P53 Mutation Status Research Articles

Abstract B014: Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma

Abstract Introduction: Mutations of the tumor suppressor p53 are detected up to 80% of esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates and poor prognosis. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer harboring Trp53R172H/-, in which we have identified Colony stimulating factor 1 (Csf1) to be upregulated. Our goal is to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote tumor invasion and lung metastasis in ESCC. Methods: We have utilized novel L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP mice and isolated tumor cells to model metastatic ESCC. We conducted ChIP-Seq analysis for p53 on tumor cells derived from lung metastases in our mouse models. Furthermore, we have genetically and pharmacologically targeted CSF1/CSF1R signaling axis to assess its role in ESCC tumor invasion and lung metastasis. In addition to studying tumor intrinsic mechanisms, we have established quantitative multiplex immunofluorescence (qmIF) and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Results And Discussion: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors, and this is dependent upon p53 mutation status, which is reinforced by the TCGA data and patient-derived tissue microarrays (TMAs). The overlay of the RNA-Seq with the ChIP-Seq analysis indicates that Csf1 is a direct gene target of p53-R172H with enriched binding motifs. Furthermore, based on the proximity ligation assay (PLA) with murine ESCC tumor cells, bromodomain and extra-terminal motif (BET) protein BRD4 interacts with p53-R172H to induce Csf1 expression. We show that the BRD4-CSF1 axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in a mutant p53 background. In accordance, upon inhibiting this signaling pathway in the tail-vein injection lung metastasis models, we have identified reduced expression of CD31+ cells and decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of the esophageal cancer datasets reveals that specific p53 mutations are associated with markedly differential overall survival rates and Csf1 expression. Based upon these results, we are generating human and murine esophageal cells with distinct DNA contact and conformational p53 mutations via base editing. Conclusion: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1/CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other squamous cell cancers. We believe this can open up new avenues for therapeutic applications. Citation Format: Gizem Efe, Qiaosi Tang, Katherine M. Cunningham, Kensuke Sugiura, Karen Dunbar, Kausik Regunath, Kensuke Suzuki, Andres J. Klein-Szanto, Lois Resnick-Silverman, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. Mutant p53-mediated CSF1/CSF1R signaling promotes tumor invasion and lung metastasis in esophageal squamous cell carcinoma [abstract]. In: Proceedings of the AACR Special Conference: Cancer Metastasis; 2022 Nov 14-17; Portland, OR. Philadelphia (PA): AACR; Cancer Res 2022;83(2 Suppl_2):Abstract nr B014.

The role of p53 and ki67 in predicting clinical outcome in breast cancer patients.

">ki67 may be used as a proliferative index in addition to estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor receptor 2 (HER2) negative status. p53 gene expression is a well-known biomarker in breast cancer and its role in predicting clinical outcome remains unclear. The current study aimed to determine the relationship between p53 gene mutation and ki67 expression, their clinical characteristics, and overall survival (OS), and to differentiate the significance of p53 and ki67 as the prognostic value in breast cancer patients. ">In this study, 135 patients were enrolled in the study from December 2015 to May 2017. Medical records for all patients were reviewed prospectively. The inclusion criteria included age more than 18 years with histologically proven breast cancer and willingness to be enrolled in p53 genetic study. Exclusion criteria included dual malignancy, male breast cancer, with a loss to follow-up during the study. ">The mean survival of patients with ki67 ≤20 index was 42.7 months (95% confidence interval [CI] 38.7-46.7) and 129 months (95% CI 101.3-157.2) in patients with ki67 >20. The mean OS was 145 months (95% CI 105.6-185.5) in the p53 wild-type group and 106 months (95% CI 78.0-133.0) in the p53 mutated group, as illustrated. ">Our results indicated that p53 mutational status and high ki67 might have an essential impact on overall survival, with p53 mutated patients having a poorer outcome than p53 wild type patients.

Identification of Potential Key Genes and Prognostic Biomarkers of Lung Cancer Based on Bioinformatics.

To analyze and identify the core genes related to the expression and prognosis of lung cancer including lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) by bioinformatics technology, with the aim of providing a reference for clinical treatment. Five sets of gene chips, GSE7670, GSE151102, GSE33532, GSE43458, and GSE19804, were obtained from the Gene Expression Omnibus (GEO) database. After using GEO2R to analyze the differentially expressed genes (DEGs) between lung cancer and normal tissues online, the common DEGs of the five sets of chips were obtained using a Venn online tool and imported into the Database for Annotation, Visualization, and Integrated Discovery (DAVID) database for Gene Ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses. The protein-protein interaction (PPI) network was constructed by STRING online software for further study, and the core genes were determined by Cytoscape software and KEGG pathway enrichment analysis. The clustering heat map was drawn by Excel software to verify its accuracy. In addition, we used the University of Alabama at Birmingham Cancer (UALCAN) website to analyze the expression of core genes in P53 mutation status, confirmed the expression of crucial core genes in lung cancer tissues with Gene Expression Profiling Interactive Analysis (GEPIA) and GEPIA2 online software, and evaluated their prognostic value in lung cancer patients with the Kaplan-Meier online plotter tool. CHEK1, CCNB1, CCNB2, and CDK1 were selected. The expression levels of these four genes in lung cancer tissues were significantly higher than those in normal tissues. Their increased expression was negatively correlated with lung cancer patients (including LUAD and LUSC) prognosis and survival rate. CHEK1, CCNB1, CCNB2, and CDK1 are the critical core genes of lung cancer and are highly expressed in lung cancer. They are negatively correlated with the prognosis of lung cancer patients (including LUAD and LUSC) and closely related to the formation and prediction of lung cancer. They are valuable predictors and may be predictive biomarkers of lung cancer.

