested, 35 (3.4%) were diagnosed with EBVaGC. Notably, the EBVaGC group exhibited a distinct predominance of males and younger patients, significantly higher Ki67 and PD-L1 expression levels, and a lower prevalence of pericancerous nerve invasion than the EBVnGC group (P < 0.01). In the 35 EBVaGC cases, Ki67 expression was negatively correlated with age (P < 0.05), suggesting that a younger onset age was associated with higher Ki67 expression. In addition, PD-L1 expression was correlated with the degree of differentiation, T-stage, and clinical stage of the patient. Furthermore, PD-L1 expression was elevated in tumors with lower differentiation or at later stages (P < 0.05). Using univariate analysis, Ki67, PD-L1, and clinical stage were identified as significant factors influencing the overall survival (OS) of EBVaGC patients (P < 0.05). Moreover, multivariate survival analysis revealed that clinical stage and Ki67 expression were independent risk factors for the OS of the patients (P < 0.05), and the three-year OS rate of EBVaGC patients was 64.2%. EBV-ISH is a practical and valuable method to identify EBVaGC. Owing to its unique etiological, pathological, and clinical characteristics, patients with EBVaGC might benefit from immune checkpoint blockade therapy.

Construction and validation of a prediction model for preoperative prediction of Ki-67 expression in endometrial cancer patients by apparent diffusion coefficient

Ki-67 is a marker of cell proliferation and is increasingly being used as a primary outcome measure in preoperative window studies of endometrial cancer (EC). This study explored the feasibility of using apparent diffusion coefficient (ADC) values in noninvasive prediction of Ki-67 expression levels in EC patients before surgery, and constructs a nomogram by combining clinical data. This study retrospectively analyzed 280 EC patients who underwent preoperative magnetic resonance imaging (MRI) diffusion-weighted imaging (DWI) in our hospital from January 2017 to February 2023. Evaluate the potential nonlinear relationship between ADC values and Ki-67 expression using the nomogram. The included patients were randomized into a training set (n = 186) and a validation set (n = 84). Using a combination of logistic regression and LASSO regression results, from which the four best predictors were identified for the construction of the nomogram. The accuracy and clinical applicability of the nomogram were assessed using the receiver operating characteristic curve (ROC), calibration curve, and decision curve analysis (DCA). The results of this study showed a nonlinear correlation between ADCmin and Ki-67 expression (nonlinear P = 0.019), and the nonlinear correlation between ADCmean and Ki-67 expression (nonlinear P = 0.019). In addition, this study constructed the nomogram by incorporating ADCmax, International Federation of Gynecology and Obstetrics (FIGO), and chemotherapy. The area under the curve (AUC) values of the ROC for nomogram, ADCmax, FIGO, chemotherapy and grade in the training set were 0.783, 0.718, 0.579, 0.636, and 0.654, respectively. In the validation set, the AUC values for nomogram, ADCmax, FIGO, chemotherapy, and grade were 0.820, 0.746, 0.558, 0.542, and 0.738, respectively. In addition, the calibration curves and the DCA curves suggested a better predictive efficacy of the model. A nomogram prediction model constructed on the basis of ADCmax values combined with clinical data can be used as an effective method to noninvasively assess Ki-67 expression in EC patients before surgery.

Interaction of Sex Hormones and Dopamine D2 Receptor Polymorphisms in Human Renal Proximal Tubule Cells

