Mining the interpretable prognostic features from pathological image of intrahepatic cholangiocarcinoma using multi-modal deep learning.

BACKGROUND: The advances in deep learning-based pathological image analysis have invoked tremendous insights into cancer prognostication. Still, lack of interpretability remains a significant barrier to clinical application. METHODS: We established an integrative prognostic neural network for intrahepatic cholangiocarcinoma (iCCA), towards a comprehensive evaluation of both architectural and fine-grained information from whole-slide images. Then, leveraging on multi-modal data, we conducted extensive interrogative approaches to the models, to extract and visualize the morphological features that most correlated with clinical outcome and underlying molecular alterations. RESULTS: The models were developed and optimized on 373 iCCA patients from our center and demonstrated consistent accuracy and robustness on both internal (n = 213) and external (n = 168) cohorts. The occlusion sensitivity map revealed that the distribution of tertiary lymphoid structures, the geometric traits of the invasive margin, the relative composition of tumor parenchyma and stroma, the extent of necrosis, the presence of the disseminated foci, and the tumor-adjacent micro-vessels were the determining architectural features that impacted on prognosis. Quantifiable morphological vector extracted by CellProfiler demonstrated that tumor nuclei from high-risk patients exhibited significant larger size, more distorted shape, with less prominent nuclear envelope and textural contrast. The multi-omics data (n = 187) further revealed key molecular alterations left morphological imprints that could be attended by the network, including glycolysis, hypoxia, apical junction, mTORC1 signaling, and immune infiltration. CONCLUSIONS: We proposed an interpretable deep-learning framework to gain insights into the biological behavior of iCCA. Most of the significant morphological prognosticators perceived by the network are comprehensible to human minds.

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.

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.

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.

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.

Inferring the progression of multifocal liver cancer from spatial and temporal genomic heterogeneity.

Multifocal tumors developed either as independent tumors or as intrahepatic metastases, are very common in primary liver cancer. However, their molecular pathogenesis remains elusive. Herein, a patient with synchronous two hepatocellular carcinoma (HCC, designated as HCC-A and HCC-B) and one intrahepatic cholangiocarcinoma (ICC), as well as two postoperative recurrent tumors, was enrolled. Multiregional whole-exome sequencing was applied to these tumors to delineate the clonality and heterogeneity. The three primary tumors showed almost no overlaps in mutations and copy number variations. Within each tumor, multiregional sequencing data showed varied intratumoral heterogeneity (21.6% in HCC-A, 20.4% in HCC-B, 53.2% in ICC). The mutational profile of two recurrent tumors showed obvious similarity with HCC-A (86.7% and 86.6% respectively), rather than others, indicating that they originated from HCC-A. The evolutionary history of the two recurrent tumors indicated that intrahepatic micro-metastasis could be an early event during HCC progression. Notably, FAT4 was the only gene mutated in two primary HCCs and the recurrences. Mutation prevalence screen and functional experiments showed that FAT4, harboring somatic coding mutations in 26.7% of HCC, could potently inhibit growth and invasion of HCC cells. In HCC patients, both FAT4 expression and FAT4 mutational status significantly correlated with patient prognosis. Together, our findings suggest that spatial and temporal dissection of genomic alterations during the progression of multifocal liver cancer may help to elucidate the basis for its dismal prognosis. FAT4 acts as a putative tumor suppressor that is frequently inactivated in human HCC.

Mitogen-activated protein kinase kinase kinase 4 deficiency in intrahepatic cholangiocarcinoma leads to invasive growth and epithelial-mesenchymal transition.

