Clinical practice guidelines and real-life practice in hepatocellular carcinoma: A Chinese perspective.

Liver cancer is the fourth most prevalent and the second most lethal cancer in China. Hepatitis B virus (HBV) infection represents a major risk factor for hepatocellular carcinoma (HCC). Liver ultrasonography plus alpha-fetoprotein every 6 months continues to be the predominant surveillance modality. The age-Male-ALBI-Platelets score was recommended in the recent 2022 Chinese guidelines to predict HCC occurrence. The Chinese liver cancer (CNLC) staging system proposed in the 2017 guidelines continues to be the standard model for staging with modifications in the treatment allocations. Considering the aggressive nature of HBV-associated HCC, multimodal and high-intensity strategies like the addition of immunotherapy-based systemic treatment to local therapies, including resection, ablation, and intra-arterial therapies, have been adopted in real-life practices in China. The latest Chinese guidelines recommend atezolizumab plus bevacizumab, suntilimab plus a bevacizumab analog, lenvatinib, sorafenib, donafenib, and FOLFOX (folinic acid, fluorouracil, and oxaliplatin) chemotherapy as first-line treatment without priority. Regorafenib, apatinib, camrelizumab, and tislelizumab have been added as second-line systemic therapies for patients who progressed on sorafenib. Systemic therapies adopted in real-life practice are sophisticated with various combination modalities and different sequences.

The influence of 18F-fluorodeoxyglucose positron emission tomography/computed tomography on the N- and M-staging and subsequent clinical management of intrahepatic cholangiocarcinoma.

Background: Intrahepatic cholangiocarcinoma (ICC) is a highly metastatic cancer. 18F-fluorodeoxyglucose positron emission tomography/computed tomography (18F-FDG PET/CT) enables sensitive tumor and metastasis detection. Our aim is to evaluate the influence of pre-treatment PET/CT on the N- and M-staging and subsequent clinical management in ICC patients. Methods: Between August 2010 and August 2018, 660 consecutive ICC patients, without prior anti-tumor treatments nor other malignancies, were enrolled. The diagnostic performance of PET/CT on the N- and M-staging was compared with conventional imaging, and the preoperative staging accuracy and treatment re-allocation by PET/CT were retrospectively calculated. Survival difference was compared between patients receiving PET/CT or not after propensity score matching. Results: Patients were divided into group A (n=291) and group B (n=369) according to whether PET/CT was performed. Among 291 patients with both PET/CT and conventional imaging for staging in group A, PET/CT showed significantly higher sensitivity (83.0% vs. 70.5%, P=0.001), specificity (88.3% vs. 74.9%, P<0.001) and accuracy (86.3% vs. 73.2%, P<0.001) than conventional imaging in diagnosing regional lymph node metastasis, as well as higher sensitivity (87.8% vs. 67.6%, P<0.001) and accuracy (93.5% vs. 89.3%, P=0.023) in diagnosing distant metastasis. Overall, PET/CT improved the accuracy of preoperative staging from 60.1% to 71.8% (P<0.001), and modified clinical treatment strategy in 5.8% (17/291) of ICC patients, with unique roles in different tumor-node-metastasis (TNM) stages. High tumor-to-non-tumor ratio (TNR) predicted poor overall survival [hazard ratio (HR) = 2.17; 95% confidence interval (CI): 1.49-3.15; P<0.001]. Furthermore, patients performing PET/CT had longer overall survival compared with those without PET/CT (HR =0.74; 95% CI: 0.58-0.93; P=0.011) after propensity score matching. Conclusions: PET/CT was valuable for diagnosing regional lymph node metastasis and distant metastasis in ICC patients, and facilitated accurate tumor staging and optimal treatment allocation.

NLRC3 High Expression Represents a Novel Predictor for Positive Overall Survival Correlated With CCL5 and CXCL9 in HCC Patients.

