Percentage Of Ki-67 Positive Cells Research Articles

PATH-60. AUTOMATED QUANTIFICATION OF KI-67 PROLIFERATION INDEX BASED ON WHOLE SLIDE IMAGES IN PEDIATRIC BRAIN TUMORS

Abstract The use of AI methods with histopathological images (pathomics) has strong potential to aid pathologists in pediatric brain cancer diagnosis and prognosis given the morphological and molecular heterogeneity of these tumors. However, use of pathomics in this context has been relatively limited. One example is the IHC-based tumor proliferation index (percentage of Ki-67 positive cells amongst all malignant cells) which is often routinely reviewed by pathologists clinically but remains to be objectively quantified or reported in a standardized manner. As a result, analysis of this marker across patients at-scale and in data-driven methods remains largely hindered. Here, we explore the extension of an automated deep learning AI tool (DeepLIIF), originally trained and validated with adult breast/prostate/liver data, for quantification of Ki-67 index from digitized whole slide images of a cohort of pediatric brain tumors from the Children’s Brain Tumor Network (N=834). We find that derived Ki-67 scores largely agreed with neuropathologist-reported Ki-67 activity in original clinical notes and could be used to successfully stratify glioma/astrocytoma patients (N=200) into low- and high-grade groups (LGG: M=11.6; SEM=1.2; HGG: M=18.7; SEM=2; p=0.001). Ki-67 scores were also related to the survival status of the patients (Alive: M=11.8; SEM=1.1; Deceased: M=18.8; SEM=2.2; p=0.003). Summary statistics across tumor types (glioma, astrocytoma, medulloblastoma, ATRT, ependymoma, etc.) and molecularly defined subtypes are also compared, providing direct insights into distributional properties between these groups that has not previously been feasible. Our results show the ability of AI-powered methods to quantify routine histopathology metrics with little human input, allowing objective comparisons between patients and opening the door for precision medicine approaches with predictive analytics in clinical contexts.

P-629 In vivo follicular activation (IVFA) through repeated ovarian punction: results of an experimental study in a premature ovarian insufficiency rodent model

Abstract Study question Can the new IVFA therapy effectively promote ovarian follicle development and increase oocyte number in a premature ovarian insufficiency (POI) animal model. Summary answer The IVFA technique reveals favourable reproductive outcomes in the animal model, demonstrating increased oocyte retrieval without compromising quality, making a promising advancement in fertility restoration. What is known already POI is characterized by the loss of the primordial follicular pool before the age of 40. Ovarian fragmentation for in vitro follicular activation (IVA) is a technique based on the disruption of the ovarian extracellular matrix, subsequently promoting actin polymerization and interfering with the evolutionarily conserved tumour suppressor Salvador-Hippo-Warts (SHW) signalling pathway. This has been proven to increase expression of downstream growth factors, thus promoting the primordial follicle growth and the generation of mature oocytes. Yet, this procedure is invasive and expensive. Consequently, a demand exists for less invasive in vivo therapies. Study design, size, duration Experimental animal study. Sixty 6-week-old C57BL/6 female mice were divided into control (CT) and POI groups. Each group, was further divided into three subsets (n = 10/group): without ovarian punctures (CT-0 and POI-0), 15 punctures (CT-15 and POI-15), and 22 punctures (CT-22 and POI-22). Twenty-one days after POI induction, the new IVFA technique was performed on the anaesthetized animals. Subsequently, controlled ovarian stimulation (COS) was initiated, followed by euthanasia for the oocyte retrieval and histological ovarian evaluation. Participants/materials, setting, methods POI condition was induced with intraperitoneally injections of 120 mg/kg of cyclophosphamide and 12 mg/kg of busulfan. The IVFA technique involved ovarian punctures using a 30-gauge needle. COS was induced with 10 IU of PMSG and 10 IU of hCG 48 hours later, leading to euthanasia for oocytes and ovaries collection. Ovaries (n = 6/group) were fixed for follicular counts and immunohistochemistry. Oocyte quality assessment included reactive oxygen species (ROS) levels, mitochondrial fluorescence (Mitotracker), and spindle analysis. Main results and the role of chance The control groups subjected to the IVFA technique (CT-15 and CT-22) demonstrated significantly higher mean total oocyte counts compared to the non-intervention group (CT-0) (30.0±9.8 and 27.7±7.9 vs 18.1±11.7, respectively; p-value<0.05 in both comparisons). Similarly, within the POI model, interventions (POI-15 and POI-22) showed significantly elevated mean total oocyte counts than the non-intervention group (POI-0) (8.1±4.4 and 7.2±2.8 vs 3.8±3.6, respectively; p-value<0.05). The intensity of ROS fluorescence, Mitotracker fluorescence, and spindle analysis in retrieved oocytes were not affected by the IVFA therapy, thereby preserving oocyte quality. Notably, the percentage of growing follicles was significantly increased in the intervention groups compared to the non-intervention groups in the control (60±5.7% and 58.1±4.7% vs 41.5±4.5%; p-value<0.001). In the POI model, statistically significant differences were observed for POI-15 vs POI-0 (57.8±9% vs 41.6±13.3%; p-value<0.05), but not for POI-22 vs POI-0 (51.5±14.2% vs 41.6±13.3%, p=n.s.) Moreover, the global Ki67 proliferation marker demonstrated a significant increase in the percentage of proliferative cells in the CT-15 intervention group compared to the CT-0 group (53±11.2% vs 46.1±15.1; p < 0.01). Finally, in the POI model, the percentage of Ki-67 positive cells was also significantly higher in both IVFA groups (39.4±18.5% and 38.1±17.3% vs 32±17.5%, respectively; p < 0.05). Limitations, reasons for caution Since this is an animal model of POI, further validation of the new therapy with human ovarian samples would be required. The magnitude of the effect could vary due to inter-species variability. Wider implications of the findings The positive results of the new therapy in promoting follicle development and increasing oocyte retrieval, without compromising quality, have significant implications for developing new fertility treatments. If validated in humans, this approach could offer a promising solution for women with POI. Trial registration number not applicable

Characteristics of quiescent adult neural stem cells induced by the bFGF/BMP4 combination or BMP4 alone in vitro.

