ObjectiveTumour pathology contains rich information, including tissue structure and cell morphology, that reflects disease progression and patient survival. However, phenotypic information is subtle ...and complex, making the discovery of prognostic indicators from pathological images challenging.DesignAn interpretable, weakly supervised deep learning framework incorporating prior knowledge was proposed to analyse hepatocellular carcinoma (HCC) and explore new prognostic phenotypes on pathological whole-slide images (WSIs) from the Zhongshan cohort of 1125 HCC patients (2451 WSIs) and TCGA cohort of 320 HCC patients (320 WSIs). A ‘tumour risk score (TRS)’ was established to evaluate patient outcomes, and then risk activation mapping (RAM) was applied to visualise the pathological phenotypes of TRS. The multi-omics data of The Cancer Genome Atlas(TCGA) HCC were used to assess the potential pathogenesis underlying TRS.ResultsSurvival analysis revealed that TRS was an independent prognosticator in both the Zhongshan cohort (p<0.0001) and TCGA cohort (p=0.0003). The predictive ability of TRS was superior to and independent of clinical staging systems, and TRS could evenly stratify patients into up to five groups with significantly different prognoses. Notably, sinusoidal capillarisation, prominent nucleoli and karyotheca, the nucleus/cytoplasm ratio and infiltrating inflammatory cells were identified as the main underlying features of TRS. The multi-omics data of TCGA HCC hint at the relevance of TRS to tumour immune infiltration and genetic alterations such as the FAT3 and RYR2 mutations.ConclusionOur deep learning framework is an effective and labour-saving method for decoding pathological images, providing a valuable means for HCC risk stratification and precise patient treatment.
Chemokines play a key role in orchestrating the recruitment and positioning of myeloid cells within the tumor microenvironment. However, the tropism regulation and functions of these cells in ...hepatocellular carcinoma (HCC) are not completely understood. Herein, by scrutinizing the expression of all chemokines in HCC cell lines and tissues, we found that CCL15 was the most abundantly expressed chemokine in human HCC. Further analyses showed that CCL15 expression was regulated by genetic, epigenetic, and microenvironmental factors, and negatively correlated with patient clinical outcome. In addition to promoting tumor invasion in an autocrine manner, CCL15 specifically recruited CCR1+ cells toward HCC invasive margin, approximately 80% of which were CD14+ monocytes. Clinically, a high density of marginal CCR1+CD14+ monocytes positively correlated with CCL15 expression and was an independent index for dismal survival. Functionally, these tumor‐educated monocytes directly accelerated tumor invasion and metastasis through bursting various pro‐tumor factors and activating signal transducer and activator of transcription 1/3, extracellular signal‐regulated kinase 1/2, and v‐akt murine thymoma viral oncogene homolog signaling in HCC cells. Meanwhile, tumor‐derived CCR1+CD14+ monocytes expressed significantly higher levels of programmed cell death‐ligand 1, B7‐H3, and T‐cell immunoglobulin domain and mucin domain‐3 that may lead to immune suppression. Transcriptome sequencing confirmed that tumor‐infiltrating CCR1+CD14+ monocytes were reprogrammed to upregulate immune checkpoints, immune tolerogenic metabolic enzymes (indoleamine and arginase), inflammatory/pro‐angiogenic cytokines, matrix remodeling proteases, and inflammatory chemokines. Orthotopic animal models confirmed that CCL15‐CCR1 axis forested an inflammatory microenvironment enriched with CCR1+ monocytes and led to increased metastatic potential of HCC cells. Conclusion: A complex tumor‐promoting inflammatory microenvironment was shaped by CCL15‐CCR1 axis in human HCC. Blockade of CCL15‐CCR1 axis in HCC could be an effective anticancer therapy.