The renal inflammatory response can be modulated by the dopamine D2 receptor (D2R) subtype. The DRD2 gene is highly polymorphic in humans, and single nucleotide polymorphisms (SNPs), such as rs6276 and rs6277 ( DRD2 SNPs), decrease its expression and function. We hypothesized that the response of human renal proximal tubular cells (hRPTCs) to hormones in the culture medium differs by sex and DRD2 SNPs, leading to differences in the inflammatory response. We determined the effects of dihydrotestosterone (DHT) and estradiol (ES) on hRPTCs genotyped for the presence or absence of DRD2 SNPs. We studied four cell lines from males (M) and females (F) with wild-type (WT) DRD2 (M- DRD2 WT and F- DRD2 WT) and DRD2 SNPs (M- DRD2 SNPs and F- DRD2 SNPs). The cells were cultured in a medium containing charcoal-stripped fetal bovine serum (FBS) for 24 hours, without (control group, CG) and with dihydrotestosterone (DHT, 5 nM) or estradiol (ES, 20 nM). We quantified the renal protein expression (protein of interest/GAPDH) of proinflammatory and profibrotic factors and markers of injury and proliferation. DHT decreased the expression of transforming growth factor (TGF-β) by ≍50% in both M- DRD2 WT (0.4±0.1 vs. 1.0±0.2) and M- DRD2 SNPs (1.0±0.1 vs. 1.6±0.1) (n=5-6, p&lt;0.05). However, there was no significant effect of DHT or ES on the expression of TGF-β in F- DRD2 WT and F- DRD2 SNPs. DHT did not affect the fibronectin 1 (FN1) expression in M- DRD2 WT and M- DRD2 SNPs. In F- DRD2 WT, DHT did not affect FN1 expression but decreased in F- DRD2 SNPs (0.9±0.1 vs. 1.7±0.1, n=6, p&lt;0.05). Similar to DHT, ES did not affect FN1 expression in M- DRD2 WT. However, ES decreased FN1 by 50% in M- DRD2 SNPs (0.5±0.1 vs. 1.1±0.1, n=6, p&lt;0.05). Similar to the studies in males, ES did not affect FN1 expression in F- DRD2 WT but also decreased FN1 (1.0±0.1 vs. 1.7±0.1, n=6, p&lt;0.05) in F-DRD2 SNPs. Neither DHT nor ES affected the expression of Kidney Injury Molecule-1 (KIM-1) in M- DRD2 WT. However, in M- DRD2 SNPs, both DHT and ES decreased KIM-1 (1.4±0.2 vs. 1.8±0.2 and 1.1±0.1 vs. 1.8±0.2, respectively; n=5, p&lt;0.05). Unlike in M- DRD2-WT, in F- DRD2 WT, ES increased KIM-1 expression (1.0±0.2 vs. 0.52±0.05, n=5, p&lt;0.05), while DHT had no significant effect. Unlike in M- DRD2 SNPs, where both DHT and ES decreased KIM-1, in F- DRD2 SNPs, neither DHT nor ES affected KIM-1. DHT did not affect the expression of Ki67 in M- DRD2 WT. However, in M- DRD2 SNPs, DHT decreased Ki67 expression (0.60±0.08 vs. 1.6±0.2, n=6, p&lt;0.05). Similar to M- DRD2-WT, DHT did not affect Ki67 expression in F- DRD2-WT. ES also did not affect Ki67 expression in M- DRD2 WT but decreased Ki67 expression in M-DRD2 SNPs (0.91± 0.18 vs. 1.6±0.2, n=6, p&lt;0.05). Similar to the male data, the treatment with ES did not affect the Ki67 expression in F- DRD2 WT but decreased Ki67 expression in F- DRD2 SNPs (1.5±0.2 vs. 0.9±0.1, n=6, p&lt;0.05). Our results demonstrate similarities (FN1 and Ki67) and differences (TGF-β and KIM-1) in profibrotic and proliferation markers expressed in hRPTCs between males and females. These differences are more pronounced in cell lines expressing DRD2 SNPs than those expressing the DRD2 WT. While both DHT and ES decreased KIM-1 expression in males with DRD2 polymorphisms, they had no effect in females with DRD2 polymorphisms. These results indicate a complex interaction of the effect of hormones and DRD2 polymorphisms in regulating the inflammatory response in hRPTCs. Their roles in regulating renal physiology and blood pressure remain to be defined. R01 DK039308 P01 HL068686 R01 HL023081 R37 HL023081 R01 DK119652/ NIH HH/ US. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Diacylglycerol kinase alpha is a proliferation marker of intrahepatic cholangiocarcinoma associated with the prognosis.

Intrahepatic cholangiocarcinoma (ICC) has a high recurrence rate and a poor prognosis. Thus, the development of effective treatment and prognostic biomarkers is required. High expression of diacylglycerol kinase alpha (DGKα) is a prognostic factor for the recurrence of hepatocellular carcinoma. However, the relationship between DGKα expression and prognosis in ICC has not been reported. Immunohistochemistry (IHC) with anti-DGKα antibody was performed on surgical specimens of ICC (n = 69). First, DGKα expression in cancer cells was qualitatively classified into four groups (-, 1+, 2+, 3+) and divided into two groups (DGKα- and DGKα+1 + to 3+). The relationship between clinical features and DGKα expression was analyzed. Second, Ki-67 expression was evaluated as a cell proliferation marker. The number of Ki-67-positive cells was counted, and the relationship with DGKα expression was examined. DGKα IHC divided the patients into a DGKα+ group (1+: n = 15; 2+: n = 5; 3+: n = 5) and a DGKα- group (-: n = 44). In the DGKα+ group, patients were older and had advanced disease. Both overall survival and recurrence-free survival (RFS) were significantly worse in the DGKα+ patients. DGKα+ was identified as an independent prognostic factor for RFS by multivariate analysis. Furthermore, the number of Ki-67-positive cells increased in association with the staining levels of DGKα. Pathological DGKα expression in ICC was a cancer proliferation marker associated with recurrence. This suggests that DGKα may be a potential therapeutic target for ICC.