Isocitrate Dehydrogenase (IDH1) and p53 Mutations in Gliomas: A Cross-sectional Study from a Tertiary Care Hospital, Karnataka, India

Introduction: Gliomas, being the most common Central Nervous System (CNS) neoplasms, are broadly divided into astrocytomas, oligodendrogliomas and ependymomas. World Health Organisation (WHO) 2016 criteria incorporated Immunohistochemistry (IHC), molecular diagnostic techniques and histological tumour grading into its diagnostic criteria. Isocitrate Dehydrogenase (IDH1) mutations are commonly seen in young people with good prognostic implications. The detection of Tumor Protein53 (p53) mutations in gliomas and its association with the grade of the tumour can be used as a diagnostic, prognostic and therapeutic tool for better survival in affected individuals. IHC can be used as surrogate markers in detecting the mutational status of IDH1 and p53. Aim: To study the expression of IDH1 and p53 mutations in gliomas and to evaluate its association with known clinicopathological variables. Materials and Methods: This cross-sectional, retrospective, analytical study was conducted in the Department of Pathology at JSSAHER, Mysuru, Karnataka, India. The duration of the study was three years and 11 months, from October 2018 to September 2022. All histopathologically diagnosed glioma cases, who underwent surgery, were appropriately reviewed and graded. IHC was performed for the detection of IDH1mutational status and p53 expression. The tumour was labelled as, IDH1 positive with cytoplasmic staining and p53 positive when nuclear staining was observed in more than 5% of tumour cells. The p53 expression was further quantified in scores as low ≤8 and high >8 score. The associations of clinicopathological variables and the comparison of the scores of p53 mutation across various categories of gliomas were analysed statistically. The data was analysed using Statistical package for Social Sciences (SPSS) version 26.0. Results: The mean age of the study participants was 15.3 years. A total of 35 cases (20 males and 15 females) were analysed. The maximum number of cases was in the age group of 31-40 years (n=13) followed by 41-50 years of age group (n=11). Three of them were seen in age less than 20 years. The male: female ratio was 1.33:1. Out of total cases, 20 cases showed IDH1 positivity, 12 cases showed high p53 expression and six cases demonstrated both, IDH1 positivity and high expression of p53. A statistically significant association (p-value=0.014) was seen between the grade of the tumour and p53 immunoreaction. Conclusion: The present study of the detection of IDH1 and p53 mutations, could provide reliable information for improved tailoring of patient therapy as study revealed that, there was a significant association of p53 expression with the grade of the tumour with low expression in low grade gliomas and high p53 expression in high grade gliomas.

P53-Bad* Fusion Gene Therapy Induces Apoptosis In Vitro and Reduces Zebrafish Tumor Burden in Hepatocellular Carcinoma.

With few curative treatments and a global yearly death rate of over 800,000, hepatocellular carcinoma (HCC) desperately needs new therapies. Although wild-type p53 gene therapy has been shown to be safe in HCC patients, it has not shown enough efficacy to merit approval. This work aims to show how p53 can be re-engineered through fusion to the pro-apoptotic BH3 protein Bcl-2 antagonist of cell death (Bad) to improve anti-HCC activity and potentially lead to a novel HCC therapeutic, p53-Bad*. p53-Bad* is a fusion of p53 and Bad, with two mutations, S112A and S136A. We determined mitochondrial localization of p53-Bad* in liver cancer cell lines with varying p53 mutation statuses via fluorescence microscopy. We defined the apoptotic activity of p53-Bad* in four liver cancer cell lines using flow cytometry. To determine the effects of p53-Bad* in vivo, we generated and analyzed transgenic zebrafish expressing hepatocyte-specific p53-Bad*. p53-Bad* localized to the mitochondria regardless of the p53 mutation status and demonstrated superior apoptotic activity over WT p53 in early, middle, and late apoptosis assays. Tumor burden in zebrafish HCC was reduced by p53-Bad* as measured by the liver-to-body mass ratio and histopathology. p53-Bad* induced significant apoptosis in zebrafish HCC as measured by TUNEL staining but did not induce apoptosis in non-HCC fish. p53-Bad* can induce apoptosis in a panel of liver cancer cell lines with varying p53 mutation statuses and induce apoptosis/reduce HCC tumor burden in vivo in zebrafish. p53-Bad* warrants further investigation as a potential new HCC therapeutic.

P53 Modulates Radiosensitivity in Head and Neck Cancers-From Classic to Future Horizons.

p53, initially considered a tumor suppressor, has been the subject of research related to cancer treatment resistance in the last 30 years. The unfavorable response to multimodal therapy and the higher recurrence rate, despite an aggressive approach, make HNSCC a research topic of interest for improving therapeutic outcomes, even if it is only the sixth most common malignancy worldwide. New advances in molecular biology and genetics include the involvement of miRNA in the control of the p53 pathway, the understanding of mechanisms such as gain/loss of function, and the development of different methods to restore p53 function, especially for HPV-negative cases. The different ratio between mutant p53 status in the primary tumor and distant metastasis originating HNSCC may serve to select the best therapeutic target for activating an abscopal effect by radiotherapy as a "booster" of the immune system. P53 may also be a key player in choosing radiotherapy fractionation regimens. Targeting any pathway involving p53, including tumor metabolism, in particular the Warburg effect, could modulate the radiosensitivity and chemo-sensitivity of head and neck cancers.

