Cell Proliferation Biomarkers Research Articles

Ubiquitin ligase TRIM22 inhibits ovarian cancer malignancy via TCF4 degradation.

Ovarian cancer (OC) is one of the most common malignancies in women. Tripartite motif-containing protein 22 (TRIM22) plays an important role in the initiation and progression of malignant tumors. Similarly, the transcription factor 4 (TCF4) is an essential factor involved in the initiation and progression of many tumors. However, it is still unclear whether TRIM22 can affect TCF4 in OC. Therefore, this study aims to investigate the mechanism related to TRIM22 and TCF4 in OC. TRIM22 protein and mRNA levels were analyzed in samples from both clinical and cell lines. The effects of TRIM22 knockdown and overexpression on cell proliferation, colony formation, migration, invasion, and related biomarkers were evaluated. In addition, the role of ubiquitination-mediated degradation of TCF4 was investigated by qRT-PCR and Western blotting. The association between TRIM22 and TCF4 was evaluated by Western blotting, co-immunoprecipitation, proliferation, colony formation, invasion, migration, and related biomarkers. The results showed that the expression of TRIM22 was minimal in OC tissues. Furthermore, upregulation of TRIM22 significantly inhibited OC cell proliferation, colony formation, migration, and invasion. In addition, TRIM22 was observed to regulate the degradation of TCF4 through the ubiquitination pathway. TCF4 can reverse the effects of TRIM22 on proliferation, colony formation, migration, and invasion in OC cells. TRIM22-mediated ubiquitination of TCF4 at K48 is facilitated by the RING domain. Implications: In conclusion, ubiquitination of TCF4 protein in OC is regulated by TRIM22, which has the potential to limit the proliferation, migration, and invasion of OC.

Abstract PO3-13-02: Genomic and intrinsic subtype correlates of serum thymidine kinase activity in patients with metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial

Abstract Background: Serum thymidine kinase activity (sTKa) is a novel non-invasive proliferation biomarker providing prognostic, predictive and monitoring information in patients (pts) with metastatic breast cancer (MBC). We have shown in PYTHIA that high baseline sTKa and lack of sTKa clearance at day 15 of the first cycle of treatment with palbociclib (P)+ fulvestrant (F) are poor prognostic factors in terms of PFS. Here we characterized the molecular features of tumor samples from PYTHIA, and their relation with sTKa. Methods: PYTHIA (IBCSG 53- 14/BIG 14-04; NCT02536742) is a phase II biomarker discovery trial downstream of the AURORA molecular program (BIG 14-01; NCT02102165) which enrolled 122 pts with endocrine-resistant, ER+ and HER2 - MBC receiving P+F. Intrinsic subtypes of metastatic samples from 24 pts were estimated using RNA-Seq. Somatic mutations (mut) from 66 pts were identified using an NGSpanel of 411 cancer-related genes (36 primary and 48 metastatic samples). sTKa was measured using DiviTumÒ TKa (Biovica International) before treatment (D0) and at day 15 (D15) of cycle 1 of P+F. sTKa clearance at D15 was defined as sTKa below the limit of detection (LOD). Intrinsic subtypes and ESR1 mut were assessed in metastatic samples, PIK3CA and TP53 mut were derived from a combined cohort, wheremetastatic samples were used when available and primary samples otherwise. Association of mut and continuous sTKa with PFS was assessed using Cox regression. Results: In metastatic samples, sTKa levels at D0 tended to be higher in luminal B and HER2-enriched subtypes. Lack of sTKa clearance at D15 was observed in 14% of the cases, all classified as luminal B. PIK3CA and TP53 were the most frequently altered genes (38% and 27%, respectively). ESR1 mut were found in 8% of primary and 25% of metastatic samples. Only TP53 was associated with worse PFS. sTKa levels at D0 tended to be higher in pts with TP53 mut vs wild-type (wt) and lower in PIK3CA mut vs wt. Interestingly, lack of sTKa clearance at D15 was more frequent in TP53 mut vs wt and less frequent in PIK3CA mut vs wt. Levels of sTKa were similar in pts with ESR1 mut vs wt both at D0 and at D15 (Table 1). The inclusion of TP53 as a predictor of PFS in bivariate Cox models with sTKa at either D0 (likelihood ratio test p=0.44) or D15 (p=0.21) did not add to the prognostic value provided by sTKa alone. Conclusions: Tumors with high proliferation and poor prognostic features (luminal B, HER2-enriched and TP53 mut) tend to have higher baseline sTKa and less sTKa response on-treatment, in line with sTKa being a circulating dynamic biomarker of tumor cell proliferation. After accounting for sTKa values, TP53mutational status did not contribute to prognosis. This suggests that high sTKa at D0 and lack of sTKa clearance at D15 might be associated with a worse prognosis independently of TP53 status. Further validation in larger datasets is warranted. Table 1: Results summary Citation Format: Svitlana Tyekucheva, Thayane Crestani, Michail Ignatiadis, Patrick Neven, Marco Colleoni, Stéphanie Henry, Konstantinos Papadimitriou, Antonio Bernardo, Elena Seles, Francois Duhoux, Iain Macpherson, Alastair Thomson, David Mark Davies, Mattias Bergqvist, Ilenia Migliaccio, Gabriele Zoppoli, Roswitha Kammler, Heidi De Swert, Barbara Ruepp, Angel Guerrero, Arnau Llinas, Danai Fimereli, Amal Arahmani, David Cameron, Sherene Loi, Martine Piccart, Meredith Regan, Matteo Benelli, Luca Malorni. Genomic and intrinsic subtype correlates of serum thymidine kinase activity in patients with metastatic breast cancer treated with palbociclib and fulvestrant in the PYTHIA trial [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 PO3-13-02.

