Cetuximab plus FOLFOXIRI versus cetuximab plus FOLFOX as conversion regimen in RAS/BRAF wild-type patients with initially unresectable colorectal liver metastases (TRICE trial): A randomized controlled trial.

BACKGROUND: It remains unclear whether intensification of the chemotherapy backbone in tandem with an anti-EGFR can confer superior clinical outcomes in a cohort of RAS/BRAF wild-type colorectal cancer (CRC) patients with initially unresectable colorectal liver metastases (CRLM). To that end, we sought to comparatively evaluate the efficacy and safety of cetuximab plus FOLFOXIRI (triplet arm) versus cetuximab plus FOLFOX (doublet arm) as a conversion regimen (i.e., unresectable to resectable) in CRC patients with unresectable CRLM. METHODS AND FINDINGS: This open-label, randomized clinical trial was conducted from April 2018 to December 2022 in 7 medical centers across China, enrolling 146 RAS/BRAF wild-type CRC patients with initially unresectable CRLM. A stratified blocked randomization method was utilized to assign patients (1:1) to either the cetuximab plus FOLFOXIRI (n = 72) or cetuximab plus FOLFOX (n = 74) treatment arms. Stratification factors were tumor location (left versus right) and resectability (technically unresectable versus ≥5 metastases). The primary outcome was the objective response rate (ORR). Secondary outcomes included the median depth of tumor response (DpR), early tumor shrinkage (ETS), R0 resection rate, progression-free survival (PFS), overall survival (not mature at the time of analysis), and safety profile. Radiological tumor evaluations were conducted by radiologists blinded to the group allocation. Primary efficacy analyses were conducted based on the intention-to-treat population, while safety analyses were performed on patients who received at least 1 line of chemotherapy. A total of 14 patients (9.6%) were lost to follow-up (9 in the doublet arm and 5 in the triplet arm). The ORR was comparable following adjustment for stratification factors, with 84.7% versus 79.7% in the triplet and doublet arms, respectively (odds ratio [OR] 0.70; 95% confidence intervals [CI] [0.30, 1.67], Chi-square p = 0.42). Moreover, the ETS rate showed no significant difference between the triplet and doublet arms (80.6% (58/72) versus 77.0% (57/74), OR 0.82, 95% CI [0.37, 1.83], Chi-square p = 0.63). Although median DpR was higher in the triplet therapy group (59.6%, interquartile range [IQR], [50.0, 69.7] versus 55.0%, IQR [42.8, 63.8], Mann-Whitney p = 0.039), the R0/R1 resection rate with or without radiofrequency ablation/stereotactic body radiation therapy was comparable with 54.2% (39/72) of patients in the triplet arm versus 52.7% (39/74) in the doublet arm. At a median follow-up of 26.2 months (IQR [12.8, 40.5]), the median PFS was 11.8 months in the triplet arm versus 13.4 months in the doublet arm (hazard ratio [HR] 0.74, 95% CI [0.50, 1.11], Log-rank p = 0.14). Grade ≥ 3 events were reported in 47.2% (35/74) of patients in the doublet arm and 55.9% (38/68) of patients in the triplet arm. The triplet arm was associated with a higher incidence of grade ≥ 3 neutropenia (44.1% versus 27.0%, p = 0.03) and diarrhea (5.9% versus 0%, p = 0.03). The primary limitations of the study encompass the inherent bias in subjective surgical decisions regarding resection feasibility, as well as the lack of a centralized assessment for ORR and resection. CONCLUSIONS: The combination of cetuximab with FOLFOXIRI did not significantly improve ORR compared to cetuximab plus FOLFOX. Despite achieving an enhanced DpR, this improvement did not translate into improved R0 resection rates or PFS. Moreover, the triplet arm was associated with an increase in treatment-related toxicity. TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT03493048.

Comprehensive analysis of m6A modification in immune infiltration, metabolism and drug resistance in hepatocellular carcinoma.