Bone morphogenetic protein-4 (BMP4) is involved in regulation of neural stem cells (NSCs) proliferation, differentiation, migration and survival. It was previously thought that the treatment of NSCs with BMP4 alone induces astrocytes, whereas the treatment of NSCs with the bFGF/BMP4 combination induces quiescent neural stem cells (qNSCs). In this study, we performed bulk RNA sequencing (RNA-Seq) to compare the transcriptome profiles of BMP4-treated NSCs and bFGF/BMP4-treated NSCs, and found that both NSCs treated by these two methods were Sox2 positive qNSCs which were able to generate neurospheres. However, NSCs treated by those two methods exhibited different characteristics in state and the potential for neuronal differentiation based on transcriptome analysis and experimental results. We found that BMP4-treated NSCs tended to be in a deeper quiescent state than bFGF/BMP4-treated NSCs as the percentage of ki67-positive cells were lower in BMP4-treated NSCs. And after exposure to differentiated environment, bFGF/BMP4-treated NSCs generated more DCX-positive immature neurons and MAP2-positive neurons than BMP4-treated NSCs. Our study characterized qNSCs treated with BMP4 alone and bFGF/BMP4 combination, providing a reference for the scientific use of BMP4 and bFGF/BMP4-induced qNSCs models.

Heart failure stimulates colorectal tumour growth through gut microbial alterations of specific populations

Abstract Background/purpose The field of reverse cardio-oncology has received growing attention in the last years, since epidemiological studies showed that patients with HF are at increased risk of developing cancer. In addition, HF can stimulate tumour growth directly in distant organs, including colon cancer. The exact mechanisms underlying this phenomenon, however, remain largely unknown. The connection between the microbiome and colorectal cancer (CRC) has been studied extensively and microbial dysbiosis in CRC is linked to cancer progression. Recent studies have also shown that HF patients display microbial dysbiosis. We hypothesized that microbial dysbiosis might contribute to HF-induced CRC tumour growth. Methods C57BL/6J mice were subjected to myocardial infarction (MI), with sham surgery as control. After six weeks faeces were collected, processed for 16s rRNA sequencing, and pooled for faecal microbial transplantation. CRC tumour growth was provoked in germ-free mice by treating them with Azoxymethane (AOM)/Dextran sodium sulphate (DSS). The CRC mice were transplanted with faeces from MI or sham mice. Disease progression was monitored using the disease severity score and endoscopy. After ten weeks mice were sacrificed, and tumour number and proliferation were quantified. All experimental procedures were performed in accordance with the European Union guidelines (CCD_8141, the Netherlands, LA_1400637, Belgium). Results MI-induced HF resulted in a decrease in microbial diversity (Shannon diversity p=0.018), characterized by a decrease in short chain fatty acid-producing bacteria, including the genera Blautia (p<0.01) and Roseburia (p<0.01). In addition, caecal butyrate levels were decreased in MI mice (p=0.029). These changes were largely comparable to microbial changes observed in HF patients and CRC patients. Interestingly, faecal transplantation with faeces from MI mice resulted in faster growth of CRC tumours in mice treated with AOM/DSS. This was demonstrated by a higher endoscopy score (7.9 ±2.5 vs. 10 ±2.4, p=0.038), indicative of worse disease progression, increased tumour counts (22 ±5.3 vs. 29 ±6, p<0.01), and a higher percentage of Ki67 positive cells in tumour tissue (p=0.012), relative to AOM/DSS treated mice transplanted with faeces from sham mice. Sequencing data revealed several differences in the microbial composition of mice transplanted with faeces from MI mice compared to mice transplanted with faeces from sham mice. Conclusion Our results demonstrate that HF induces microbial dysbiosis in mice. In addition, we show, for the first time, using a faecal transplantation model, that HF-induced microbial dysbiosis is causally involved in CRC tumour growth in a mouse model of CRC. Targeting the microbiome may present a tool to mitigate HF associated co-morbidities, especially cancer.Graphical abstract

Abstract 12858: Myocardial Infarction Promotes Breast Cancer Growth Through the Nerve Growth Factor/Tropomyosin Receptor Kinase A Pathway