Evolution of Treatment Patterns and Prognostic Biomarkers in Latin American Patients with Chronic Lymphocytic Leukemia: A Study By the Grupo De Estudio Latinoamericano De Linfoproliferativos (GELL)

Introduction: Chronic Lymphocytic Leukemia (CLL), the most frequent leukemia in adult patients in Western countries, has an incidence that varies according to the geographical region. LATAM is a very heterogeneous region regarding ethnicity and access to care. This translates into differences in the availability of diagnostic and prognostic tools as well as therapeutic options for patients throughout the continent. In the last years, the treatment of CLL has shifted toward the use of targeted agents in first- and second-line therapy. The differences in the access to such agents results in inequality when it comes to offer treatment options. In this observational, retrospective, international study we aim to describe the epidemiological data of patients with CLL in Latin America, the access to and use of biomarkers and treatment patterns. Methods: Patients with 18 years or older, diagnosed with or treated for CLL between 2005-2022 in centers participating in the GELL were included. Patients treated in clinical trials were excluded. Results: 470 patients were analyzed from 6 countries (Peru, Mexico, Paraguay, Chile, Venezuela and Uruguay). 78% were treated at private institutions. The median age was 69 years (35-97), and 193 (41%) were women. According to race, 416 (89%) were white, 2 (0.4%) were black, 2 (0.4%) were indigenous, and 50 (11%) were of mixed race. 337 (72%) had ECOG 0-1. Stage distribution was Rai 0: 183 (39%), I: 120 (26%), II: 61 (13%), III: 34 (7%), IV 26 (6%), and unclassified 46 (9%). Regarding prognostic factors: 11% were CD38 positive. B2-microglobulin data were available in 233 patients (49.5%) and in 124 was high (53%), IgVH mutational status in 41 (9%) of whom 27 (6%) were mutated and 14 (3%) were unmutated. 355 (76%) of patients were observed at time of diagnosis. 220 (47%) patient required treatment with median time to first line treatment of 6 months (range 0-204). Among patients who received treatment, 50 (23%) had cytogenetic and 57 (26%) FISH studies performed. Del17p was detected in 5 patients. P53 mutational status was studied in 12 patients, 1 was found mutated. First line treatment was as follows: Chemotherapy: (Chlorambucil +/- prednisone 46 patients, FC 15, CHOP-like 8), Chemoimmunotherapy (FCR 56, R-CHOP-like 40, BR 24, Chlorambucil + AntiCD20 MoAb 17, FR 1), Ibrutinib 9, Venetoclax + Obinutuzumab 3. Treatment strategies according to date of treatment is shown in Figure 1. 146 (78%) of patients who received chemotherapy or chemoimmunotherapy did not have a previous cytogenetic analysis (Figure 2). According to the country, Uruguay 37%, Mexico 74%, Chile 82%, Peru 83%, Paraguay 89% and Venezuela 96% did not have genetic studies before therapy. Conclusions: Our study shows real-world data in CLL patients from LATAM. The median age at diagnosis is comparable to those reported in developed countries moreover, we report a high percentage of patients with early-stage disease at diagnosis related to the higher diagnosis of asymptomatic patients in routine exams.Most patients treated with chemotherapy or chemoimmunotherapy did not have a cytogenetic testing to guide treatment selection. Also, there are differences in access to cytogenetic testing between the countries analyzed. We show a slight change in treatment patterns in the last years, chemoimmunotherapy remains the most used first line treatment, and a suboptimal access to novel agents is evident. We plan to expand our study cohort with data from the other LATAM countries to have a deeper understanding of the reality of our region and help overcome inequalities. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Treatment Patterns and Outcomes of Patients with Primary or Secondary Acute Myeloid Leukemia By Type of Site (Academic or Community/Government): A CONNECT® Myeloid Registry Study