Pigment epithelium‑derived factor inhibits proliferation, invasion and angiogenesis, and induces ferroptosis of extravillous trophoblasts by targeting Wnt‑β‑catenin/VEGF signaling in placenta accreta spectrum.

Placenta accreta spectrum (PAS) is one of the most dangerous complications in obstetrics, which can lead to severe postpartum bleeding and shock, and even necessitate uterine removal. The abnormal migration and invasion of extravillous trophoblast cells (EVTs) and enhanced neovascularization occurring in an uncontrolled manner in time and space are closely related to the abnormal expression of pro‑angiogenic and anti‑angiogenic factors. The pigment epithelium‑derived factor (PEDF) is a multifunctional regulatory factor that participates in several important biological processes and is recognized as the most efficient inhibitor of angiogenesis. The present study aimed to explore the effects of PEDF on EVT phenotypes and the underlying mechanisms in PAS. HTR‑8/SVneo cells were transfected to overexpress or knock down PEDF. Cell proliferation and invasion were assessed using Cell Counting Kit‑8, 5‑ethynyl‑2'‑deoxyuridine and Transwell assays. In vitro angiogenesis was analyzed using tube formation assays. The degree of ferroptosis was assessed by evaluating the levels of lipid reactive oxygen species, total iron, Fe2+, malondialdehyde and reduced glutathione using commercial kits. The expression levels of biomarkers of ferroptosis, angiogenesis, cell proliferation and Wnt signaling were examined by western blotting. PEDF overexpression decreased the proliferation, invasion and angiogenesis, and induced ferroptosis of EVTs. Activation of Wnt signaling with BML‑284 and overexpression of vascular endothelial growth factor (VEGF) reversed the PEDF overexpression‑induced suppression of cell proliferation, invasion and tube formation. PEDF overexpression‑induced ferroptosis was also decreased by Wnt agonist treatment and VEGF overexpression. It was predicted that PEDF suppressed the proliferation, invasion and angiogenesis, and increased ferroptosis in EVTs by decreasing Wnt‑β‑catenin/VEGF signaling. The findings of the present study suggested a novel regulatory mechanism of the phenotypes of EVTs and PAS.

Dendritic cell subpopulations in carcinoma ex-pleomorphic adenoma: A multicenter study.

Carcinoma ex-pleomorphic adenoma (CEXPA) represents a malignant transformation from a recurrent or primary pleomorphic adenoma (PA), and the immune response may be essential in this process. Therefore, in this study, we aimed to identify and quantify subpopulations of dendritic cells (DCs) in CEXPA, residual PA in CEXPA (rPA), and PA. A multicenter study was performed collecting salivary gland tumor (SGT) samples from three Oral and Maxillofacial Pathology Centers. A tissue microarray containing 41 samples of CEXPA and 22 samples of PA was included in this study and submitted to immunohistochemical reactions against CD1a, CD83, CD207, and Ki67 antibodies. Both PA and rPA showed a higher quantification of CD207+ and CD83+ cells when compared to CEXPA (p < 0.001 and p < 0.01, respectively). There was also a difference when comparing the cell proliferation index between PA/rPA and CEXPA using the Ki-67 marker (p = 0.043). However, there was no difference in the DC population regarding clinical parameters such as sex, anatomical location, size, and metastases (p > 0.06). Immunohistochemical profile of DC subpopulations and cell proliferation biomarkers in SGTs can contribute as an important tool in the differentiation of benign and malignant tumors or detection of initial areas with malignant transformation.

OralImmunoAnalyser: a software tool for immunohistochemical assessment of oral leukoplakia using image segmentation and classification models.

Oral cancer ranks sixteenth amongst types of cancer by number of deaths. Many oral cancers are developed from potentially malignant disorders such as oral leukoplakia, whose most frequent predictor is the presence of epithelial dysplasia. Immunohistochemical staining using cell proliferation biomarkers such as ki67 is a complementary technique to improve the diagnosis and prognosis of oral leukoplakia. The cell counting of these images was traditionally done manually, which is time-consuming and not very reproducible due to intra- and inter-observer variability. The software presently available is not suitable for this task. This article presents the OralImmunoAnalyser software (registered by the University of Santiago de Compostela-USC), which combines automatic image processing with a friendly graphical user interface that allows investigators to oversee and easily correct the automatically recognized cells before quantification. OralImmunoAnalyser is able to count the number of cells in three staining levels and each epithelial layer. Operating in the daily work of the Odontology Faculty, it registered a sensitivity of 64.4% and specificity of 93% for automatic cell detection, with an accuracy of 79.8% for cell classification. Although expert supervision is needed before quantification, OIA reduces the expert analysis time by 56.5% compared to manual counting, avoiding mistakes because the user can check the cells counted. Hence, the SUS questionnaire reported a mean score of 80.9, which means that the system was perceived from good to excellent. OralImmunoAnalyser is accurate, trustworthy, and easy to use in daily practice in biomedical labs. The software, for Windows and Linux, with the images used in this study, can be downloaded from https://citius.usc.es/transferencia/software/oralimmunoanalyser for research purposes upon acceptance.