N6-methyladenosine (m6A) is important in regulating mRNA stability, splicing, and translation, and it also contributes to tumor development. However, there is still limited understanding of the comprehensive effects of m6A modification patterns on the tumor immune microenvironment, metabolism, and drug resistance in hepatocellular carcinoma (HCC). In this study, we utilized unsupervised clustering based on the expression of 23 m6A regulators to identify m6A clusters. We identified differential m6A modification patterns and characterized m6A-gene-cluster A, which exhibited poorer survival rates, a higher abundance of Treg cells, and increased expression of TGFß in the tumor microenvironment (TME). Additionally, m6A-gene-cluster A demonstrated higher levels of glycolysis activity, cholesterol metabolism, and fatty acid biosynthesis. We also found that the m6A score was associated with prognosis and drug resistance. Patients with a low m6A score experienced worse prognoses, which were linked to an abundance of Treg cells, upregulation of TGFß, and increased metabolic activity. HCC patients with a higher m6A score showed improved prognosis following sorafenib treatment and immunotherapy. In conclusion, we reveals the association between m6A modification patterns and the tumor immune microenvironment, metabolism, and drug resistance in HCC. Furthermore, the m6A score holds potential as a predictive factor for the efficacy of targeted therapy and immunotherapy in HCC.

Correction: Systemic Delivery of MicroRNA-101 Potently Inhibits Hepatocellular Carcinoma In Vivo by Repressing Multiple Targets.

[This corrects the article DOI: 10.1371/journal.pgen.1004873.].

KLF4 Suppresses the Progression of Hepatocellular Carcinoma by Reducing Tumor ATP Synthesis through Targeting the Mir-206/RICTOR Axis.

To address the increased energy demand, tumor cells undergo metabolic reprogramming, including oxidative phosphorylation (OXPHOS) and aerobic glycolysis. This study investigates the role of Kruppel-like factor 4 (KLF4), a transcription factor, as a tumor suppressor in hepatocellular carcinoma (HCC) by regulating ATP synthesis. Immunohistochemistry was performed to assess KLF4 expression in HCC tissues. Functional assays, such as CCK-8, EdU, and colony formation, as well as in vivo assays, including subcutaneous tumor formation and liver orthotopic xenograft mouse models, were conducted to determine the impact of KLF4 on HCC proliferation. Luciferase reporter assay and chromatin immunoprecipitation assay were utilized to evaluate the interaction between KLF4, miR-206, and RICTOR. The findings reveal low KLF4 expression in HCC, which is associated with poor prognosis. Both in vitro and in vivo functional assays demonstrate that KLF4 inhibits HCC cell proliferation. Mechanistically, it was demonstrated that KLF4 reduces ATP synthesis in HCC by suppressing the expression of RICTOR, a core component of mTORC2. This suppression promotes glutaminolysis to replenish the TCA cycle and increase ATP levels, facilitated by the promotion of miR-206 transcription. In conclusion, this study enhances the understanding of KLF4's role in HCC ATP synthesis and suggests that targeting the KLF4/miR-206/RICTOR axis could be a promising therapeutic approach for anti-HCC therapeutics.

Characterization of sandalwood (E,E)-α-farnesene synthase whose overexpression enhances cold tolerance through jasmonic acid biosynthesis and signaling in Arabidopsis.

MAIN CONCLUSION: Santalum album (E,E)-α-farnesene synthase catalyzes FPP into (E,E)-α-farnesene. Overexpression of the SaAFS gene positively improved cold stress tolerance through JA biosynthesis and signaling pathways in Arabidopsis. Volatile terpenoids are released from plants that suffer negative effects following exposure to various biotic and abiotic stresses. Recent studies revealed that (E,E)-α-farnesene synthase (AFS) plays a significant role in a plant's defence against biotic attack. However, little is known about whether AFS contributes to plant resistance to cold stress. In this study, a SaAFS gene was isolated from Indian sandalwood (Santalum album L.) and functionally characterized. The SaAFS protein mainly converts farnesyl diphosphate to (E,E)-α-farnesene. SaAFS was clustered into the AFS clade from angiosperms, suggesting a highly conserved enzyme. SaAFS displayed a significant response to cold stress and methyl jasmonate. SaAFS overexpression (OE) in Arabidopsis enhanced cold tolerance by increasing proline content, reducing malondialdehyde content, electrolyte leakage, and accumulating reactive oxygen species. Transcriptomic analysis revealed that upregulated genes related to stress response and JA biosynthesis and signaling were detected in SaAFS-OE lines compared with wild type plants that were exposed to cold stress. Endogenous JA and jasmonoyl-isoleucine content increased significantly in SaAFS-OE lines exposed to cold stress. Collectively considered, these results suggest that the SaAFS gene is a positive regulator during cold stress tolerance via JA biosynthesis and signaling pathways.

A novel NHEJ gene signature based model for risk stratification and prognosis prediction in hepatocellular carcinoma.

