Characterization of tumor microbiome and associations with prognosis in intrahepatic cholangiocarcinoma.

BACKGROUND: The tumor microbiome has been characterized in several malignancies; however, no previous studies have investigated its role in intrahepatic cholangiocarcinoma (ICC). Hence, we explored the tumor microbiome and its association with prognosis in ICC. METHODS: One hundred and twenty-one ICC tumor samples and 89 adjacent normal tissues were profiled by 16S rRNA sequencing. Microbial differences between tumor and adjacent nontumoral liver tissues were assessed. Tumor microbial composition was then evaluated to detect its association with prognosis. Finally, a risk score calculated by the tumor microbiota was accessed by the least absolute shrinkage and selector operator method (Lasso) to predict prognosis of ICC. RESULTS: The tumor microbiome displayed a greater diversity than that in adjacent nontumoral liver tissues. Tumor samples were characterized by a higher abundance of Firmicutes, Actinobacteria, Bacteroidetes, and Acidobacteriota. Higher tumor microbial α diversity was associated with lymph node metastasis and predicted shortened overall survival (OS) and recurrence-free survival (RFS). A total of 11 bacteria were selected to generate the risk score by Lasso. This score showed potential in predicting OS, and was an independent risk factor for OS. CONCLUSION: In conclusion, our study characterized the tumor microbiome and revealed its role in predicting prognosis in ICC.

Whole-genome sequencing reveals the evolutionary trajectory of HBV-related hepatocellular carcinoma early recurrence.

Patients with hepatocellular carcinoma (HCC) have poor long-term survival following curative resection because of the high rate of tumor early recurrence. Little is known about the trajectory of genomic evolution from primary to early-recurrent HCC. In this study, we performed whole-genome sequencing (WGS) on 40 pairs of primary and early-recurrent hepatitis B virus (HBV)-related HCC tumors from patients who received curative resection, and from four patients whose primary and recurrent tumor were extensively sampled. We identified two recurrence patterns: de novo recurrence (18/40), which developed genetically independently of the primary tumor and carried different HCC drivers, and ancestral recurrence (22/40), which was clonally related to the primary tumor and progressed more rapidly than de novo recurrence. We found that the recurrence location was predictive of the recurrence pattern: distant recurrence tended to display the de novo pattern, whereas local recurrence tended to display the ancestral pattern. We then uncovered the evolutionary trajectories based on the subclonal architecture, driver-gene mutations, and mutational processes observed in the primary and recurrent tumors. Multi-region WGS demonstrated spatiotemporal heterogeneity and polyclonal, monophyletic dissemination in HCC ancestral recurrence. In addition, we identified recurrence-specific mutations and copy-number gains in BCL9, leading to WNT/ß-catenin signaling activation and an immune-excluded tumor microenvironment, which suggests that BCL9 might serve as a new therapeutic target for recurrent HCC. Collectively, our results allow us to view with unprecedented clarity the genomic evolution during HBV-related HCC early recurrence, providing an important molecular foundation for enhanced understanding of HCC with implications for personalized therapy to improve patient survival.

Genomic evolution and the impact of SLIT2 mutation in relapsed intrahepatic cholangiocarcinoma.

BACKGROUND AND AIMS: Intrahepatic cholangiocarcinoma (ICC) is aggressive and has high rates of relapse, conferring poor long-term survival after curative resection. Little is known about the genomic evolution that occurs during ICC relapse. APPROACH AND RESULTS: We conducted whole-exome sequencing of 30 paired primary and relapsed tumors from 10 patients with ICC who received curative resection. We sought to identify frequently altered genes, infer tumor subclonal architectures, and track genomic evolution from primary to relapsed tumors. We examined functional effects and the mechanism of action of SLIT2, a gene specifically mutated in relapsed tumors, on tumor growth and metastasis and the tumor microenvironment. Our results indicated that relapsed ICCs were genetically derived from intrahepatic dissemination of primary tumors. However, they acquired additional mutations while maintaining most drivers, such as TP53 and IDH1. Multiregion sequencing suggested polyclonal seeding of ICC dissemination. Four of 10 relapsed ICCs acquired SLIT2 mutations that were not present in the corresponding primary tumors. Validation in an expanded sample revealed SLIT2 mutations in 2.3% (1/44) of primary ICCs and 29.5% (13/44) of relapsed ICCs. Biofunctional investigations revealed that inactivating mutation of SLIT2 resulted in activation of PI3K-Akt signaling in ICC cells, directly enhanced neutrophil chemotaxis, mediated tumor-associated neutrophil infiltration, and contributed to ICC growth and metastasis. CONCLUSIONS: We characterized genomic evolution during ICC relapse and identified SLIT2 as a driver of tumor dissemination and tumor-associated neutrophil infiltration.