NLRC3 (NLR family caspase recruitment domain containing 3) has been reported as a factor of inhibiting inflammatory responses. It's role in HCC (hepatocellular carcinoma) is still unknown. In this study we firstly used the GEO (Gene Expression Omnibus) database and mIHC (multiple immunohistochemical analysis) with TMAs (tumor tissue microarrays) of HCC patients to evaluate NLRC3 levels. The tumor-bearing mouse models were also established with NLRC3 over-expressing and knock-down Hepal-6 cells to assess its effect. The data showed high NLRC3 expression was related with favorable overall survival (P=0.0386) and disease-free survival (P=0.0458). In addition, NLRC3 expression showed a positive correlation between CD8+ T cells infiltration. In vivo, NLRC3-overexpressing Hepal-6 tumors showed increased CD8+ T cell infiltration. NLRC3-knockdown Hepa1-6 tumors displayed decreased CD8+ T cell infiltration. At the same time, we also found the positive correlations between NLRC3 and CCL5 (C-C motif chemokine ligand 5, P<0.0001, R2 = 0.2372) as well as CXCL9 (C-X-C motif chemokine ligand 9, P<0.0001, R2 = 0.2338) expressions. So NLRC3 high expression represents a novel predictor for positive survival outcomes in HCC patients, and NLRC3 is involved in CD8+ T cell infiltration, which is correlated with increased CCL5 and CXCL9 in TME (tumor microenvironment). This study implies that boosting NLRC3 is a promising treatment to enhance survival in HCC patients.

Distribution and density of tertiary lymphoid structures predict clinical outcome in intrahepatic cholangiocarcinoma.

BACKGROUND & AIMS: The prognostic value and clinical relevance of tertiary lymphoid structures (TLSs) in intrahepatic cholangiocarcinoma (iCCA) remain unclear. Thus, we aimed to investigate the prognostic value and functional involvement of TLSs in iCCA. METHODS: We retrospectively included 962 patients from 3 cancer centers across China. The TLSs at different anatomic subregions were quantified and correlated with overall survival (OS) by Cox regression and Kaplan-Meier analyses. Multiplex immunohistochemistry (mIHC) was applied to characterize the composition of TLSs in 39 iCCA samples. RESULTS: A quaternary TLS scoring system was established for the intra-tumor region (T score) and peri-tumor region (P score) respectively. T scores positively correlated with favorable prognosis (p <0.001), whereas a high P score signified worse survival (p <0.001). mIHC demonstrated that both T follicular helper and regulatory T cells were significantly increased in intra-tumoral TLSs compared to peri-tumoral counterparts (p <0.05), and regulatory T cell frequencies within intra-tumoral TLSs were positively associated with P score (p <0.05) rather than T score. Collectively, the combination of T and P scores stratified iCCAs into 4 immune classes with distinct prognoses (p <0.001) that differed in the abundance and distribution pattern of TLSs. Patients displaying an immune-active pattern had the lowest risk, with 5-year OS rates of 68.8%, whereas only 3.4% of patients with an immune-excluded pattern survived at 5 years (p <0.001). The C-index of the immune class was statistically higher than the TNM staging system (0.73 vs. 0.63, p <0.001). These results were validated in an internal and 2 external cohorts. CONCLUSIONS: The spatial distribution and abundance of TLSs significantly correlated with prognosis and provided a useful immune classification for iCCA. T follicular helper and regulatory T cells may play a critical role in determining the functional orientation of spatially different TLSs. LAY SUMMARY: Tertiary lymphoid structures (TLSs) are associated with favorable prognosis in a number of cancers. However, their role in intrahepatic cholangiocarcinoma (iCCA) remains unclear. Herein, we comprehensively evaluated the spatial distribution, abundance, and cellular composition of TLSs in iCCA, and revealed the opposite prognostic impacts of TLSs located within or outside the tumor. This difference could be mediated by the different immune cell subsets present within the spatially distinct TLSs. Based on our analysis, we were able to stratify iCCAs into 4 immune subclasses associated with varying prognoses.