Introduction Up to 30% of acute myeloid leukemias (AML) evolve from prior hematologic disorders and are classified as secondary AML. Secondary AML is traditionally associated with worse prognosis than de novo, or primary, AML, resulting in inferior response rates and poorer survival. The treatment landscape for AML has evolved recently with the introduction of venetoclax (VEN) and the availability of targeted agents. To better understand evolution of the management of secondary AML, we evaluated treatment patterns and outcomes in patients (pts) with primary AML vs secondary AML treated in real-world clinical practice and enrolled in the CONNECT® Myeloid Disease Registry (NCT01688011) by type of site (academic or community/government). Methods Pts were classified as having either primary or secondary AML based on history of myelodysplastic syndrome (MDS) or progression from MDS to AML. Overall survival (OS) from the date of AML diagnosis, estimated by the Kaplan-Meier method, was evaluated in each group according to type of site (academic or community/government), utilization of allogeneic hematopoietic stem cell transplantation (alloHSCT), and tumor protein p53 (TP53) mutational status. Time to adoption of a VEN-based therapy, defined as first use of VEN-based therapy following Food and Drug Administration (FDA) approval in this setting, was evaluated using a Cox model with factors for age (< 75 and ≥ 75 y), type of site, and geographic region. Results A total of 891 pts in the Registry with a diagnosis of AML were assessed (primary AML, n = 676; secondary AML, n = 215). Median (range) ages were 70 (55-97) y in the primary AML group and 74 (19-93) y in the secondary AML group. Most pts were male (primary AML, 61.6%; secondary AML, 61.5%) and most were treated at community or government centers (primary AML, 60.2%; secondary AML, 66.0%). Pts with primary AML had significantly longer survival than pts with secondary AML; median OS (95% confidence interval [CI]) was 15.6 (13.6-17.4) mo and 5.1 (3.6-6.7) mo, respectively (P < 0.001). Median OS for pts with primary AML and secondary AML treated at academic centers was 17.0 mo and 5.6 mo, respectively (hazard ratio [HR] [95% CI], 0.41 [0.30-0.54], P < 0.001) and in community/government centers was 14.1 mo and 5.2 mo, respectively (HR [95% CI], 0.53 [0.43-0.66], P < 0.001). There was no significant difference in median OS in pts treated at academic centers compared with pts treated at community/government centers for both primary AML (academic vs community/government centers: 17.0 mo [95% CI, 14.7-20.7] vs 14.1 mo [11.0-16.9]; Figure 1A) and secondary AML (5.0 mo [95% CI, 3.0-7.2] vs 5.1 mo [3.7-7.3]; Figure 1B). Significantly longer median OS in pts with primary AML vs secondary AML was observed regardless of alloHSCT status (no alloHSCT: 11.7 mo vs 4.5 mo; HR [95% CI], 0.53 [0.44-0.64], P < 0.001; with alloHSCT: 64.3 mo [35.8-NC] vs 24.9 mo [6.8-48.9], P = 0.002]. Longer median OS was observed among pts who received alloHSCT compared with those without alloHSCT: primary AML, 41.1 mo vs 12.8 mo; secondary AML, 24.9 mo vs 4.4 mo; additional analyses are warranted to account for immortal time bias. Regardless of TP53 mutation status, pts with primary AML had significantly longer median OS than pts with secondary AML (no mutation: 22.0 mo [17.6-26.1] vs 6.2 mo [2.5-12.6], P < 0.001; with mutation: 8.8 mo [3.9-14.2] vs 3.8 mo [1.4-6.8], P = 0.014). VEN-based therapies were adopted more quickly by pts aged ≥ 75 y vs < 75 y (P < 0.001) and those treated at community or government centers vs academic centers (P = 0.045); no association between geographic region and time to adoption of VEN-based therapies was observed. Conclusions Overall, outcomes of pts with AML in the CONNECT® Myeloid Disease Registry are consistent with previously reported outcomes. Median OS is longer in primary AML vs secondary AML regardless of whether pts receive alloHSCT or have mutations in TP53. This analysis suggests that contrary to commonly held perceptions, site of care does not affect overall pt outcome and that VEN adoption occurs sooner at community centers. The clinically meaningful increase in OS in pts receiving alloHSCT, when compared with pts without alloHSCT, regardless of primary or secondary AML further emphasizes the importance of providing pts with the opportunity for transplant. These observations merit further study into potential differences in treatment patterns and outcomes. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

EXTH-19. OPTIMIZING MDM2 INHIBITION FOR THE TREATMENT OF GLIOBLASTOMA

Abstract Over 60% of glioblastomas retain wild-type p53, resulting in potential susceptibility to MDM2 inhibitors. These molecules disrupt the interaction between p53 and its negative regulator MDM2, allowing downstream pathway signaling and cell fate decisions including growth inhibition and death resulting from cellular stress. We have analyzed clinical samples from 10 patients newly diagnosed with glioblastoma multiforme (GBM) and treated with the MDM2 inhibitor KRT-232. We detected upregulation of CDKN1A transcript (a target of p53) in all p53 wild-type tumors (8 of 10), but not in p53-mutant tumors (2 of 10). In patient-derived cell lines, treatment with KRT-232 at the clinically detected concentration was only sufficient to stall tumor growth. Cell death via apoptosis can be achieved when MDM2 inhibition is combined with the chemotherapeutic agent temozolomide. As the effects and resistance mechanisms of MDM2 inhibition remain poorly understood in GBM, we have additionally performed genomic and transcriptomic analyses in patient-derived cell lines to better characterize sensitive tumors and identify putative biomarkers of drug response. Dose response curves and growth assays showed that tumors with inactivating p53 mutations are highly resistant to treatment, but those that retain wild-type p53 exhibit various degrees of drug sensitivity. This suggests that other factors, in addition to p53 mutational status, mediate response to MDM2 inhibition in gliomas. Transcriptional analyses of patient samples following drug treatment suggest that cell division, chromatin reorganization, cell differentiation state and immune response programs become deregulated under MDM2 inhibition and in the absence of p53-inactivating mutations.