Multifactorial risk prediction analysis of liver metastasis in colorectal cancer: incorporating programmed cell death ligand 1 combined positive score and other factors

BackgroundThe occurrence of liver metastasis significantly impacts the prognosis of colorectal cancer. Existing research indicates that primary tumor location, vascular invasion, lymph node metastasis, and abnormal preoperative tumor markers are risk factors for colorectal cancer liver metastasis. Positive expression of PD-L1(Programmed Cell Death 1 Ligand 1) may serve as a favorable prognostic marker for nasopharyngeal and gastric cancers, where CPS（Combined Positive Score） quantifies the level of PD-L1 expression. Based on previous studies, exploring CPS as a potential risk factor for colorectal cancer liver metastasis and integrating other independent risk factors to establish a novel predictive model for colorectal cancer liver metastasis. MethodsA retrospective analysis was conducted on 437 colorectal cancer patients pathologically diagnosed at Xiangya Second Hospital, Central South University, from January 1, 2019, to December 31, 2021. Data were collected, including CPS, age, gender, primary tumor location, Ki-67 expression, pathological differentiation, neural invasion, vascular invasion, lymph node metastasis, and preoperative tumor markers.The optimal cut-off point for the continuous variable CPS was determined using the Youden index, and all CPS were dichotomized into high and low-risk groups based on this threshold (scores below the threshold were considered high-risk, and those above it were considered low-risk). Univariate logistic regression analysis was employed to identify risk factors for colorectal cancer liver metastasis, followed by the application of multivariate logistic regression analysis to integrate the selected risk factors.The predictive model established was validated through the construction of ROC (Receiver Operating Characteristic) curves, calibration curves, and DCA (Decision Curve Analysis). A nomogram was constructed for visualization purposes. ResultsThe determined cut-off point for PD-L1 CPS was 4.5, with scores below this threshold indicating high risk for colorectal cancer liver metastasis. Additionally, primary tumor origin other than the rectum, presence of pericolonic lymph node metastasis, and abnormal levels of tumor markers CEA and CA199 were identified as independent risk factors for colorectal cancer liver metastasis. The constructed clinical prediction model demonstrated good predictive ability and accuracy, with an area under the ROC curve of 0.871 (95% CI 0.838-0.904). ConclusionThe exploration and validation of CPS as a novel predictor for colorectal cancer liver metastasis were conducted. Based on this, a new clinical prediction model for colorectal cancer liver metastasis was developed by integrating other independent risk factors. The DCA, clinical impact curve, and nomogram graph constructed based on this model have significant clinical implications and provide guidance for clinical practice.

Neonatal epithelial reparative capacity is enhanced by acellular placental extract in porcine models

Necrotizing enterocolitis (NEC) is a devastating condition with a ~30% mortality rate in pre-term, very low birth-weight infants. Premature infants lack vital in utero ingestion of amniotic fluid which accelerates intestinal maturation and reduces inflammation. Therefore, an acellular placental extract (PE) akin to amniotic fluid may be therapeutic for NEC. The objective of this study was to evaluate PE’s capacity to accelerate repair following or prevent the induction of NEC-like injury. We hypothesized that PE would enhance neonatal porcine intestinal epithelial cells’ capacity to repair in vitro and in vivo. NEC was induced in 12 piglets via pre-term cesarean delivery and hyperosmolar formula feeding. Prior to formula feeding, piglets were nil per os or enterally supplemented with PE. Tissue, collected at euthanasia, up to 72H after formula initiation, was evaluated grossly and histologically and with immunohistochemical staining for proliferation (Ki67), stem cell identity (Sox9), and apoptosis (CC3). Numerical outputs were assessed with Kolmogorov-Smirnov testing. PE’s effect on epithelial restitution was assessed with scratch assays performed on confluent monolayers derived from ~1-day old piglet ileum. Monolayers were pre-treated with either actin polymerization inhibitor, Latrunculin A, or a cell cycle inhibitor, hydroxyurea. After scratch, either media or PE in media was applied. Scratch-closure was monitored for 24H; percentage-closure was calculated and analyzed using 2-way ANOVA and Tukey multiple comparison. Scratch-wound leading-edge cells were examined for EdU and Ki67 immunofluorescent positivity which was evaluated using Kolmogorov-Smirnov analysis. Tight junction recovery was assessed using identically derived confluent monolayers grown on Transwell inserts and exposed to hypoxia (18H, 1%O2, 5%CO2). Following hypoxia, PE or media alone was added to the apical chamber. Transepithelial electrical resistance (TEER) was measured every 6-12H for 48H using a symmetrical disc electrode chamber. TEER rate of change was analyzed using a two-way ANOVA. For all analyses, significance was set at p&lt;0.05. In vivo PE administration resulted in reduced gross and histological NEC damage in treated compared to untreated piglets (p=0.02). Ki67 and Sox9 expression increased (p=0.71; p=0.93) and CC3 expression decreased with PE treatment (p=0.65). Evaluation of these markers in additional animals is pending. In vitro, PE expedited scratch wound closure (p&lt;0.05) and expanded proliferative cell number along scratch edges (EdU/DAPI, p&lt;0.05; Ki67+EdU/DAPI, p&lt;0.01). Other analyses are pending. PE application prevented gross NEC development in vivo and accelerated recovery in vitro. NIH #R44HD100243; #T32562967-39806. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