Association of KRAS Variant Subtypes With Survival and Recurrence in Patients With Surgically Treated Intrahepatic Cholangiocarcinoma.

Importance: KRAS variants are associated with tumor progression; however, the prevalence of KRAS variant subtypes and their association with survival and recurrence in patients with intrahepatic cholangiocarcinoma (ICC) after curative resection are largely unknown. Objective: To explore the prognostic association of KRAS variant subtypes with survival and recurrence in patients with ICC. Design, Setting, and Participants: In this cohort study, patients who underwent curative resection for ICC from January 2009 through December 2016 at a single hospital in China were recruited, and whole-exome sequencing, targeted sequencing, and Sanger sequencing were performed to identify KRAS variants. Kaplan-Meier and log-rank tests were used to compare overall survival (OS) and disease-free survival (DFS). Univariate and multivariate analyses were performed using the Cox proportional hazards regression model. Data were analyzed from April 2020 to January 2021. Interventions: Hepatectomy in patients with ICC. Main Outcomes and Measures: The association of KRAS variant subtypes with OS and DFS. Results: Of 1024 included patients with ICC, 621 (60.6%) were male, and the mean (SD) age was 59.2 (10.2) years. A total of 14 different subtypes of KRAS somatic variants affecting 127 patients (12.4%) were identified. G12D was the most frequent allele in this cohort, accounting for 55 of 127 identified KRAS variants (43.3%), followed by G12V (25 [19.7%]), G12C (9 [7.1%]), and G13D (8 [6.3%]). Compared with patients with wild-type KRAS, patients with variant KRAS were more likely to have high levels of carbohydrate antigen 19-9 (92 of 127 [72.4%] vs 546 of 897 [60.9%]; P = .01) and γ-glutamyltransferase (72 of 127 [56.7%] vs 420 of 897 [46.8%]; P = .04). Multivariable analysis revealed that G12 KRAS variants but not non-G12 KRAS variants were independently associated with worse OS (hazard ratio [HR], 1.69; 95% CI, 1.31-2.18; P < .001) and DFS (HR, 1.47; 95% CI, 1.16-1.88; P = .002). Among the patients with G12 KRAS variants, the G12V KRAS variant was the strongest prognostic determinant for the worst OS (HR, 3.05; 95% CI, 1.94-4.79; P < .001) and DFS (HR, 1.79; 95% CI, 1.13-2.85; P = .01). Conclusions and Relevance: In this cohort study, the distribution of KRAS variant subtypes was characterized in a large cohort of patients with ICC from China. The presence of G12 KRAS variants but not non-G12 KRAS variants was associated with worse survival and increased risk of recurrence. Patients with the G12V variant exhibited the worst outcomes in the whole cohort.

LINC01133 promotes hepatocellular carcinoma progression by sponging miR-199a-5p and activating annexin A2.

