New evidence: Metformin unsuitable as routine adjuvant for breast cancer: a drug-target mendelian randomization analysis.

PURPOSE: The potential efficacy of metformin in breast cancer (BC) has been hotly discussed but never conclusive. This genetics-based study aimed to evaluate the relationships between metformin targets and BC risk. METHODS: Metformin targets from DrugBank and genome-wide association study (GWAS) data from IEU OpenGWAS and FinnGen were used to investigate the breast cancer (BC)-metformin causal link with various Mendelian Randomization (MR) methods (e.g., inverse-variance-weighting). The genetic association between type 2 diabetes (T2D) and the drug target of metformin was also analyzed as a positive control. Sensitivity and pleiotropic tests ensured reliability. RESULTS: The primary targets of metformin are PRKAB1, ETFDH and GPD1L. We found a causal association between PRKAB1 and T2D (odds ratio [OR] 0.959, P = 0.002), but no causal relationship was observed between metformin targets and overall BC risk (PRKAB1: OR 0.990, P = 0.530; ETFDH: OR 0.986, P = 0.592; GPD1L: OR 1.002, P = 0.806). A noteworthy causal relationship was observed between ETFDH and estrogen receptor (ER)-positive BC (OR 0.867, P = 0.018), and between GPD1L and human epidermal growth factor receptor 2 (HER2)-negative BC (OR 0.966, P = 0.040). Other group analyses did not yield positive results. CONCLUSION: The star target of metformin, PRKAB1, does not exhibit a substantial causal association with the risk of BC. Conversely, metformin, acting as an inhibitor of ETFDH and GPD1L, may potentially elevate the likelihood of developing ER-positive BC and HER2-negative BC. Consequently, it is not advisable to employ metformin as a standard supplementary therapy for BC patients without T2D.

Down-regulation of ALDOB during metabolic reprogramming mediates malignant behavior in hepatocellular carcinoma and insensitivity to postoperative adjuvant transarterial chemoembolization.

BACKGROUND: Postoperative transarterial chemoembolization (PA-TACE) is an effective adjuvant therapy for preventing early postoperative recurrence of hepatocellular carcinoma (HCC); however, many patients are insensitive to it. Therefore, the present study aimed to explore the in-depth reasons for PA-TACE resistance and provide a reliable basis for selecting patients who will benefit the most from PA-TACE. METHODS: The unique gene expression profiles of primary tumors from PA-TACE-sensitive or -insensitive patients were analyzed using microarray data. Combined differential expression analysis, gene set enrichment analysis (GSEA), and weighted correlation network analysis (WGCNA) were used to screen for potential drivers of PA-TACE insensitivity. The expression of ALDOB was silenced or overexpressed in hepatoma cell lines, and changes in glycolytic activity, cycle, apoptosis, and malignant biological phenotypes were observed under normoxia and hypoxia. Finally, an animal model was constructed to verify the effects of ALDOB dysregulation on the tumorigenic ability of HCC cells in vivo. RESULTS: The inhibition of ALDOB promoted the up-regulation of Ki67 expression, and glycolytic activity was significantly enhanced. Moreover, the proliferation, invasion, and migration capabilities were increased in HCC cells and even worse in hypoxia. This advantage of malignant behavior was also validated using in vivo models. CONCLUSION: Down-regulation of ALDOB may underlie the metabolic reprogramming observed in HCC by promoting the malignant behavior of HCC cells. Hypoxia and ALDOB down-regulation acted additively, which was closely related to PA-TACE insensitivity. The use of ALDOB and Ki67 as a combined marker has the potential to identify the 'PA-TACE beneficiary population'.

Clinical implications and biological features of a novel postoperative recurrent HCC classification: A multi-centre study.

