Developing survival prediction models in colorectal cancer using epigenome-wide DNA methylation data from whole blood.

While genome-wide association studies are valuable in identifying CRC survival predictors, the benefit of adding blood DNA methylation (blood-DNAm) to clinical features, including the TNM system, remains unclear. In a multi-site population-based patient cohort study of 2116 CRC patients with baseline blood-DNAm, we analyzed survival predictions using eXtreme Gradient Boosting with a 5-fold nested leave-sites-out cross-validation across four groups: traditional and comprehensive clinical features, blood-DNAm, and their combination. Model performance was assessed using time-dependent ROC curves and calibrations. During a median follow-up of 10.3 years, 1166 patients died. Although blood-DNAm-based predictive signatures achieved moderate performances, predictive signatures based on clinical features outperformed blood-DNAm signatures. The inclusion of blood-DNAm did not improve survival prediction over clinical features. M1 stage, age at blood collection, and N2 stage were the top contributors. Despite some prognostic value, incorporating blood DNA methylation did not enhance survival prediction of CRC patients beyond clinical features.

Lymphotoxin beta-activated LTBR/NIK/RELB axis drives proliferation in cholangiocarcinoma.

BACKGROUND AND AIMS: Cholangiocarcinoma (CCA) is an aggressive malignancy arising from the intrahepatic (iCCA) or extrahepatic (eCCA) bile ducts with poor prognosis and limited treatment options. Prior evidence highlighted a significant contribution of the non-canonical NF-κB signalling pathway in initiation and aggressiveness of different tumour types. Lymphotoxin-ß (LTß) stimulates the NF-κB-inducing kinase (NIK), resulting in the activation of the transcription factor RelB. However, the functional contribution of the non-canonical NF-κB signalling pathway via the LTß/NIK/RelB axis in CCA carcinogenesis and progression has not been established. METHODS: Human CCA-derived cell lines and organoids were examined to determine the expression of NF-κB pathway components upon activation or inhibition. Proliferation and cell death were analysed using real-time impedance measurement and flow cytometry. Immunoblot, qRT-PCR, RNA sequencing and in situ hybridization were employed to analyse gene and protein expression. Four in vivo models of iCCA were used to probe the activation and regulation of the non-canonical NF-κB pathway. RESULTS: Exposure to LTα1/ß2 activates the LTß/NIK/RelB axis and promotes proliferation in CCA. Inhibition of NIK with the small molecule inhibitor B022 efficiently suppresses RelB expression in patient-derived CCA organoids and nuclear co-translocation of RelB and p52 stimulated by LTα1/ß2 in CCA cell lines. In murine CCA, RelB expression is significantly increased and LTß is the predominant ligand of the non-canonical NF-κB signalling pathway. CONCLUSIONS: Our study confirms that the non-canonical NF-κB axis LTß/NIK/RelB drives cholangiocarcinogenesis and represents a candidate therapeutic target.

Combined analysis of albumin in situ hybridisation and C reactive protein immunohistochemistry for the diagnosis of intrahepatic cholangiocarcinoma: towards a molecular classification paradigm.

AIMS: Intrahepatic cholangiocarcinoma (iCCA) is a diagnosis of exclusion that can pose a challenge to the pathologist despite thorough clinical workup. Although several immunohistochemical markers have been proposed for iCCA, none of them reached clinical practice. We here assessed the combined usage of two promising diagnostic approaches, albumin in situ hybridisation (Alb-ISH) and C reactive protein (CRP) immunohistochemistry, for distinguishing iCCA from other adenocarcinoma primaries. METHODS: We conducted Alb-ISH and CRP immunohistochemistry in a large European iCCA cohort (n=153) and compared the results with a spectrum of other glandular adenocarcinomas of different origin (n=885). In addition, we correlated expression patterns with clinicopathological information and mutation data. RESULTS: Alb-ISH was highly specific for iCCA (specificity 98.8%) with almost complete negativity in perihilar CCA and only rare positives among other adenocarcinomas (sensitivity 69.5%). CRP identified the vast majority of iCCA cases (sensitivity 84.1%) at a lower specificity of 86.4%. Strikingly, the combination of CRP and Alb-ISH boosted the diagnostic sensitivity to 88.0% while retaining a considerable specificity of 86.1%. Alb-ISH significantly correlated with CRP expression, specific tumour morphologies and small or large duct iCCA subtypes. Neither Alb-ISH nor CRP was associated with iCCA patient survival. 16 of 17 recurrent mutations in either IDH1, IDH2 and FGFR2 affected Alb-ISH positive cases, while the only KRAS mutation corresponded to an Alb-ISH negative case. CONCLUSIONS: In conclusion, we propose a sequential diagnostic approach for iCCA, integrating CRP immunohistochemistry and Alb-ISH. This may improve the accuracy of CCA classification and pave the way towards a molecular-guided CCA classification.