BACKGROUND: Non-homologous DNA end joining (NHEJ) is the predominant DNA double-strand break (DSB) repair pathway in human. However, the relationship between NHEJ pathway and hepatocellular carcinoma (HCC) is unclear. We aimed to explore the potential prognostic role of NHEJ genes and to develop an NHEJ-based prognosis signature for HCC. METHODS: Two cohorts from public database were incorporated into this study. The Kaplan-Meier curve, the Least absolute shrinkage and selection operator (LASSO) regression analysis, and Cox analyses were implemented to determine the prognostic genes. A NHEJ-related risk model was created and verified by independent cohorts. We derived enriched pathways between the high- and low-risk groups using Gene Set Enrichment Analysis (GSEA). CIBERSORT and microenvironment cell populations-counter algorithm were used to perform immune infiltration analysis. XRCC6 is a core NHEJ gene and immunohistochemistry (IHC) was further performed to elucidate the prognostic impact. In vitro proliferation assays were conducted to investigate the specific effect of XRCC6. RESULTS: A novel NHEJ-related risk model was developed based on 6 NHEJ genes and patients were divided into distinct risk groups according to the risk score. The high-risk group had a poorer survival than those in the low-risk group (P < 0.001). Meanwhile, an obvious discrepancy in the landscape of the immune microenvironment also indicated that distinct immune status might be a potential determinant affecting prognosis as well as immunotherapy reactiveness. High XRCC6 expression level associates with poor outcome in HCC. Moreover, XRCC6 could promote HCC cell proliferation in vitro. CONCLUSIONS: In brief, this work reveals a novel NHEJ-related risk signature for prognostic evaluation of HCC patients, which may be a potential biomarker of HCC immunotherapy.

A hepatocyte differentiation model reveals two subtypes of liver cancer with different oncofetal properties and therapeutic targets.

Clinical observation of the association between cancer aggressiveness and embryonic development stage implies the importance of developmental signals in cancer initiation and therapeutic resistance. However, the dynamic gene expression during organogenesis and the master oncofetal drivers are still unclear, which impeded the efficient elimination of poor prognostic tumors, including human hepatocellular carcinoma (HCC). In this study, human embryonic stem cells were induced to differentiate into adult hepatocytes along hepatic lineages to mimic liver development in vitro. Combining transcriptomic data from liver cancer patients with the hepatocyte differentiation model, the active genes derived from different hepatic developmental stages and the tumor tissues were selected. Bioinformatic analysis followed by experimental assays was used to validate the tumor subtype-specific oncofetal signatures and potential therapeutic values. Hierarchical clustering analysis revealed the existence of two subtypes of liver cancer with different oncofetal properties. The gene signatures and their clinical significance were further validated in an independent clinical cohort and The Cancer Genome Atlas database. Upstream activator analysis and functional screening further identified E2F1 and SMAD3 as master transcriptional regulators. Small-molecule inhibitors specifically targeting the oncofetal drivers extensively down-regulated subtype-specific developmental signaling and inhibited tumorigenicity. Liver cancer cells and primary HCC tumors with different oncofetal properties also showed selective vulnerability to their specific inhibitors. Further precise targeting of the tumor initiating steps and driving events according to subtype-specific biomarkers might eliminate tumor progression and provide novel therapeutic strategy.

Unique molecular characteristics of NAFLD-associated liver cancer accentuate ß-catenin/TNFRSF19-mediated immune evasion.

BACKGROUND & AIMS: The hepatic manifestation of the metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), can lead to the development of hepatocellular carcinoma (HCC). Despite a strong causative link, NAFLD-HCC is often underrepresented in systematic genome explorations. METHODS: Herein, tumor-normal pairs from 100 patients diagnosed with NAFLD-HCC were subject to next-generation sequencing. Bioinformatic analyses were performed to identify key genomic, epigenomic and transcriptomic events associated with the pathogenesis of NAFLD-HCC. Establishment of primary patient-derived NAFLD-HCC culture was used as a representative human model for downstream in vitro investigations of the underlying CTNNB1 S45P driver mutation. A syngeneic immunocompetent mouse model was used to further test the involvement of CTNNB1mutand TNFRSF19 in reshaping the tumor microenvironment. RESULTS: Mutational processes operative in the livers of patients with NAFLD inferred susceptibility to tumor formation through defective DNA repair pathways. Dense promoter mutations and dysregulated transcription factors accentuated activated transcriptional regulation in NAFLD-HCC, in particular the enrichment of MAZ-MYC activities. Somatic events common in HCCs arising from NAFLD and viral hepatitis B infection underscore similar driver pathways, although an incidence shift highlights CTNNB1mut dominance in NAFLD-HCC (33%). Immune exclusion correlated evidently with CTNNB1mut. Chromatin immunoprecipitation-sequencing integrated with transcriptome and immune profiling revealed a unique transcriptional axis, wherein CTNNB1mut leads to an upregulation of TNFRSF19 which subsequently represses senescence-associated secretory phenotype-like cytokines (including IL6 and CXCL8). This phenomenon could be reverted by the Wnt-modulator ICG001. CONCLUSIONS: The unique mutational processes in the livers of patients with NAFLD and NAFLD-HCC allude to a "field effect" involving a gain-of-function role of CTNNB1 mutations in immune exclusion. LAY SUMMARY: The increasing prevalence of metabolic syndrome in adult populations means that NAFLD is poised to be the major cause of liver cancer in the 21st century. We showed a strong "field effect" in the livers of patients with NAFLD, wherein activated ß-catenin was involved in reshaping the tumor-immune microenvironment.