MDS and AML show elevated fractions of CD34-positive blast cell populations with a high anti-apoptotic versus proliferation ratio

This study investigates the intertwined processes of (anti-)apoptosis and cell proliferation in myelodysplastic syndromes (MDS) and acute myeloid leukemia (AML). Utilizing antibodies to Bcl-2 and Ki-67, the CD34-positive blast cell compartments in bone marrow aspirates from 50 non-malignant cases, 25 MDS patients, and 25 AML patients were analyzed for their anti-apoptotic and proliferative cell fractions through ten-color flow cytometry. MDS patients exhibited a significantly increased anti-apoptotic (p=0.0014) and reduced proliferative cell fraction (p=0.0030) in their blast cell population as compared to non-malignant cases. AML patients showed an even more exacerbated trend than MDS patients. The resulting Bcl-2:Ki-67 cell fraction ratios in MDS and AML were significantly increased as compared to the non-malignant cases (p=0.0004 and p<0.0001, respectively). AML patients displayed, however, a high degree of variability in their anti-apoptotic and proliferation index, attributed to heterogeneity in maturation stage and severity of the disease at diagnosis. Using double-labeling for Bcl-2 and Ki-67 it could be shown that besides blast cells with a mutually exclusive Ki-67 and Bcl-2 expression, also blast cells concurrently exhibiting anti-apoptotic and proliferative marker expression were found.Integrating these two dynamic markers into MDS and AML diagnostic workups may enable informed conclusions about their biological behavior, facilitating individualized therapy decisions for patients.

Correlation of the expression of Ki-67 with the histopathological features and grade of meningioma

Correlation of the expression of Ki-67 with the histopathological features and grade of meningioma

Novel cancer-fighting role of ticagrelor inhibits GTSE1-induced EMT by regulating PI3K/Akt/NF-κB signaling pathway in malignant glioma

BackgroundGlioma is the most common malignant brain tumor of the central nervous system. Despite of the improvement of therapeutic strategy, the prognosis of malignant glioma patients underwent by STUPP strategy is still unexpected. Previous studies have suggested that ticagrelor exerted chemotherapeutic effects by inhibition of epithelial-mesenchymal transition (EMT) in various diseases including tumors. However, whether ticagrelor can exhibit the antitumor efficiency in glioma by affecting the EMT process is still unclear. In this study, we investigated the cancer-fighting role of ticagrelor and demonstrated its chemotherapeutic mechanism in glioma. Materials and methodsThe MTT assay was performed to detect the cytotoxicity of ticagrelor in glioma cells. We evaluated the expression of Ki67 in glioma cells by immunofluorescence assay after ticagrelor treatment. We conducted wound healing assay and transwell assay to determine the effects of ticagrelor on the migration and invasion of glioma cells. RNA-seq analysis was conducted to examine potential target genes and alternative signaling pathways for ticagrelor treatment. The expression levels of key EMT -related proteins were examined by Western blot experiment. ResultsTicagrelor inhibited the proliferation, migration and invasion of glioma cells with a favorable toxicity profile in vitro. Ticagrelor downregulated the expression of GTSE1 in glioma cells. RNA-seq analysis explored that GTSE1 acted as the potential target gene for ticagrelor treatment. Upregulation of GTSE1 antagonized the inhibitory effect of ticagrelor on the invasion of glioma and EMT progression by regulation of PI3K/Akt/NF-κB signaling pathway. And ticagrelor also exhibited the similar chemotherapeutic effect of glioma in vivo. ConclusionsTicagrelor as a potential chemotherapeutic option induced the inhibition of the GTSE1-induced EMT progression by regulation of PI3K/AKT/NF-κB signaling pathway.

Outcome and Factors Related to Isolated Liver Metastasis due to Breast Cancer.

