Assessing clinical response in early oncology development with a predictive biomarker.

In early oncology clinical trials there is often limited data for biomarkers and their association with response to treatment. Thus, it is challenging to decide whether a biomarker should be used for patient selection and enrollment. Most evidence about any potential predictive biomarker comes from preclinical research and, sometimes, clinical observations. How to translate the preclinical predictive biomarker data to clinical study remains an active field of research. Here, we propose a method to incorporate existing knowledge about a predictive biomarker - its prevalence, association with response and the performance of the assay used to measure the biomarker - to estimate the response rate in a clinical study designed with or without using the predictive biomarker. Importantly, we quantify the uncertainty associated with the biomarker and its predictability in a probabilistic model. This model estimates the distribution of the clinical response when a predictive biomarker is used to select patients and compares it to unselected cohort. We applied this method to two real world cases of approved biomarker-guided therapies to demonstrate its utility and potential value. This approach helps to make a data-driven decision whether to select patients with a predictive biomarker in early oncology clinical development.

Experimental design considerations and statistical analyses in preclinical tumor growth inhibition studies.

Animal models are used in cancer pre-clinical research to identify drug targets, select compound candidates for clinical trials, determine optimal drug dosages, identify biomarkers, and ensure compound safety. This tutorial aims to provide an overview of study design and data analysis from animal studies, focusing on tumor growth inhibition (TGI) studies used for prioritization of anticancer compounds. Some of the experimental design aspects discussed here include the selection of the appropriate biological models, the choice of endpoints to be used for the assessment of anticancer activity (tumor volumes, tumor growth rates, events, or categorical endpoints), considerations on measurement errors and potential biases related to this type of study, sample size estimation, and discussions on missing data handling. The tutorial also reviews the statistical analyses employed in TGI studies, considering both continuous endpoints collected at single time-point and continuous endpoints collected longitudinally over multiple time-points. Additionally, time-to-event analysis is discussed for studies focusing on event occurrences such as animal deaths or tumor size reaching a certain threshold. Furthermore, for TGI studies involving categorical endpoints, statistical methodology is outlined to compare outcomes among treatment groups effectively. Lastly, this tutorial also discusses analysis for assessing drug combination synergy in TGI studies, which involves combining treatments to enhance overall treatment efficacy. The tutorial also includes R sample scripts to help users to perform relevant data analysis of this topic.

Cystine-glutamate antiporter xCT deficiency suppresses tumor growth while preserving antitumor immunity.

T cell-invigorating cancer immunotherapies have near-curative potential. However, their clinical benefit is currently limited, as only a fraction of patients respond, suggesting that these regimens may benefit from combination with tumor-targeting treatments. As oncogenic progression is accompanied by alterations in metabolic pathways, tumors often become heavily reliant on antioxidant machinery and may be susceptible to increases in oxidative stress. The cystine-glutamate antiporter xCT is frequently overexpressed in cancer and fuels the production of the antioxidant glutathione; thus, tumors prone to redox stress may be selectively vulnerable to xCT disruption. However, systemic inhibition of xCT may compromise antitumor immunity, as xCT is implicated in supporting antigen-induced T cell proliferation. Therefore, we utilized immune-competent murine tumor models to investigate whether cancer cell expression of xCT was required for tumor growth in vivo and if deletion of host xCT impacted antitumor immune responses. Deletion of xCT in tumor cells led to defective cystine uptake, accumulation of reactive oxygen species, and impaired tumor growth, supporting a cancer cell-autonomous role for xCT. In contrast, we observed that, although T cell proliferation in culture was exquisitely dependent on xCT expression, xCT was dispensable for T cell proliferation in vivo and for the generation of primary and memory immune responses to tumors. These findings prompted the combination of tumor cell xCT deletion with the immunotherapeutic agent anti-CTLA-4, which dramatically increased the frequency and durability of antitumor responses. Together, these results identify a metabolic vulnerability specific to tumors and demonstrate that xCT disruption can expand the efficacy of anticancer immunotherapies.

miR-20b-5p attenuates hypoxia-induced apoptosis in cardiomyocytes via the HIF-1α/NF-κB pathway.