Exploring prognostic indicators in the pathological images of hepatocellular carcinoma based on deep learning.

OBJECTIVE: Tumour 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. DESIGN: An 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. RESULTS: Survival 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. CONCLUSION: Our 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.

Autoantibody signature in hepatocellular carcinoma using seromics.

BACKGROUND: Alpha-fetoprotein (AFP) is a widely used biomarker for hepatocellular carcinoma (HCC) early detection. However, low sensitivity and false negativity of AFP raise the requirement of more effective early diagnostic approaches for HCC. METHODS: We employed a three-phase strategy to identify serum autoantibody (AAb) signature for HCC early diagnosis using protein array-based approach. A total of 1253 serum samples from HCC, liver cirrhosis, and healthy controls were prospectively collected from three liver cancer centers in China. The Human Proteome Microarray, comprising 21,154 unique proteins, was first applied to identify AAb candidates in discovery phase (n = 100) and to further fabricate HCC-focused arrays. Then, an artificial neural network (ANN) model was used to discover AAbs for HCC detection in a test phase (n = 576) and a validation phase (n = 577), respectively. RESULTS: Using HCC-focused array, we identified and validated a novel 7-AAb panel containing CIAPIN1, EGFR, MAS1, SLC44A3, ASAH1, UBL7, and ZNF428 for effective HCC detection. The ANN model of this panel showed improvement of sensitivity (61.6-77.7%) compared to AFP (cutoff 400 ng/mL, 28.4-30.7%). Notably, it was able to detect AFP-negative HCC with AUC values of 0.841-0.948. For early-stage HCC (BCLC 0/A) detection, it outperformed AFP (cutoff 400 ng/mL) with approximately 10% increase in AUC. CONCLUSIONS: The 7-AAb panel provides potentially clinical value for non-invasive early detection of HCC, and brings new clues on understanding the immune response against hepatocarcinogenesis.

The distribution of immune cells within combined hepatocellular carcinoma and cholangiocarcinoma predicts clinical outcome.

BACKGROUND: This study aimed to investigate the clinical relevance of the immune microenvironment in patients with combined hepatocellular carcinoma and cholangiocarcinoma (cHCC-ICC). PATIENTS AND METHODS: The density of tumor-infiltrating CD3+ , CD8+ , CD163+ , and Foxp3+ immune cells, as well as Programmed cell death 1, Programmed cell death-ligand 1, and Tumor necrosis factor receptor superfamily member 4, was measured in the peritumor liver, tumor invasive margin, and intratumor subregions of 56 cHCC-ICC by immunohistochemistry. The immune index was established to stratify patients. Prognostic significance of immune cell subsets and immune indices was evaluated. RESULTS: The distribution of immune cells was highly heterogeneous among different subregions of cHCC-ICC. As compared with the hepatocellular carcinoma (HCC) component, the lower density of CD8+ T cells and higher intensity of Foxp3+ Tregs and immune checkpoints in the intrahepatic cholangiocarcinoma (ICC) component may indicate a stronger immune evasive ability of ICC. Based on clustering classification or a combination of random forest and lasso-cox, two models of immune indices were established and both were identified as independent prognostic factors for cHCC-ICC patients. The selected immune variables in the immune prognostic models derived from both HCC and ICC subregions, indicating that the prognosis of cHCC-ICC patients was a complex interaction of both components. CONCLUSIONS: The immune contexture was heterogeneous among different subregions of cHCC-ICC patients and contributed differently to patient prognosis. Immune score based on the densities of immune cells might serve as a promising prognostic predictor for cHCC-ICC patients.

Landscape of infiltrating B cells and their clinical significance in human hepatocellular carcinoma.