To determine the associated risk factors for isolated liver metastasis in breast cancer patients and to detect the prognostic factors related to survival. Analytical study. Place and Duration of the Study: Department of General Surgery, The University of Health Sciences, Istanbul, Turkiye, from January 2011 to November 2020. Patients with breast cancer liver metastasis who experienced surgery were retrospectively analysed for breast cancer and metastases-related characteristics. Descriptive statistical methods were used in the evaluation of data. Survival analyses were estimated by the Kaplan-Meier method. Log-rank and univariable Cox regression tests were utilised to search for prognostic factors' impact on survival. Out of 12 patients, 11 had recurrent disease after a median of 36 months of disease-free survival (DFS) and one patient had de novo metastasis. Grade 3 tumours and increased expression of Ki-67 had a negative effect on DFS. The median follow-up period was 66 months. Survival analysis showed 2- and 3-year progression-free survival (PFS); overall survival rates were 82%, 69%, 92%, and 82%, respectively. Development of liver metastasis in 3 years following breast cancer treatment was linked to worse PFS (p = 0.040). Long-term survival is possible for breast cancer survivors with liver metastasis. Disease-free interval is an important determinant. Longer progression-free survival was detected in patients who had developed metastasis after three years of breast cancer treatment. Breast cancer, Liver metastasis, Hepatic surgery.

H3K27ac-induced RHOXF2 activates Wnt2/β-catenin pathway by binding to HOXC13 to aggravate the malignant progression of triple negative breast cancer

Triple negative breast cancer (TNBC) is insensitive to conventional targeted therapy and endocrine therapy, and is characterized by high invasiveness and high recurrence rate. This study aimed to explore the role and mechanism of RHOXF2 and HOXC13 on the malignant progression of TNBC. RT-qPCR and western blot were used to detect RHOXF2 and HOXC13 expression in TNBC cells. The proliferation, colony formation, invasion, migration, apoptosis and cell cycle of TNBC cells after transfection were analyzed by CCK-8 assay, colony formation assay, transwell assay, wound healing assay and flow cytometry analysis. Co-Immunoprecipitation and GST pull-down assays were used to analyze the combination between RHOXF2 and HOXC13. ChIP-PCR and luciferase reporter gene assay were used to examine the regulation of H3K27ac on RHOXF2. Besides, the expression of Ki67 and cleaved Caspase3 in tumor tissues of nude mice was determined by immunofluorescence. Results revealed that RHOXF2 and HOXC13 expression was increased in TNBC cells. RHOXF2 knockdown suppressed the proliferation, invasion and migration, as well as induced G0/G1 cell cycle arrest and apoptosis of TNBC cells. Besides, RHOXF2 could bind to HOXC13 and RHOXF2 knockdown suppressed HOXC13 expression in TNBC cells. Furthermore, HOXC13 overexpression reversed the impacts of RHOXF2 downregulation on the proliferation, invasion, migration, G0/G1 cell cycle arrest and apoptosis of TNBC cells. In addition, RHOXF2 silencing limited the tumor volume in nude mice, which was reversed by HOXC13 overexpression. Moreover, RHOXF2 knockdown interfered with Wnt2/β-catenin pathway in vitro and in vivo by binding to HOXC13. Importantly, H3K27ac acetylation could activate the expression of RHOXF2 promoter region. In conclusion, RHOXF2 activated by H3K27ac functioned as a tumor promoter in TNBC via mediating Wnt2/β-catenin pathway by binding to HOXC13, which provided promising insight into exploration on TNBC therapy.

Effects of silencing NLRP3 gene on proliferation of psoriasis-like HaCaT cells and expressions of cytokines.

We aimed to explore the effects of silencing NOD-like receptor protein 3 (NLRP3) on proliferation of psoriasis-like HaCaT cells and expressions of cytokines. HaCaT cells were treated with human keratinocyte growth factor (KGF) and were divided into KGF group, negative control group, NLRP3-RNAi group and control group. Cells proliferation was detected by CCK8, cell clone formation rate was detected by clone formation assay, distribution of cells cycle was detected by flow cytometry, expressions of cyclin B1 (Cyclin B1), cyclin-dependent kinase 2 (CDK2), Ki67 and proliferating cell nuclear antigen (PCNA) proteins were detected by Western blot, and levels of interleukin (IL)-17, IL-23, IL-6 and tumor necrosis factor α (TNF-α) were detected by enzyme-linked immunosorbent assay. Compared with control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were increased in KGF group, percentage of cells in G0/G1 phase was decreased, percentage of cells in S phase was increased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were increased, and levels of IL-17, IL-23, IL-6 and TNF-α were increased. Compared with negative control group, expressions of NLRP3 mRNA and protein, proliferation rate and clonal formation rate were decreased in NLRP3-RNAi group, percentage of cells in G0/G1 phase was increased, percentage of cells in S phase was decreased, expressions of Cyclin B1, CDK2, Ki67 and PCNA proteins were decreased, and levels of IL-17, IL-23, IL-6 and TNF-α were decreased. Silencing NLRP3 gene can inhibit the proliferation of psoriasis-like HaCaT cells, arrest cell cycle, inhibit the expressions of cell proliferation-related proteins and reduce levels of pro-inflammatory factors.

