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Ki-67 expression in BP-NEN. The boxplots are depicting the Ki-67 protein (left) and mRNA (right) levels in the different BP-NEN entities as evaluated by immunohistochemistry and by RT-qPCR. The different Ki-67-IHC evlauation methods are represented by different shades of grey: The Ki-67-Average is depicted by the first box in light grey, the Ki-67-Hotspots are illustrated in slightly darker grey and the last dark grey box stands for the IHC Ki-67 levels as determined by the fully automated digital image analysis. Beneath the respective Kaplan-Meier-Analyses are shown. Blue indicates low, green moderate and red high Ki-67 expression levels in each evaluation method, according to the cut-off limits of the ROC analysis between TC – AC and AC – SCLC. High Ki-67 expression levels correlate with a poor survival of the patients.
Source publication
The classification of bronchopulmonary neuroendocrine neoplasms (BP-NEN) into four tumor entities (typical carcinoids (TC), atypical carcinoids (AC), small cell lung cancers (SCLC), large cell neuroendocrine lung carcinomas (LCNEC)) is difficult to perform accurately, but important for prognostic statements and therapeutic management decisions. In...
Contexts in source publication
Context 1
... can be seen from the boxplots of Figure 2 and as confirmed by the Mann-Whitney-Test, Ki-67 protein levels increased highly significantly from TC to AC (Ki-67- Average: U = −4.337, p < 0.001, Ki-67-Hotspots: U = −5.041, ...
Context 3
... on the cut-off limits for TC vs. AC and AC vs. SCLC obtained by means of the ROC analysis, all three proliferation markers were subdivided into low, moderate and high expression levels and Kaplan-Meier-Analyses were performed. All three proliferation markers displayed a strong prognostic value in all methods used (see Figures 2 and 3): high Ki-67, TOP2A and RacGAP1 protein and mRNA levels correlated with poor patient survival. These data were highly significant according to the Log-Rank and Breslow Test. ...
Citations
... Cells were clustered based on specific cell markers. Using the established expression markers, such as for endothelial cells (KLF2) (31), M2 macrophages (SLC2A1) (32), TNBC cancer cells (TRPS1) (33), proliferating cells (TOP2A) (34), myeloid cells (FTH1) (35), progenitor cells (NES) (36), adipocytes (CLEC11A) (37), mesenchymal cells (S100A4) (38), angiogenic cells (RPL35A) (39), breast cancer stem cells (FXYD3) (40), dendritic cells (CD68) (41), cancerassociated fibroblast (COL1A2) (42), epithelial cells (EPCAM) (43), cycling cells (CDK4) (44), and B-cells (CD83) (45), we found that CRD altered different cell populations in the TME (Fig. 3B). This showed that CRD induced a higher number of M2-macrophage, TNBC cancer cells, cycling cells, adipocytes, cancer-associated fibroblasts (CAFs) and proliferating cells but a lower number of M1 macrophages, B-cells, and dendritic cells in the tumor (Fig. 3B). ...
Epidemiological studies have shown that circadian rhythm disruption (CRD) caused by shift work or frequent jet lag is associated with the risk of breast cancer development. However, the role of CRD in mammary gland morphology and aggressive mammary tumorigenesis and the molecular mechanisms underlying CRD and cancer risk remain unknown. We found that chronic CRD disrupted mouse mammary gland morphology and increased tumor burden and lung metastasis in a genetically engineered mouse model of aggressive breast cancer and induced an immunosuppressive tumor microenvironment by enhancing leukocyte immunoglobulin-like receptor 4a (LILRB4a or LILRB4) expression. Moreover, CRD increased the M2 macrophage (anti-inflammatory) and regulatory T-cell populations but decreased the M1 macrophage (proinflammatory) populations. These findings identify and implicate LILRB4a as a link between CRD and aggressive mammary tumorigenesis.
Teaser
Circadian rhythm disruption enhances aggressive mammary tumorigenesis by elevating LILRB4a expression.
... The differential diagnosis of SCLC includes other primary NE tumors (typical carcinoid, atypical carcinoid, and large cell NE carcinoma), basaloid squamous cell carcinoma, combined adenocarcinoma, small cell carcinoma, small round cell sarcomas both in the Ewing sarcoma family (e.g., Ewing sarcoma) and recently described morphologically similar tumors lacking EWSR1 gene rearrangement (e.g., CIC-DUX4-rearranged and BCOR-CCNB3-rearranged tumors), Merkel cell carcinoma, lymphomas, thoracic SMARCA4-deficient undifferentiated tumors (SMARCA4-UT) with small round cell morphology, NUT carcinoma, and metastasis from small cell carcinoma of HPV-related sites (cervix, head, and neck) [15][16][17]. Ki-67 immunostaining is often very helpful in differentiating NE tumor types, especially in small biopsies [18]. In the context of negative TTF-1 staining, keratin positivity, NE marker positivity, and clinical history are important to make an accurate diagnosis. ...