BACKGROUND & AIMS: The multiplicity of hepatocellular carcinoma (HCC) recurrence patterns is the most important determinant of patients' postsurgical survival. A systematic HCC recurrence classification is needed to help prevent and treat postoperative HCC recurrence in the era of precision medicine. METHODS: A total of 1319 patients with recurrent HCC from four hospitals were enrolled and divided into a development cohort (n = 916), internal validation cohort (n = 225) and external validation cohort (n = 178). A comprehensive study of patients' clinicopathological factors and biological features was conducted. RESULTS: Four subtypes of recurrence were identified, which integrated recurrence features, survival, effects on systemic and liver function and potential therapeutics after recurrence: type I (solitary-intrahepatic oligorecurrence); type II (multi-intrahepatic oligorecurrence); type III (progression recurrence) and type IV (hyper-progression recurrence). Type III~IV recurrence indicated exceptionally poor prognosis. Subsequently, two nomogram models were established for type III~IV recurrence prediction, and both demonstrated excellent predictive performance and applicability of pre and postoperative strategy formulation. Multiple biological analyses revealed that HCC cases with type III~IV recurrence were characterized by enrichment in p53 mutations, CCND1 amplification, high proliferation/metastasis potential, inactive metabolism and immune exhaustion features. Over-expression of high mobility group protein 2 (HMGA2) enhanced the highly malignant behaviour of HCC through multiple molecular pathways, making it a potential prognostic predictor and therapeutic target. CONCLUSIONS: This 'recurrent HCC classification' has important potential value in identifying patients with surgical benefit, predicting postsurgical survival and guiding treatment strategies. Multidimensional biological insights also increased knowledge of factors associated with HCC recurrence.

Outcomes of postoperative adjuvant transarterial chemoembolization for hepatocellular carcinoma according to the Ki67 index.

Aim: To assess whether Ki67 is related to the efficacy of postoperative adjuvant transarterial chemoembolization (PA-TACE) in hepatocellular carcinoma patients at high risk of postsurgical recurrence. Methods: A total of 716 patients undergoing surgical resection with or without PA-TACE were retrospectively enrolled. Immunohistochemistry was used to analyze Ki67 expression. Results: There was no significant difference in tumor-free survival between patients who underwent resection with or without chemoembolization. However, chemoembolization was associated with significantly higher tumor-free survival rates among patients with 'low' (<30%) or 'moderate' (30-59%) levels of Ki67. Patients highly expressing Ki67 displayed higher rates of overall recurrence, earlier recurrence, multiple intrahepatic recurrence and extrahepatic metastasis. Conclusion: In patients with relatively high Ki67 levels, PA-TACE does not appear to improve outcomes.

Postoperative adjuvant transarterial chemoembolization (PA-TACE), as an adjuvant treatment to surgery, is widely recommended in patients with high-risk factors for recurrence. Nevertheless, some studies challenge whether it actually improves prognosis, thus the influence of PA-TACE on prognosis remains controversial. The present research indicated that the ability of PA-TACE to help inhibit hepatocellular carcinoma recurrence is conditionally restrictive, and it appears to be beneficial only in those patients with a low or moderate Ki67 index (<60%). For patients with high Ki67 expression, compared with PA-TACE, 'adjuvant immunotherapy' may be a potential alternative option. This finding suggests a valuable reference to identify the best beneficiaries of PA-TACE for individualized treatment.

Nomograms for postsurgical extrahepatic recurrence prediction of hepatocellular carcinoma based on presurgical circulating tumor cell status and clinicopathological factors.

BACKGROUND AND AIMS: Extrahepatic recurrence (EHR) is one of the major reasons for the poor prognosis of hepatocellular carcinoma (HCC). The present study aimed to develop and assess the performance of predictive models by using a combination of presurgical circulating tumor cell (CTCs) data and clinicopathological features to screen patients at high risk of EHR to achieve precise decision-making. PATIENTS AND METHODS: A total of 227 patients with recurrent HCC and preoperative CTC data from January 2014 to August 2019 were enrolled. All patients were randomly assigned to one of two cohorts: development or validation. Two preoperative and postoperative nomogram models for EHR prediction were developed and multi-dimensionally validated. RESULTS: Patients with EHR had generally lower recurrence-free survival (p < 0.001), and overall survival (p < 0.001), and significantly higher CTC counts (epithelial CTCs, epithelial/mesenchymal hybrid CTCs, and mesenchymal CTCs count, all p < 0.05) than those without EHR. Univariate and multivariate analyses revealed that EHR was associated with four risk factors in the development cohort: total CTC count (p = 0.014), tumor size (p = 0.028), node number (p = 0.045), and microvascular invasion (p = 0.035). These factors were incorporated into two nomogram models (preoperative and postoperative), which reliably predicted EHR through multidimensional verification (e.g., calibration plot, receiver operating characteristic analysis, decision curve analysis, and clinical impact curve analysis) in the development and validation cohorts, respectively. With threshold of scores of 100.3 and 176.8 before and after surgery respectively, both nomograms were able to stratify patients into two distinct prognostic subgroups (all p < 0.05). CONCLUSION: The present study proposed two nomogram models integrating presurgical CTC counts and clinicopathological risks and showed relatively good predictive performance of EHR, which may be beneficial to the clinical practice of HCC recurrence. Further multicenter studies are needed to assess its general applicability.