Coupling of the expression of proliferation and epithelialmesenchymal transition markers with the histidine-rich glycoprotein HRG mRNA expression in breast diseases

Non-malignant breast diseases (NMBD) may increase the risk of developing a malignant neoplasm. Therefore, it seems relevant to search for criteria for cell malignancy in NMBD. Aim of the study was to investigate the relationship between expression of proliferation and epithelial-mesenchymal transition (EMT) markers and histidine-rich glycoprotein (HRG) mRNA in breast diseases. Material and methods. In breast biopsy specimens of 37 patients with invasive carcinoma of a non-specific type (ICNT) and 17 patients with NMBD expression of proliferation markers (Ki-67, cyclin D1 (CCND1)) and EMT markers (E-cadherin (CDH1), type II collagen (CII) and β1-integrin (CD29)) was determined immunohistochemically. HRG mRNA expression was estimated using real time PCR. Results. HRG mRNA expression was detected in 91.9 % cases (34 of 37) in ICNT, 82.4 % (14 of 17) in NMBD and in the latter case was inversely related to the expression of CDH1, CD29 and Ki-67. A direct relationship has been established between the presence of Ki-67 and CCND1, CII, between CCND1 and CD29 in NMBD. In patients with ICNT, a direct correlation was found between the HRG mRNA expression and the presence of CII, and an inverse correlation between the number of cells containing CII and CD29. It was found that in ICNT and NMBD with the presence of HRG mRNA expression, the CDH1 expression is less than in its absence. Conclusions. Indicators of HRG mRNA expression in NMBD, combined with the assessment of proliferation and EMT markers, can be useful in developing criteria for cell malignancy in benign breast diseases.

Genetically engineered mouse model of HPV16 E6-E7 with vaginal-cervical intraepithelial neoplasia and decreased immunity

ObjectiveTo construct models of high-risk human papillomavirus (HPV) infection with precancerous lesions or cervical cancer and explore the immune function. MethodsUsing CRISPR/Cas9, the expression vector HPV16-E6-E7-Rosa26 was microinjected into fertilized eggs of C57BL/6 N mice using homologous recombination, and the F0 generation was obtained for reproduction. Then, the formation of precancerous lesions was promoted via intramuscular injection of estradiol. Presence of precancerous cervical-vaginal intraepithelial lesions, Ki67 and p16 expression levels, and CD8+ T cell proportions in the spleen were evaluated. ResultsTwo F0 generation mice exhibited correct the homologous recombination. Seven positive mice were identified in the F1 generation. After breeding and mating, 25 homozygous and 11 heterozygous HPV16-E6-E7-engineered mice were obtained from the F2 generation. After estradiol benzoate treatment, the cervical-vaginal epithelium appeared as precancerous lesions with positive Ki67 and p16 expression. The percentage of CD8+ T cells decreased. ConclusionHPV16-E6-E7-Rosa26 induced low immune function in mice, and provides a good model for the basic research of the mechanisms of action of HPV infection-associated precancerous lesions or cervical cancer.

CCT6A Regulates Cervical Cancer Cell Glycolysis and Proliferation under Hypoxic Conditions via the Telomerase Cajal Body Protein 1/Telomerase Reverse Tranase.