As a major cellular component in tumor microenvironment, the distribution, frequency, and prognostic significance of infiltrating B cell subsets in hepatocellular carcinoma (HCC) remain controversial. Using tyramide signal amplification (TSA) based fluorescent multiplexed immunohistochemistry in situ, we evaluated the distribution and frequency of B cell subsets in two independent HCC cohorts (n = 619). The results were further confirmed by flow cytometry. Correlations of B cell subsets with clinicopathologic features and patient prognosis were analyzed. Five B cell subsets were defined by multiplexed immunohistochemistry and each subset was clearly separated by t-SNE dimension reduction analysis. Notably, the densities of all B cell subsets were significantly decreased in the tumor. The frequency of plasma cells within B cells was most abundant in the tumor. In training cohort (n = 258), high densities of tumor-infiltrating CD20+ B cells, naive B cells, IgM+ memory B cells, CD27- isotype-switched memory B cells, and plasma cells were associated with superior survival. Multivariate analysis further identified CD20+ B cells, naive B cells, and CD27- isotype-switched memory B cells as independent prognosticators for survival. Unsupervised cluster analysis confirmed increased B cell subsets harbored superior survival. In addition, high density of B cells was correlated with smaller tumor size and well differentiation. The results were validated in the independent cohort of 361 HCC patients. Intratumor infiltration of B cells is significantly impaired during HCC progression. High densities of tumor-infiltrating B cells imply a better clinical outcome. Therapies designed to target B cells may be a novel strategy in HCC.

Activated and Exhausted MAIT Cells Foster Disease Progression and Indicate Poor Outcome in Hepatocellular Carcinoma.

PURPOSE: Innate immunity is an indispensable arm of tumor immune surveillance, and the liver is an organ with a predominance of innate immunity, where mucosal-associated invariant T (MAIT) cells are enriched. However, little is known about the phenotype, functions, and immunomodulatory role of MAIT cells in hepatocellular carcinoma (HCC).Experimental Design: The distribution, phenotype, and function of MAIT cells in patients with HCC were evaluated by both flow cytometry (FCM) and in vitro bioassays. Transcriptomic analysis of MAIT cells was also performed. Prognostic significance of tumor-infiltrating MAIT cells was validated in four independent cohorts of patients with HCC. RESULTS: Despite their fewer densities in HCC tumor than normal liver, MAIT cells were significantly enriched in the HCC microenvironment compared with other mucosa-associated organs. Tumor-derived MAIT cells displayed a typical CCR7-CD45RA-CD45RO+CD95+ effector memory phenotype with lower costimulatory and effector capabilities. Tumor-educated MAIT cells significantly upregulated inhibitory molecules like PD-1, CTLA-4, TIM-3, secreted significantly less IFNγ and IL17, and produced minimal granzyme B and perforin while shifting to produce tumor-promoting cytokines like IL8. Transcriptome sequencing confirmed that tumor-derived MAIT cells were reprogrammed toward a tumor-promoting direction by downregulating genes enriched in pathways of cytokine secretion and cytolysis effector function like NFKB1 and STAT5B and by upregulating genes like IL8, CXCL12, and HAVCR2 (TIM-3). High infiltration of MAIT cells in HCC significantly correlated with an unfavorable clinical outcome, revealed by FCM, qRT-PCR, and multiplex IHC analyses, respectively. CONCLUSIONS: HCC-infiltrating MAIT cells were functionally impaired and even reprogrammed to shift away from antitumor immunity and toward a tumor-promoting direction.See related commentary by Carbone, p. 3199.

CCL15 Recruits Suppressive Monocytes to Facilitate Immune Escape and Disease Progression in Hepatocellular Carcinoma.

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.

Clinical significance of PD-1/PD-Ls gene amplification and overexpression in patients with hepatocellular carcinoma.