Combination of in silico and cell culture strategies to predict biomaterial performance: Effects of sintering temperature on the biological properties of hydroxyapatite.

Advances in methodologies to evaluate biomaterials brought an explosive growth of data, ensuing computational challenges to better analyzing them and allowing for high-throughput profiling of biological systems cost-efficiently. In this sense, we have applied bioinformatics tools to better understand the biological effect of different sintering temperatures of hydroxyapatite (abbreviated HA; at 1100, 1150, and 1250°C) on osteoblast performance. To do, we have better analyzed an earlier deposited study, in which the access code is E-MTAB-7219, which the authors have explored different in silico tools on this purpose. In this study, differential gene expression analyses were performed using the gene set variation analysis (GSVA) algorithm from the transcriptomes respecting the thermal changes of HA, which were validated using exclusively in vitro strategies. Furthermore, in silico approaches elected biomarkers during cell behavior in response to different sintering temperatures of HA, and it was further validated using cell culture and qPCR technologies. Altogether, the combination of those strategies shows the capacity of sintered HA at 1250°C to present a better performance in organizing an adequate microenvironment favoring bone regeneration, angiogenesis and material resorption stimulus once it has promoted higher involvement of genes such as CDK2, CDK4 (biomarkers of cell proliferation), p15, Osterix gene (related with osteogenic differentiation), RANKL (related with osteoclastogenesis), VEGF gene (related with angiogenesis), and HIF1α (related with hypoxia microenvironment). Altogether, the combination of in silico and cell culture strategies shows the capacity of sintered HA at 1250°C in guaranteeing osteoblast differentiation and it can be related in organizing an adequate microenvironment favoring bone regeneration, angiogenesis, and material resorption stimulus.

Abstract C067: Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC

Abstract Background: Pancreatic ductal adenocarcinoma (PDAC) shows variable degrees of differentiation, with both intratumoral, regional, and case to case heterogeneity, including glandular elements, sheet-like aggregates, cell clusters, and single diffusely infiltrative tumor cells. Prior research from our lab and others have established keratin 17 (K17) to be a defining hallmark of the basal molecular subtype of PDAC. In addition to its role as a component of the cytoskeleton, soluble forms of K17 undergo nuclear translocation to drive cell cycle progression and chemoresistance to first line chemotherapy. While K17 is heterogeneously expressed in the gland-forming elements of PDACs, it is most consistently expressed in tumor cells which are either in small groups or are single diffusely infiltrative. The aims of the current study are to define the correlation between K17 expression and tumor cell differentiation, with the focus on glandular versus single diffusely infiltrative components of PDAC, and to relate these findings to established biomarkers of cell proliferation and invasion. Methods: Archival human PDACs were provided by the Stony Brook Cancer Center Biobank and K17 immunohistochemistry was performed by indirect immunoperoxidase method, as previously described, to select cases with both glandular and diffusely infiltrative cell components and heterogeneous K17 expression. Multiplex immunofluorescence with a panel of antibodies to K17 (Cell Signaling Technology), pan-cytokeratin, Ki-67 and vimentin [KS1] (Miltenyi Biotec) was performed on 5-micron FFPE sections using the MACSima system (Miltenyi Biotec). Distribution patterns of K17 were evaluated in glandular and diffusely invasive components and were related to the Ki-67 status. Results: Cytoplasmic staining for K17 was found in the minority of tumor cells in glands and displayed intraglandular heterogeneity but was found in most diffusely infiltrative tumor cells. Also, nuclear K17 was rarely detected in the glandular components but was found in a high proportion of the diffusely infiltrative cells. Ki-67 was detected mainly in glandular cells, unrelated to K17 status, and rarely in single diffusely infiltrative cells, never co-localizing with nuclear K17. Of note, only a small fraction of tumor cells co-expressed K17 and vimentin, suggesting that majority of diffusely infiltrative tumor cells were not engaged in epithelial to mesenchymal transition. Conclusion: While cytoplasmic K17 is heterogeneously expressed in the glandular components of PDAC, strong staining for K17 in both the cytoplasm and the nucleus is a hallmark of diffusely infiltrative, Ki-67-negative tumor cells. These observations support the paradigm that tumor cells are committed to either cell division in the glandular components, or to diffuse single cell invasion, highlighted by increased cytoplasmic and nuclear K17 expression. Therefore, the results emphasize the importance of understanding the expression of PDAC biomarkers in both gland-forming elements and diffusely infiltrative tumor cells. Citation Format: Shayan Sarkar, Lyanne Oblein, Michael Horowitz, Luisa Escobar-Hoyos, Natalia Marchenko, Kenneth Shroyer. Correlation of keratin17 expression with tumor cell proliferation and invasion in PDAC [abstract]. In: Proceedings of the AACR Special Conference in Cancer Research: Pancreatic Cancer; 2023 Sep 27-30; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(2 Suppl):Abstract nr C067.

Emerging Role of [18F]FLT PET/CT in Lymphoid Malignancies: A Review of Clinical Results.