Background & Aims The pathogenesis of intrahepatic cholangiocarcinoma (ICC), the second most common hepatic cancer, is poorly understood, and the incidence of ICC is increasing worldwide. We searched ...for mutations in human ICC tumor samples and investigated how they affect ICC cell function. Methods We performed whole exome sequencing of 7 pairs of ICC tumors and their surrounding nontumor tissues to detect somatic alterations. We then screened 124 pairs of ICC and nontumor samples for these mutations, including 7 exomes. We compared mutations in PTPN3 with tumor recurrence in 124 patients and PTPN3 expression levels with recurrence in 322 patients (the combination of both in 86 patients). The functional effects of PTPN3 variations were determined by RNA interference and transgenic expression in cholangiocarcinoma cell lines (RBE, HCCC-9810, and Huh28). Results Based on exome sequencing, pathways that regulate protein phosphorylation were among the most frequently altered in ICC samples and genes encoding protein tyrosine phosphatases (PTPs) were among the most frequently mutated. We identified mutations in 9 genes encoding PTPs in 4 of 7 ICC exomes. In the prevalence screen of 124 paired samples, 51.6% of ICCs contained somatic mutations in at least 1 of 9 PTP genes; 41.1% had mutations in PTPN3 . Transgenic expression of PTPN3 in cell lines increased cell proliferation, colony formation, and migration. PTPN3 L232R and PTPN3 L384H , which were frequently detected in ICC samples, were found to be gain-of-function mutations; their expression in cell lines further increased cell proliferation, colony formation, and migration. ICC-associated variants of PTPN3 altered phosphatase activity. Patients whose tumors contained activating mutations or higher levels of PTPN3 protein than nontumor tissues had higher rates of disease recurrence than patients whose tumors did not have these characteristics. Conclusions Using whole exome sequencing of ICC samples from patients, we found that more than 40% contain somatic mutations in PTPN3 . Activating mutations in and high expression levels of PTPN3 were associated with tumor recurrence.
It has been reported that tetraspanin CD151 acts as a promoter of metastasis in several tumors and plays an important role in c‐Met/hepatocyte growth factor signaling. However, the role of CD151 ...alone and coexpression of CD151/c‐Met in hepatocellular carcinoma (HCC) remains unclear. We found that expression of CD151 was positively related to metastatic potential of HCC cell lines, and modified cells with CD151high showed higher secretion of matrix metalloproteinase 9 and aggressiveness in vitro and higher metastatic ability in vivo. Furthermore, HCC patients with vascular invasion, large tumors, multiple tumors, high tumor‐node‐metastasis stage, and undifferentiated tumor were prone to have higher CD151 expression. The postoperative 3‐, 5‐, and 7‐year overall survival (OS) of patients in HCCs with CD151high were significantly lower than those in the CD151low group, and correspondingly cumulative recurrence rates in HCCs with CD151high were significantly higher than those in the CD151low group. Both CD151 and c‐Met were remarkably overexpressed in HCCs, compared with adjacent nontumorous and normal liver tissues. Pearson correlation analysis showed a slight correlation between CD151 and c‐Met in HCCs. Importantly, the 5‐ and 7‐year OS rates in CD151high/c‐Methigh patients were 50.5% and 37.8%, respectively, significantly lower than those of CD151low/c‐Metlow patients (63.9% and 54.6%, respectively). Five‐ and 7‐year cumulative recurrence rates in CD151high/c‐Methigh patients were 53.3% and 71.9%, respectively, markedly higher than those of CD151low/c‐Metlow patients (39.0% and 52.5%, respectively). Multivariate analysis revealed that CD151 and combination of CD151/c‐Met were independent prognostic indicators for OS and cumulative recurrence. Conclusion: CD151 is positively associated with invasiveness of HCC, and CD151 or combination of CD151/c‐Met is a novel marker in predicting the prognosis of HCC and a potential therapeutic target. (HEPATOLOGY 2008.)
MiRNA-30a (miR-30a) was previously reported as one of metastatic hepatocellular carcinoma (HCC)-related microRNAs. However, the function of miR-30a on enhancing our biological understanding of HCC ...metastasis is not clear. This study demonstrated that miR-30a was significantly down-regulated in HCC tissues and cell lines, and was associated with vascular invasion, metastasis potential and recurrent disease in HCC. Functional studies confirmed that miR-30a could inhibit the metastasis of HCC in a well-established nude mouse model of lung metastasis. Moreover, miR-30a was proved to prevent anoikis inhibition of HCC cells in vivo and in vitro. Mechanically, autophagy related protein Beclin 1 and Atg5 were direct downstream targets of miR-30a, and mediated autophagy activity influence of miR-30a in HCC. Taken together, downregulated miR-30a in metastatic HCC mediates Beclin 1 and Atg5-dependent autophagy, which confers anoikis resistance in HCC cells. The molecular basis of autophagy action during this process partly contributes to the HCC metastasis, suggesting that targeting autophagy via miR-30a may have therapeutic implications for the prevention of HCC recurrence/metastasis.