Background: Epidemiological studies have suggested that patients with heart failure due to myocardial infarction (MI) have a higher cancer incidence. Nerve growth factor (NGF) plays a pivotal role in sympathetic nerve remodeling after MI, but several studies have suggested that NGF also plays a significant role in cancer progression. However, the impact of NGF on cancer progression in the setting of MI remains to be elucidated. Methods and Results: MI was induced by permanent ligation of the left anterior descending artery in female BALB/c mice. 4T1 cells, mammary tumor cells, were injected into the right mammary fat pad 2 weeks after surgery. Levels of NGF expression in the myocardium 3 days after surgery (1.40±0.17 AU vs. 1.00±0.09 AU, P=0.049) and serum NGF at 2 weeks after surgery (279±34 pg/ml vs. 173±16 pg/ml, P=0.044) were higher in the MI group than in the sham group. In addition, the tumor volume was significantly larger in the MI group than in the sham group 3 weeks after 4T1 cell injection (465.3±32.6 mm 3 vs. 292.1±28.3 mm 3 , P<0.001). The percentage of Ki67-positive cells in tumor tissue was higher in the MI group than in the sham group (37.1±5.7% vs. 16.5±3.6%, P=0.010). Moreover, phosphorylation activities of membranous tropomyosin receptor kinase A (TRKA, 1.54±0.04 AU vs. 1.00±0.09 AU, P<0.001) and AKT (1.56±0.18 AU vs. 1.00±0.08 AU, P=0.046) were higher in the MI group than in the sham group. Inhibition of TRKA by a TRKA inhibitor, GW441756 (20 mg/kg/week, I.P.), suppressed tumor volume in MI mice compared to DMSO-treated MI mice (281.6±34.8 mm 3 vs. 443.9±50.8 mm 3 , P<0.001). The percentage of Ki67-positive cells (31.9.1±2.8% vs. 42.2±2.0%, P=0.010), phosphorylation levels of TRKA (0.99±0.07 AU vs. 1.68±0.09 AU, P<0.001) and AKT (0.90±0.01 AU vs. 1.19±0.05 AU, P=0.001) in tumor tissues were decreased in GW441756-treated MI mice than in DMSO-treated MI mice. Conclusion: The NGF/TRKA pathway is involved in accelerated tumor cell growth under MI-induced heart failure.

Use of a Novel Deep Learning Open-Source Model for Quantification of Ki-67 in Breast Cancer Patients in Pakistan: A Comparative Study between the Manual and Automated Methods.

Introduction: Breast cancer is the most common cancer in women; its early detection plays a crucial role in improving patient outcomes. Ki-67 is a biomarker commonly used for evaluating the proliferation of cancer cells in breast cancer patients. The quantification of Ki-67 has traditionally been performed by pathologists through a manual examination of tissue samples, which can be time-consuming and subject to inter- and intra-observer variability. In this study, we used a novel deep learning model to quantify Ki-67 in breast cancer in digital images prepared by a microscope-attached camera. Objective: To compare the automated detection of Ki-67 with the manual eyeball/hotspot method. Place and duration of study: This descriptive, cross-sectional study was conducted at the Jinnah Sindh Medical University. Glass slides of diagnosed cases of breast cancer were obtained from the Aga Khan University Hospital after receiving ethical approval. The duration of the study was one month. Methodology: We prepared 140 digital images stained with the Ki-67 antibody using a microscope-attached camera at 10×. An expert pathologist (P1) evaluated the Ki-67 index of the hotspot fields using the eyeball method. The images were uploaded to the DeepLiif software to detect the exact percentage of Ki-67 positive cells. SPSS version 24 was used for data analysis. Diagnostic accuracy was also calculated by other pathologists (P2, P3) and by AI using a Ki-67 cut-off score of 20 and taking P1 as the gold standard. Results: The manual and automated scoring methods showed a strong positive correlation as the kappa coefficient was significant. The p value was <0.001. The highest diagnostic accuracy, i.e., 95%, taking P1 as gold standard, was found for AI, compared to pathologists P2 and P3. Conclusions: Use of quantification-based deep learning models can make the work of pathologists easier and more reproducible. Our study is one of the earliest studies in this field. More studies with larger sample sizes are needed in future to develop a cohort.

The lipid peroxidation-derived DNA adduct γ-OHPdG as a diagnostic and prognostic biomarker in hepatocellular carcinoma.

Chronic inflammation and lipid peroxidation (LPO) are associated with the pathogenesis of hepatocellular carcinoma (HCC), and γ-hydroxy-1, N2-propanodeoxyguanosine (γ-OHPdG) is a promutagenic DNA adduct derived from LPO. This study aimed to examine the relationship between γ-OHPdG and the progression of liver carcinogenesis. Primary HCC specimens were obtained from 228 patients and cirrhosis specimens from 46 patients. The patients were followed up with after surgery via outpatient visits and telephone calls. The levels of γ-OHPdG were determined by immunohistochemical analysis in the carcinomatous tissues together with adjacent and cirrhosis tissues. γ-OHPdG levels in the cancerous tissues were significantly higher compared to adjacent tissues (P < 0.001) and also higher than the ones from the tissues of cirrhosis patients. Along with tumor size, histological grade, MVI grade, T stage, the percentage of ki67-positive cells and HCC progression, γ-OHPdG levels in cancerous tissues showed a gradually increasing trend. Moreover, prognostic analysis showed that higher γ-OHPdG levels in cancerous tissues were strongly correlated with lower overall survival (P < 0.001), lower intrahepatic recurrence-free survival (P < 0.001) and lower distant metastasis-free survival (P < 0.05). There was a trend, although not statistically significant, of increased levels of γ-OHPdG in cirrhosis cases that advanced to HCC, whereas γ-OHPdG levels reversely correlated with the period of time observed for cirrhosis advanced to HCC. These results suggest that γ-OHPdG is a prognostic biomarker for predicting outcomes in HCC, and may serve as a prospective indicator for predicting HCC in cirrhosis patients.