Prognostic factors and an innovative nomogram model for patients with hepatocellular carcinoma treated with postoperative adjuvant transarterial chemoembolization.

PURPOSE: The purpose of this study was to estimate the clinical efficacy and identify the best beneficiaries of postoperative adjuvant transcatheter arterial chemoembolization (PA-TACE) in hepatocellular carcinoma (HCC). PATIENTS AND METHODS: A total of 749 HCC patients who underwent surgical resection (380 underwent PA-TACE, 369 had resection only) with a high risk of recurrence were reviewed retrospectively. Patients receiving PA-TACE were randomly split into development and validation cohorts. Univariate and multivariate analyses were performed in the development cohort. A novel model for PA-TACE-insensitivity prediction was built based on univariate and multivariate analysis and was multi-dimensionally validated in the validation set and all samples. RESULTS: After propensity score matching (PSM), in the early-recurrence group, no significant improvement in RFS was achieved with PA-TACE compared to radical hepatic resection alone. PA-TACE insensitive patients were considered as the PA-TACE non-benefit population and were associated with six clinicopathological factors: AFP, node number, tumor capsule, Ki-67 index, MVI, and complications in the development cohort. These factors were incorporated into a nomogram model, which reliably predicted PA-TACE insensitivity, with concordance indices of 0.874 and 0.897 for the development and validation cohort, respectively. In the overall sample, PA-TACE did not significantly improve patients' RFS and OS in the high-score group, while the low-score group had statistical significance. Recurrence pattern diversity was also found to be a factor leading to PA-TACE insensitivity. CONCLUSION: We constructed a new PA-TACE-insensitivity prediction model with potential clinical value. The good predictive performance and availability would allow this model to effectively screen PA-TACE beneficiaries.KEY MESSAGESThe independent influencing factors of PA-TACE insensitivity in patients who received PA-TACE were analyzed to construct a predictive model and its clinical application performance was verified with multi-dimensional methods.PA-TACE treatment should be avoided for patients with high scores according to this model, while it should be cautiously recommended for patients with low scores after multiple considerations.Compared with other related models, this model has obvious advantages in versatility and effectiveness. It can effectively screen the best benefit population of PA-TACE and provide a reliable reference for the selection of precise treatment plans for patients after radical resection of hepatocellular carcinoma.

Specific gut microbiome signature predicts hepatitis B virus-related hepatocellular carcinoma patients with microvascular invasion.

BACKGROUND: We aimed to assess differences in intestinal microflora between patients with operable hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) with microvascular invasion (MVI) and those without MVI. Additionally, we investigated the potential of the microbiome as a non-invasive biomarker for patients with MVI. METHODS: We analyzed the preoperative gut microbiomes (GMs) of two groups, the MVI (n = 46) and non-MVI (n = 56) groups, using 16S ribosomal RNA gene sequencing data. At the operational taxonomic unit level, we employed random forest models to predict MVI risk and validated the results in independent validation cohorts [MVI group (n = 17) and non-MVI group (n = 15)]. RESULTS: ß diversity analysis, utilizing weighted UniFrac distances, revealed a significant difference between the MVI and non-MVI groups, as indicated by non-metric multidimensional scaling and principal coordinate analysis. We also observed a significant correlation between the characteristic intestinal microbial communities at the genus level and their main functions. Nine optimal microbial markers were identified, with an area under the curve of 79.76% between 46 MVI and 56 non-MVI samples and 79.80% in the independent verification group. CONCLUSION: This pioneering analysis of the GM in patients with operable HBV-HCC with and without MVI opens new avenues for treating HBV-HCC with MVI. We successfully established a diagnostic model and independently verified microbial markers for patients with MVI. As preoperative targeted biomarkers, GM holds potential as a non-invasive tool for patients with HBV-HCC with MVI.