Fluorine-18 fluorodeoxyglucose ([18F]FDG) is nowadays the leading positron emission tomography (PET) tracer for routine clinical work-ups in hematological malignancies; however, it is limited by false positive findings. Notably, false positives can occur in inflammatory and infective cases or in necrotic tumors that are infiltrated by macrophages and other inflammatory cells. In this context, 3'-deoxy-3'-[18F]fluorothymidine ([18F]FLT) has been shown to be a promising imaging biomarker of hematological malignant cell proliferation. In this review, a total of 15 papers were reviewed to collect literature data regarding the clinical application of [18F]FLT PET/CT in hematological malignancies. This imaging modality seems to be a suitable tool for noninvasive assessment of tumor grading, also showing a correlation with Ki-67 immunostaining. Moreover, [18F]FLT PET/CT demonstrated high sensitivity in detecting aggressive lymphoma lesions, especially when applying a standardized uptake value (SUV) cutoff of 3. At baseline, the potential of [18F]FLT imaging as a predictive tool is demonstrated by the low tracer uptake in patients with a complete response. However, its use is limited in evaluating bone diseases due to its high physiological uptake in bone marrow. Interim [18F]FLT PET/CT (iFLT) has the potential to identify high-risk patients with greater precision than [18F]FDG PET/CT, optimizing risk-adapted therapy strategies. Moreover, [18F]FLT uptake showed a greater ability to differentiate tumor from inflammation compared to [18F]FDG, allowing the reduction of false-positive findings and making the first one a more selective tracer. Finally, FLT emerges as a superior independent predictor of PFS and OS compared to FDG and ensures a reliable early response assessment with greater accuracy and predictive value.

Leptin secreted by adipocytes promotes EMT transition and endometrial cancer progression via the JAK2/STAT3 signalling pathway

ABSTRACT Background Endometrial cancer is a malignant tumour with a high incidence and mortality rate, and obesity is one of the most significant risk factors for the disease. However, it remains unclear whether leptin affects cell activity, proliferation, migration, invasion, and epithelial-mesenchymal transition (EMT). Materials and methods Samples of endometrial cancer tissue were obtained from clinical patients and nude mice Enzyme-linked immunosorbent assays (ELISAs) were performed to assess leptin levels. Western blotting, immunohistochemical (IHC) and immunofluorescence (IF) analyses were conducted to detect EMT, JAK2/STAT3 signalling pathway proteins, and cell proliferation biomarkers. Cell Counting Kit-8 (CCK-8) assays, 5-ethynyl-2’-deoxyuridine (EdU) staining, and Transwell assays were used to evaluate cell activity, proliferation, migration, and invasion, respectively. Results ELISA, western blot and immunohistochemistry (IHC) analyses showed that leptin was highly expressed, and the JAK2/STAT3 signalling pathway was activated in endometrial cancer patients. Cell-based experiments showed that adipocytes secreted leptin, which increased the levels of leptin, and also promoted cell migration and invasion, EMT transition, and cell activity and proliferation. Leptin accelerated cell progression and promoted EMT via the JAK2/STAT3 signalling pathway in a dose-dependent manner. The tumour-promoting effect of leptin on endometrial cancer cells was further verified by in vivo experiments, in which leptin promoted tumour growth and activated the JAK2/STAT3 signalling pathway. Conclusion Leptin secreted by adipocytes promotes EMT transition and endometrial cancer progression via the JAK2/STAT3 signalling pathway in a dose-dependent manner. Highlights Endometrial cancer patients have high levels of leptin Leptin promotes EMT transition via the JAK2/STAT3 signalling pathway Leptin promotes endometrial cancer progression via the JAK2/STAT3 signalling pathway Leptin promotes endometrial cancer in a dose-dependent manner

A monoclonal antibody-based sandwich ELISA for measuring canine Thymidine kinase 1 protein and its role as biomarker in canine lymphoma.

Dogs play an important role in society, which increased during the covid epidemics. This has led to a much higher workload for the veterinarians. Therefore, there is a need for efficient diagnostic tools to identify risk of malignant diseases. Here the development of a new test that can solve some of these problems is presented. It is based on serum Thymidine Kinase 1 (TK1), which is a biomarker for cell proliferation and cell lysis. Anti-TK1 monoclonal antibodies were produced against two different epitopes, the active site of the TK1 protein and the C-terminal region of canine TK1. The antibodies were developed with hybridoma technology and validated using dot blot, Quartz Crystal Microbalance (QCM) technology, western blots, immunoprecipitation (IP), and enzyme-linked immunosorbent assay (ELISA). Clinical evaluation of Canine TK1 ELISA was done by using sera from 131 healthy dogs and 93 dogs with lymphoma. The two selected Anti-TK1 monoclonal antibodies have Kd values in the range of 10-9 M and further analysis with dot and western blots confirmed the high affinity binding of these antibodies. A sandwich Canine TK1 ELISA was developed using the anti-TK1 antibodies, and TK1 concentrations in serum samples were determined using dog recombinant TK1 as a standard. Serum TK1 protein levels were significantly higher in dogs with lymphoma compared to those in healthy dogs (p < 0.0001). Receiver operating curve analysis showed that the canine TK1-ELISA obtain a sensitivity of 0.80, at a specificity of 0.95. Moreover, the Canine TK1 ELISA has a positive predictive value (PPV) of 97%, and the negative predictive value (NPV) of 83%, reflecting the proportion of test results that are truly positive and negative. Furthermore, Canine TK1 ELISA had significantly higher capacity to differentiate dogs with T-cell lymphoma from those with B-cell lymphoma compared to earlier used TK1 activity assays. These results demonstrate that the Canine TK1 ELISA can serve as an efficient tool in the diagnosis and management of dogs with lymphomas.