Myofibroblast-Specific Msi2 Knockout Inhibits HCC Progression in a Mouse Model.

BACKGROUND AND AIMS: Myofibroblasts play a pivotal role in the development and progression of HCC. Here, we aimed to explore the role and mechanism of myofibroblast Musashi RNA binding protein 2 (MSI2) in HCC progression. APPROACH AND RESULTS: Myofibroblast infiltration and collagen deposition were detected and assessed in the tissues from 117 patients with HCC. Transgenic mice (Msi2ΔCol1a1 ) with floxed Msi2 allele and collagen type I alpha 1 chain (Col1a1)-ligand inducible Cre recombinases (CreER) were constructed to generate a myofibroblast-specific Msi2 knockout model. Mouse HCC cells were orthotopically transplanted into the Msi2ΔCol1a1 or the control mice (Msi2F/F ). We found that the deposition of collagen fibers, the main product of myofibroblasts, predicted a poor prognosis for HCC; meanwhile, we detected high MSI2 expression in the peritumoral infiltrated myofibroblasts. Conditional deletion of Msi2 in myofibroblasts significantly inhibited the growth of orthotopically implanted HCC, reduced both intrahepatic and lung metastasis, and prolonged the overall survival of tumor-bearing mice (P = 0.002). In vitro analysis demonstrated that myofibroblasts promoted cell proliferation, invasion, and epithelial-mesenchymal transformation of HCC cells, whereas Msi2 deletion in myofibroblasts reversed these effects. Mechanically, Msi2 knockout decreased myofibroblast-derived IL-6 and IL-11 secretion by inhibiting the extracellular signal-regulated kinase 1/2 pathway, and thus attenuated the cancer stem cell-promoting effect of myofibroblasts. Interestingly, we found that the simultaneous knockout of Msi2 in myofibroblasts and knockdown of Msi2 in HCC cells could not further attenuate the implanted HCC progression. CONCLUSIONS: Myofibroblast-specific Msi2 knockout abrogated the tumor-promoting function of myofibroblasts and inhibited HCC progression in mouse models. Targeting myofibroblast MSI2 expression may therefore prove to be a therapeutic strategy for HCC treatment in the future.

Radiomics-Based Preoperative Prediction of Lymph Node Metastasis in Intrahepatic Cholangiocarcinoma Using Contrast-Enhanced Computed Tomography.

BACKGROUND: Lymph node (LN) metastasis is significantly associated with worse prognosis for patients with intrahepatic cholangiocarcinoma (ICC). Improvement in preoperative assessment on LN metastasis helps in treatment decision-making. We aimed to investigate the role of radiomics-based method in predicting LN metastasis for patients with ICC. METHODS: A total of 296 patients with ICC who underwent curative-intent hepatectomy and lymphadenectomy at two centers in China were analyzed. Radiomic features, including histogram- and wavelet-based features, shape and size features, and texture features were extracted from four-phase computerized tomography (CT) images. The clinical and conventional radiological variables which were independently associated with LN metastasis were also identified. A combined nomogram predicting LN metastasis was developed, and its performance was determined by discrimination, calibration, and stratification of long-term prognosis. The results were validated by the internal and external validation cohorts. RESULTS: Twenty-four radiomic features were selected into the nomogram. The established nomogram demonstrated good discrimination and calibration, with areas under the curve (AUCs) of 0.98 [95% confidence interval (CI) 0.96-0.99], 0.93 (0.88-0.98), and 0.89 (0.81-0.96) in the training and two validation cohorts, respectively. The 5-year overall survival (OS) and recurrence-free survival (RFS) rates of patients with high risk of LN metastasis as grouped by nomogram were poorer than those of patients with low risk in the training cohort (OS 28.8% versus 53.9%, p < 0.001; RFS 26.3% versus 44.2%, p = 0.001). Similar results were observed in the two validation cohorts. CONCLUSIONS: Radiomics-based method provided accurate prediction of LN metastasis and prognostic assessment for ICC patients, and might aid the preoperative surgical decision.