A new biomarker panel for differential diagnosis of cholangiocarcinoma: Results from an exploratory analysis.

INTRODUCTION: Diagnosis of cholangiocarcinoma (CCA) can be challenging due to unclear imaging criteria and difficulty obtaining adequate tissue biopsy. Although serum cancer antigen 19-9 and carcinoembryonic antigen have been proposed as potential diagnostic aids, their use remains limited by insufficient sensitivity and specificity. This exploratory analysis aimed to identify individual- and combinations of serum biomarkers to distinguish CCA from hepatocellular carcinoma (HCC) and chronic liver disease (CLD) controls using samples from a published study. METHODS: This prospective, multicenter, case-control study included patients aged ≥18 years at high-risk of HCC. Serum and ethylene diamine tetraacetic acid-plasma samples were collected prior to any treatment and confirmed diagnosis of HCC or CCA. Fourteen biomarkers (measured by electrochemiluminescence immunoassays or enzyme-linked immunosorbent assays) were subjected to univariate analysis and 13 included in a multivariate analysis (per selected combinations and exhaustive search). RESULTS: Overall, 55 CCA, 306 HCC, and 733 CLD control samples were analyzed. For distinguishing CCA from HCC, alpha-fetoprotein and matrix metalloproteinase-2 (MMP-2) showed the best individual performance (area under the curve (AUC) 86.6% and 84.4%, respectively); tissue inhibitor of metalloproteinase-1 (TIMP-1) was most able to distinguish CCA from CLD (AUC 94.5%) and from HCC + CLD (AUC 88.6%). The combination of MMP-2 and TIMP-1 was the best-performing two-marker panel, with AUC >90% for all comparisons. CONCLUSION: MMP-2 and TIMP-1 are promising biomarkers that could support differential diagnosis of CCA. Incorporating these assays into the diagnostic algorithm could provide additional diagnostic information in a non-invasive, rapid manner, and could supplement existing diagnostic methods.

Discovery of a haptoglobin glycopeptides biomarker panel for early diagnosis of hepatocellular carcinoma.

Background: There is a need for new serum biomarkers for early detection of hepatocellular carcinoma (HCC). Haptoglobin (Hp) N-glycosylation is altered in HCC, but the diagnostic value of site-specific Hp glycobiomarkers is rarely reported. We aimed to determine the site-specific glycosylation profile of Hp for early-stage HCC diagnosis. Method: Hp glycosylation was analyzed in the plasma of patients with liver diseases (n=57; controls), early-stage HCC (n=50) and late-stage HCC (n=32). Hp phenotype was determined by immunoblotting. Hp was immunoisolated and digested into peptides. N-glycopeptides were identified and quantified using liquid chromatography-mass spectrometry. Cohort samples were compared using Wilcoxon rank-sum (Mann-Whitney U) tests. Diagnostic performance was assessed using receiver operating characteristic (ROC) curves and area under curve (AUC). Results: Significantly higher fucosylation, branching and sialylation of Hp glycans, and expression of high-mannose glycans, was observed as disease progressed from cirrhosis to early- and late-stage HCC. Several glycopeptides demonstrated high values for early diagnosis of HCC, with an AUC of 93% (n=1), >80% (n=3), >75% (n=13) and >70% (n=11), compared with alpha-fetoprotein (AFP; AUC of 79%). The diagnostic performance of the identified biomarkers was only slightly affected by Hp phenotype. Conclusion: We identified a panel of Hp glycopeptides that are significantly differentially regulated in early- and late-stage HCC. Some glycobiomarkers exceeded the diagnostic value of AFP (the most commonly used biomarker for HCC diagnosis). Our findings provide evidence that glycobiomarkers can be effective in the diagnosis of early HCC - individually, as a panel of glycopeptides or combined with conventional serological biomarkers.