Identifying overall survival in 98 glioblastomas using VASARI features at 3T

PurposeThis study aims to evaluate qualitative and quantitative imaging metrics along with clinical features affecting overall survival in glioblastomas and to classify them into high survival and low survival groups based on 12, 19, and 24 months thresholds using machine learning. MethodsThe cohort consisted of 98 adult glioblastomas. A standard brain tumor magnetic resonance (MR) imaging protocol, was performed on a 3T MR scanner. Visually Accessible REMBRANDT Images (VASARI) features were assessed. A Kaplan-Meier survival analysis followed by a log-rank test and multivariate Cox regression analysis were used to investigate the effects of VASARI features along with the age, gender, the extent of resection, pre- and post-KPS, ki67 and P53 mutation status on overall survival. Supervised machine learning algorithms were employed to predict the survival of glioblastoma patients based on 12, 19, and 24 months thresholds. ResultsTumor location (p<0.001), the proportion of non-enhancing component (p=0.0482), and proportion of necrosis (p=0.02) were significantly associated with overall survival based on Kaplan-Meier analysis. Multivariate Cox regression analysis revealed that increases in proportion of non-enhancing component (p=0.040) and proportion of necrosis (p=0.039) were significantly associated with overall survival. Machine-learning models were successful in differentiating patients living longer than 12 months with 96.40% accuracy (sensitivity=97.22%, specificity=95.55%). The classification accuracies based on 19 and 24 months survival thresholds were 70.87% (sensitivity=83.02%, specificity=60.11%) and 74.66% (sensitivity=67.58%, specificity=82.08%), respectively. ConclusionEmploying clinical and VASARI features together resulted in a successful classification of glioblastomas that would have a longer overall survival.

Distinct expression and function of breast cancer metastasis suppressor 1 in mutant P53 glioblastoma.

Glioblastoma (GBM) is the most malignant subtype of astrocytic tumors with the worst prognosis in all its progressive forms. Breast cancer metastasis suppressor 1 (BRMS1) is a metastasis suppressor gene that controls malignancy in multiple tumors. As yet, however, its clinical and functional significance in mutant P53 GBM remains inconclusive. Here, we attempted to study the importance of BRMS1 in mutant P53 GBM. BRMS1 expression was evaluated in 74 human astrocytoma tissues by qRT-PCR, Western blotting and immunohistochemistry. BRMS1 expression in the astrocytoma tissues was correlated with clinicopathological parameters, the P53 mutation status and BRMS1 downstream targets, and compared with TCGA and NCI-60 datasets. siRNA-mediated knockdown of BRMS1 was performed in selected GBM cell lines to evaluate the functional role of BRMS1. Our study revealed an enhanced expression of BRMS1 in GBM which was associated with a poor patient survival, and this observation was corroborated by the TCGA dataset. We also found a positive correlation between BRMS1 expression and a mutant P53 status in GBM which was associated with a poor prognosis. In vitro BRMS1 silencing reduced the growth of mutant P53 GBM cells and repressed their colonization and migration/invasion by modulating EGFR-AKT/NF-κB signaling. Transcriptional profiling revealed a positive and negative correlation of uPA and ING4 expression with BRMS1 expression, respectively. Our data indicate upregulation of BRMS1 in high grade astrocytomas which correlates positively with mutant P53 and a poor patient survival. Silencing of BRMS1 in mutant P53 GBM cell lines resulted in a reduced cellular growth and migration/invasion by suppressing the EGFR-AKT/NF-kB signaling pathway. BRMS1 may serve as a predictive biomarker and therapeutic target in mutant P53 GBM.

Competing endogenous RNA networks related to prognosis in chronic lymphocytic leukemia: comprehensive analyses and construction of a novel risk score model

BackgroundChronic lymphocytic leukemia (CLL) is a heterogeneous B-cell malignancy that lacks specific biomarkers and drug targets. Competing endogenous RNAs (ceRNAs) play vital roles in oncogenesis and tumor progression by sponging microRNAs (miRNAs). Nevertheless, the regulatory mechanisms of survival-related ceRNA networks in CLL remain to be uncovered.MethodsWe included 865 de novo CLL patients to investigate RNA expression profiles and Illumina sequencing was performed on four CLL patients, two CLL cell lines and six healthy donors in our center. According to univariate Cox regression, LASSO regression as well as multivariate Cox regression analyses, we established a novel risk score model in CLL patients. Immune signatures were compared between the low- and high-risk groups with CIBERSORT and ESTIMATE program. Afterwards, we analyzed the relationship between differentially expressed miRNAs (DEmiRNAs) and IGHV mutational status, p53 mutation status and del17p. Based on the survival analyses and differentially expressed RNAs with targeting relationships, the lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed. In addition, the circRNA circ_0002078/miR-185-3p/TCF7L1 axis was verified and their interrelations were delineated by dual-luciferase reporter gene assay.ResultsTotally, 57 differentially expressed mRNAs (DEmRNAs) and 335 DEmiRNAs were identified between CLL patient specimens and normal B cells. A novel risk score model consisting of HTN3, IL3RA and NCK1 was established and validated. The concordance indexes of the model were 0.825, 0.719 and 0.773 in the training, test and total sets, respectively. The high-risk group was related to del(13q14) as well as shorter overall survival (OS). Moreover, we identified DEmiRNAs that related to cytogenetic abnormality of CLL patients, which revealed that miR-324-3p was associated with IGHV mutation, p53 mutation and del17p. The survival-related lncRNA/circRNA-miRNA-mRNA ceRNA networks were constructed to further facilitate the development of potential predictive biomarkers. Besides, the expression of circ_0002078 and TCF7L1 were significantly elevated and miR-185-3p was obviously decreased in CLL patients. Circ_0002078 regulated TCF7L1 expression by competing with TCF7L1 for miR-185-3p.ConclusionsThe comprehensive analyses of RNA expression profiles provide pioneering insights into the molecular mechanisms of CLL. The novel risk score model and survival-related ceRNA networks promote the development of prognostic biomarkers and potential therapeutic vulnerabilities for CLL.