Dopamine receptor D3 is related to prognosis in human hepatocellular carcinoma and inhibits tumor growth.

BACKGROUND: Dopamine receptors have been reported to play important roles in cancer progression. However, the role of dopamine receptor D3 (DRD3) in hepatocellular carcinoma (HCC) remains unclear. METHODS: The expression of DRD3 was detected by immunohistochemistry and real-time qPCR. The prognostic value of DRD3 in patients was investigated by analyzing selected databases, including cBioPortal and Kaplan-Meier plotter. Cell growth was tested by CCK8 assay, and Transwell assays were performed to assess cancer cell migration and invasion. The cAMP/ERK/CREB signaling pathway was evaluated by Western blot analysis and ELISA. An HCC xenograft model was established for in vivo experiments. RESULTS: DRD3 mRNA expression was significantly higher in nontumor tissues than in tumor tissues. Lower protein expression of DRD3 was related to poor recurrence-free survival (RFS) and overall survival (OS). Kaplan-Meier plotter analysis showed that higher expression of DRD3 mRNA was associated with better OS, RFS, disease-specific survival (DSS), and progression-free survival (PFS). cBioPortal analysis revealed that the alteration group, which harbored genetic mutations in DRD3, exhibited poor OS, RFS, DSS and PFS. According to CCK8 and Transwell assays, stable DRD3 overexpression cell line (ex-DRD3-SK-HEP-1) showed weaker proliferation, migration and invasion behaviors. PD128907, a DRD3 agonist, suppressed proliferation, migration and invasion in HCC cell lines, while U99194, a DRD3 antagonist, enhanced proliferation, migration and invasion in HCC cell lines. Western blot analysis and ELISA revealed that stable DRD3 knock-down cell line (sh-DRD3-PLC/PRF/5) and U99194 both increased the protein levels of cAMP, p-ERK and p-CREB; on the other hand, ex-DRD3-SK-HEP-1 and PD128907 decreased the protein levels of cAMP, p-ERK and p-CREB. SCH772984, an ERK antagonist, abolished the effect of U99194 on the malignant biological behaviors of HCC cells. In vivo, PD128907 suppressed tumor growth, and U99194 enhanced tumor growth. CONCLUSION: Our results suggest that down-regulation of DRD3 is strongly involved in the progression of HCC, and DRD3 might be consider as an independent prognostic factor for HCC. Furthermore, DRD3 agonists may be a promising strategy for HCC therapy.

PARP inhibitor Olaparib overcomes Sorafenib resistance through reshaping the pluripotent transcriptome in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide with very poor prognosis. Resistance to targeted therapeutic drugs such as sorafenib remains one of the major challenges in clinical treatment. In the present study, PARP1 was found to be highly expressed in human embryonic stem cells, but progressively decreased upon specified hepatic differentiation. Reactivation of PARP1 expression was also detected in HCC residual tumors after sorafenib treatment in xenograft mouse model, indicating the potential important roles of PARP1 in stem cell pluripotency and HCC sorafenib treatment resistance. Overexpression of PARP1 was frequently observed in HCC patients, and closely associated with poor clinical outcome. Treatment of Sorafenib induced activation of DNA damage repair signaling, which is highly active and essential for maintenance of stem cell pluripotency in HCC residual tumors. PARP inhibitor Olaparib extensively suppressed the DNA damage repair signaling, and significantly inhibited the global pluripotent transcriptional network. The repression of key pluripotent transcriptional factors and DNA damage repair signaling by Olaparib was mainly through CHD1L-mediated condensation of the chromatin structure at their promotor regions. The global reshaping of the pluripotent transcriptome by Olaparib might reinforce Sorafenib in eliminating HCC residual tumors and enhance therapeutic efficiency.

Long-term outcome for colorectal liver metastases: combining hepatectomy with intraoperative ultrasound guided open microwave ablation versus hepatectomy alone.