Prognostic Biomarkers of Cell Proliferation in Colorectal Cancer (CRC): From Immunohistochemistry to Molecular Biology Techniques.

Colorectal cancer (CRC) is one of the most common and severe malignancies worldwide. Recent advances in diagnostic methods allow for more accurate identification and detection of several molecular biomarkers associated with this cancer. Nonetheless, non-invasive and effective prognostic and predictive testing in CRC patients remains challenging. Classical prognostic genetic markers comprise mutations in several genes (e.g., APC, KRAS/BRAF, TGF-β, and TP53). Furthermore, CIN and MSI serve as chromosomal markers, while epigenetic markers include CIMP and many other candidates such as SERP, p14, p16, LINE-1, and RASSF1A. The number of proliferation-related long non-coding RNAs (e.g., SNHG1, SNHG6, MALAT-1, CRNDE) and microRNAs (e.g., miR-20a, miR-21, miR-143, miR-145, miR-181a/b) that could serve as potential CRC markers has also steadily increased in recent years. Among the immunohistochemical (IHC) proliferative markers, the prognostic value regarding the patients' overall survival (OS) or disease-free survival (DFS) has been confirmed for thymidylate synthase (TS), cyclin B1, cyclin D1, proliferating cell nuclear antigen (PCNA), and Ki-67. In most cases, the overexpression of these markers in tissues was related to worse OS and DFS. However, slowly proliferating cells should also be considered in CRC therapy (especially radiotherapy) as they could represent a reservoir from which cells are recruited to replenish the rapidly proliferating population in response to cell-damaging factors. Considering the above, the aim of this article is to review the most common proliferative markers assessed using various methods including IHC and selected molecular biology techniques (e.g., qRT-PCR, in situ hybridization, RNA/DNA sequencing, next-generation sequencing) as prognostic and predictive markers in CRC.

Isorhapontigenin (ISO) inhibits malignant cell transformation, migration, and invasion through MEG3/NEDD9 signaling in Cr(VI)-transformed cells

Cr(VI) compounds are confirmed human carcinogens. Maternally expression 3 (MEG3) is the first long non-coding RNA to be identified as a tumor suppressor. MEG3 is frequently downregulated or lost in various primary human tumor tissues and cancer cell lines. Downregulation of MEG3 is associated with cancer initiation, progression, and metastasis. Our previous study has revealed that MEG3 was lost and NEDD9 was upregulated in Cr(VI)-transformed cells compared to those in passage-matched normal BEAS-2B cells. Overexpression of MEG3 reduced NEDD9. β-Catenin was activated in Cr(VI)-transformed cells, overexpression of MEG3 or knockdown of NEDD9 inhibited the activation of β-Catenin. The results from the present study showed that isorhapontigenin (ISO) treatment is able to suppress cell proliferation, migration, and invasion of Cr(VI)-transformed cells. Further study showed that ISO treatment in Cr(VI)-transformed cells decreases the levels of Ki67, a biomarker for cell proliferation, and of cyclin D1, a regulator for the cell cycle. ISO elevated the MEG3 expression level in Cr(VI)-transformed cells. The DNA methylation transferases DNMT3a, DNMT3b, and DNMT1 levels were reduced upon ISO treatment. ISO treatment decreased both mRNA and protein levels of NEDD9. In addition, ISO treatment reduced the activation of β-catenin. Slug was upregulated and E-Cadherin was downregulated in Cr(VI)-transformed cells, treatment with ISO decreased Slug and increased E-Cadherin. This study demonstrated that ISO is a potent therapeutical agent against lung cancer induced by Cr(VI).

Study of Ki67 in odontogenic benign tumors

The aim of the present study was to investigate the Ki67 expression level and to measure the cell proliferation index (Ki67) in odontogenic benign tumors.This was an analytical cross-sectional study of odontogenic benign tumors. The sampling was non-probabilistic with an exhaustive recruitment including all biopsy or surgical excision specimen of odontogenic benign tumors. Ki67 immunohistochemistry was performed on histological sections of paraffin-fixed tissues of 3µ thickness with the Ki67 (MM1) monoclonal antibody. The studied variables were sociodemographic, clinical and histopathological. The data have been analysed with SPSS 20.0 software.: Ameloblastoma represented 50.9% of cases and cemento-osseous dysplasia 36.4%. Among the 28 ameloblastoma cases, the 15 (53.6%) were plexiform type, the 7 (25%) follicular type and the 6 (21.4%) unicystic type. The percentage of Ki67 labelled cells was less than 1% in 74.6% (n=41) of the tumors and was equal to 5% in 12.7% (n=7) of cases. A Ki67 law labelling was found in 87.3% (n=48) of tumours and a negative labelling in 12.7% (n=7) of cases. The odontogenic benign tumors had a weak Ki67 expression level and a low epithelial cell proliferation index. The Ki67 could not therefore be used as a predictive biomarker of tumor cell proliferation and the risk of recurrence in odontogenic benign tumors.