Background
The prevalence of thyroid transcription factor-1 (TTF-1) and napsin A expression are poorly characterized in lung core biopsies of small cell carcinoma. Locally, the TTF-1 clone is 8G7G3/1 (Agilent/Dako), and the napsin A clone is IP64 (Leica Biosystems).
Methods
All in-house lung core biopsy reports for cases accessioned at a regional laboratory from January 2011 to December 2020 were retrieved and analyzed using a validated hierarchical free-text string matching algorithm (HFTSMA) to establish the diagnosis. TTF-1 and napsin A were manually coded with the assistance of a logical text parsing tool. All TTF-1-negative small cell lung carcinoma (SCLC) cases had a full report review by pathologists.
Results
The cohort had 5,867 lung core biopsies, and 232 cases were confirmed as small cell carcinoma on pathologist review. TTF-1 immunostain results were available in 173 SCLC cases, and 16 cases of TTF-1-negative SCLC were confirmed on full report review. These 16 cases had at least one positive neuroendocrine (NE) marker and positive keratin staining; cases with mixed histology or positive CK5/6 staining were excluded. Ki-67 was done in 10/16 cases; the average Ki-67 was 75%. Napsin A was negative in 50/51 small cell carcinomas, and 0/3 TTF-1-negative SCLC had napsin A positivity.
Conclusions
Standardized immunostain reporting would simplify such analyses. Based on the cohort, approximately 9% (16/173) of SCLC is TTF-1 negative. Napsin A positivity in suspected small cell carcinoma should prompt consideration of an alternate diagnosis or explanation.
... However, this was found only at the sequencing level, and the mechanism has not been further verified and explored. In addition, RACGAP1 is overexpressed in NE small cell lung cancer (SCLC) that mirrors NEPC [36]. AURKA is a cell cycle kinase that impedes N-Myc degradation [8,26,37]. ...
Neuroendocrine prostate cancer (NEPC) is a lethal subtype of prostate cancer. It is characterized by the loss of androgen receptor (AR) signaling in neuroendocrine transdifferentiation, and finally, resistance to AR-targeted therapy. With the application of a new generation of potent AR inhibitors, the incidence of NEPC is gradually increasing. The molecular mechanism of neuroendocrine differentiation (NED) after androgen deprivation therapy (ADT) remains largely unclear. In this study, using NEPC-related genome sequencing database analyses, we screened RACGAP1, a common differentially expressed gene. We investigated RACGAP1 expression in clinical prostate cancer specimens by IHC. Regulated pathways were examined by Western blotting, qRT-PCR, luciferase reporter, chromatin immunoprecipitation, and immunoprecipitation assays. The corresponding function of RACGAP1 in prostate cancer was analyzed by CCK-8 and Transwell assays. The changes of neuroendocrine markers and AR expression in C4-2-R and C4-2B-R cells were detected in vitro. We confirmed that RACGAP1 contributed to NE transdifferentiation of prostate cancer. Patients with high tumor RACGAP1 expression had shorter relapse-free survival time. The expression of RACGAP1 was induced by E2F1. RACGAP1 promoted neuroendocrine transdifferentiation of prostate cancer by stabilizing EZH2 expression in the ubiquitin-proteasome pathway. Moreover, overexpression of RACGAP1 promoted enzalutamide resistance of castration-resistant prostate cancer (CRPC) cells. Our results showed that the upregulation of RACGAP1 by E2F1 increased EZH2 expression, which drove NEPC progression. This study explored the molecular mechanism of NED and may provide novel methods and ideas for targeted therapy of NEPC.
... TOP2A and Ki67 are 2 common markers of cell proliferation. 41,42 Of note, reduced gene expression of Mki67 detected by single-cell RNA sequencing (supplemental Figure 3A-B) was consistent with the decreased protein levels of Ki67 by flow cytometry ( Figure 2E). Production of reactive oxygen species (ROS) and reactive nitrogen species is the main mechanism used by G-MDSCs to suppress T-cell proliferation. ...