Chronic hypoxia is a common inducer of end-stage cardiovascular disease. In cells under hypoxia, the hypoxia-inducible factor-1 (HIF-1) plays a vital role in regulating downstream target genes. However, the mechanism of hypoxia in cardiomyocytes is still unclear. In this study, we aimed to identify novel downstream epigenetic targets of HIF-1α in cardiomyocytes under hypoxia. H9c2 cells were exposed to hypoxia condition, and quantitative real-time PCR analysis was performed to evaluate the expression of miR-20b-5p. The results indicated that the expression of miR-20b-5p was down-regulated in H9c2 cells under low oxygen condition. Meanwhile, HIF-1α overexpression further down-regulated the miR-20b-5p expression in H9c2 cells transfected with HIF-1α plasmids. In addition, Annexin-V-FITC/PI flow cytometry analysis suggested that overexpression of miR-20b-5p attenuated cell apoptosis under hypoxia condition in H9c2 cells. Western blot analysis showed that the hypoxia apparently increased Bax and cleaved-caspase-3, but decreased Bcl-2 expression in H9c2 cells, indicating that hypoxia-induced NF-κB signaling pathway activation is mediated by miR-20b-5p. Hypoxia-induced H9c2 cell apoptosis was reduced after HIF-1α knockdown as shown by the flow cytometry analysis. In conclusion, we identified that miR-20b-5p plays an important role in mediating cardiomyocytes apoptosis under hypoxia, which is mediated by the HIF-1/NF-κB signaling pathway.

Traumatic occlusion aggravates bone loss during periodontitis and activates Hippo-YAP pathway.

AIM: This study aimed at exploring changes in YAP expression and their effect on periodontitis (PD) combined with traumatic occlusion (TO). MATERIALS AND METHODS: BALB/cJ mice were used to establish a PD model by local administration of Porphyromonas gingivalis (P.g, ATCC 33277) and a TO model by occlusal elevation (OE) using composite resin bonding on the bilateral maxillary molar. The mouse fibroblast cell line (L929) and pre-osteoblast cell line (MC3T3-E1) were subjected to cyclic tensile/compressive stress and inflammatory stimuli (lipopolysaccharide from Escherichia coli) to verify in vivo results. RESULTS: Severe bone resorption was observed by microCT scanning in OE with P.g group, when compared to OE only and P.g only groups. Mechanical stress caused by OE activated the Hippo-YAP pathway in periodontal tissues and upregulated the expression of JNK/AP-1. OE with P.g further promoted the expression of YAP and JNK/AP1, leading to the upregulation of the JNK/AP-1 related inflammatory cytokines TNF-α and IL6. Similar results were obtained when osteoblasts were subjected to mechanical stress in vitro. CONCLUSIONS: Our study demonstrated that periodontitis with TO caused severe inflammation-induced bone resorption. Activation of YAP and upregulation of JNK/AP-1 induced by TO potentially aggravated the symptoms of PD.

TNER: a novel background error suppression method for mutation detection in circulating tumor DNA.

BACKGROUND: Ultra-deep next-generation sequencing of circulating tumor DNA (ctDNA) holds great promise as a tool for the early detection of cancer and for monitoring disease progression and therapeutic responses. However, the low abundance of ctDNA in the bloodstream coupled with technical errors introduced during library construction and sequencing complicates mutation detection. RESULTS: To achieve high accuracy of variant calling via better distinguishing low-frequency ctDNA mutations from background errors, we introduce TNER (Tri-Nucleotide Error Reducer), a novel background error suppression method that provides a robust estimation of background noise to reduce sequencing errors. The results on both simulated data and real data from healthy subjects demonstrate that the proposed algorithm consistently outperforms a current, state-of-the-art, position-specific error polishing model, particularly when the sample size of healthy subjects is small. CONCLUSIONS: TNER significantly enhances the specificity of downstream ctDNA mutation detection without sacrificing sensitivity. The tool is publicly available at https://github.com/ctDNA/TNER .