Cervical cancer is one of the most common female malignancies worldwide, a hypoxic microenvironment usually causes enhanced viability and glycolytic capacity of cervical cancer cells. The aim of this study was to investigate the association between chaperonin containing t-complex polypeptide 1 subunit 6A (CCT6A) and cell proliferation as well as hypoxic glycolysis. Bioinformatics analysis was conducted to explore the association between cervical cancer and its partner protein under hypoxic conditions, namely, CCT6A. Subsequently, the expression of CCT6A was silenced, and the effects of CCT6A silencing on cervical cancer cell proliferation, cell cycle, glycolysis-related proteins, and telomerase activity were examined. Bioinformatics analysis was performed to investigate the expression of CCT6A in cervical cancer under hypoxic conditions. The expression of CCT6A was silenced in cervical cancer cells, HeLa and SiHa, to study its effects on cell proliferation and hypoxic glycolysis. The localization of telomerase activity-related proteins, telomerase CTAB1 and TERT, was detected using immunofluorescence, and their interaction was assessed using immunoprecipitation. A cellular hypoxia model was established, and the products of the glycolysis reaction were detected. A nude mouse tumor model was constructed, and the changes in glycolysis-related proteins in tumor tissues were examined using Western blot, while Ki67 expression in tumor tissues was evaluated by immunohistochemistry. Telomerase activity was found to be enhanced in CCT6A-silenced cervical cancer cells, along with an increase in telomerase TCAB1 and TERT protein binding associated with telomerase activity. Additionally, the proportion of cells in the Gap 2/mitosis (G2/M) stage and the 5-ethynyl-2'-deoxyuridine (EdU) positivity rate were decreased in CCT6A-silenced cells, indicating a reduction in cell proliferation. The expression of cell cycle-related proteins, including cyclin E, CCNA2, and cyclin-dependent kinase 2 (CDK2), was suppressed. Furthermore, under a hypoxic environment, silencing CCT6A led to a significant reduction in cell viability and downregulation of glycolysis-related proteins, such as lactate dehydrogenase A (LDHA) and hexokinase 2 (HK2). Mechanistically, silencing CCT6A may reduce telomerase activity by inhibiting the TCAB1/TERT interaction. Additionally, TERT was found to activate the promoter region of the HK2 gene, and inhibition of TERT activity reduced the transcriptional level of HK2. The study primarily explored the involvement of CCT6A in cervical cancer, yet it did not account for the myriad of other elements potentially influencing cell proliferation and glycolysis. It is essential to recognize that cervical cancer's etiology is multifaceted, shaped by an array of genetic variations, environmental influences, and protein interactions. Silencing CCT6A could effectively attenuate the upregulation of cell proliferation and glycolytic function mediated by TCAB1/TERT in cervical cancer cells.

Exosomes from MicroRNA-125b-Modified Adipose-Derived Stem Cells Promote Wound Healing of Diabetic Foot Ulcers.

Exosomes derived from Adipose-Derived Stem Cells (ADSCs-Exo) have been implicated in the enhancement of wound repair in Diabetic Foot Ulcers (DFU). The current research was designed to explore the therapeutic potential and underlying mechanisms of ADSCs-Exo modified with microRNA-125b (miR-125b) in the context of DFU. Rat models with DFU and human umbilical vein endothelial cells (HUVECs) subjected to high glucose (HG) conditions served as experimental systems and were administered miR-125b-engineered ADSCs-Exo. Then, the expressions of CD34, Ki-67, angiogenesis-related factors (VEGF and TGFβ-1), angiogenesis inhibitor DLL-4, and inflammation-related proteins (TLR-4 and IL-6) were detected. MiR-125b was upregulated in ADSCs-Exo. MiR-125b-mimics transfection in ADSCs- Exo reduced inflammatory infiltration and promoted granulation formation and wound healing in wound tissues. MiR-125b-mimics-modified ADSCs-Exo injection increased the expression of CD34, Ki-67, VEGF, and TGFβ-1, whereas decreased the expression of DLL-4, TLR-4, and IL-6 in wound tissues of DFU rats. In addition, miR-125b-mimics-ADSCs-Exo injection reversed the negative effects of HG on the proliferation, migration, and angiogenesis of HUVECs, as well as the positive effects of cell apoptosis. Moreover, miR-125b-inhibitor-ADSCs-Exo injection had the opposite effects to miR-125b-mimics-ADSCs-Exo. ADSCs-Exo promoted wound healing of DFU rats, especially when overexpressing miR-125b.

P179 CD19hi B cells express T-bet and display activated metabolism and function in rheumatoid arthritis and systemic lupus erythematosus patient samples