BACKGROUND: Long noncoding RNAs (lncRNAs) are functionally associated with cancer development and progression. Although gene copy number variation (CNV) is common in hepatocellular carcinoma (HCC), it is not known how CNV in lncRNAs affects HCC progression and recurrence. We aimed to identify a CNV-related lncRNA involved in HCC progression and recurrence and illustrate its underlying mechanisms and prognostic value. METHODS: We analyzed the whole genome sequencing (WGS) data of matched cancerous and noncancerous liver samples from 49 patients with HCC to identify lncRNAs with CNV. The results were validated in another cohort of 238 paired HCC and nontumor samples by TaqMan copy number assay. We preformed Kaplan-Meier analysis and log-rank test to identify lncRNA CNV with prognostic value. We conducted loss- and gain-of-function studies to explore the biological functions of LINC01133 in vitro and in vivo. The competing endogenous RNAs (ceRNAs) mechanism was clarified by microRNA sequencing (miR-seq), quantitative real-time PCR (qRT-PCR), western blot, and dual-luciferase reporter assays. We confirmed the binding mechanism between lncRNA and protein by RNA pull-down, RNA immunoprecipitation, qRT-PCR, and western blot analyses. RESULTS: Genomic copy numbers of LINC01133 were increased in HCC, which were positively related with the elevated expression of LINC01133. Increased copy number of LINC01133 predicted the poor prognosis in HCC patients. LINC01133 overexpression in HCC cells promoted proliferation and aggressive phenotypes in vitro, and facilitated tumor growth and lung metastasis in vivo, whereas LINC01133 knockdown had the opposite effects. LINC01133 sponged miR-199a-5p, resulting in enhanced expression of SNAI1, which induced epithelial-to-mesenchymal transition (EMT) in HCC cells. In addition, LINC01133 interacted with Annexin A2 (ANXA2) to activate the ANXA2/STAT3 signaling pathway. CONCLUSIONS: LINC01133 promotes HCC progression by sponging miR-199a-5p and interacting with ANXA2. LINC01133 CNV gain is predictive of poor prognosis in patients with HCC.

Associations among the mutational landscape, immune microenvironment, and prognosis in Chinese patients with hepatocellular carcinoma.

Recent studies suggested that the immune microenvironment and mutational landscape are associated with the response to immune-based therapy in several types of cancer. The roles of those factors in Chinese HCC remain largely unknown. In this study, we obtained 182 FFPE samples of HCC cohort that were previously subjected to NGS (49 WGS, 18 WES, and 115 targeted sequencing). We performed immunohistochemistry to detect CD3, CD4, CD8, CD57, Foxp3, CD68, CD66b, and PD-L1 expression in the samples. We identified diverse associations between the mutational landscape and the immune microenvironment in the HCC samples. High mutational burden and an aristolochic acid-dominated mutational signature were both correlated with elevated tumoral PD-L1 expression and CD3+ T-cell infiltration and high numbers of CD68+ TAMs and CD66b+ TANs. CD4+ and CD8+ T cells exhibited lower infiltration levels in tumors with mutations in AXIN1/CTNNB1 and in tumors with aflatoxin-dominant mutational signatures. Moreover, tumors with TP53 mutations had less CD8+ T-cell infiltration and more Foxp3+ Treg-cell infiltration than those without TP53 mutations. Kaplan-Meier survival analysis revealed that the presence of CD8+, Foxp3+, CD66b+, or CD68+ immune cells; tumoral PD-L1 expression alone; or the presence of CD8+ or Foxp3+ cells combined with TP53 mutation were predictive of recurrence and poor overall survival after curative resection. In conclusion, the association between the mutational landscape and the immune microenvironment warrants further analysis to determine its impact on patient outcomes to guide personalized immune-based therapy for Chinese patients with HCC.

Peritumoral plasmacytoid dendritic cells predict a poor prognosis for intrahepatic cholangiocarcinoma after curative resection.

BACKGROUND: Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in intrahepatic cholangiocarcinoma is not clear. METHODS: To evaluate pDCs' distributions in and around tumors as well as their potential function and predictive value for prognosis in patients undergoing curative resection, we performed immunohistochemistry to examine the expression of pDC marker BDCA2, and CD3, CD4, CD8 and Foxp3 in intratumoral and peritumoral tissues from 359 patients with intrahepatic cholangiocarcinoma and compared with prognostic and clinicopathologic factors. RESULTS: Results showed that patients with high numbers of BDCA2+ pDCs in peritumoral tissues were more likely to have elevated levels of carbohydrate antigen 19-9 and gamma-glutamyl transferase, larger and more tumors, advanced tumor-node-metastasis staging, more vascular/bile duct invasion, and lymphatic metastasis in association with greater chance of recurrence and shorter overall survival. Peritumoral tissues with larger numbers of pDCs also showed increased Foxp3+ regulatory T cell infiltration, both of which were found to be independent factors for predicting time to recurrence and overall survival. By contrast, patient outcomes were not associated with the presence of intratumoral pDCs. CONCLUSIONS: Peritumoral pDC infiltration may indicate an immune tolerogenic peritumor microenvironment and can be used to predict a poor prognosis for patients undergoing curative resection for intrahepatic cholangiocarcinoma.