Background: The remarkable clinical activity of PD-1 antibody in advanced hepatocellular carcinoma (HCC) highlights the importance of PD-1/PD-L1-mediated immune escape as therapeutic target in HCC. However, the frequency and prognostic significance of PD-Ls genetic alterations in HCC remain unknown. Methods: Fluorescence in situ hybridization were used to determine PD-Ls genetic alterations, and qPCR data coupled with immunofluorescence were used to measure the mRNA and protein levels of PD-Ls. Clinical relevance and prognostic value of 9p24.1 genetic alterations were investigated on tissue microarray containing three independent cohorts of 578 HCC patients. The results were further validated in an independent cohort of 442 HCC patients from The Cancer Genome Atlas (TCGA) database. Results: In total, 7.1%-15.0% for amplification and 15.8%-31.3% for polysomy of 9p24.1 were revealed in three cohorts of HCC patients, similar to the objective response rate of PD-1 antibody in HCC. Patients with 9p24.1 genetic alterations significantly and independently correlated with unfavorable outcomes than those without. FISH and qPCR data coupled with immunofluorescence revealed that genetic alterations of 9p24.1 robustly contributed to PD-L1 and PD-L2 upregulation. In addition, increased expression of PD-L1 instead of PD-L2 also predicted poor survival by multivariate analyses. Meanwhile, high infiltration of PD-1+ immune cells also indicated dismal survival in HCC. Conclusions: Amplification or higher expression of PD-L1 significantly and independently correlated with unfavorable survival in HCC patients, authenticating the PD-1/PD-L1 axis as rational immunotherapeutic targets for HCC.

Radiomics score: a potential prognostic imaging feature for postoperative survival of solitary HCC patients.

BACKGROUND: 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). METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.

Diverse modes of clonal evolution in HBV-related hepatocellular carcinoma revealed by single-cell genome sequencing.

Hepatocellular carcinoma (HCC) is a cancer of substantial morphologic, genetic and phenotypic diversity. Yet we do not understand the relationship between intratumor heterogeneity and the associated morphologic/histological characteristics of the tumor. Using single-cell whole-genome sequencing to profile 96 tumor cells (30-36 each) and 15 normal liver cells (5 each), collected from three male patients with HBV-associated HCC, we confirmed that copy number variations occur early in hepatocarcinogenesis but thereafter remain relatively stable throughout tumor progression. Importantly, we showed that specific HCCs can be of monoclonal or polyclonal origins. Tumors with confluent multinodular morphology are the typical polyclonal tumors and display the highest intratumor heterogeneity. In addition to mutational and copy number profiles, we dissected the clonal origins of HCC using HBV-derived foreign genomic markers. In monoclonal HCC, all the tumor single cells exhibit the same HBV integrations, indicating that HBV integration is an early driver event and remains extremely stable during tumor progression. In addition, our results indicated that both models of metastasis, late dissemination and early seeding, have a role in HCC progression. Notably, early intrahepatic spreading of the initiating clone leads to the formation of synchronous multifocal tumors. Meanwhile, we identified a potential driver gene ZNF717 in HCC, which exhibits a high frequency of mutation at both single-cell and population levels, as a tumor suppressor acting through regulating the IL-6/STAT3 pathway. These findings highlight multiple distinct tumor evolutionary mechanisms in HCC, which suggests the need for specific treatment strategies.

Efficacy and Safety of Transcatheter Arterial Chemoembolization and Transcatheter Arterial Chemotherapy Infusion in Hepatocellular Carcinoma: A Systematic Review and Meta-Analysis.