UBE2T promotes glioblastoma malignancy through ubiquitination-mediated degradation of RPL6.

Glioblastoma (GBM) is the most frequent and aggressive malignant glioma. Due to patients' poor prognosis, it is of great clinical significance to determine new targets that may improve GBM treatment. In the present study, we showed that ubiquitin (Ub)-conjugating enzyme E2T (UBE2T) was significantly overexpressed in GBM and could promote proliferation, invasion, and inhibit apoptosis of GBM cells. Mechanistically, UBE2T functioned as the Ub enzyme of ribosomal protein L6 (RPL6) and induced the ubiquitination and degradation of RPL6 in an E3 ligase-independent manner through direct modification by K48-linked polyubiquitination, thus contributing to the malignant progression of GBM cells. Furthermore, inhibiting the expression of RPL6 by UBE2T could not only reduce the expression of wild-type p53,but also enhance the gain-of-function of mutant p53. Moreover, knockdown of UBE2T in LN229 cells obviously suppressed tumor growth in LN229 xenograft mouse models. Collectively, our study demonstrated that UBE2T promotes GBM malignancy through ubiquitination-mediated degradation of RPL6 regardless of the p53 mutation status. It will provide new candidates for molecular biomarkers and therapeutic targets for clinical application in GBM.

Immunohistochemical Markers and TILs Evaluation for Endometrial Carcinoma.

Objective: The molecular classification for endometrial cancer (EC) introduced by The Cancer Genome Atlas Research Network (TCGA) and the Proactive Molecular Risk Classifier for Endometrial Cancer (ProMisE) proved the existence of four molecular prognostic subtypes; however, both classifications require costly technology. We suggest a prognostic model for EC based on immunohistochemistry (IHC) and tumor-infiltrating lymphocytes (TILs). Study design: One hundred patients were included. We retrospectively investigated IHC prognostic parameters: mismatch repair (MMR)-deficient tumors, p53 mutation status, progesterone receptors (PgRs), and estrogen receptors (ERs). We further evaluated TILs. These parameters were related to the clinical and morphological features and to the outcome. Results: We classified tumors into three groups (IHC analysis): MMR-deficient, p53-mutated, p53 wild-type. MMR-deficient tumors had a good prognosis, p53 wild-type tumors an intermediate one, and p53-mutated tumors had the poorest outcomes. Disease-free (DFS) and overall survival (OS) were significantly better among PgR+ tumors (respectively p = 0.011 and p = 0.001) and PgR expression is an independent prognostic factor for a better DFS frommultivariate analysis (OR = 0.3; CI: 0.1–0.9; p = 0.03).No significant correlation was observed between DFS and TILs. However, among MMR-deficient tumors, the mean value of TILs was higher than among the other tumors(111 versus 71, p = 0.01) Conclusions: The prognostic model based on IHC markers could potentially be a valid and applicable alternative to the TCGA one. The PgR determination could represent an additional prognostic factor for EC.

Glioblastoma Multiforme: A Review

In recent years, many research groups have begun to utilize bioengineered in vitro models of cancer to study mechanisms of disease progression, test drug candidates, and develop platforms to advance personalized drug treatment options. Due to advances in cell and tissue engineering over the last few decades, there are now a myriad of tools that can be used to create such in vitro systems. In this review, we describe the considerations one must take when developing model systems that accurately mimic the in vivo tumor microenvironment (TME) and can be used to answer specific scientific questions. We will summarize the importance of cell sourcing in models with one or multiple cell types and outline the importance of choosing biomaterials that accurately mimic the native extracellular matrix (ECM) of the tumor or tissue that is being modeled. We then provide examples of how these two components can be used in concert in a variety of model form factors and conclude by discussing how biofabrication techniques such as bioprinting and organ-on-a-chip fabrication can be used to create highly reproducible complex in vitro models. Since this topic has a broad range of applications, we use the final section of the review to dive deeper into one type of cancer, glioblastoma, to illustrate how these components come together to further our knowledge of cancer biology and move us closer to developing novel drugs and systems that improve patient outcomes. Glioblastomas are universally fatal cancers and contain self-renewing glioblastoma stem cells (GSCs) that initiate tumors. Traditional anticancer drug development based on in vitro cultures tends to select targets with low therapeutic indices and fails to effectively represent the tumour microenvironment's impacts. Glioblastoma multiforme (GBM) is the most common and aggressive form of brain cancer, with treatment options often constrained due to inherent resistance of malignant cells to conventional therapy. We investigated the impact of triggering programmed cell death (PCD) by using BH3 mimetic drugs in human GBM cell lines. Glioblastoma multiforme (GBM) has been characterized by the high incidence, therapy tolerance and relapse. The molecular events controlling GBM resistant to chemotherapy temozolomide (TMZ) remain to be elusive. Here, we identified WNT signaling was amplified by TMZ and mediated drug response in GBM. We found O6-methylguanine DNA methyltransferase (MGMT) was redundant to WNT-mediated chemoresistance, which was highly associated with p53 mutation status. In GBM with p53 mutation, loss of function of p53 downregulated miR-34a expression, which represses transcription of WNT ligand 6 (WNT6) by directly binding to 3′ UTR of WNT6 mRNA, leading to activation of WNT signaling, and the eventual WNT-mediated chemoresistance to TMZ. Combined treatment of TMZ with WNT inhibitor or miR34a mimic induced drug sensitivity of p53-mutant GBM cells and extended survival in xenograft mice in vivo. Our findings provide insight into understanding the molecular mechanism of GBM chemoresistance to TMZ and facilitating to develop novel treatment strategy to combat p53-mutant GBM by targeting miR-34a/WNT6 axis. Advantages and drawbacks of dexamethasone in glioblastoma multiforme: Dexamethasone has been used for many years to treat brain edema and inflammation caused by GBM.Several investigations have shown that dexamethasone also exerts antitumoral effects against GBM.Unfortunately, steroids are associated with various undesirable side effects. Herein, we review pre-clinical and clinical applications of Dexamethasone in GBM. The most widespread, malignant, and deadliest type of glial tumor is glioblastoma multiforme (GBM). Despite radiation, chemotherapy, and radical surgery, the median survival of afflicted individuals is about 12 months. Unfortunately, existing therapeutic interventions are abysmal. Dexamethasone (Dex), a synthetic glucocorticoid, has been used for many years to treat brain edema and inflammation caused by GBM. Several investigations have recently shown that Dex also exerts antitumoral effects against GBM. On the other hand, more recent disputed findings have questioned the long-held dogma of Dex treatment for GBM. Unfortunately, steroids are associated with various undesirable side effects, including severe immunosuppression and metabolic changes like hyperglycemia, which may impair the survival of GBM patients. Current ideas and concerns about Dex's effects on GBM cerebral edema, cell proliferation, migration, and its clinical outcomes were investigated in this study. Glioblastoma multiforme (GBM) is one of the most common, most formidable, and deadliest malignant types of primary astrocytoma with a poor prognosis. At present, the standard of care includes surgical tumor resection, followed by radiation therapy concomitant with chemotherapy and temozolomide. New developments and significant advances in the treatment of GBM have been achieved ..........