MACROD2 deficiency promotes hepatocellular carcinoma growth and metastasis by activating GSK-3ß/ß-catenin signaling.

Structural variations (SVs) influence the development and progression of multiple types of cancer. The genes affected by SVs in hepatocellular carcinoma (HCC) and their contribution to tumor growth and metastasis remain unknown. In this study, through whole-genome sequencing (WGS), we identified MACROD2 as the gene most frequently affected by SVs, which were associated with low MACROD2 expression levels. Low MACROD2 expression was predictive of tumor recurrence and poor overall survival. MACROD2 expression was decreased in HCC cell lines, especially those with high metastatic potential. MACROD2 knockdown in HCC cells markedly enhanced proliferation and invasiveness in vitro and tumor progression in vivo and promoted epithelial-mesenchymal transition (EMT). By contrast, MACROD2 overexpression reversed EMT and inhibited HCC growth and metastasis. Mechanistically, MACROD2 deficiency suppressed glycogen synthase kinase-3ß (GSK-3ß) activity and activated ß-catenin signaling, which mediated the effect of MACROD2 on HCC. In clinical HCC samples, decreased MACROD2 expression was correlated with the activation of GSK-3ß/ß-catenin signaling and the EMT phenotype. Overall, our results revealed that MACROD2 is frequently affected by SVs in HCC, and its deficiency promotes tumor growth and metastasis by activating GSK-3ß/ß-catenin signaling.

TGM3 promotes epithelial-mesenchymal transition and hepatocellular carcinogenesis and predicts poor prognosis for patients after curative resection.

BACKGROUND: Prognosis of hepatocellular carcinoma (HCC) remains poor despite significant recent improvement in therapy. Recent studies have reported that transglutaminase 3 (TGM3) plays an important role in several human cancer types. However, the role of TGM3 in HCC have not been previously elucidated. METHODS: We evaluated the role of TGM3 in regulating HCC cell proliferation, migration, and invasion. We also investigated the prognostic significance of TGM3 in an HCC cohort. Finally, we explored the signalling pathways that TGM3 regulates in HCC. RESULTS: We identified TGM3 to be overexpressed in HCC compared to normal tissues. Higher expression of TGM3 predicts poor prognosis in HCC patients. TGM3 knockdown led to decreased HCC cell proliferation, invasion, and xenograft tumour growth. TGM3 depletion inhibited AKT, extracellular signal-regulated kinase (ERK), p65, and glycogen synthase kinase 3ß (GSK3ß)/ß-catenin activation, but promoted levels of cleaved caspase 3. Moreover, TGM3 knockdown cells had increased E-cadherin levels and decreased vimentin levels, suggesting that TGM3 contributes to epithelial-mesenchymal transition (EMT) in HCC. CONCLUSION: Our results suggest that TGM3 controls multiple oncogenic pathways in HCC, thereby contributing to increased cell proliferation and EMT, and TGM3 potentially enhances HCC metastasis. TGM3 may serve as a novel therapeutic target in HCC.

Circular RNA Sequencing Identifies CircASAP1 as a Key Regulator in Hepatocellular Carcinoma Metastasis.