Selection of optimal quantile protein biomarkers based on cell-level immunohistochemistry data

BackgroundProtein biomarkers of cancer progression and response to therapy are increasingly important for improving personalized medicine. Advanced quantitative pathology platforms enable measurement of protein expression in tissues at the single-cell level. However, this rich quantitative cell-by-cell biomarker information is most often not exploited. Instead, it is reduced to a single mean across the cells of interest or converted into a simple proportion of binary biomarker-positive or -negative cells.ResultsWe investigated the utility of retaining all quantitative information at the single-cell level by considering the values of the quantile function (inverse of the cumulative distribution function) estimated from a sample of cell signal intensity levels in a tumor tissue. An algorithm was developed for selecting optimal cutoffs for dichotomizing cell signal intensity distribution quantiles as predictors of continuous, categorical or survival outcomes. The proposed algorithm was used to select optimal quantile biomarkers of breast cancer progression based on cancer cells’ cell signal intensity levels of nuclear protein Ki-67, Proliferating cell nuclear antigen, Programmed cell death 1 ligand 2, and Progesterone receptor. The performance of the resulting optimal quantile biomarkers was validated and compared to the standard cancer compartment mean signal intensity markers using an independent external validation cohort. For Ki-67, the optimal quantile biomarker was also compared to established biomarkers based on percentages of Ki67-positive cells. For proteins significantly associated with PFS in the external validation cohort, the optimal quantile biomarkers yielded either larger or similar effect size (hazard ratio for progression-free survival) as compared to cancer compartment mean signal intensity biomarkers.ConclusionThe optimal quantile protein biomarkers yield generally improved prognostic value as compared to the standard protein expression markers. The proposed methodology has a broad application to single-cell data from genomics, transcriptomics, proteomics, or metabolomics studies at the single cell level.

MiR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy

ObjectiveIntestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R). MethodsCaco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, β-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2’-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells. ConclusionKnockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

Acinar Cell Proliferation Promoted by BMP2 in Injured Mouse Parotid Gland: BMP2 Promotes Cell Proliferation in Parotid Gland.

To identify factors that affect salivary gland recovery, we investigated the expression and function of bone morphogenetic protein 2 (BMP2) in mice. Using a micro clip, mice parotid glands were removed 7 days after the ligation of the unilateral parotid excretory duct. Thereafter, they were weighed and stained with hematoxylin and eosin, and BMP2 expression was examined via real-time reverse transcription-polymerase chain reaction. Primary cultures of parotid glands were prepared, and BMP2 protein was added to the culture medium for 48 hr to examine its effect on cell proliferation. E-cadherin and vimentin expression was examined using western blotting. Finally, immunohistochemical staining using an anti-Ki67 antibody was performed. Duct-ligated parotid glands weighed less than those that were collected after sham surgery and showed acinar cell atrophy. They also showed higher BMP2 expression than control glands. Primary-cultured parotid acinar cells supplemented with BMP2 showed higher proliferative potential than control cells. Furthermore, they showed E-cadherin, but not vimentin, expression, and their percentage of Ki67-positive cells were higher than that corresponding to the controls. Injury to salivary glands by excretory duct ligation increased BMP2 expression, which may be involved in maintaining salivary gland function by inducing acinar cell proliferation.

A Novel Radiographic Pattern Related to Poor Prognosis in Patients with Prostate Cancer with Metastatic Spinal Cord Compression

BackgroundProstate cancer spinal bone metastases can have a radiographic profile that mimics multiple myeloma. ObjectiveTo analyse the presence and prognostic value of myeloma-like prostate cancer bone metastases and its relation to known clinical, molecular, and morphological prognostic markers. Design, setting, and participantsA cohort of 110 patients with prostate cancer who underwent surgery for metastatic spinal cord compression (MSCC) was analysed. Spinal bone metastases were classified as myeloma like (n = 20) or non–myeloma like (n = 90) based on magnetic resonance imaging prior to surgery. An immunohistochemical analysis of metastasis samples was performed to assess tumour cell proliferation (percentage of Ki67-positive cells) and the expression levels of prostate-specific antigen (PSA) and androgen receptor (AR). The metastasis subtypes MetA, MetB, and MetC were determined from transcriptomic profiling. Outcome measurements and statistical analysisSurvival curves were compared with the log-rank test. Univariate and multivariate Cox proportional hazard models were used to assess the effects of prognostic variables. Groups were compared using the Mann-Whitney U test for continuous variables and the chi-square test for categorical variables. Results and limitationsPatients with the myeloma-like metastatic pattern had median survival after surgery for MSCC of 1.7 (range 0.1–33) mo, while the median survival period of those with the non–myeloma-like pattern was 13 (range 0–140) mo (p < 0.001). The myeloma-like appearance had an independent prognostic value for the risk of death after MSCC surgery (adjusted hazard ratio 2.4, p = 0.012). Postoperative neurological function was significantly reduced in the myeloma-like group. No association was found between the myeloma-like pattern and morphological markers of known relevance for this patient group: the transcriptomic subtypes MetA, MetB, and MetC; tumour cell proliferation; and AR and PSA expression. ConclusionsA myeloma-like metastatic pattern identifies an important subtype of metastatic prostate cancer associated with poor survival and neurological outcomes after surgery for MSCC. Patient summaryThis study describes a novel radiographic pattern of prostate cancer bone metastases and its relation to poor patient prognosis.