Myeloid-derived suppressor cells (MDSCs) are immature myeloid cells which originate in the bone marrow (BM) and have immunoregulatory functions. MDSCs have been implicated in the pathogenesis of several autoimmune diseases but have not been investigated in immune aplastic anemia (AA). We examined the roles of granulocytic-MDSCs (G-MDSCs) in murine models of human AA and bone marrow failure (BMF). As both prophylaxis and therapy, BM-derived G-MDSCs improved pancytopenia and BM cellularity and suppressed BM T cell infiltration in major histocompatibility complex (MHC)-matched C.B10 BMF mice. These effects were not obtained in MHC-mismatched CByB6F1 AA model, likely due to MHC disparity between G-MDSCs and donor T cells. Single cell RNA sequencing demonstrated that G-MDSCs downregulated cell cycle related genes in BM infiltrated T cells, consistent with suppression of T cell proliferation by G-MDSCs through reactive oxygen species pathway. Clearance of G-MDSCs in MHC-mismatched CByB6F1 model using anti-Ly6G antibody facilitated T cell-mediated BM destruction, suggesting an intrinsic immunosuppressive property of G-MDSCs. However, the same anti-Ly6G antibody mildly mitigated marrow failure due to expansion of an intermediate Ly6G population in the BM of MHC-matched C.B10 AA model. Our results demonstrate that G-MDSC eradication and therapeutic efficacy are immune context-dependent.
... unannotated (UA) 1/2; Figure 2A) did not have signatures indicative for a given cell state or type, a previously observed common characteristic of GBM. 28,29 Gene transcription and cell proliferation (TP1/2) signatures were marked by upregulated TOP2A and H1-2, 3 and 5 ( Figure 2B), 30 and split in two clusters based on genes relating to extracellular matrix, COL3A1 and COL1A2 (for TP2, Figure 2B). 31 Both clusters were enriched for proliferative/progenitor cells as classified according to Garofano et al. ...
Background
Glioblastoma (GBM) is the most aggressive primary brain tumor. Its cellular composition is very heterogeneous, with cells exhibiting stem-cell characteristics (GSCs) that co-determine therapy resistance and tumor recurrence. Bone Morphogenetic Protein (BMP)-4 promotes astroglial and suppresses oligodendrocyte differentiation in GSCs, processes associated with superior patient prognosis. We characterized variability in cell viability of patient-derived GBM cultures in response to BMP4 and, based on single-cell transcriptome profiling, propose predictive positive and early-response markers for sensitivity to BMP4.
Methods
Cell viability was assessed in 17 BMP4-treated patient-derived GBM cultures. In two cultures, one highly sensitive to BMP4 (high therapeutic efficacy) and one with low sensitivity, response to treatment with BMP4 was characterized. We applied single-cell RNA-sequencing, analyzed the relative abundance of cell clusters, searched for and identified the aforementioned two marker types, and validated these results in all 17 cultures.
Results
High variation in cell viability was observed after treatment with BMP4. In three cultures with highest sensitivity for BMP4, a substantial new cell subpopulation formed. These cells displayed decreased cell proliferation and increased apoptosis. Neuronal differentiation was reduced most in cultures with little sensitivity for BMP4. OLIG1/2 levels were found predictive for high sensitivity to BMP4. Activation of ribosomal translation (RPL27A, RPS27) was upregulated within one day in cultures that were very sensitive to BMP4.
Conclusion
The changes in composition of patient-derived GBM cultures obtained after treatment with BMP4 correlate with treatment efficacy. OLIG1/2 expression can predict this efficacy, and upregulation of RPL27A and RPS27 are useful early-response markers.
... et al., 2016;Wang et al., 2011Wang et al., , 2018aYang et al., 2018b;Yin et al., 2019). RacGAP1 is associated with poor patient survival and resistance to the chemotherapy drug doxorubicin in HNSCCs, early recurrence and poor prognosis in breast cancer (especially in luminal (ER+) tumors), and poor survival in bronchopulmonary neuroendocrine neoplasms (BP-NEN) (Hazar- Rethinam et al., 2015;Milde-Langosch et al., 2013;Neubauer et al., 2016;Pliarchopoulou et al., 2013;Sahin et al., 2016). Similarly, expression of RacGAP1 at the invasive front in GC correlates with tumor size, lymph node metastasis, lymphatic invasion, vascular invasion, advanced stage, and poor prognosis . ...
Cancer progression and metastasis are processes that involve significant cellular changes. Many of these changes include alterations in the activity of the Rho GTPase family of proteins. Rho GTPases are signaling proteins that function as molecular switches and are involved in the regulation of most major cellular processes. Cancer development is often associated with abnormalities in Rho GTPase signaling. Rho GTPase signaling is regulated by two families of proteins, guanine nucleotide-exchange factors (RhoGEFs) and GTPase activating proteins (RhoGAPs), that function upstream of the Rho proteins to regulate their activation and inactivation, respectively. While initial work has focused on the role of RhoGEFs in cancer, the RhoGAP family members are rapidly being established as key regulators of cancer development and progression. The aim of this review is to summarize our advances in understanding the role of RhoGAPs in cancer and to discuss their significance in the development of therapeutics.