Biological activities of extracts and compounds from Thai Kae-Lae (Maclura cochinchinensis (Lour.) Corner)

Background and aimsThe purpose of this study was to investigate the biological properties of Kae-Lae (Maclura cochinchinensis (Lour.) Corner), a traditional medicinal plant used in Ayurvedic recipes in Thailand. To achieve this objective, heartwood samples were collected from 12 sources across Thailand. Fractional extracts (n-hexane, ethyl acetate, and ethanol) and the dominant compounds (morin, resveratrol, and quercetin) were examined for their abilities on cytotoxicity, antioxidant, anti-inflammation, and antileukaemic activity (Wilms’ tumour 1 protein was used as a well-known biomarker for leukaemic cell proliferation).MethodsThe study used MTT to assess cytotoxicity in leukaemic cells (K562, EoL-1, and KG-1a). Antioxidant activities were evaluated using ABTS, DPPH, and FRAP assays. The anti-inflammatory activity was investigated by detecting IL-2, TNF-α, and NO using appropriate detection kits. Wilms’ tumour 1 protein expression was measured by Western blotting to determine the anti-leukaemic activity. The inhibition of cell migration was also analyzed to confirm anticancer progression.ResultsAmong the tested extract fraction, ethyl acetate No. 001 displayed strong cytotoxicity specifically in EoL-1 cells, while n-hexane No. 008 demonstrated this effect in three cell lines. Resveratrol, on the other hand, displayed cytotoxicity in all the tested cells. Additionally, the three major compounds, morin, resveratrol, and quercetin, exhibited significant antioxidant and anti-inflammatory properties. In particular, resveratrol demonstrated a noteworthy decreased Wilms’ tumour 1 protein expression and a reduction in cell proliferation across all cells. Moreover, ethyl acetate No. 001, morin, and resveratrol effectively inhibited MCF-7 cell migration. None of these compounds showed any impact on red blood cell haemolysis.ConclusionBased on these findings, it can be concluded that Kae-Lae has promising chemotherapeutic potential against leukaemic cells, with fractional extracts (ethyl acetate and n-hexane) and resveratrol exhibiting the most potent cytotoxic, antioxidant, anti-inflammatory, and anti-cell migration activities.

Could Apparent Diffusion coefficient (ADC) be used as an imaging marker for proliferative activity in breast carcinoma?

&amp;lt;p&amp;gt;Introduction. Multiparametric magnetic resonance mammography (mMRM) has an important role in detection, evaluation and follow-up of breast lesions. The aim of this study was to explore whether imaging parameters, in particular ADC, can be used as a biomarker of cell proliferation in breast cancer. Methods. This cohort-study included 67 lesions in 50 female patients who underwent mMRM on a 3T scanner. Percutaneous biopsies and surgical excisions were performed after imaging in period up to 3 weeks. The Ki67 index was assessed microscopically. Seven Ki67 categories were defined: 0&amp;amp;ndash;5%, 10&amp;amp;ndash;20%, 20&amp;amp;ndash;40%, 40&amp;amp;ndash;50%, 50&amp;amp;ndash;80% and over 80%. Methods of descriptive statistics were used; correlations were determined using Pearson&amp;amp;rsquo;s correlation test. ROC curve was constructed and analyzed for determination of &amp;amp;ldquo;cut-off&amp;amp;rdquo; values for diagnostic potential. Statistical significance was set at p &amp;amp;lt; 0.05. Results. Different subtypes of breast carcinomas were involved: ductal carcinoma (59.9%), lobular carcinoma (17.9%), metastatic carcinoma (10.5%), ductal carcinoma in situ (9%), and tubular carcinoma (3%). It was not possible to determine whether there was significant difference in values of ADC for histological subtypes of breast carcinoma because of small number of samples in some groups. The cut off value of ADC for breast carcinoma was 0.792 (sensitivity 98.6%, specificity 65.7%). There was no significant difference in values of ADC for categories of Ki67. There was no significant correlation between ADC mean and Ki67 for all histological subtypes of breast carcinomas (r = 0.156, p = 0.243). Conclusion. ADC cannot be used as a reliable imaging marker for proliferative activity in breast cancer.&amp;lt;/p&amp;gt;

Secretory and Membrane-Associated Biomarkers of Mast Cell Activation and Proliferation.

Mast cells (MCs) are immune cells distributed in many organs and tissues and involved in the pathogenesis of allergic and inflammatory diseases as a major source of pro-inflammatory and vasoactive mediators. MC-related disorders are heterogeneous conditions characterized by the proliferation of MC within tissues and/or MC hyper-reactivity that leads to the uncontrolled release of mediators. MC disorders include mastocytosis, a clonal disease characterized by tissue MC proliferation, and MC activation syndromes that can be primary (clonal), secondary (related to allergic disorders), or idiopathic. Diagnosis of MC disorders is difficult because symptoms are transient, unpredictable, and unspecific, and because these conditions mimic many other diseases. Validation of markers of MC activation in vivo will be useful to allow faster diagnosis and better management of MC disorders. Tryptase, being the most specific MC product, is a widely used biomarker of proliferation and activation. Other mediators, such as histamine, cysteinyl leukotrienes, and prostaglandin D2, are unstable molecules and have limitations in their assays. Surface MC markers, detected by flow cytometry, are useful for the identification of neoplastic MC in mastocytosis but, so far, none of them has been validated as a biomarker of MC activation. Further studies are needed to identify useful biomarkers of MC activation in vivo.