OBJECTIVE: To compare the long-term outcome of combining hepatectomy with intraoperative ultrasound (IOUS)-guided open microwave ablation (MWA) versus hepatectomy alone in patients with colorectal cancer liver metastases (CRLM). METHOD: A retrospective analysis of patients with CRLM who underwent hepatectomy alone (HT group; 380 patients) or hepatectomy combined with IOUS-guided open MWA (HT + MWA group; 57 patients) from April 2002 to September 2018 was conducted at our center. A propensity score-matched (PSM) analysis was used to reduce data bias between the two groups. RESULTS: The overall survival (OS) and disease-free survival (DFS) were not significantly different between the two groups after matching. Although intrahepatic recurrence was more frequent in the HT + MWA group in both the whole and matched cohort, the two groups exhibited similar rates of extrahepatic recurrence as well as concomitant intra- and extrahepatic recurrence. A higher number of CRLM (>3), larger maximum-size and absence of response to induction chemotherapy were independent risk factors for OS. CONCLUSION: The oncological outcomes of hepatectomy combined with intraoperative open ablation was not significantly different to hepatectomy alone and should be considered as a safe and fair option for patients with difficultly resectable CRLM.

Surgical Resection versus Re-Ablation for Intrahepatic Recurrent Hepatocellular Carcinoma after Initial Ablation Therapy.

BACKGROUND AND AIMS: Whether surgical resection or repeated ablation should be recommended for intrahepatic recurrent hepatocellular carcinoma (HCC) conforming to the Milan criteria after initial ablation remains unclear. In this study, we compared the outcomes of patients who underwent surgical resection with those who underwent re-ablation for recurrent HCC after initial curative-intent ablation. METHODS: The data of 28 and 98 patients who underwent surgical resection and re-ablation, respectively, for recurrent HCC after initial ablation between January 2003 and 2017 were analyzed using propensity score matching. RESULTS: Before matching, the 1-, 3-, and 5-year overall survival (OS) rates were 95.7, 83.0, and 74.4% for the ablation group, compared to 92.9, 89.1, and 70.9% for the resection group (p = 0.490). The corresponding disease-free survival (DFS) rates were 67.5, 40.1, and 25.6% for the ablation group and were 85.4, 59.9, and 53.3% for the resection group (p = 0.018). After matching, the 1-, 3-, and 5-year OS rates for the ablation and resection group were 95.2, 85.5 and 81.8% versus 96.0, 96.0, and 76.4%, respectively (p = 0.550). The 1-, 3-, and 5-year DFS rates were 58.0, 39.5, and 29.9% for the ablation group and were 95.8, 67.2, and 59.8% for the resection group (p = 0.004). Cox proportional hazards model identified surgical resection as the only significant prognostic factor for DFS but not for OS. CONCLUSION: For intrahepatic recurrent HCC patients after initial ablation, surgical resection could provide better DFS than re-ablation, while no difference in OS was observed between the 2 treatment groups.

Histopathological growth patterns correlate with the immunoscore in colorectal cancer liver metastasis patients after hepatectomy.

Various scoring systems have been proposed to predict the postoperative prognosis of colorectal liver metastasis (CRLM), including the clinical risk score (CRS), the immunoscore and so on. Recently, histopathological growth patterns (HGPs) have been recognized. However, the correlation between HGPs and the immunoscore, and their prognostic values in patients with CRLM after liver resection remain undetermined. In this study, HGPs were retrospectively evaluated in H&E-stained slides from 166 CRLM patients. The immunoscore was calculated according to the densities of immunostained CD3 + and CD8 + cells. A risk score combining HGPs, the immunoscore and the CRS was defined and divided patients into the low-, medium- and high-risk group. Our results showed that the densities of CD3 + and CD8 + cells were higher in the desmoplastic HGP (dHGP) group than in the non-dHGP group, and the proportion of high immunoscores was also higher in the dHGP group (51.9% vs. 33.0%, respectively, P = 0.020). Patients with the dHGP had significantly longer relapse-free survival (RFS) and overall survival (OS) than those with the non-HGP. The low-risk group showed significantly higher 2-year RFS and 5-year OS rates than the other two groups (RFS: 76.2%, 43.7% and 33.1%, respectively; P < 0.001; OS: 89.7%, 54.4% and 33.3%, respectively; P < 0.001). In conclusion, the dHGP correlates with relatively high immunoscores, predicting a favorable prognosis independent of the immunoscore and CRS. A novel risk score combining HGPs, the immunoscore and the CRS may be used for the stratification of CRLM patients' survival.

Comparison of the prognostic value of inflammation-based scores in early recurrent hepatocellular carcinoma after hepatectomy.