•miR-30a expression is correlated with metastasis in HCC.•miR-30a inhibits the metastasis of HCC in vivo.•miR-30a prevents anoikis inhibition of HCC cells in vivo and in vitro.•Downregulated miR-30a in metastatic HCC mediates Beclin 1 and Atg5-dependent autophagy.
Radiomics is an emerging field in oncological research. In this study, we aimed at developing a radiomics score (rad-score) to estimate postoperative recurrence and survival in patients with solitary ...hepatocellular carcinoma (HCC).
A total of 319 solitary HCC patients (training cohort: n = 212; validation cohort: n = 107) were enrolled. Radiomics features were extracted from the artery phase of preoperatively acquired computed tomography (CT) in all patients. A rad-score was generated by using the least absolute shrinkage and selection operator (lasso) logistic model. Kaplan-Meier and Cox's hazard regression analyses were used to evaluate the prognostic significance of the rad-score. Final nomograms predicting recurrence and survival of solitary HCC patients were established based on the rad-score and clinicopathological factors. C-index and calibration statistics were used to assess the performance of nomograms.
Six potential radiomics features were selected out of 110 texture features to formulate the rad-score. Low rad-score positively correlated with aggressive tumor phenotypes, like larger tumor size and vascular invasion. Meanwhile, low rad-score was significantly associated with increased recurrence and reduced survival. In addition, multivariate analysis identified the rad-score as an independent prognostic factor (recurrence: Hazard ratio (HR): 2.472, 95% confident interval (CI): 1.339-4.564, p = 0.004;survival: HR: 1.558, 95%CI: 1.022-2.375, p = 0.039). Notably, the nomogram integrating rad-score had a better prognostic performance as compared with traditional staging systems. These results were further confirmed in the validation cohort.
The preoperative CT image based rad-score was an independent prognostic factor for the postoperative outcome of solitary HCC patients. This score may be complementary to the current staging system and help to stratify individualized treatments for solitary HCC patients.
Metastasis is one of the main causes of poor prognosis for hepatocellular carcinoma (HCC), which has been linked to cell-death resistance. Autophagy is an important survival mechanism under ...conditions of cell stress. We hypothesized that autophagy may play a role in HCC metastasis due to its prosurvival effect. Highly metastatic HCC cell lines with stable autophagy inhibition were established via lentivirus-mediated silencing of BECN1 and ATG5 genes. Mouse models of pulmonary metastasis were then developed using the cells with or without autophagy inhibition. The analysis of lung metastasis by histopathological examination and small animal imaging showed that autophagy inhibition significantly decreased the incidence of pulmonary metastases in vivo. Further invasion, migration, detachment, lung colonization, and epithelial-mesenchymal transition (EMT) assays indicated that autophagy inhibition did not affect cell invasiveness, migration or EMT but attenuated the anoikis-resistance and lung colonization of HCC cells. Investigation of the molecular mechanisms underlying showed that the autophagy-inhibition-mediated anoikis-resistance attenuation was associated with the regulation of apoptotic signaling. As autophagy inhibition was shown to be able to suppress HCC metastasis, an autophagy-based HCC tissue-specific target therapy system (AFP-Cre/LoxP-shRNA) was constructed. In vitro and in vivo analyses showed that the system was able to efficiently inhibit autophagy of HCC cells and tissue in a tissue-specific manner. Further in vivo metastasis assay showed that intratumoral administration of the system could significantly suppress lung metastasis. Together, our findings suggest that autophagy may be involved in HCC metastasis through facilitating anoikis resistance and lung colonization of HCC cells. Autophagy-based HCC tissue-specific target therapy may be a new strategy for the management of HCC metastasis.