Abstract 10584: Nerve Growth Factor/Tropomyosin Receptor Kinase A Axis Plays a Pivotal Role in Breast Cancer Progression After Myocardial Infarction

Background: Several studies have shown that cancer incidence is increasing in patients with chronic heart failure. We have reported that nerve growth factor (NGF) plays a pivotal role in sympathetic nerve remodeling after myocardial infarction (MI). However, the impact of NGF on cancer progression in the setting of MI-induced heart failure remains to be elucidated. Methods and Results: NGF phosphorylated tropomyosin receptor kinase A (TrkA) in murine breast carcinoma 4T1 cells, and NGF increased the percentage of Ki-67 positive 4T1 cells. MI was induced by permanent ligation of the left anterior descending artery of female Balb/c mice. Then, 4T1 cells were injected into the right fourth mammary fat pad at two weeks after MI surgery. Serum levels of NGF were higher in the MI group than in the sham group at the time of 4T1 cell injection (279±107 pg/ml vs. 172±36 pg/ml, P=0.014). The size of breast cancer was significantly larger in the MI group than in the sham group at three weeks after 4T1 cell injection (451.8±137 mm 3 vs. 304.3±136 mm 3 , P=0.0014, figure). The percentage of Ki-67 positive cells in cancer tissue was higher in the MI group compared to the sham group (37.1±6.8% vs. 16.5±3.2%, P&lt;0.001). Furthermore, membranous TrkA was more phosphorylated in the MI group than in the sham group (1.56±0.11 AU vs. 1.00±0.11 AU, P&lt;0.001). Knockdown of TrkA with siRNA in 4T1 cells suppressed the increase of Ki-67 positive cells by NGF stimulation (51.9±1.2% vs. 24.9±3.7%, P=0.044). A TrkA inhibitor, GW441756, suppressed the NGF-induced phosphorylation of TrkA in 4T1 cells. Moreover, the percentage of Ki-67 positive cells in cancer tissue was lower in the MI mice treated with a TrkA inhibitor (10 mg/kg/week, I.P.) than in the MI mice without a TrkA inhibitor (26.3±3.6% vs. 45.6±3.7%, P&lt;0.001). Conclusion: Increased NGF caused by MI is associated with breast cancer progression through NGF/TrkA axis.

Abstract 12398: CD151 Expression Reflects Different Features in Cardiac Subtype Specification From Human Pluripotent Stem Cells

Introduction: In conventional cardiac differentiation methods, a heterogeneous cardiac population is generated from human induced pluripotent stem cells (hiPSCs). Purifying ventricular- or atrial cardiomyocytes (VCMs or ACMs, respectably) is valuable for drug discovery for region-specific heart diseases. Previously, we have identified a novel marker, CD151, which can separate advanced specified subtypes; CD151 high VCMs in ventricular inducing condition (VIC) and CD151 low ACMs in atrial inducing condition (AIC). Although CD151 is known as a cell adhesion molecule, the function in cardiac subtype specification is unclear. In this study, we addressed the mechanisms of how CD151 distinguishes cardiomyocyte subtypes in cardiac differentiation from hiPSCs. Methods: We performed RNA sequencing in VIC- and AIC- CD151 high/low CMs. Pathway analysis using differential expression genes between CD151 high/low CMs was conducted to identify prominent pathways in subtype specification. Results: In VIC, mitosis-related genes were enriched in CD151 high VCMs. To clarify whether those genes affect cell proliferation or multi-nucleation, we performed the flow cytometric analysis of CD151 high/low VCMs with KI-67 proliferation marker and Hoechst staining. Although the percentage of KI67-positive cells was almost the same, binucleated cells were significantly more in CD151 high VCMs than in CD151 low VCMs. In AIC, transcriptome analysis suggested that Notch signaling was activated in CD151 high ACMs. To reveal that Notch signaling inhibits atrial specification, we analyzed ACMs treated with LY411575, a Notch signaling inhibitor. LY411575 induced atrial marker gene expression and increased the number of cells showing action potentials of ACMs. CD151 low ACMs after LY411575 treatment contained ACMs with the highest efficiency. Conclusion: Our results demonstrate that the CD151 expression indicates mitotic activity in VCMs. CD151 high VCMs are more advanced specified VCMs with binucleation without cell proliferation. On the other hand, in ACMs, CD151 works as an indicator of Notch signaling, which suppresses atrial specification. Our study provides a novel approach to generating and purifying hiPSCs-ACMs by Notch inhibition and CD151 separation.

Establishment of a novel mouse model of adenomyosis suitable for longitudinal and quantitative analysis and perinatal outcome studies