Etiology-independent activation of the LTß-LTßR-RELB axis drives aggressiveness and predicts poor prognosis in HCC.

BACKGROUND AND AIMS: HCC is the most common primary liver tumor, with an increasing incidence worldwide. HCC is a heterogeneous malignancy and usually develops in a chronically injured liver. The NF-κB signaling network consists of a canonical and a noncanonical branch. Activation of canonical NF-κB in HCC is documented. However, a functional and clinically relevant role of noncanonical NF-κB and its downstream effectors is not established. APPROACH AND RESULTS: Four human HCC cohorts (total n = 1462) and 4 mouse HCC models were assessed for expression and localization of NF-κB signaling components and activating ligands. In vitro , NF-κB signaling, proliferation, and cell death were measured, proving a pro-proliferative role of v-rel avian reticuloendotheliosis viral oncogene homolog B (RELB) activated by means of NF-κB-inducing kinase. In vivo , lymphotoxin beta was identified as the predominant inducer of RELB activation. Importantly, hepatocyte-specific RELB knockout in a murine HCC model led to a lower incidence compared to controls and lower maximal tumor diameters. In silico , RELB activity and RELB-directed transcriptomics were validated on the The Cancer Genome Atlas HCC cohort using inferred protein activity and Gene Set Enrichment Analysis. In RELB-active HCC, pathways mediating proliferation were significantly activated. In contrast to v-rel avian reticuloendotheliosis viral oncogene homolog A, nuclear enrichment of noncanonical RELB expression identified patients with a poor prognosis in an etiology-independent manner. Moreover, RELB activation was associated with malignant features metastasis and recurrence. CONCLUSIONS: This study demonstrates a prognostically relevant, etiology-independent, and cross-species consistent activation of a lymphotoxin beta/LTßR/RELB axis in hepatocarcinogenesis. These observations may harbor broad implications for HCC, including possible clinical exploitation.

Deep Learning-Enabled Diagnosis of Liver Adenocarcinoma.

BACKGROUND & AIMS: Diagnosis of adenocarcinoma in the liver is a frequent scenario in routine pathology and has a critical impact on clinical decision making. However, rendering a correct diagnosis can be challenging, and often requires the integration of clinical, radiologic, and immunohistochemical information. We present a deep learning model (HEPNET) to distinguish intrahepatic cholangiocarcinoma from colorectal liver metastasis, as the most frequent primary and secondary forms of liver adenocarcinoma, with clinical grade accuracy using H&E-stained whole-slide images. METHODS: HEPNET was trained on 714,589 image tiles from 456 patients who were randomly selected in a stratified manner from a pool of 571 patients who underwent surgical resection or biopsy at Heidelberg University Hospital. Model performance was evaluated on a hold-out internal test set comprising 115 patients and externally validated on 159 patients recruited at Mainz University Hospital. RESULTS: On the hold-out internal test set, HEPNET achieved an area under the receiver operating characteristic curve of 0.994 (95% CI, 0.989-1.000) and an accuracy of 96.522% (95% CI, 94.521%-98.694%) at the patient level. Validation on the external test set yielded an area under the receiver operating characteristic curve of 0.997 (95% CI, 0.995-1.000), corresponding to an accuracy of 98.113% (95% CI, 96.907%-100.000%). HEPNET surpassed the performance of 6 pathology experts with different levels of experience in a reader study of 50 patients (P = .0005), boosted the performance of resident pathologists to the level of senior pathologists, and reduced potential downstream analyses. CONCLUSIONS: We provided a ready-to-use tool with clinical grade performance that may facilitate routine pathology by rendering a definitive diagnosis and guiding ancillary testing. The incorporation of HEPNET into pathology laboratories may optimize the diagnostic workflow, complemented by test-related labor and cost savings.