Macrophages are a major component of the leukocyte infiltrate of tumors and play a pivotal role in the progression of hepatocellular carcinoma (HCC). However, the molecular mechanisms by which ...macrophages promote HCC invasion are poorly understood. The present study was undertaken to investigate the relationship between macrophages and epithelial-mesenchymal transition (EMT) of HCC. Double-staining immunohistochemistry was used to observe the association between macrophages and EMT markers in clinical HCC samples and it showed that EMT primarily occurred at the edge of the tumor nest, in which infiltrating macrophages were always observed. This indicated that CD68 which is a marker of macrophages, was correlated with EMT marker levels. In addition, after being cultured with macrophages for 24 h, the ability of HCC cells to migrate and invade increased, Snail and N-Cadherin expression was upregulated, and E-Cadherin was downregulated. An antibody array assay was applied to analyze the supernatant of these cultures and it demonstrated IL-8 increased significantly in the macrophage co-culture system. Finally, the role of macrophage-derived IL-8 in the invasion of HCC cells was assayed, and downstream signaling pathways were also investigated. We found that IL-8: i) may induce EMT and promote HCC cell migration and invasion and ii) is associated with the JAK2/STAT3/Snail signaling pathway. Taking together, these findings revealed that macrophages that have infiltrated tumors may induce epithelial-mesenchymal transition of HCC cells via the IL-8 activated JAK2/STAT3/Snail pathway. Thus, this may offer a potential target for developing new HCC therapies.
The overall survival of patients with hepatocellular carcinoma (HCC) remains poor, and the molecular pathogenesis remains incompletely defined in HCC. Here we report that increased expression of ...αB‐Crystallin in human HCC predicts poor survival and disease recurrence after surgery. Multivariate analysis identifies αB‐Crystallin expression as an independent predictor for postoperative recurrence and overall survival. We show that elevated expression of αB‐Crystallin promotes HCC progression in vivo and in vitro. We demonstrate that αB‐Crystallin overexpression fosters HCC progression by inducing epithelial‐mesenchymal transition (EMT) in HCC cells through activation of the extracellular‐regulated protein kinase (ERK) cascade, which can counteract the effect of sorafenib. αB‐Crystallin complexes with and elevates 14‐3‐3ζ protein, leading to up‐regulation of ERK1/2 activity. Moreover, overexpression of αB‐Crystallin in HCC cells induces EMT progression through an ERK1/2/Fra‐1/slug signaling pathway. Clinically, our data reveal that overexpression of both αB‐Crystallin and 14‐3‐3ζ correlates with the HCC poorest survival outcomes, and sorafenib response is impaired in patients with αB‐Crystallin overexpression. Conclusion: These data suggest that the αB‐Crystallin‐14‐3‐3ζ complex acts synergistically to promote HCC progression by constitutively activating ERK signaling. This study reveals αB‐Crystallin as a potential therapeutic target for HCC and a biomarker for predicting sorafenib treatment response. (HEPATOLOGY 2013)
We studied the value of circulating tumor DNA (ctDNA) in predicting early postoperative tumor recurrence and monitoring tumor burden in patients with hepatocellular carcinoma (HCC). Plasma‐free DNA, ...germline DNA, and tissue DNA were isolated from 41 patients with HCC. Serial ctDNAs were analyzed by next‐generation sequencing before and after operation. Whole‐exome sequencing was used to detect the DNA of HCC and adjacent tissues. In total, 47 gene mutations were identified in the ctDNA of the 41 patients analyzed before surgery. ctDNA was detected in 63.4% and 46% of the patient plasma pre‐ and postoperation, respectively. The preoperative ctDNA positivity rate was significantly lower in the nonrecurrence group than in the recurrence group. With a median follow‐up of 17.7 months, nine patients (22%) experienced tumor recurrence. ctDNA positivity at two time‐points was associated with significantly shorter recurrence‐free survival (RFS). Tumors with NRAS, NEF2L2, and MET mutations had significantly shorter times to recurrence than those without mutations and showed high recurrence prediction performance by machine learning. Multivariate analyses showed that the median variant allele frequency (VAF) of mutations in preoperative ctDNA was a strong independent predictor of RFS. ctDNA is a real‐time monitoring indicator that can accurately reflect tumor burden. The median VAF of baseline ctDNA is a strong independent predictor of RFS in individuals with HCC.
Here, we studied the value of circulating tumor DNA (ctDNA), in combination with germline and tissue DNA by whole‐exome sequencing in patients withhepatocellular carcinoma (HCC). Our analysis demonstrated that ctDNA may serve as a real‐time monitoring indicator by accurately reflecting tumor burden, and as a strong independent predictor of recurrence‐free survival in HCC.
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