Combination of Preoperative Circulating Tumor Cell Count and Neutrophil-Lymphocyte Ratio for Prognostic Prediction in Hepatocellular Carcinoma Patients after Curative Hepatectomy.

Background: Both the preoperative neutrophil-lymphocyte ratio (NLR) and circulating tumor cell count (CTC) are associated with poor prognosis in hepatocellular carcinoma (HCC). The purpose of this study was to explore the prognostic value of these two indices (CTC-NLR) in HCC. Methods: We retrospectively collected demographic and clinical data, including NLR and CTC, from 97 patients with HCC who underwent curative hepatectomy at our institution from March 2014 to May 2017. X-Tile software was used to confirm the optimal cut-off value of NLR and CTC for predicting overall survival (OS) in this study. OS were also analyzed using Kaplan-Meier and Cox regression methods. Based on preoperative CTC and NLR, patients were divided into three groups: CTC-NLR (0), CTC-NLR (1), and CTC-NLR (2). Relationships of CTC-NLR with clinicopathological factors and survival were evaluated. Results: Preoperatively, CTC positively correlated with NLR. Patients with NLR and CTC higher than the cut-offs had shorter OS than patients with low NLR and CTC. Kaplan-Meier analysis, and log-rank tests revealed significantly lower OS among patients with CTC-NLR scores of 0, 1, and 2. Uni- and multivariate analyses showed that CTC-NLR (hazard ratio 2.050, P = 0.005), CTC (hazard ratio 2.285, P = 0.032), and NLR (hazard ratio 1.902, P = 0.048) were independent predictor of OS. A time-dependent ROC curve indicated that the prognostic efficacy of the CTC-NLR at 1 year (0.714) was better than that of NLR (0.687) and CTC (0.590); the prognostic efficacy of the CTC-NLR at 2 years (0.746) was better than that of NLR (0.711) and CTC (0.601); the prognostic efficacy of the CTC-NLR at 3 years (0.742) was better than that of NLR (0.694) and CTC (0.629). Conclusions: HCC patients with higher NLR and CTC tend to show shorter OS. Preoperative CTC-NLR may be associated with poor survival and might be a reliable prognostic predictor in HCC after curative hepatectomy.

Gut microbiome dysbiosis in patients with hepatitis B virus-related hepatocellular carcinoma after extended hepatectomy liver failure.

Background: Hepatitis B virus-related hepatocellular carcinoma (B-HCC) negatively affects the gut microbiome. This study aimed to investigate the gut microbiome profiles and functions post-hepatectomy liver failure (PHLF) after extended hepatectomy (e-PHLF) to obtain valuable insights, identify potential diagnostic biomarkers, and assist in the treatment of this disease. Methods: B-HCC patients who underwent extended hepatectomy were consecutively recruited and divided into Group A (n=15) and Group B (n=15) based on the presence and absence of e-PHLF, respectively. The relationships between gut microbiota and extended hepatectomy liver failure were explored using 16S ribosomal RNA (16S rRNA) gene sequencing data. Results: Following extended hepatectomy, the α-diversity of Group A was significantly higher than that of Group B (Shannon P=0.034 or Simpson P=0.031), and the ß-diversity differed significantly between Groups A and B (P=0.004, R=0.100). At the genus level, 10 bacterial genera (Bacteroides, Pantoea, Methylobacterium-Methylorubrum, Inquilinus, Mycobacterium, Allisonella, Helicobacter, GCA-900066575, IS-44, and Faecalibacterium) were significantly enriched in Group A, whereas five genera (Papillibacter, Scardovia, Turicibacter, Catabacter, and Senegalimassilia) were significantly enriched in Group B. The highly abundant genera Bacteroides, Pantoea, Faecalibacterium, and Turicibacter participated in multiple amino acid metabolism pathways, organic acid metabolism pathways, pyrimidine metabolism pathways, palmitate biosynthesis, and stearate biosynthesis. Redundancy analysis showed that four environmental factors (total bilirubin, international normalized ratio, prealbumin, and albumin) were significantly correlated with intestinal microorganisms. The formation of interaction networks between different gut microbiomes revealed important correlations between the gut microbiome, and there was a significant correlation between the highly abundant gut microbiome and main functions. Conclusions: The gut microbiota characteristics in B-HCC patients after extended hepatectomy liver failure might allow for the use of non-invasive biomarkers for disease diagnosis and treatment.