Spatiotemporal analysis of tumour-infiltrating immune cells in biliary carcinogenesis.

BACKGROUND: Intraductal papillary neoplasms (IPN) and biliary epithelial neoplasia (BilIN) are well-defined precursor lesions of biliary tract carcinoma (BTC). The aim of this study was to provide a comprehensive characterisation of the inflammatory microenvironment in BTC precursor lesions. METHODS: Immunohistochemistry was employed to assess tumour-infiltrating immune cells in tissue samples from patients, for whom precursor lesions were identified alongside invasive BTC. The spatiotemporal evolution of the immune microenvironment during IPN-associated carcinogenesis was comprehensively analysed using triplet sample sets of non-neoplastic epithelium, precursor lesion and invasive BTC. Immune-cell dynamics during IPN- and BilIN-associated carcinogenesis were subsequently compared. RESULTS: Stromal CD3+ (P = 0.002), CD4+ (P = 0.007) and CD8+ (P < 0.001) T cells, CD20+ B cells (P = 0.008), MUM1+ plasma cells (P = 0.012) and CD163+ M2-like macrophages (P = 0.008) significantly decreased in IPN compared to non-tumorous biliary epithelium. Upon transition from IPN to invasive BTC, stromal CD68+ (P = 0.001) and CD163+ (P < 0.001) macrophages significantly increased. In contrast, BilIN-driven carcinogenesis was characterised by significant reduction of intraepithelial CD8+ T-lymphocytic infiltration from non-tumorous epithelium via BilIN (P = 0.008) to BTC (P = 0.004). CONCLUSION: IPN and BilIN are immunologically distinct entities that undergo different immune-cell variations during biliary carcinogenesis. Intraepithelial CD8+ T-lymphocytic infiltration of biliary tissue decreased already at the IPN-precursor stage, whereas BilIN-associated carcinogenesis showed a slowly progressing reduction towards invasive carcinoma.

Comprehensive evaluation of microRNA as a biomarker for the diagnosis of hepatocellular carcinoma.

BACKGROUND: Hepatocellular carcinoma (HCC) is the most common type of primary liver cancer. Current guidelines for HCC management recommend surveillance of high-risk patients every 6 mo using ultrasonography. Serum biomarkers, like alpha-fetoprotein (AFP), protein induced by vitamin K absence/antagonist-II (PIVKA-II) and lectin-reactive AFP, show suboptimal performance for detection of HCC, which is crucial for successful resection or treatment. Thus, there is a significant need for new biomarkers to aid early diagnosis of HCC. Studies have shown that the expression level of human microRNAs (miRNAs), a small, non-coding RNA species released into the blood, can serve as an early marker for various diseases, including HCC. AIM: To evaluate the diagnostic role of miRNAs in HCC as single markers, signatures or in combination with known protein biomarkers. METHODS: Our prospective, multicenter, case-control study recruited 660 participants (354 controls with chronic liver disease and 306 participants with HCC) and employed a strategy of initial screening by two independent methods, real-time quantitative PCR (n = 60) and next-generation sequencing (n = 100), to assess a large number of miRNAs. The results from the next-generation sequencing and real-time quantitative PCR screening approaches were then combined to select 26 miRNAs (including two putative novel miRNAs). Those miRNAs were analyzed for their diagnostic potential as single markers or in combination with other miRNAs or established protein biomarkers AFP and PIVKA-II via real-time quantitative PCR in training (n = 200) and validation cohorts (n = 300). RESULTS: We identified 26 miRNAs that differentiated chronic liver disease controls from (early) HCC via two independent discovery approaches. Three miRNAs, miR-21-5p (miR-21), miR-320a and miR-186-5p, were selected by both methods. In the training cohort, only miR-21, miR-320d and miR-423 could significantly distinguish (Q < 0.05) between the HCC and chronic liver disease control groups. In the multivariate setting, miR-21 with PIVKA-II was selected as the best combination, resulting in an area under the curve of 0.87 for diagnosis and area under the curve of 0.74 for early diagnosis of HCC. In the validation cohort, only miR-21 and miR-423 could be confirmed as potential HCC biomarkers. A combination of miRNAs did not perform better than any single miRNA. Improvement of PIVKA-II performance through combination with miRNAs could not be confirmed in the validation panel. Two putative miRs, put-miR-6 and put-miR-99, were tested in the training and validation panels, but their expression could only be detected in very few samples and at a low level (cycle threshold between 31.24 and 34.97). CONCLUSION: miRNAs alone or as a signature in combination with protein biomarkers AFP and PIVKA-II do not improve the diagnostic performance of the protein biomarkers.