... Ki-67 antigen and mitotic count represent the consensus proliferation markers for prognosis stratification among neuroendocrine neoplasms (NENs) [16], with Ki-67 recently shown to outperform mitotic count on a large cohort of lung NENs [14]. However, Ki-67, mitotic count and other morphological or immunohistochemical prognostic markers rely on either the simple evaluation of their prevalence on a narrow, subjectively selected tissue region-by counting the percentage of cells positive to the marker in that region-or a merely qualitative inspection, rather than a quantitative assessment [17][18][19][20][21]. These evaluations therefore lack repeatability, and carry inevitable observer bias [19,[22][23][24]. ...
... However, Ki-67, mitotic count and other morphological or immunohistochemical prognostic markers rely on either the simple evaluation of their prevalence on a narrow, subjectively selected tissue region-by counting the percentage of cells positive to the marker in that region-or a merely qualitative inspection, rather than a quantitative assessment [17][18][19][20][21]. These evaluations therefore lack repeatability, and carry inevitable observer bias [19,[22][23][24]. Ki-67 is also known for intratumoural heterogeneity of distribution due to the presence of differentially regulated sets of tumour cells [10,23,25,26], particularly in higher grade carcinoids [10]. ...
Lung neuroendocrine neoplasms (lung NENs) are categorised by morphology, defining a classification sometimes unable to reflect ultimate clinical outcome. Subjectivity and poor reproducibility characterise diagnosis and prognosis assessment of all NENs. Here, we propose a machine learning framework for tumour prognosis assessment based on a quantitative, automated and repeatable evaluation of the spatial distribution of cells immunohistochemically positive for the proliferation marker Ki-67, performed on the entire extent of high-resolution whole slide images. Combining features from the fields of graph theory, fractality analysis, stochastic geometry and information theory, we describe the topology of replicating cells and predict prognosis in a histology-independent way. We demonstrate how our approach outperforms the well-recognised prognostic role of Ki-67 Labelling Index on a multi-centre dataset comprising the most controversial lung NENs. Moreover, we show that our system identifies arrangement patterns in the cells positive for Ki-67 that appear independently of tumour subtyping. Strikingly, the subset of these features whose presence is also independent of the value of the Labelling Index and the density of Ki-67-positive cells prove to be especially relevant in discerning prognostic classes. These findings disclose a possible path for the future of grading and classification of NENs.
... Another important proliferation marker KI67 encodes a nuclear antigen during the G1, S, and G2-M phases of proliferating cells, meaning that it is present during all active phases of the cell cycle, except the G0 phase [60,61]. The expression of TOP2α is cell cycle-dependent and encodes DNA topoisomerase, which is an enzyme that controls and alters the topologic states of DNA during transcription [60,62]. The results showed that all three studied genes, TOP2A, PCNA, and KI67, were time-dependently downregulated compared to 2 days old monolayer culture ( Figure 3A). ...
In genetic toxicology, there is a trend against the increased use of in vivo models as highlighted by the 3R strategy, thus encouraging the development and implementation of alternative models. Two-dimensional (2D) hepatic cell models, which are generally used for studying the adverse effects of chemicals and consumer products, are prone to giving misleading results. On the other hand, newly developed hepatic three-dimensional (3D) cell models provide an attractive alternative, which, due to improved cell interactions and a higher level of liver-specific functions, including metabolic enzymes, reflect in vivo conditions more accurately. We developed an in vitro 3D cell model from the human hepatocellular carcinoma (HepG2) cell line. The spheroids were cultured under static conditions and characterised by monitoring their growth, morphology, and cell viability during the time of cultivation. A time-dependent suppression of cell division was observed. Cell cycle analysis showed time-dependent accumulation of cells in the G0/G1 phase. Moreover, time-dependent downregulation of proliferation markers was shown at the mRNA level. Genes encoding hepatic markers, metabolic phase I/II enzymes, were time-dependently deregulated compared to monolayers. New knowledge on the characteristics of the 3D cell model is of great importance for its further development and application in the safety assessment of chemicals, food products, and complex mixtures.
... [28][29][30][31] Current efforts increasingly focus on molecular biomarkers, which may make the histologic classification of tumours more accurate and facilitate us in identifying more aggressive cancers, promoting staging revisions, and creating personalized therapy and better clinical outcomes. [32][33][34][35][36] The goal of this study was to identify the prognostic value of certain clotting markers for patients with colorectal cancer. ...