BACKGROUND AND AIMS: There is growing evidence that single-stranded, circular RNA (circRNA) plays a key role in the development of certain cancers, including hepatocellular carcinoma (HCC). It is less clear, however, what role circRNA plays in HCC metastasis. APPROACH AND RESULTS: In this study, through circRNA sequencing, we identified a circRNA: circASAP1 (a circRNA derived from exons 2 and 3 of the ASAP1 gene, hsa_circ_0085616), which is associated with pulmonary metastasis after curative resection in patients with HCC. CircASAP1 was overexpressed in HCC cell lines with high metastatic potential and in metastatic HCCs. In vitro, circASAP1 promoted cell proliferation, colony formation, migration, and invasion, and in vivo, it enhanced tumor growth and pulmonary metastasis. Mechanism studies showed that circASAP1 acts as a competing endogenous RNA for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), both of which are tumor suppressors in HCC. We found that mitogen-activated protein kinase (MAPK) 1 and colony stimulating factor (CSF)-1 were direct common targets for microRNA 326 (miR-326) and microRNA 532-5p (miR-532-5p), which were regulated by circASAP1. CircASAP1 promotes HCC cell proliferation and invasion by regulating miR-326/miR-532-5p-MAPK1 signaling and, furthermore, mediates tumor-associated macrophage infiltration by regulating the miR-326/miR-532-5p-CSF-1 pathway. Clinical HCC samples exhibited a positive correlation between circASAP1 expression and levels of CSF-1, MAPK1, and CD68+ tumor-associated macrophages, all of which were predictive of patient outcomes. CONCLUSION: We identified circASAP1 as a key regulator of HCC metastasis that acts on miR-326/miR-532-5p-MAPK1/CSF-1 signaling and serves as a prognostic predictor in patients with HCC.

Genomic sequencing identifies WNK2 as a driver in hepatocellular carcinoma and a risk factor for early recurrence.

BACKGROUND & AIMS: Early recurrence of hepatocellular carcinoma (HCC) after curative resection is common. However, the association between genetic mechanisms and early HCC recurrence, especially in Chinese patients, remains largely unknown. METHODS: We performed whole-genome sequencing (49 cases), whole-exome sequencing (18 cases), and deep targeted sequencing (115 cases) on 182 primary HCC samples. Focusing on WNK2, we used Sanger sequencing and qPCR to evaluate all the coding exons and copy numbers of that gene in an additional 554 HCC samples. We also explored the functional effect and mechanism of WNK2 on tumor growth and metastasis. RESULTS: We identified 5 genes (WNK2, RUNX1T1, CTNNB1, TSC1, and TP53) harboring somatic mutations that correlated with early tumor recurrence after curative resection in 182 primary HCC samples. Focusing on WNK2, the overall somatic mutation and copy number loss occurred in 5.3% (39/736) and 27.2% (200/736), respectively, of the total 736 HCC samples. Both types of variation were associated with lower WNK2 protein levels, higher rates of early tumor recurrence, and shorter overall survival. Biofunctional investigations revealed a tumor-suppressor role of WNK2: its inactivation led to ERK1/2 signaling activation in HCC cells, tumor-associated macrophage infiltration, and tumor growth and metastasis. CONCLUSIONS: Our results delineate genomic events that characterize Chinese HCCs and identify WNK2 as a driver of early HCC recurrence after curative resection. LAY SUMMARY: We applied next-generation sequencing and conducted an in-depth genomic analysis of hepatocellular carcinomas from a Chinese patient cohort. The results delineate the genomic events that characterize hepatocellular carcinomas in Chinese patients and identify WNK2 as a driver associated with early tumor recurrence after curative resection.

Intratumoral plasmacytoid dendritic cells as a poor prognostic factor for hepatocellular carcinoma following curative resection.

Plasmacytoid dendritic cells (pDCs) are present in various primary and metastatic human neoplasms; however, their clinical significance in hepatocellular carcinoma (HCC) is unclear. In this study, we investigated the distribution, prognostic value, and potential function of pDCs in HCC patients undergoing curative resection. We performed immunohistochemical analyses of whole tumor sections from 224 patients to assess the expression of BDCA2, CD3, CD4, CD8, Foxp3, granzyme B, IL-17, and CD34. The findings were validated using tissue microarrays from another two independent cohorts totaling 841 HCC patients undergoing curative resection. Our results demonstrated that high numbers of BDCA2+ pDCs within tumors correlated with high alpha-fetoprotein levels, greater vascular invasion, advanced tumor-node-metastasis stage, shorter overall survival, and a higher recurrence rate. However, patient outcomes were not associated with pDCs in peritumoral stromal or nontumor tissues. Furthermore, an increase in intratumoral pDCs was associated with increased intratumoral infiltration of Foxp3+ regulatory T cells and IL-17-producing cells and correlated with tumor vascular density. Univariate and multivariate analyses revealed that the presence of intratumoral pDCs alone or in combination with regulatory T and/or IL-17-producing cells was an independent predictor of time to recurrence and overall survival. In conclusion, our study demonstrated that intratumoral infiltration by pDCs is a novel indicator for poor prognosis in patients with HCC, possibly through the induction of an immune tolerogenic and inflammatory tumor microenvironment comprising regulatory T and IL-17-producing cells. An assessment of the combination of these cells represents a superior predictor of patient outcome.