Abstract Background/Aims CD19, a co-receptor of the B cell receptor, fine-tunes the threshold for B-cell activation with implications for cell metabolism and function. Activating receptors such as CD40, IFNγR and TLRs also influence B cell activation. B cell subpopulation, double negative (DN, IgD-CD27-) T-bet+ B cells, is considered pathogenic in rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Here, we explored whether CD19hi B cells differed from CD19lo B cells with regards to proliferation, metabolism, and function. Methods Peripheral blood samples from nine healthy participants, 16 RA and 20 SLE patients were analysed. Subgroup analysis of rituximab-treated (RTX-T), n = 6 versus rituximab naïve (RTX-N), n = 6, was performed. B cells or peripheral blood mononuclear cells were stimulated with anti-IgG, anti-IgM and anti-human CD40 antibodies and a TLR9 agonist for 20 hours prior to flow cytometric analysis of B cell expression of phosphorylated AMPK (pAMPK, the master regulator of metabolism), intracellular expression of Ki67 (proliferation marker), and cytokines IL-6 and IL-10. Statistical analysis was performed using GraphPad Prism v10.0.1. Results Compared to CD19+CD20+ B cells, CD19+CD20- B cells: 1) were predominantly composed of DN (55% versus 19%, p &amp;lt; 0.01) and IgD-CD27+ memory subpopulations (37% versus 25%, p = 0.1094); 2) were detectable at a hierarchical frequency, RTX-T&amp;gt;RTX-N&amp;gt;HC cohorts; 3) had a greater frequency of Ki67+ cells (38% versus 5%, p &amp;lt; 0.01); 4) displayed higher CD38 MFI (41478.0 versus 10589.0, p &amp;lt; 0.01). Approximately 50% of CD19hi B cells were T-bet+, compared to 28% of CD19lo B cells (n = 33, p &amp;lt; 0.0001). Compared to CD19lo, CD19hi B cells 1) expressed (MFI) significantly greater TLR7, TLR9, IFNγR and pAMPK (n = 7 for all except pAMPK, n = 4), and 2) had greater frequency of IL6+ and IL10+ B cells (n = 7, p &amp;lt; 0.05) (Table). Conclusion CD20- B cell subpopulations with potent proliferative capacity express CD19. Further, CD19hi B cells: 1) upregulate the TLR-IFNγ-T-bet pathway; 2) have significantly greater expression of pAMPK; 3) have higher cytokine expression compared to CD19lo B cells. These data suggest the activation pathway, metabolic activity, and functional profile of CD19hi cells. Therefore, the potential of CD19hi cells to promote inflammation provides strong rationale for targeting CD19 in autoimmune disease with emerging therapies such as CAR-T cells and T cell engagers. Disclosure K. Shah: Grants/research support; KS has received funding for research from Roche. M.J. Leandro: Consultancies; MJL acted as a consultant for Roche and Genentech. Other; MJL received funds less than $10000 and support to attend educational conferences. M.S. Cragg: Consultancies; Consultant for BioInvent International, Boehringer Ingelheim, GSK, Radiant, iteos, Surrozen, Hanall and Mestag. Honoraria; He has received honoraria/speakers fees from Boehringer Ingelheim, Amgen, Roche and Symphogen. Grants/research support; BioInvent International, GSK and UCB. F. Kollert: Corporate appointments; FK is employed by Roche. Shareholder/stock ownership; FK has stock ownership at Roche. F. Schuler: Corporate appointments; FS is employed by Roche. Shareholder/stock ownership; FS has patents and stock ownership at Roche. D.A. Isenberg: None. A. Akbar: None. C. Klein: Corporate appointments; CK is employed by Roche. Shareholder/stock ownership; CK has patents and stock ownership at Roche. V. Reddy: Grants/research support; VR has received a research grant from Roche.

Concordance between ER, PR, Ki67, and HER2-low expression in breast cancer by MammaTyper RT-qPCR and immunohistochemistry: implications for the practising pathologist.

There are limited data on the role of multigene tests and their correlation with immunohistochemistry (IHC), especially on core biopsy. MammaTyper is a quantitative conformite Europeeanne (CE) marked, National Institute for Health and Care excellence (NICE) approved, in in vitro diagnostic quantitative real-time polymerase chain reaction (RT-qPCR) test for assessment of mRNA expression of four biomarkers (ESR1, PGR, ERBB2, MKI67). We evaluated the concordance of MammaTyper with oestrogen receptor (ER), progesterone receptor(PR), HER2, and Ki67 by IHC on 133 core needle biopsies of breast cancer. HER2 was positive if IHC 3+ or 2+ and fluorescence in situ hybridization (FISH)-amplified. Global and hotspot Ki67 expression was analysed using a cutoff of ≥20% assessed manually and by digital image analysis. Agreements were expressed as overall percent agreement (OPA), positive percent agreement (PPA), negative percent agreement (NPA), and Cohen's kappa. RT-qPCR results of ESR1 were highly concordant with IHC with OPA of 94.7% using 1% cutoff and 91.7% when the low ER-positive category was included. The PPA and NPA between RT-qPCR and IHC for PR was 91.5% and 88.0%, respectively, when using the 1% cutoff. For ERBB2/HER2, the OPA was 95% and the PPA was 84.6%. 40 of 72 HER2 IHC score 0 tumours were classified as ERBB2 low. Best concordance between MKI67 by MammaTyper and Ki67 IHC was achieved using hotspot digital image analysis (OPA: 87.2%, PPA: 90.6%, NPA: 80%). RT-qPCR-based assessment of the mRNA expression of ESR1, PGR, ERBB2, and MKI67 showed high concordance with IHC, suggesting that the MammaTyper test on core needle biopsies represents a reliable, efficient, and reproducible alternative for breast cancer classification and refining HER2 low categorisation.