Integrated omics analysis: the relationship between significantly increased Klebsiella post-hepatectomy and decreased hub-metabolite 3-methyl-2-oxobutanoic acid is associated with induced liver failure.

Background: This study sought to evaluate the association between intestinal Klebsiella and post-hepatectomy liver failure (PHLF) in patients with hepatitis B virus (HBV)-related hepatocellular carcinoma (B-HCC), and identify the inner relationship. Methods: Patients with B-HCC were divided into Groups A and B based on the presence or absence of PHLF. 16S ribosomal ribonucleic acid surveys were used to identify gut microbiome alterations. PICRUST2 was used to examine the metagenomic data in PHLF patients. Fecal and serum samples were processed by chromatography-mass spectrometry based non-targeted metabonomics, then comprehensively analyzed to obtain hub metabolites. A Spearman correlation analysis was then conducted to find any associations between fecal differential metabolites and the relative abundance of differential microbes. Results: Hepatectomies were significantly associated with a gut microbial imbalance in B-HCC patients, and a significant elevation of Klebsiella abundance was observed in PHLF patients. Klebsiella appears to act on 13 amino acid-related pathways, especially significantly observed in branched-chain amino acid (BCAA) metabolic pathways. Additionally, Klebsiella was found to be highly correlated with 3-methyl-2-oxobutanoic acid shared by feces and serum in the BCAA metabolic pathway. Conclusions: Hepatectomy can lead to an imbalance of intestinal microflora in B-HCC patients. Due to its potential connections with 3-methyl-2-oxobutanoic acid in the BCAA pathway, significantly increased Klebsiella has the potential to be an evaluation indicator of PHLF in B-HCC patients. Moreover, 3-methyl-2-oxobutanoic acid has research value in PHLF-targeted treatments.

Operable hepatitis B virus-related hepatocellular carcinoma: gut microbiota profile of patients at different ages.

Background: Age was important prognostic factors for operable hepatocellular carcinoma patients. The aim of the present study was to assess the difference in gut microbiota in patients with operable hepatitis B virus-related hepatocellular carcinoma (HBV-HCC) at different ages ; to investigate the features of the microbiota and its function associated with different ages; to provide a preliminary look at effects of the gut microbiota dimension on prognostic. Methods: From September 2020 to May 2021, patients with HBV-HCC were able to undergo liver resection and were recruited consecutively and divided into the younger age group (age <45 years) (Y.AG) (n=20), middle age group (age from 45 to 65 years) (M.AG) (n=13) 45-65 years, and older age group (age >65 years) (O.AG) (n=20). The relationships between gut microbiota and different ages were explored using 16S rRNA gene sequencing data. PICRUST2 was used to examine the metagenomic data in PHLF patients. Fisher's exact and Mann-Whitney U-test were used for the data analysis. Results: Pairwise comparison between the three groups showed that the α-diversity of Y.AG was significantly higher than that of O.AG (ACE Index, P=0.017; chao1 Index, P=0.031; observed_species Index, P=0.011; and goods_coverage Index, P=0.041). The ß-diversity in the 3 groups differed significantly (stress =0.100), while the composition (ß-diversity) differed significantly between the Y.AG and the M.AG (stress =0.090), the M.AG and the O.AG (stress =0.095), and the Y.AG and the O.AG (stress =0.099). At the genus level, 7 bacterial genera were significantly enriched in the O.AG compared with the Y.AG, of which Streptococcus, Blautia, Erysipelotrichaceae_UCG-003, and Fusicatenibacter represented the major variances in O.AG microbiomes. Eleven genera were significantly increased in the O.AG, of which Prevotella, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Ruminiclostridium, and Phascolarctobacterium represented the major variances in the O.AG. The Y.AG and the O.AG were predicted by PICRUSt2 analysis, which found 72 pathways related to differential gut microbiome at the genus level. Redundancy analysis showed that 7 environmental factors were significantly correlated with intestinal microorganisms, especially in the Y.AG compared with the O.AG. Conclusions: Analysis of gut microbiota characteristics in patients of different ages could ultimately contribute to the development of novel avenues for the treatment of HCC at different ages.