Abstract 5255: Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models

Abstract Bladder cancer (BC) is a lethal genitourinary malignancy associated with frequent recurrence and poor survival due to metastatic potential. Identification of key cancer cell signaling networks and developing promising agents is critical for effectively inhibiting tumor growth and progression. In many cancers, including bladder cancer (BC), signal transducer and activator of transcription 3 (STAT3) has emerged as an important molecular pathway due to its role in promoting proliferation, invasion, and chemoresistance. Thus, developing STAT3 targeting, orally bioavailable small molecule inhibitors may be helpful for the prevention of BC progression and improving the survival rate of patients with muscle invasive BC. Monolayer culture has limitations for drug testing. Therefore, spheroid and organoid culture are used extensively as they may mimic in-vivo drug response more accurately. The aim of our study is to examine the preclinical anticancer efficacy of STAT3 inhibitors [TTI-101 (C188-9) and SH5-07] in 3D (spheroid and tumoroid) invitro models of BC. We optimized the spheroid growth using various BC cell lines [human (J82), rat (NBT-II), and mouse (MB49) BC cells]. Similarly, tumoroids from rat (BBN-induced bladder tumors) and transgenic mice (UPII-SV40T) bladder tumors were developed. These spheroids and tumoroids were treated with various concentrations (0 - 50 μM range) of STAT3 inhibitors and evaluated for their viability [Calcein AM (CA) and EtBr staining], ATP production (CellTiter-Glo™ 3D), and ROS production (MitoSOXTM). Effect of drug treatment on biomarkers of cell proliferation, apoptosis, stemness, STAT3 signaling, and immune modulation was determined using western blotting and immunofluorescence. Treatment with TTI-101 (0 - 50 μM or SH5-07 (0 - 50 μM) for 144 hrs resulted in significant reduction in the spheroids size (39-45% smaller Vs untreated; p&amp;lt;0.0001), along with decreased ATP levels (20%-40%, p&amp;lt;0.05). MitoSOXTM staining suggested that STAT3 inhibitors treatment increased ROS production in BC cells. CA and EtBr staining revealed that TTI-101 and SH5-07 treatment resulted in increased cell death in BC spheroids compared to control. Decreased spheroids and organoids size also correlated with increased apoptotic marker (cleaved caspase-3) along with decreased cyclin D1, PCNA, and pSTAT3 protein expression. Drug treated BC spheroids/tumoroids also showed reduction in CD44 (BC stemness and invasion marker) and induction of cGAS-STING pathway (cGAS, STING, TBK1, and IRF3) in comparison to the control. These findings indicate that STAT3 inhibitors, TTI-101 and SH5-07, could inhibit bladder cancer by suppressing STAT3 pathway activation and therefore warrant further study in vivo. (Supported by P30 CA225520 and Kerley-Cade Endowed Chair) Citation Format: Surya P. Singh, Gopal Pathuri, Adam Asch, Brian Cholewa, Robert Shoemaker, Chinthalapally V. Rao, Venkateshwar Madka. Effect of STAT3 inhibitors, TTI-101 and SH5-07, against bladder cancer in preclinical 3D tumor models. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5255.

Serum thymidine kinase activity in patients with HR-positive/HER2-negative advanced breast cancer treated with ribociclib plus letrozole: Results from the prospective BioItaLEE trial

BackgroundThymidine kinase 1 (TK1) is an enzyme downstream of the CDK4/6 pathway, with a critical role in DNA synthesis; serum TK1 activity (sTKa) is a novel liquid biopsy biomarker of tumour cell proliferation. MethodsThe phase IIIb, BioItaLEE trial (NCT03439046) collected sera from postmenopausal patients with hormone receptor–positive (HR+), HER2-negative (HER2–) advanced breast cancer (ABC) treated with first-line ribociclib plus letrozole at baseline, day 15 of cycle 1 (C1D15), day 1 of cycle 2 (C2D1), and at first imaging. Associations between sTKa assessed at different time points or sTKa dynamic patterns, and progression-free survival (PFS) were evaluated using multivariate Cox models. ResultsOverall, 287 patients were enroled. Median follow-up was 26.9 months. High sTKa (>median) at baseline was associated with higher risk of progression (hazard ratio [HR], 2.21; 95% confidence interval [95% CI], 1.45, 3.37; P = 0.0002); similar results were observed for patients with high sTKa levels at C1D15 and C2D1. Early sTKa dynamic patterns were strongly predictive of PFS. The pattern with high sTKa levels at C2D1 following initial decrease at C1D15 was associated with higher risk of progression versus the pattern with low sTKa levels at both time points (HR, 2.89; 95% CI, 1.57, 5.31; P = 0.0006), while the pattern with high sTKa levels at C1D15 was associated with the shortest PFS (HR, 5.65; CI: 2.84, 11.2; P < 0.0001). Baseline and dynamic sTKa changes provided independent information. ConclusionssTKa appears to be a new promising prognostic and pharmacodynamic biomarker in patients with HR+/HER2– ABC treated with ribociclib plus letrozole as first-line therapy.