Molecular Characteristics of Metastatic Lesions Have Superior Prognostic Impact on Endometrial Cancer

Background/Aim: In endometrial cancer (EC), lymph node (LN) metastasis significantly impacts prognosis. Thus far, no studies have reported the molecular genetics of each metastatic lesion. This study aimed to investigate the molecular characteristics of primary and metastatic LNs and their association with clinical outcomes. Patients and Methods: The clinicopathological and molecular characteristics of 33 patients with EC with regional LN metastasis (FIGO stage IIIC) were investigated; we evaluated the mutational status of p53 and DNA mismatch repair (MMR) proteins in the primary lesion, all the positive LNs (102 lesions), mutational variation between primary and paired metastatic lesions, inter-lesion heterogeneity, and their association with clinical outcomes. Results: Immunohistochemically, 12 patients (36.4%) displayed aberrant p53 expression in metastatic lesions, and a concordant rate of 93.4% was observed between primary and metastatic lesions. Inter-lesion heterogeneity was observed in 20 cases (60.6%). In Kaplan–Meier analysis, patients with aberrant p53 expression in metastatic LNs exhibited worse progression-free survival (PFS) than those with wild-type p53 expression (p=0.008). Wild-type p53 expression in primary lesion with inter-lesion heterogeneity had a significantly worse PFS (p=0.049) than those without heterogeneity. In the Cox univariate analysis, p53 expression in metastatic LNs was significantly associated with recurrence (p=0.013). Genetic diversity between primary and metastatic lesions and among metastases was validated by evaluating the p53 and MMR proteins using immunohistochemistry analysis. Conclusion: The molecular characteristics of metastatic lesions in addition to those of primary lesions could provide beneficial prognostic information in patients with EC with regional LN metastasis.

Clinicopathological features and CT findings of papillary gastric adenocarcinoma.

This study aimed to analyze the clinicopathological and computed tomography (CT) findings of papillary gastric adenocarcinoma and to evaluate the feasibility of the multivariate model based on clinical information and CT findings for discriminating papillary gastric adenocarcinomas. This retrospective study included 22 patients with papillary gastric adenocarcinoma and 88 patients with tubular adenocarcinoma. The demographic data, tumor markers, histopathological information, CT morphological characteristics, and CT value-related parameters of all patients were collected and analyzed. The multivariate model based on regression analysis was performed to improve the diagnostic efficacy for discriminating papillary gastric adenocarcinomas preoperatively. The diagnostic performance of the established nomogram was evaluated by receiver operating characteristic curve analysis. The distribution of age, carcinoembryonic antigen, differentiation degree, neural invasion, human epidermal growth factor receptor 2 overexpression, P53 mutation status, 4 CT morphological characteristics, and 10 CT valued-related parameters differed significantly between papillary gastric adenocarcinoma and tubular adenocarcinoma groups (all p < 0.05). The established multivariate model based on clinical information and CT findings for discriminating papillary gastric adenocarcinomas preoperatively achieved the area under the curve of 0.920. There existed differences in clinicopathological features and CT findings between papillary gastric adenocarcinomas and tubular adenocarcinomas. The combination of demographic data, tumor markers, CT morphological characteristics, and CT value-related parameters could discriminate papillary gastric adenocarcinomas preoperatively with satisfactory diagnostic efficiency.