UNLABELLED: The molecular pathogenesis of intrahepatic cholangiocarcinoma (iCCA) is poorly understood, and its incidence continues to increase worldwide. Deficiency of mitogen-activated protein kinase kinase kinase 4 (MAP3K4) has been reported to induce the epithelial-mesenchymal transition (EMT) process of placental and embryonic development, yet its role in human cancer remains unknown. MAP3K4 has somatic mutation in iCCA so we sequenced all exons of MAP3K4 in 124 iCCA patients. We identified nine somatic mutations in 10 (8.06%) patients, especially in those with lymph node metastasis and intrahepatic metastasis. We also showed that messenger RNA and protein levels of MAP3K4 were significantly reduced in iCCA versus paired nontumor tissues. Furthermore, knockdown of MAP3K4 in cholangiocarcinoma cells markedly enhanced cell proliferation and invasiveness in vitro and tumor progression in vivo, accompanied by a typical EMT process. In contrast, overexpression of MAP3K4 in cholangiocarcinoma cells obviously reversed EMT and inhibited cell invasion. Mechanistically, MAP3K4 functioned as a negative regulator of EMT in iCCA by antagonizing the activity of the p38/nuclear factor κB/snail pathway. We found that the tumor-inhibitory effect of MAP3K4 was abolished by inactivating mutations. Clinically, a tissue microarray study containing 322 iCCA samples from patients revealed that low MAP3K4 expression in iCCA positively correlated with aggressive tumor characteristics, such as vascular invasion and intrahepatic or lymph node metastases, and was independently associated with poor survival and increased recurrence after curative surgery. CONCLUSIONS: MAP3K4, significantly down-regulated, frequently mutated, and potently regulating the EMT process in iCCA, was a putative tumor suppressor of iCCA.

Protein tyrosine phosphatase receptor S acts as a metastatic suppressor in hepatocellular carcinoma by control of epithermal growth factor receptor-induced epithelial-mesenchymal transition.

UNLABELLED: Hepatocellular carcinoma (HCC) is the third-most lethal cancer worldwide. Understanding the molecular pathogenesis of HCC recurrence and metastasis is the key to improve patients' prognosis. In this study, we report that protein tyrosine phosphatase receptor S (PTPRS) is significantly down-regulated in nearly 80% of HCCs, and its expression negatively correlates with aggressive pathological features, such as larger tumor size and advanced stage. In addition, PTPRS deficiency is independently associated with shorter survival and increased recurrence in patients, although 16.7% of HCCs show intratumor heterogeneous expression of PTPRS. Restoration of wild-type, but not mutant, PTPRS expression significantly inhibits HCC cell migration and invasion in vitro as well as lung metastasis in vivo, whereas knockdown of its expression significantly promotes invasion and metastasis. Notably, PTPRS-regulated HCC invasiveness is accompanied by typical changes of epithelial-mesenchymal transition (EMT). Moreover, PTPRS forms a complex with epithermal growth factor receptor (EGFR) and regulates its tyrosine residues' phosphorylation. Ectopic expression of EGFR reverses the metastasis-inhibiting effects of PTPRS, whereas silencing of EGFR or inhibiting phosphorylation of key molecules in EGFR downstream pathways reinhibits EMT and metastasis caused by PTPRS down-regulation. Meanwhile, promoter hypermethylation of PTPRS is frequently detected in HCC samples and cell lines. Treatment with a demethylation agent, 5-aza-2'-deoxycytidine, recovers PTPRS expression in a dose-dependent manner. CONCLUSIONS: Epigenetic inactivation of PTPRS may increase phosphorylation and activity of EGFR signaling to promote EMT and metastasis in HCC.

Genetic polymorphisms of the multidrug resistance 1 gene MDR1 and the risk of hepatocellular carcinoma.

A possible association between multiple drug resistance 1 gene (MDR1) polymorphisms and the risk of developing hepatocellular carcinoma (HCC) is currently under debate, and evidence from various epidemiological studies has yielded controversial results. To derive a more precise estimation of the association between MDR1 polymorphisms and HCC risk, the present meta-analysis was performed. A total of 8 studies containing 11 cohorts with 4407 cases and 4436 controls were included by systematic literature search of EMBASE, PubMed, Web of Science, and CNKI. All polymorphisms were classified as mutant/wild-type alleles. In particular, the variation type, functional impact, and protein domain location of the polymorphisms were assessed and used as stratified indicators. The pooled odds ratio (OR) with 95 % confidence interval (CI) was calculated to evaluate the association. Overall, our results suggested that the mutant alleles of the MDR1 gene were associated with a significantly increased risk for HCC under all genetic models (allelic model: OR = 1.28, 95 % CI = 1.20-1.36, P < 0.001; dominant model: OR = 1.27, 95 % CI = 1.16-1.38, P < 0.001; recessive model: OR = 1.59, 95 % CI = 1.36-1.85, P < 0.001). Furthermore, increased risks for HCC were also revealed in stratified analyses by ethnicity, sample size, and quality scores of cohorts as well as variation type, functional impact, and protein domain location of polymorphisms. In conclusion, the present meta-analysis suggested that the presence of MDR1 mutant alleles might be a risk factor for HCC.