Hepatocellular carcinoma (HCC) is a worldwide health threat with increasing incidence and a high mortality rate. Most HCC patients are diagnosed at an advanced stage and are unable to undergo potential curative surgery. Transcatheter arterial chemoembolization (TACE) and transcatheter arterial chemotherapy infusion (TACI) are two of the main palliative treatments for advanced HCC patients. The clinical efficacy and safety of TACE and TACI are controversial. For this reason, we conducted a systematic review and meta-analysis to summarize the current evidence. We searched for randomized controlled trials (RCTs) and cohort studies that compared the clinical outcomes and adverse effects in HCC patients who received TACE or TACI treatments. The database search was performed and last updated on November 1, 2016. Overall survival and clinical response were compared using a hazard ratio (HR) with a 95% confidence interval (CI). A total of 11 clinical studies that included 13,090 patients were included based on the inclusion/exclusion criteria, of which 9 were cohort studies and 2 were RCTs. TACE was associated with a 23% lower hazard of death compared to TACI (pooled HR = 0.77, 95% CI = 0.67-0.88, p = 0.0002). Patients receiving TACE had a 28% higher disease control rate (DCR) and 162% higher objective response rate (ORR). Only the increase in ORR associated with TACE was statistically significant [DCR: odds ratio (OR) = 1.28, 95% CI = 0.35-4.64, p = 0.71; ORR: OR = 2.62, 95% CI = 1.33-5.15, p = 0.002]. TACE is associated with more favorable survival and response rate than TACI in patients with intermediate or advanced HCC.

A New Preoperative Prognostic System Combining CRP and CA199 For Patients with Intrahepatic Cholangiocarcinoma.

OBJECTIVES: In this study, we aimed at investigating the preoperatively available prognostic factors for intrahepatic cholangiocarcinoma (ICC) patients and proposing a new preoperative prognostic scoring system for ICC. METHODS: A total of 246 consecutive ICC patients who underwent curative hepatectomy were enrolled retrospectively and were randomly divided into training (n=164) and validation cohorts (n=82) at a ratio of 2:1. The prognostic factors were investigated in both cohorts using multivariate Cox's proportional hazards regression model. RESULTS: Multivariate analyses identified that two preoperative factors (serum C-reactive protein (CRP) levels >4.1 mg/l (hazard ratio (HR): 2.75, 95% CI: 1.65-4.73, P<0.001) and carbohydrate antigen 19-9 (CA19-9) levels >300 mg/ml (HR: 3.76, 95% CI: 2.18-6.49)) were independent prognostic factors for postoperative survival in the training cohort. The results were further confirmed in the validation cohort. On the basis of these data, a preoperative prognostic score (PPS) was established by allocating 0 or 1 point to the two factors, respectively. Then, both in the training and validation cohorts, the PPS showed the power to stratify patients into three distinct groups (groups with scores 2, 1, and 0) with significant difference in the risk of postoperative death. CONCLUSIONS: A new preoperative scoring system consisting of preoperative CRP and CA19-9 levels could effectively predict postoperative survival of ICC patients.

FOXP3 Is a HCC suppressor gene and Acts through regulating the TGF-ß/Smad2/3 signaling pathway.

BACKGROUND: FOXP3 has been discovered to be expressed in tumor cells and participate in the regulation of tumor behavior. Herein, we investigated the clinical relevance and biological significance of FOXP3 expression in human hepatocellular carcinoma (HCC). METHODS: Expression profile of FOXP3 was analyzed using real-time RT-PCR, western blotting and immunofluorescence on HCC cell lines, and immunostaing of a tissue microarray containing of 240 primary HCC samples. The potential regulatory roles of FOXP3 were dissected by an integrated approach, combining biochemical assays, analysis of patient survival, genetic manipulation of HCC cell lines, mouse xenograft tumor models and chromatin immunoprecipitation (ChIP) sequencing. RESULTS: FOXP3 was constitutively expressed in HCC cells with the existence of splice variants (especially exon 3 and 4 deleted, Δ3,4-FOXP3). High expression of FOXP3 significantly correlated with low serum α-fetoprotein (AFP) level, absence of vascular invasion and early TNM stage. Survival analyses revealed that increased FOXP3 expression was significantly associated with better survival and reduced recurrence, and served as an independent prognosticator for HCC patients. Furthermore, FOXP3 could potently suppress the proliferation and invasion of HCC cells in vitro and reduce tumor growth in vivo. However, Δ3,4-FOXP3 showed a significant reduction in the tumor-inhibiting effect. The inhibition of FOXP3 on HCC aggressiveness was acted probably by enhancing the TGF-ß/Smad2/3 signaling pathway. CONCLUSION: Our findings suggest that FOXP3 suppresses tumor progression in HCC via TGF-ß/Smad2/3 signaling pathway, highlighting the role of FOXP3 as a prognostic factor and novel target for an optimal therapy against this fatal malignancy.