Background and Objective
Tools for the non-invasive assessment of colorectal cancer (CRC) prognosis have profound significance. Although plasma coagulation tests have been investigated in a variety of tumours, the prognostic value of the prothrombin time (PT) and activated partial thromboplastin time (APTT) in CRC has not been discussed. Our study objective was to explore the prognostic significance of preoperative PT and APTT in CRC patients.
Patients and Methods
A retrospective analysis of preoperative coagulation indexes including PT, PTA, INR, APTT, FIB, TT, PLT, NLR and PLR in 250 patients with CRC was performed. Kaplan–Meier and multivariate Cox regression analysis were used to demonstrate the prognostic value of these preoperative coagulation indexes.
Results
The overall survival (OS, p<0.05) and disease-free survival (DFS, p<0.05) of CRC patients with lower PT and APTT levels were significantly prolonged. Based on univariate analysis, PT levels (p<0.001, p<0.001), PTA levels (p=0.001, p=0.001), APTT levels (p=0.001, p<0.001), INR levels (p<0.001, p<0.001), fibrinogen levels (p=0.032, p=0.036), tumour status (p=0.005, p=0.003), nodal status (p<0.001, p<0.001), metastasis status (p<0.001, p<0.001) and TNM stages (p<0.001, p<0.001) were remarkably associated with DFS and OS. Multivariate Cox regression analysis suggested that the levels of PT (HR: 2.699, p=0.006) and APTT (HR: 1.942, p=0.015), metastasis status (HR: 2.091, p= 0.015) and TNM stage (HR: 7.086, p=0.006) were independent predictors of survival in CRC. In the whole cohort, the enrolled patients were then divided into three groups according to their PT and APTT levels. The OS and DFS differed notably among the low-risk (PT<11.85 sec and APTT<25.85 sec), medium-risk (PT≥11.85 sec or APTT≥25.85 sec), and high-risk (PT≥11.85 sec and APTT≥25.85 sec) groups.
Conclusion
Elevated levels of preoperative PT and APTT were predictors of poor outcomes in CRC patients. Moreover, the combination of preoperative PT and APTT can be a new prognostic stratification approach for more precise clinical staging of CRC.
... 8 Although Ki-67 is not currently used in the WHO grading scheme for pulmonary carcinoids, many studies have shown that ACTs have increased Ki-67, and that increased Ki-67 is generally associated with poorer prognosis. 2,[9][10][11][12][13][14][15][16][17][18][19][20][21][22][23][24] However, the use of Ki-67 labeling index in the evaluation of pulmonary carcinoid tumors is controversial and not standardized, because there are conflicting data regarding the added value to the current WHO system. A Ki-67 cutoff value to divide TCT from ACT also has not been clearly defined, which is likely in part due to the highly variable methods used to determine Ki-67 index, and the wide range of cutoffs that have been proposed. ...
Context.—
Pulmonary carcinoids are classified as typical or atypical by assessing necrosis and mitoses, which usually cannot be adequately assessed on small biopsies. Ki-67 is not currently used to grade pulmonary carcinoids, but it may be helpful to determine preliminary grade in biopsies. However, the rate at which Ki-67 could underestimate or overestimate grade on small biopsies has not been well studied.
Objective.—
To compare Ki-67 labeling obtained on small biopsies to subsequent resection.
Design.—
Ki-67 was performed on paired biopsy and resection specimens from 55 patients. Slides were scanned using Aperio ScanScope. Labeling index was determined using automated hot spot and tumor tracing methods.
Results.—
The study included 41 typical and 14 atypical carcinoids. Atypical carcinoids were larger and had more distant metastases. Death from disease occurred in 3 patients (all had atypical carcinoids). Median hot spot Ki-67 labeling index was greater in resection compared with biopsy by 0.7% ( P = .02). Median tumor tracing Ki-67 was lower in resection compared with biopsy by 0.5% ( P < .001). Receiver-operating characteristic analysis showed similar hot spot Ki-67 cutoffs to predict atypical histology (3.5% for biopsy, 3.6% for resection; area under the curve [AUC], 0.75 and 0.74, respectively). Different optimal cutoffs were needed for tracing method based on biopsy (2.1%; AUC, 0.75) compared with resection (1.0%; AUC, 0.67).
Conclusions.—
Hot spot Ki-67 tends to underestimate grade on small biopsies, whereas grade is overestimated by tumor tracing. Hot spot Ki-67 cutoff of 3.5% predicted atypical histology for both biopsy and resection. Different biopsy and resection cutoffs were necessary for tumor tracing, which would make clinical implementation more difficult.