Micro-RNA29b enhances the sensitivity of glioblastoma multiforme cells to temozolomide by promoting autophagy.

To explore whether or not aberrant expression of miR-29b in glioblastoma multiforme (GBM) cells was associated with temozolomide (TMZ) resistance and to elucidate potential underlying mechanisms. Upregulation of miR-29 in GBM cells was achieved by transfecting miR-29b mimics. Changes in cell viability were measured by using CCK-8 assays. Flow cytometry and TUNEL assays were used to quantify the number of apoptotic cells. The expression levels of apoptosis-related proteins as well as autophagy-associated proteins, and the expression levels of both apoptotic and autophagic genes were determined by Western blotting. Autophagy flux was monitored by transfecting mRFP-GFP-LC3 adenovirus. We halted autophagy by introducing Atg 5-specific siRNA or the autophagy inhibitor Bafilomycin A1 (Baf-A1). We also employed a GBM xenograft mice model to confirm the role of miR-29b in vivo. miR-29b overexpression induced inhibition of cell viability, and also induced apoptosis and autophagy in U251 and U87MG cells. Furthermore, upregulation of miR-29b was able to potentiate the level of antitumor activity of TMZ against tested cells. We also found that autophagy induced by miR-29b, at least partially, contributed to the increase of TMZ sensitivity in GBM cells. As was evidenced by blockade of autophagy, the application of Atg 5 siRNA or Baf-A1 was able to significantly reverse these effects. Consistent with observations in vitro, findings of in vivo assessment also confirmed that overexpression of miR-29b was able to effectively halt tumor growth and enhance the antitumor activity of TMZ. miR-29b potentiates TMZ sensitivity against GBM cells by inducing autophagy and the combined use of miR-29 mimic and TMZ might represent a potential therapeutic strategy for GBM patients.

Mucinous adenocarcinoma: A unique clinicopathological subtype in colorectal cancer.

Mucinous adenocarcinoma (MAC) is a unique clinicopathological subtype of colorectal cancer, which is characterized by extracellular mucinous components that comprise at least 50% of the tumor tissue. The clinical characteristics, molecular features, response to chemo-/radiotherapy, and prognosis of MAC are different from that of non-MAC (NMAC). MAC is more common in the proximal colon, with larger volume, higher T-stage, a higher proportion of positive lymph nodes, poorer tumor differentiation, and a higher proportion of peritoneal implants compared to NMAC. Although biopsy is the main diagnostic method for MAC, magnetic resonance imaging is superior in accuracy, especially for rectal carcinoma. The aberrant expression of mucins, including MUC1, MUC2 and MUC5AC, is a notable feature of MAC, which may be related to tumor invasion, metastasis, inhibition of apoptosis, and chemo-/radiotherapy resistance. The genetic origin of MAC is mainly related to BRAF mutation, microsatellite instability, and the CpG island methylator phenotype pathway. In addition, the poor prognosis of rectal MAC has been confirmed by various studies, and that of colonic MAC is still controversial. In this review, we summarize the epidemiology, clinicopathological characteristics, molecular features, methods of diagnosis, and treatments of MAC in order to provide references for further fundamental and clinical research.