Telomere length variation in tumor cells and cancer-associated fibroblasts: potential biomarker for hepatocellular carcinoma.

The role of telomere dysfunction and aberrant telomerase activities in hepatocellular carcinoma (HCC) has been overlooked for many years. This study aimed to delineate the variation and prognostic value of telomere length in HCC. Telomere-specific fluorescence in situ hybridization (FISH) and qPCR were used to evaluate telomere length in HCC cell lines, tumor tissues, and isolated non-tumor cells within the tumor. Significant telomere attrition was found in tumor cells and cancer-associated fibroblasts (CAFs) compared to their normal counterparts, but not in intratumor leukocytes or bile duct epithelial cells. Clinical relevance and prognostic value of telomere length were investigated on tissue microarrays of 257 surgically treated HCC patients. Reduced intensity of telomere signals in tumor cells or CAFs correlated with larger tumor size and the presence of vascular invasion (p < 0.05). Shortened telomeres in tumor cells or CAFs associated with reduced survival and increased recurrence, and were identified as independent prognosticators for HCC patients (p < 0.05). These findings were validated in an independent HCC cohort of 371 HCC patients from The Cancer Genome Atlas (TCGA) database, confirming telomere attrition and its prognostic value in HCC. We also showed that telomerase reverse transcriptase promoter (TERTp) mutation correlated with telomere shortening in HCC. Telomere variation in tumor cells and non-tumor cells within the tumor microenvironment of HCC was a valuable prognostic biomarker for this fatal malignancy. © 2017 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Cell Culture System for Analysis of Genetic Heterogeneity Within Hepatocellular Carcinomas and Response to Pharmacologic Agents.

BACKGROUND & AIMS: No targeted therapies have been found to be effective against hepatocellular carcinoma (HCC), possibly due to the large degree of intratumor heterogeneity. We performed genetic analyses of different regions of HCCs to evaluate levels of intratumor heterogeneity and associate alterations with responses to different pharmacologic agents. METHODS: We obtained samples of HCCs (associated with hepatitis B virus infection) from 10 patients undergoing curative resection, before adjuvant therapy, at hospitals in China. We collected 4-9 spatially distinct samples from each tumor (55 regions total), performed histologic analyses, isolated cancer cells, and carried them low-passage culture. We performed whole-exome sequencing, copy-number analysis, and high-throughput screening of the cultured primary cancer cells. We tested responses of an additional 105 liver cancer cell lines to a fibroblast growth factor receptor (FGFR) 4 inhibitor. RESULTS: We identified a total of 3670 non-silent mutations (3192 missense, 94 splice-site variants, and 222 insertions or deletions) in the tumor samples. We observed considerable intratumor heterogeneity and branched evolution in all 10 tumors; the mean percentage of heterogeneous mutations in each tumor was 39.7% (range, 12.9%-68.5%). We found significant mutation shifts toward C>T and C>G substitutions in branches of phylogenetic trees among samples from each tumor (P < .0001). Of note, 14 of the 26 oncogenic alterations (53.8%) varied among subclones that mapped to different branches. Genetic alterations that can be targeted by existing pharmacologic agents (such as those in FGF19, DDR2, PDGFRA, and TOP1) were identified in intratumor subregions from 4 HCCs and were associated with sensitivity to these agents. However, cells from the remaining subregions, which did not have these alterations, were not sensitive to these drugs. High-throughput screening identified pharmacologic agents to which these cells were sensitive, however. Overexpression of FGF19 correlated with sensitivity of cells to an inhibitor of FGFR 4; this observation was validated in 105 liver cancer cell lines (P = .0024). CONCLUSIONS: By analyzing genetic alterations in different tumor regions of 10 HCCs, we observed extensive intratumor heterogeneity. Our patient-derived cell line-based model, integrating genetic and pharmacologic data from multiregional cancer samples, provides a platform to elucidate how intratumor heterogeneity affects sensitivity to different therapeutic agents.