The purpose of this study was to establish a novel mouse model of adenomyosis suitable for longitudinal and quantitative analyses and perinatal outcome studies. Using a 30 G needle, the entire uterine wall of one horn was mechanically punctured at a frequency of 100 times/1 cm (adenomyosis horn). The other horn was left unpunctured (control horn). Balb/c mice were sacrificed on day 14 (D14) or day 65 (D65) (n = 3 each). The uterus was fixed, paraffin-embedded, sliced, and stained. Lesions were detected and counted, and their volumes were measured. Cell proliferation and fibrosis were assessed by Ki67 and Masson’s Trichrome staining, respectively. Blood vessels were detected using CD31 immunostaining. Some of the mice (n = 4), were mated and the date of delivery, litter size, number of implantations, and number and volume of postpartum lesions were measured. The number of lesions per horn did not differ between D14 and D65. The volume of the entire lesion was significantly greater on D65 than on D14 (p < 0.0001). The volume of the epithelial part of the lesion was significantly greater in D65 (p < 0.0001). The volume of the stromal part of the lesion was also greater on D65 (p < 0.0001). The percentage of Ki67 positive cells in the epithelial part of the lesion was significantly higher on D14 (p < 0.05). In contrast, the percentage of Ki67-positive cells in the stromal part was significantly higher on D65 (p < 0.01). Vascular density in the lesions was higher in on D65 (p < 0.05). The percentage of fibrotic area was significantly higher on D65 (p < 0.01). The date of delivery was slightly earlier than that reported for healthy mice of the same strain. The litter size was smaller than that reported in previous research. The number of implantation sites did not differ between the control and the adenomyosis horn. The number and volume of lesions did not differ between the non-pregnant and postpartum groups. This model can be applied to evaluate the pathogenesis of adenomyosis, validate the efficacy of therapeutic agents, and evaluate the effect of adenomyosis on pregnancy and vice versa.

Reduced inflammation following human endometrial stromal/stem cell injection into male Wistar rats with cisplatin-induced acute kidney injury.

Introduction: Inflammation is one of the most important mechanisms involved in cisplatin-induced acute kidney injury (AKI). Mesenchymal stromal/stem cells (MSCs) exhibit anti-inflammatory and immunomodulatory abilities. Human endometrial stromal/stem cells (hEnSCs) exhibit similar properties to MSCs. These cells secrete immunoregulators, so we investigated the inflammatory aspect of hEnSCs in the treatment of cisplatin-induced AKI in Wistar rats. Methods: Each group consisted of 6 male Wistar rats. Groups were as follows: sham, model (5 mg/kg cisplatin, IP), and treatment (1 million hEnSCs, IV, 3 hours after cisplatin). Renal function, histopathology, proliferation rate, infiltration of CD3+ T cell, and expression of Il-10 and cystatin c (Cst3) were assessed on day 5. DiI-labeled cells were tracked in kidney and liver on days 4 and 14. Results: HEnSC transplantation improved cisplatin-induced injuries such as renal dysfunction and tissue damage. The highest levels of pathologic scores and hyaline cast formation were observed in the model group while hEnSCs transplantation resulted in their reduction (154.00 ± 14.95, 8.00 ± 1.41 vs. 119.40 ± 5.43, 2.50 ± 1.05). The percentage of Ki-67 positive cells in the treatment group increased while cisplatin decreased proliferation (39.91 ± 5.33 vs. 23.91 ± 3.57 in glomeruli and 39.07 ± 2.95 vs. 16.61 ± 3.25 in tubules). The expression of Cst3 and Il-10 was higher in the model and treatment groups, respectively. DiI-labeled cells were observed in the renal tubules and liver lobes on days 4 and 14. Conclusion: HEnSCs may ameliorate cisplatin-induced AKI through anti-inflammatory and immunomodulatory effects and/or through paracrine effects.

Bile Duct Regeneration with an Artificial Bile Duct Made of Gelatin Hydrogel Nonwoven Fabrics.

Although choledochojejunostomy is the standard technique for biliary reconstruction, there are various associated problems that need to be solved such as reflux cholangitis. Interposition with an artificial bile duct (ABD) to replace the resected bile duct maintains a physiological conduit for bile and may solve this problem. This study investigated the usefulness of an ABD made of gelatin hydrogel nonwoven fabric (GHNF). GHNF was prepared by the solution blow spinning method. The migration and activity of murine fibroblast L929 cells were examined in GHNF sheets. L929 cells migrated into GHNF sheets, where they proliferated and synthesized collagen, suggesting GHNF is a promising scaffold for bile duct regeneration. ABDs made of GHNF were implanted in place of resected bile duct segments in rats. The rats were killed at 2, 6, and 12 weeks postimplantation. The implantation site was histologically evaluated for bile duct regeneration. At postoperative 2 weeks, migrating cells were observed in the ABD pores. The implanted ABD was mostly degraded and replaced by collagen fibers at 6 weeks. Ki67-positive bile duct epithelial cells appeared within the implanted ABD. These were most abundant within the central part of the ABD after 6 weeks. The percentages of Ki67-positive cells were 31.7 ± 9.1% in the experimental group and 0.8 ± 0.6% in the sham operation group at 6 weeks (p < 0.05), indicating that mature biliary epithelial cells at the stump proliferated to regenerate the biliary epithelium. Biliary epithelial cells had almost completely covered the bile duct lumen at 12 weeks (epithelialization ratios: 10.4 ± 6.9% at 2 weeks, 93.1 ± 5.1% at 6 weeks, 99.2 ± 1.6% at 12 weeks). The regenerated epithelium was positive for the bile duct epithelium marker cytokeratin 19. Bile duct regeneration was accompanied by angiogenesis, as evidenced by the appearance of CD31-positive vascular structures. Capillaries were induced 2 weeks after implantation. The number of capillaries reached a maximum at 6 weeks and decreased to the same level as that of normal bile ducts at 12 weeks. These results showed that an ABD of GHNF contributed to successful bile duct regeneration in rats by facilitating the cell migration required for extracellular matrix synthesis, angiogenesis, and epithelialization. Impact Statement Development of an artificial bile duct (ABD) enables physiological biliary reconstruction and may solve clinical problems associated with choledochojejunostomy. In this study, we created ABDs with gelatin hydrogel nonwoven fabric and implanted them in place of resected bile duct in rats. We evaluated the process of bile duct regeneration as well as decomposition of the ABD and demonstrated successful regeneration of resected bile duct, highlighting the possibility of this novel biliary reconstruction method to replace choledochojejunostomy.