Immunohistochemical analyses of paraffin-embedded sections after primary surgery or trimodality treatment in esophageal carcinoma

BackgroundThe microscopic tumor extension before, during or after radiochemotherapy (RCHT) and its correlation with the tumor microenvironment (TME) are presently unknown. This information is, however, crucial in the era of image-guided, adaptive high-precision photon or particle therapy. Materials and methodsIn this pilot study, we analyzed formalin-fixed paraffin-embedded (FFPE) tumor resection specimen from patients with histologically confirmed squamous cell carcinoma (SCC; n = 10) or adenocarcinoma (A; n = 10) of the esophagus, having undergone neoadjuvant radiochemotherapy followed by resection (NRCHT + R) or resection (R)]. FFPE tissue sections were analyzed by immunohistochemistry regarding tumor hypoxia (HIF-1α), proliferation (Ki67), immune status (PD1), cancer cell stemness (CXCR4), and p53 mutation status. Marker expression in HIF-1α subvolumes was part of a sub-analysis. Statistical analyses were performed using one-sided Mann-Whitney tests and Bland-Altman analysis. ResultsIn both SCC and AC patients, the overall percentages of positive tumor cells among the five TME markers, namely HIF-1α, Ki67, p53, CXCR4 and PD1 after NRCHT were lower than in the R cohort. However, only PD1 in SCC and Ki67 in AC showed significant association (Ki67: p = 0.03, PD1: p = 0.02). In the sub-analysis of hypoxic subvolumes among the AC patients, the percentage of positive tumor cells within hypoxic regions were statistically significantly lower in the NRCHT than in the R cohort across all the markers except for PD1. ConclusionIn this pilot study, we showed changes in the TME induced by NRCHT in both SCC and AC. These findings will be correlated with microscopic tumor extension measurements in a subsequent cohort of patients.

Prognostic implications of cellular senescence in resected non-small cell lung cancer.

BackgroundCure and long-term survival for non-small cell lung cancer (NSCLC) remains hard to achieve. Cellular senescence, an emerging hallmark of cancer, is considered as an endogenous tumor suppressor mechanism. However, senescent cancer cells can paradoxically affect the surrounding tumor microenvironment (TME), ultimately leading to cancer relapse and metastasis. As such, the role of cellular senescence in cancer is highly controversial.MethodsIn 155 formalin-fixed paraffin-embedded (FFPE) samples from surgically resected NSCLC patients with pathological tumor-node-metastasis (pTNM) stages I–IV (8th edition), cellular senescence was assessed using a combination of four immunohistochemical senescence markers, i.e., lipofuscin, p16INK4a, p21WAF1/Cip1 and Ki67, and correlated to clinicopathological parameters and outcomes, including overall survival (OS) and disease-free survival (DFS).ResultsA tumoral senescence signature (SS) was present in 48 out of 155 NSCLC patients, but did not correlate to any clinicopathological parameter, except for p53 mutation status. In a histologically homogenous patient cohort of 100 patients who fulfilled the following criteria: (I) one type of histology, i.e., adenocarcinoma, (II) without known epidermal growth factor receptor (EGFR) mutation, (III) curative (R0) resection and (IV) no neoadjuvant systemic therapy or radiotherapy, the median OS and DFS for patients with a tumoral SS (n=30, 30.0%) compared to patients without a tumoral SS (n=70, 70.0%) was 53 versus 141 months (P=0.005) and 45 versus 55 months (P=0.25), respectively. In multiple Cox proportional hazards (Cox PH) model analysis correcting for age, pTNM stage I–III and adjuvant therapy, a tumoral SS remained a significant prognostic factor for OS (HR =2.03; P=0.014).ConclusionsThe presence of a tumoral SS particularly based on high p16INK4a expression significantly affects OS in NSCLC adenocarcinoma. In this light, adjuvant senolytic therapy could be an interesting strategy for NSCLC patients harboring a tumoral SS, ultimately to improve survival of these patients.

Statins inhibit proliferation and induce apoptosis in triple-negative breast cancer cells

TP53 (p53) is mutated in 80–90% of cases of triple-negative breast cancer (TNBC). Statins, which are widely used to treat elevated cholesterol, have recently been shown to degrade mutant p53 protein and exhibit anti-cancer activity. The aim of this work was to evaluate the potential of statins in the treatment of TNBC. The anti-proliferative effects of 2 widely used statins were investigated on a panel of 15 cell lines representing the different molecular subtypes of breast cancer. Significantly lower IC50 values were found in triple-negative (TN) than in non-TN cell lines (atorvastatin, p < 0.01; simvastatin p < 0.05) indicating greater sensitivity. Furthermore, cell lines containing mutant p53 were more responsive to both statins than cell lines expressing wild-type p53, suggesting that the mutational status of p53 is a potential predictive biomarker for statin response. In addition to inhibiting proliferation, simvastatin was also found to promote cell cycle arrest and induce apoptosis. Using an apoptosis array capable of detecting 43 apoptosis-associated proteins, a novel protein shown to be upregulated by simvastatin was the IGF-signalling modulator, IGBP4, a finding we confirmed by Western blotting. Finally, we found synergistic growth inhibition between simvastatin and the IGF-1R inhibitor, OSI-906 as well as between simvastatin and doxorubicin or docetaxel. Our work suggests repurposing of statins for clinical trials in patients with TNBC. Based on our findings, we suggest that these trials investigate statins in combination with either doxorubicin or docetaxel and include p53 mutational status as a potential predictive biomarker.