Protein tyrosine phosphatase PTP4A1 promotes proliferation and epithelial-mesenchymal transition in intrahepatic cholangiocarcinoma via the PI3K/AKT pathway.

The protein tyrosine phosphatase PTP4A1 is a key molecule that activates tyrosine phosphorylation, which is important for cancer progression and metastasis. However, the clinical implications and biological function of PTP4A1 in intrahepatic cholangiocarcinoma (ICC) remains unknown. Here, we showed that PTP4A1 was frequently overexpressed in ICC versus adjacent non-tumor tissues. This overexpression significantly correlated with aggressive tumor characteristics like the presence of lymph node metastasis and advanced tumor stages. Survival analysis further indicated that high PTP4A1 expression was significantly and independently associated with worse survival and increased recurrence in ICC patients. Moreover, through forced overexpression and knock-down of PTPT4A1, we demonstrated that PTP4A1 could significantly promote ICC cells proliferation, colony formation, migration, and invasion in vitro, and markedly enhance tumor progression in vivo. Mechanistically, PTP4A1 was involved in PI3K/AKT signaling and its downstream molecules, such as phosphorylation level of GSK3ß and up-regulation of CyclinD1, in ICC cells to promote proliferation. Importantly, PTP4A1 induced ICC cells invasion was through activating PI3K/AKT signaling controlled epithelial-mesenchymal transition (EMT) process by up-regulating Zeb1 and Snail. Thus, PTP4A1 may serve as a potential oncogene that was a valuable prognostic biomarker and therapeutic target for ICC.

Naive Treg-like CCR7(+) mononuclear cells indicate unfavorable prognosis in hepatocellular carcinoma.

Chemokine receptor-like 1 (CCRL1) has the potential in creating a low level of CCL19 and CCL21 to hinder CCR7(+) cell tracking to tumor tissue. Previously, we found a tumor suppressive role of CCRL1 by impairing CCR7-related chemotaxis of tumor cells in human hepatocellular carcinoma (HCC). Here, we reported a contribution of CCR7(+) mononuclear cells in the tumor microenvironment to the progression of disease. Immunohistochemistry was used to investigate the distribution and clinical significance of CCR7(+) cells in a cohort of 240 HCC patients. Furthermore, the phenotype, composition, and functional status of CCR7(+) cells were determined by flow cytometry, immunofluorescence, and in vitro co-culture assays. We found that CCR7(+) mononuclear cells were dispersed around tumor tissue and negatively related to tumoral expression of CCRL1 (P < 0.001, r = 0.391). High density of CCR7(+) mononuclear cells positively correlated with the absence of tumor capsule, vascular invasion, and poor differentiation (P < 0.05). Survival analyses revealed that increased number of CCR7(+) mononuclear cells was significantly associated with worse survival and increased recurrence. We found that CCR7(+) mononuclear cells featured a naive Treg-like phenotype (CD45RA(+)CD25(+)FOXP3(+)) and possessed tumor-promoting potential by producing TGF-ß1. Moreover, CCR7(+) cells were also composed of several immunocytes, a third of which were CD8(+) T cells. CCR7(+) Treg-like cells facilitate tumor growth and indicate unfavorable prognosis in HCC patients, but fortunately, their tracking to tumor tissue is under the control of CCRL1.