BACKGROUND & AIMS: Inflammation-based prognostic scores, such as the Glasgow Prognostic Score (GPS), modified Glasgow Prognostic Score (mGPS), Prognostic Index (PI), Prognostic Nutritional Index (PNI), neutrophil to lymphocyte ratio (NLR), platelet to lymphocyte ratio (PLR), lymphocyte to monocyte ratio (LMR) and systemic immune-inflammation index (SII), are correlated with the survival of hepatocellular carcinoma (HCC) patients, while remain unclear for recurrent HCC. This study aimed to compare the prognostic value of inflammation-based prognostic scores for post-recurrence survival (PRS) in patients with early recurrent HCC (ErHCC, within 2 years after hepatectomy). METHODS: A total of 580 patients with ErHCC were enrolled retrospectively. The association between the independent baseline and the time-dependent variables and PRS was evaluated by cox regression. The prediction accuracy of the inflammation-based prognostic scores was assessed by time-dependent receiver operating characteristic (ROC) and Harrell's concordance index (C-index) analyses. RESULTS: The GPS, mGPS, PI, PNI, NLR, PLR, LMR and SII were all related to the PRS of ErHCC patients, while only the SII (P < .001) remained an independent predictor for PRS in multivariate analysis (hazard ratio: 1.92, 95% confidence interval: 1.33-2.79). Both the C-index of the SII (0.65) and the areas under the ROC curves showed that the SII score was superior to the other inflammation-based prognostic scores for predicting the PRS of ErHCC patients. CONCLUSIONS: The SII is a useful prognostic indicator for PRS in patients with ErHCC after hepatectomy and is superior to the other inflammation-based prognostic scores in terms of prognostic ability.

Bioactive Polyhydroxanthones from Penicillium purpurogenum.

Eight new polyhydroxanthones, penicixanthones A-H (1-8), including four monomers (1-4) and four dimers (5-8), were isolated from solid cultures of Penicillium purpurogenum SC0070. Their structures were elucidated by extensive spectroscopic analysis, X-ray single-crystal diffraction, and theoretical computations of ECD spectra. Penicixanthone B (2) has a hexahydroxanthone structure featuring an unusual oxygen bridge between C-6 and C-8a. Penicixanthone D (4) is distinct from other penicixanthones in stereochemistry, and its biosynthetic mechanism was proposed based on theoretical simulations for the reaction pathway of C-10a epimerization. Penicixanthone G (6) exhibited the most potent cytotoxicity (IC50: 0.3-0.6 µM) when tested against human carcinoma A549, HeLa, and HepG2 cells, whereas it was nontoxic to the normal Vero cells (IC50 > 50 µM). It also displayed the strongest antibacterial activity (MIC: 0.4 µg/mL) against both Staphylococcus aureus and the methicillin-resistant strain MRSA.

Primary tumor location affects recurrence-free survival for patients with colorectal liver metastases after hepatectomy: a propensity score matching analysis.

BACKGROUND: Whether primary tumor location of colorectal cancer (CRC) affects survival of patients after resection of liver metastases remains controversial. This study was conducted to investigate the differences in clinicopathological characteristics and prognosis between right-sided CRC and left-sided CRC patients with liver metastases after hepatectomy. METHODS: From 2002 to 2018, 611 patients with colorectal liver metastases (CRLM) who underwent hepatectomy at our center were reviewed. Primary tumors located from the cecum to transverse colon were defined as right-sided group (n = 141); tumors located from the splenic flexure to rectum were defined as left-sided group (n = 470). Patients were compared between two groups before and after a 1:1 propensity score matching (PSM) analysis. RESULTS: Before PSM, median survival time and 5-year overall survival (OS) rate in right-sided group were 77 months and 56.3%, and those in left-sided group were 64 months and 51.1%, respectively. After PSM, median survival time and 5-year OS rate in right-sided group were 77 months and 55.9%, and those in left-sided group were 58.8 months and 47.3%, respectively. The OS rates did not differ between two groups before and after PSM (P = 0.575, P = 0.453). However, significant different recurrence-free survival (RFS) rate was found before and after PSM between right-sided and left-sided group (P = 0.028, P = 0.003). CONCLUSIONS: Compared to patients with left-sided primary tumors, patients with right-sided primary tumors had a worse RFS but similar OS. Careful preoperative evaluation, intensive preoperative chemotherapy, and frequent follow-up to detect early recurrence might be justified for CRLM patients with right-sided primary tumors.

Germline Duplication of SNORA18L5 Increases Risk for HBV-related Hepatocellular Carcinoma by Altering Localization of Ribosomal Proteins and Decreasing Levels of p53.