Abstract P2-15-01: Conversion from luminal to normal intrinsic subtype by PAM50 after neoadjuvant endocrine therapy is associate with biomarkers of good prognosis in luminal breast cancer

Abstract Background. Neoadjuvant endocrine therapy (NET) in luminal breast cancer (LBC) is the perfect scenario for real-time evaluation of the biological and molecular changes that occur after estrogenic deprivation. This evaluation is mainly performed in surgical specimens after NET and has been used as a research tool to obtain prognostic and predictive information using tumor response to decide adjuvant treatment. Nevertheless, there are not many validated biomarkers in this setting to predict response beyond Ki67 levels and modified Preoperative Prognostic Index (mPEPI score) after treatment. Hence, the aim of this study is to determine if changes in mRNA-based PAM50 analysis (intrinsic subtype) after NET in LBC correlate with such biomarkers. Methods. We collected a series of postmenopausal ER+/HER2- breast cancer patients (n=58) treated for at least 4 months with NET before surgery. Next, we performed gene expression analysis by PAM50 accompanied by pathological characterization of surgical tumor specimens and immunohistochemistry characterization of ER and Ki67 levels, both at diagnosis and in surgery specimen. Finally, we studied the association of our results with clinical and histopathological features and with validated biomarkers for endocrine response, such as mPEPI score and changes in Ki67 levels. Results. The distribution of changes in intrinsic subtype determined by PAM50 after NET is presented in Table 1. We observed that tumors that changed from luminal to a normal intrinsic subtype showed larger changes in Ki67 levels after NET and reduced percentage of Ki67 positive cells at surgery, compared to those that presented a luminal persistent status: both analysis P&amp;lt;0.0001 (Mann-Whitney test). Similarly, we found a statistical association between lower mPEPI score in those tumors that change from luminal to a normal intrinsic subtype compared to those that presented a luminal persistent status: P=0.0034 (Mann-Whitney test). Moreover, in agreement with these results, we also studied ROR-P (PAM50 risk of recurrence (ROR) of subtype plus proliferation) in tumors that change from luminal to a normal intrinsic subtype and in those that presented a luminal persistent status. Results showed the same trends as the ones observed for Ki67-related parameters and mPEPI. Conclusions. PAM50 analysis revealed that changes from luminal A and luminal B to normal intrinsic subtype are associated with response to NET, measured by different well-established parameters. This result gives us clues to further explore which genes are orchestrating these changes and their impact on clinical outcomes. However, more analyses should be performed to determine the relationship between ROR and parameters used to measure response in NET in order to validate the results presented. In conclusion, our data support that the determination of the changes in gene expression profile using PAM50, before and after NET, can be used as a tool to predict response, conjointly with other clinical parameters. Table 1.- Intrinsic subtypes determined by PAM50 in samples pre and post NETPAM50 PREPAM50 POSTNO.%PERSISTANT LUMINAL STATUSCHANGE FROM LUMINAL TO A NOLMAL INTRINSIC SUBTYPELumALumA2136YESLumANormal1526YESLumALumB35YESLumBLumA610YESLumBNormal35YESLumBLumB59YESHER2HER223NANAHER2LumA12NANALumAHER212NANANormalNormal12NANATOTAL58100Lum=LuminalNA= Not applicable Citation Format: Joanna Ines Lopez Velazco, María Otaño, Inazio Lacambra, Kepa Elorriaga, Ainhara Lahuerta, Ana Martínez, Virginia Segur, Sara Manzano, Aleix Prat, María Caffarel, Ander Urruticoechea. Conversion from luminal to normal intrinsic subtype by PAM50 after neoadjuvant endocrine therapy is associate with biomarkers of good prognosis in luminal breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-15-01.

Impact of FGF1 on human periodontal ligament fibroblast growth, osteogenic differentiation and inflammatory reaction in vitro.

To investigate in vitro the impact of fibroblast growth factor1 (FGF1) in comparison to ascorbic acid (AscA) on human periodontal ligament fibroblast (HPdLF) growth, their osteogenic differentiation, and modulation of their inflammatory reaction to mechanical stress. The influence of different concentrations of FGF1 (12.5-200 ng/mL) on growth and proliferation of HPdLF cells was analyzed over 20days by counting cell numbers and the percentage of Ki67-positive cells. Quantitative expression analysis of genes encoding the osteogenic markers alkaline phosphatase (ALPL), Runt-related transcription factor2 (RUNX2), osteocalcin (OCN), and osteopontin (OSP), as well as the fibroblast markers vimentin (VIM) and fibroblast-specific protein1 (FSP1), was performed after 2and 20days of cultivation. Metabolic activity was determined by MTT assay. For comparison with AscA, 50 ng/mL FGF1 was used for stimulation for 2and 20days. Cell number, percentage of Ki67-positive cells, and expression of osteoblast- and fibroblast-specific genes were examined. Alkaline phosphatase activity was visualized by NBT/BCIP and calcium deposits were stained with alizarin red. Cytokine (IL‑6, IL‑8, COX2/PGE2) expression and secretion were analyzed by qPCR and ELISA in 6 h mechanically compressed HPdLF cultured for 2days with FGF1 or ascorbic acid. Higher concentrations of FGF1 promoted cell proliferation upon short-term stimulation, whereas prolonged treatment induced the expression of osteogenic markers even with low concentrations. AscA promotes cell growth more markedly than FGF1 in short-term cultures, whereas FGF1 induced osteogenic cell fate more strongly in long-term culture. Both factors induced an increased inflammatory response of HPdLF to mechanical compression. Our data suggest that FGF1 promotes an osteogenic phenotype of HPdLF and limits inflammatory response to mechanical forces compared to AscA.