Identifying stem cells in the main excretory ducts of rat major salivary glands: adventures with commercial antibodies

ABSTRACT We investigated the entire length of the main excretory ducts (MED) of the major sublingual, parotid and submandibular salivary glands of mature laboratory rats for mucous (goblet) and luminal ciliated cells, biomarkers of cell proliferation, apoptosis, and five biomarkers of stem cells. Spleen and testis were used as positive controls. We used formalin fixed, paraffin embedded tissues. No mucous cells or cells with luminal cilia were observed in hematoxylin and eosin, alcian blue or periodic acid-Schiff stained sections. Immunohistochemistry using rabbit anti-rat antibodies produced anomalous reactions with cleaved caspase-3 for apoptosis, Ki-67 for proliferative activity and Sox 2. Following antigen retrieval, no primary antibody and all three negative controls, labeled macrophages appeared in the spleen. TUNEL staining revealed a few cells per section undergoing apoptosis. Reactions deemed valid occurred in MED with cytokeratin-5 and c-Kit and stem cell antigen 1 (Sca-1) mostly in the gland and middle segments. Other ducts, but not acini or myoepithelial cells, also were variably stained with c-Kit and Sca-1.

Abstract P6-04-09: Variations in diagnostic practice of Ki67 scoring suggest standard guidelines needed for pathological assessment: survey results of global Ki67 testing methods

Abstract BACKGROUND The cell proliferation biomarker Ki67 is expressed during every phase of the cell cycle and has long been used as a diagnostic tool for cancer prognosis, especially for hormone receptor positive breast cancer (HR+ BC). More recently, Ki67 has emerged as a companion diagnostic to select patients for the medicines targeting high risk HR+ BC. We report survey results of local Ki67 immunohistochemistry (IHC) testing practices across 99 pathology labs in 11 countries supporting clinical trial sites in 2020-2021 for the coopERA Breast Cancer clinical study in neoadjuvant HR+ BC (NCT04436744). METHODS The survey was disseminated to pathology labs across five continents to assess local Ki67 IHC staining, analysis, and scoring methodologies. Metrics included pre-analytical considerations (e.g. sample type and requirements) and analytical considerations (e.g. test validation status, antibody, scoring methods, reporting). RESULTS All pathology labs reported requiring formalin-fixed, paraffin-embedded (FFPE) tissue for local Ki67 testing with 89% using sections with thickness of 2-5 microns. For the Ki67 test, the majority (65%) reported using an in vitro diagnostic assay, 23% used a validated test, and 8% utilized a research-use-only assay. Ki67 antibody selection varied among the labs with 46% using the MIB-1 mouse monoclonal (Dako Agilent), followed by 33% using the 30-9 rabbit monoclonal (Ventana) and 12% reporting the SP6 rabbit monoclonal (Thermo Fisher). A majority (65%) reported using single pathologist visual assessment for scoring, and 17% reported using two or more pathologists. Use of automated digital image analysis (ADIA) was reported by 18% of labs, either alone or in combination with pathologist visual assessment. A significant portion (75%) reported using the International Ki67 in Breast Cancer Working Group (IKWG) recommendations, whereas 7% reported using only digital image analysis (e.g. Ventana Virtuoso). A minority (7%) indicated neither and instead described variations of “eyeball” or “hot spot” visual estimates. Most labs (65%) reported counting at least 500 cells with 15% of these counting more than 1000 cells. Remaining labs (30%) counted less than 500 or no cells. Predominantly, 85% reported counting cells in at least 3 or more high power fields. Most labs (96%) report Ki67 scores as a percentage of positive nuclei and the remaining minority reported using other methods (e.g. ranges [&amp;lt; 10%, 10-20%, etc.] or H-score [0-300]). CONCLUSIONS The survey results suggest high global variability of local Ki67 testing practices with the highest variability observed in the test validation status, Ki67 clone, and scoring methods. Despite efforts by the IKWG to harmonize and increase the clinical validity of Ki67 as a biomarker, many labs indicating IKWG compliance had survey answers that were discordant with the specific guidelines set forth by the working group. Taking into account the totality of all answers provided by each respondent, only 51% of the surveyed labs fully conformed to the IKWG recommendations. Moreover, a small fraction conducts global estimations without specific cell counting or use “hot spot” scoring methods, despite the high variability and low reproducibility of these scores both intra- and inter-lab. This study demonstrates the benefits of using a central assay in clinical studies to reduce the variability of local Ki67 results in identifying high risk HR+ BC patients and suggests more work is needed to streamline the analytical practices of local Ki67 methodologies, which may directly impact clinical decisions such as the use of neoadjuvant therapies in HR+ BC. Citation Format: Heather M. Moore, Wendy W. Lin, Tharu M. Fernando, Celine Lopez, Emma Kent, Karine Ellouk, Jennifer M. Giltnane. Variations in diagnostic practice of Ki67 scoring suggest standard guidelines needed for pathological assessment: survey results of global Ki67 testing methods [abstract]. In: Proceedings of the 2022 San Antonio Breast Cancer Symposium; 2022 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2023;83(5 Suppl):Abstract nr P6-04-09.