TREM-1, an inflammatory modulator, is expressed in hepatocellular carcinoma cells and significantly promotes tumor progression.

BACKGROUND: Triggering receptors expressed on myeloid cells 1 (TREM-1) is a novel molecule that modulates inflammatory responses. Hepatocellular carcinoma (HCC) is a well-known type of inflammation-related cancer. However, TREM-1 expression and its direct effects on HCC cells have not been previously determined. METHODS: Western blotting, quantitative reverse transcription-PCR (qRT-PCR), and immunofluorescence were used to detect TREM-1 expression. TREM-1 upregulation by pcDNA (mammalian expression vector with the CMV promoter) and its downregulation by shRNA (short hairpin RNA) were used to determine the function of this molecule. Transwell, CCK-8, cell cycle, and apoptosis assays were used to detect the effects of TREM-1 on HCC cells. Immunohistochemical staining of samples from a cohort of 322 HCC patients was used to determine the prognostic value of TREM-1. RESULTS: TREM-1 investigation through Western blot, qRT-PCR, and immunofluorescence analyses revealed that TREM-1 was expressed in HCC cells and tumor tissues. Functional experiments suggested that TREM-1 significantly promoted proliferation, invasion, and inhibited apoptosis of HCC cells. Inflammatory cytokine profiles under TREM-1 up- or downregulation indicated the majority of proinflammation cytokines significantly and positively correlated with TREM-1 expression, including IL-1ß, TNF-α, and MCP-1. Western blot analyses revealed that p65, STAT3, ERK, and AKT might be the downstream effectors of TREM-1 signal transduction. High TREM-1 expression correlated significantly with increased recurrence and poorer survival in HCC patients, and it was an independent prognostic factor for recurrence (P = 0.009). CONCLUSIONS: TREM-1 was found to be expressed in HCC cells and to be a prognostic factor for the clinical outcome of HCC.

CC chemokine receptor-like 1 functions as a tumour suppressor by impairing CCR7-related chemotaxis in hepatocellular carcinoma.

Atypical chemokine receptors (ACRs) have been discovered to participate in the regulation of tumour behaviour. Here we report a tumour-suppressive role of a novel ACR member, CC chemokine receptor like 1 (CCRL1), in human hepatocellular carcinoma (HCC). Both mRNA and protein expressions of CCRL1 correlated with the malignant phenotype of HCC cells and were significantly down-regulated in tumour tissue compared with paired normal liver tissue. In both the initial and validation cohorts (n = 240 and n = 384, respectively), CCRL1 deficiency was associated with advanced tumour stage and was an independent index for worse survival and increased recurrence. Furthermore, knock-down or forced expression of CCRL1 revealed that CCRL1 suppressed the proliferation and invasion of HCC cells in vitro and reduced tumour growth and lung metastasis in vivo, with depressed levels of CCL19 and CCL21. By sequestrating CCL19 and CCL21, CCRL1 reduced their binding to CCR7 and consequently mitigated the detrimental impact of CCR7, including Akt-GSK3ß pathway activation and nuclear accumulation of ß-catenin in tumour cells. Clinically, the prognostic value of the CCR7 expression in HCC depended on the expression level of CCRL1, suggesting that CCRL1 may serve as an upstream switch for the CCR7 signalling cascade. Together, our findings suggest that CCRL1 impairs chemotactic events associated with CCR7 in the progression and metastasis of HCC. Our results also show a potential interplay between typical and atypical chemokine receptors in human cancer. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.