A Positive Feedback Loop Between Cancer Stem-Like Cells and Tumor-Associated Neutrophils Controls Hepatocellular Carcinoma Progression.

Tumor-associated neutrophils (TANs) play a crucial role in tumor development and progression in the cancer microenvironment. Despite increased understanding of TAN contributions to hepatocellular carcinoma (HCC) progression and prognosis, the direct interaction between TANs and HCC cells is not fully understood. In this study, we tested the effect of TANs on HCC cells in vitro and in vivo and investigated the mechanism of interaction between them. Our results showed that TANs secreted bone morphogenetic protein 2 and transforming growth factor beta 2 and triggered microRNA 301b-3p (miR-301-3p) expression in HCC cells, subsequently suppressed gene expression of limbic system-associated membrane protein (LSAMP) and CYLD lysine 63 deubiquitinase (CYLD), and increased stem cell characteristics in HCC cells. These TAN-induced HCC stem-like cells were hyperactive in nuclear factor kappa B signaling, secreted higher levels of chemokine (C-X-C motif) ligand 5 (CXCL5), and recruited more TAN infiltration, suggesting a positive feedback loop. In clinical HCC samples, increased TANs correlated with elevated miR-301b-3p, decreased LSAMP and CYLD expression, and increased nuclear p65 accumulation and CXCL5 expression, all of which predicted patient outcome. Conclusion: Our work identified a positive feedback loop governing cancer stem-like cells and TANs in HCC that controls tumor progression and patient outcome.

Downregulation of MAGE family member H1 enhances hepatocellular carcinoma progression and serves as a biomarker for patient prognosis.

AIM: The MAGE family member H1 (MAGEH1) belongs to melanoma-associated antigen (MAGE) superfamily. The role of MAGEH1 in hepatocellular carcinoma (HCC) is largely undefined. MATERIALS & METHODS: We used quantitative reverse transcription PCR and immunohistochemistry to detect MAGEH1 expression in HCC tissues. CCK-8 assay, wound healing migration assay and Transwell Matrigel invasion assay were used to measure HCC cell proliferation, migration and invasion ability. RESULTS:  MAGEH1 expression was downregulated in HCC tumor tissues compared with adjacent normal liver tissues and in samples from patients with tumor recurrence. MAGEH1 reduced HCC cell proliferation, migration and invasion ability. Low MAGEH1 expression was significantly correlated with poor prognosis in HCC patients. CONCLUSION:  MAGEH1 may serve as a potential biomarker and a new prognostic factor for HCC.

Overexpression of semaphorin 3A promotes tumor progression and predicts poor prognosis in hepatocellular carcinoma after curative resection.

The semaphorins were originally identified as having roles as guidance cues during neural development. Class 3 semaphorins are involved in cancer progression. However, the roles of class 3 semaphorins in hepatocellular carcinoma (HCC) are unknown. We examined the expression levels of class 3 semaphorins in HCC cell lines with different metastatic potential and in carcinoma tissue samples. The results indicated that Semaphorin 3A expression was up-regulated in metastatic cell lines and in samples from patients with tumor recurrence. Cell functional studies revealed that Semaphorin 3A promoted HCC cell proliferation, migration, and invasion. Animal studies indicated that Semaphorin 3A overexpression enhanced tumor growth and lung metastasis. Semaphorin 3A also acted as a chemoattractant involved in direct recruitment of macrophages in vitro, and facilitated tumor-associated macrophage (TAM) infiltration in vivo. Multivariate analysis revealed that Semaphorin 3A expression alone, or combined with the number of TAMs, can be an independent predictor for overall survival time and time to recurrence. Overall, the results suggested that Semaphorin 3A increased TAM infiltration and promoted HCC progression. Semaphorin 3A expression alone, or combined with the number of TAMs, is a new prognostic factor and potential target for the treatment of HCC.