BACKGROUND & AIMS: Single nucleotide polymorphisms could affect risk for hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC). We performed a germline copy number variation (CNV)-based genome-wide association study (GWAS) in populations of Chinese ancestry to search for germline CNVs that increase risk of HCC. METHODS: We conducted a CNV-based GWAS of 1583 HCC cases (persons with chronic HBV infection and HCC) and 1540 controls (persons with chronic HBV infection without HCC) in Chinese populations. Identified candidates were expressed in L-02, HepG2, or TP53-/- or wild-type HCT116 cells, and knocked down with short hairpin RNAs in HepG2, Bel-7402, and SMMC-7721 cells; proliferation, colony formation, and apoptosis were measured. Formation of xenograft tumors from cell lines was monitored in nude mice. Subcellular localization of ribosome proteins and levels or activity of p53 were investigated by co-immunoprecipitation, immunofluorescence, and immunoblot analyses. Levels of small nucleolar RNA H/ACA box 18-like 5 (SNORA18L5) were quantified by quantitative reverse transcription polymerase chain reaction. RESULTS: We identified a low-frequency duplication at chromosome 15q13.3 strongly associated with risk of HBV-related HCC (overall P = 3.17 × 10-8; odds ratio, 12.02). Copy numbers of the 15q13.3 duplication correlated with the expression of SNORA18L5 in liver tissues. Overexpression of SNORA18L5 increased HCC cell proliferation and growth of xenograft tumors in mice; knockdown reduced HCC proliferation and tumor growth. SNORA18L5 overexpression in HepG2 and SMMC-7721 cells inhibited p53-dependent cell cycle arrest and apoptosis. Overexpression of SNORA18L5 led to hyperactive ribosome biogenesis, increasing levels of mature 18S and 28S ribosomal RNAs and causing the ribosomal proteins RPL5 and RPL11 to stay in the nucleolus, which kept them from binding to MDM2. This resulted in increased MDM2-mediated ubiquitination and degradation of p53. Levels of SNORA18L5 were increased in HCC tissues compared with nontumor liver tissues and associated with shorter survival times of patients. CONCLUSIONS: In a CNV-based GWAS, we associated duplication at 15q13.3 with increased risk of HBV-related HCC. We found SNORA18L5 at this location to promote HCC cell proliferation and tumor growth in mice. SNORA18L5 increases ribosome biogenesis, facilitates ribosomal RNA maturation, and alters localization of RPL5 and RPL11, allowing for increased MDM2-mediated proteolysis of p53 and cell cycle arrest.

Identification and functional characterization of three new terpene synthase genes involved in chemical defense and abiotic stresses in Santalum album.

BACKGROUND: It is well known that aromatic essential oils extracted from the heartwood of Santalum album L. have wide economic value. However, little is known about the role of terpenoids in response to various adverse environmental stresses as other plants do in the form of signals during plant-environment interactions. RESULTS: In this study, trace amounts of volatiles consisting of α-santalene, epi-ß-santalene, ß-santalene, α-santalol, ß-santalol, (E)-α-bergamotene, (E)-ß-farnesene and ß-bisabolene were found in the leaves of mature S. album trees. We identified more than 40 candidate terpene synthase (TPS) unigenes by mining publicly-available RNA-seq data and characterized the enzymes encoded by three cDNAs: one mono-TPS catalyzes the formation of mostly α-terpineol, and two multifunctional sesqui-TPSs, one of which produces (E)-α-bergamotene and sesquisabinene as major products and another which catalyzes the formation of (E)-ß-farnesene, (E)-nerolidol and (E,E)-farnesol as main products. Metabolite signatures and gene expression studies confirmed that santalol content is closely related with santalene synthase (SaSSY) transcripts in heartwood, which is key enzyme responsible for santalol biosynthesis. However, the expression of three new SaTPS genes differed significantly from SaSSY in the essential oil-producing heartwood. Increased activities of antioxidant enzymes, superoxide dismutase, catalase, peroxidase and ascorbate peroxidase, were detected in different tissues of S. album plants after applying 1 mM methyl jasmonate (MeJA) and 1 mM salicylic acid (SA), or exposure to 4°C, 38°C and high light intensity. MeJA and SA dramatically induced the expression of SaTPS1 and SaTPS2 in leaves. SaTPS1 to 3 transcripts were differentially activated among different tissues under adverse temperature and light stresses. In contrast, almost all SaSSY transcripts decreased in response to these environmental stresses, unlike SaTPS1 to 3. CONCLUSIONS: Multifunctional enzymes were biochemically characterized, including one chloroplastic mono-TPS and two cytosolic sesqui-TPSs in sandalwood. Our results suggest the ecological importance of these three new SaTPS genes in defensive response to biotic attack and abiotic stresses in S. album.