The comparison of biocompatibility and osteoinductivity between multi-walled and single-walled carbon nanotube/PHBV composites.

The applications of poly (3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) in tissue engineering have been widely studied. This study aimed to compare the biocompatibility and osteoinductivity of single-walled carbon nanotubes (SWCNTs)/PHBV composites with multi-walled CNTs (MWCNTs)/PHBV composites. CNTs were dispersed in PHBV by ultrasonication and composites were created using thermal injection moulding. In order to test their biocompatibility and osteoinductivity. Rat osteoblasts (rOBs) were then cultured and seeded on the composites. The composites were implanted in rat femoral bone defects. Our results showed that lower weight percentages of SWCNTs and MWCNTs (2-4%) improved both their mechanical and thermal decomposition properties. However, further reduction of rOBs cell death was observed in MWCNTs/PHBV. SWCNTs were shown to upregulate the expression of Runx-2 and Bmp-2 in early stage significantly, while MWCNTs showed a stronger long-term effect on Opn and Ocn. The in vivo result was that MWCNTs/PHBV composites induced intact rounding new bone, increased integration with new bone, and earlier completed bone remodeling when compared with SWCNTs. Immunohistochemistry also detected higher expression of RUNX-2 around MWCNTs/PHBV composites. In conclusion, there were no differences observed between SWCNTs and MWCNTs in the reinforcement of PHBV, while MWCNTs/PHBV composites showed better biocompatibility and osteoinductivity both in vitro and in vivo.

Whole-genome sequencing identifies recurrent mutations in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most deadly cancers worldwide and has no effective treatment, yet the molecular basis of hepatocarcinogenesis remains largely unknown. Here we report findings from a whole-genome sequencing (WGS) study of 88 matched HCC tumor/normal pairs, 81 of which are Hepatitis B virus (HBV) positive, seeking to identify genetically altered genes and pathways implicated in HBV-associated HCC. We find beta-catenin to be the most frequently mutated oncogene (15.9%) and TP53 the most frequently mutated tumor suppressor (35.2%). The Wnt/beta-catenin and JAK/STAT pathways, altered in 62.5% and 45.5% of cases, respectively, are likely to act as two major oncogenic drivers in HCC. This study also identifies several prevalent and potentially actionable mutations, including activating mutations of Janus kinase 1 (JAK1), in 9.1% of patients and provides a path toward therapeutic intervention of the disease.

Unbiased Scanning Method and Data Banking Approach Using Ultra-High Performance Liquid Chromatography Coupled with High-Resolution Mass Spectrometry for Quantitative Comparison of Metabolite Exposure in Plasma across Species Analyzed at Different Dates.

An unbiased scanning methodology using ultra high-performance liquid chromatography coupled with high-resolution mass spectrometry was used to bank data and plasma samples for comparing the data generated at different dates. This method was applied to bank the data generated earlier in animal samples and then to compare the exposure to metabolites in animal versus human for safety assessment. With neither authentic standards nor prior knowledge of the identities and structures of metabolites, full scans for precursor ions and all ion fragments (AIF) were employed with a generic gradient LC method to analyze plasma samples at positive and negative polarity, respectively. In a total of 22 tested drugs and metabolites, 21 analytes were detected using this unbiased scanning method except that naproxen was not detected due to low sensitivity at negative polarity and interference at positive polarity; and 4'- or 5-hydroxy diclofenac was not separated by a generic UPLC method. Statistical analysis of the peak area ratios of the analytes versus the internal standard in five repetitive analyses over approximately 1 year demonstrated that the analysis variation was significantly different from sample instability. The confidence limits for comparing the exposure using peak area ratio of metabolites in animal plasma versus human plasma measured over approximately 1 year apart were comparable to the analysis undertaken side by side on the same days. These statistical analysis results showed it was feasible to compare data generated at different dates with neither authentic standards nor prior knowledge of the analytes.