Alterations of DNA methylation were associated with the rapid growth of cortisol-producing adrenocortical adenoma during pregnancy

BackgroundCortisol-producing adrenocortical adenoma (CPA) during pregnancy rarely occurs in clinic. Growing evidence suggests that DNA methylation plays a key role in adrenocortical adenomas. The present study aims to examine the genome-wide DNA methylation profiles and identify the differences in DNA methylation signatures of non-pregnant and pregnant patients with CPA.ResultsFour pregnant and twelve non-pregnant patients with CPA were enrolled. The pregnant patients with CPA had higher serum cortisol, Estradiol, Progesterone, and human chorionic gonadotropin concentration, while having lower serum FSH (follicle-stimulating hormone) and luteinizing hormone concentrations (P < 0.01). Compared with the non-pregnant patients, the duration is shorter, and the growth rate of the tumor is faster in pregnant patients with CPA (P < 0.05). Morphology and cell proliferation assay showed that the percentage of Ki-67 positive cells in CPA were higher in pregnant group than non-pregnant group (8.0% vs 5.5%, P < 0.05). The DNA methylation analysis showed that Genome-wide DNA methylation signature difference between pregnant and non-pregnant with CPA, that the pregnant group had more hypermethylated DMPs (67.94% vs 22.16%) and less hypomethylated DMPs (32.93% vs 77.84%). The proportion of hypermethylated DMPs was relatively high on chromosomes 1 (9.68% vs 8.67%) and X (4.99% vs 3.35%) but lower on chromosome 2(7.98% vs 12.92%). In pregnant patients with CPA, 576 hypomethylated DMPs and 1109 hypermethylated DMPs were identified in the DNA promoter region. Bioinformatics analysis indicated that the Wnt/β-Catenin pathway, Ras/MAPK Pathway and PI3K-AKT Pathway were associated with the development of CPA during pregnancy.ConclusionsGenome-wide DNA methylation profiling of CPA in non-pregnant and pregnant patients was identified in the present study. Alterations of DNA methylation were associated with the pathogenesis and exacerbation of CPA during pregnancy.

Abstract PS5-39: Tumour cellularity size as a biomarker to predict response after neoadjuvant endocrine therapy: Correlation analysis between Ki67 expression and PEPI score

Abstract Background: Neoadjuvant endocrine therapy (NET) is an approach that allows real-time evaluation of drug efficacy as well as investigation of the biological and molecular changes that occur after estrogenic deprivation. This evaluation is mainly performed in surgical specimens after NET and it has been used as a research tool to obtain prognostic and predictive information using tumour response to decide adjuvant treatment. In this setting, there are not many validated biomarkers to predict response beyond Ki67 expression and Preoperative Prognostic Index (PEPI score) in NET. The aim of this study is to determine if the tumour cellularity size (TCS) in surgical specimen after NET correlates with PEPI score and Ki67 expression. Methods: Retrospective study of postmenopausal patients with estrogen receptor (ER) positive/HER2 negative resectable breast cancer, treated with an aromatase inhibitor for at least 4 months prior to surgery. Pathological characterization of tumour specimens: Evaluation of the percentage of residual tumour cellularity of formalin fixed paraffin embedded surgical specimens and immunohistochemistry characterization of ER and Ki67. Tumour cellularity size: calculated by combining the percentage of residual tumour cellularity and tumour pathological size. Results: N=104. Tumour characteristics at surgery and breakdown for the calculated PEPI score: table 1. Correlation between the percentage of Ki67 positive cells at surgery and TCS: (r=0.2503) p=0.04 (95% CI, 0.0014 to 0.4700). Correlation between TCS and PEPI score: (r=0.2582) p=0.05 (95% CI, -0.0131 to 0.4940). Conclusions: Tumour cellularity size is a promising biomarker to determine response and prognosis after NET. There is a need to find other biomarkers to predict response after NET. Table 1Pathology/Biomarker statusPEPI score RFS pointsNo. of patientspTNot available (NA)1pT1/T20102pT3/T431pNNA7Negative075Positive322KI67 levelNA10%-2.7%039&amp;gt;2.7%-7.3%131&amp;gt;7.3%-19.7%119&amp;gt;19.7%-53.1%213&amp;gt;53.1%31ER-status Allred scoreNA120-2303-8092PEPI scoreNA190281342533495561PEPI groupNot calculated19I (0 score)28II (1-3 score)42III (≥4 score)15 Citation Format: Joanna Ines Lopez Velazco, María Otaño, Lide Larburu, Ainhara Lahuerta, Kepa Elorriaga, Virginia Segur, Juan Carlos Irizabal, Ana Martínez, Lourdes Jáuregui, Maria M. Caffarel, Ander Urruticoechea. Tumour cellularity size as a biomarker to predict response after neoadjuvant endocrine therapy: Correlation analysis between Ki67 expression and PEPI score [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS5-39.