Bcl-xL as prognostic marker and potential therapeutic target in cholangiocarcinoma.

Intrahepatic, perihilar, and distal cholangiocarcinoma (iCCA, pCCA, dCCA) are highly malignant tumours with increasing mortality rates due to therapy resistances. Among the mechanisms mediating resistance, overexpression of anti-apoptotic Bcl-2 proteins (Bcl-2, Bcl-xL , Mcl-1) is particularly important. In this study, we investigated whether antiapoptotic protein patterns are prognostically relevant and potential therapeutic targets in CCA. Bcl-2 proteins were analysed in a pan-cancer cohort from the NCT/DKFZ/DKTK MASTER registry trial (n = 1140, CCA n = 72) via RNA-sequencing and transcriptome-based protein activity interference revealing high ranks of CCA for Bcl-xL and Mcl-1. Expression of Bcl-xL , Mcl-1, and Bcl-2 was assessed in human CCA tissue and cell lines compared with cholangiocytes by immunohistochemistry, immunoblotting, and quantitative-RT-PCR. Immunohistochemistry confirmed the upregulation of Bcl-xL and Mcl-1 in iCCA tissues. Cell death of CCA cell lines upon treatment with specific small molecule inhibitors of Bcl-xL (Wehi-539), of Mcl-1 (S63845), and Bcl-2 (ABT-199), either alone, in combination with each other or together with chemotherapeutics was assessed by flow cytometry. Targeting Bcl-xL induced cell death and augmented the effect of chemotherapy in CCA cells. Combined inhibition of Bcl-xL and Mcl-1 led to a synergistic increase in cell death in CCA cell lines. Correlation between Bcl-2 protein expression and survival was analysed within three independent patient cohorts from cancer centers in Germany comprising 656 CCA cases indicating a prognostic value of Bcl-xL in CCA depending on the CCA subtype. Collectively, these observations identify Bcl-xL as a key protein in cell death resistance of CCA and may pave the way for clinical application.

Specific Targeting of Antiapoptotic Bcl-2 Proteins as a Radiosensitizing Approach in Solid Tumors.

Avoidance of therapy-induced apoptosis is a hallmark of acquired resistance towards radiotherapy. Thus, breaking resistance still challenges modern cancer therapy. The Bcl-2 protein family is known for its regulatory role in apoptosis signaling, making Bcl-2, Mcl-1 and Bcl-xL promising targets. This study evaluates the effects of highly specific inhibitors for Bcl-xL (WEHI-539), Bcl-2 (ABT-199) and Mcl-1 (S63845) as radiosensitizers. Covering a broad spectrum of solid tumors, Non-Small-Cell Lung Cancer (NSCLC), Head and Neck Squamous Cell Carcinoma (HNSCC) and synovial sarcoma cell lines were exposed to fractionated radiation as standard therapy with or without Bcl-2 protein inhibition. Protein expression was detected by Western blot and cell death was assessed by flow cytometry measuring apoptosis. In contrast to NSCLC, a high level of Bcl-xL and its upregulation during radiotherapy indicated radioresistance in HNSCC and synovial sarcoma. Radioresistant cell lines across all entities benefited synergistically from combined therapy with Bcl-xL inhibition and fractionated radiation. In NSCLC cell lines, Mcl-1 inhibition significantly augmented radiotherapy independent of the expression level. Our data suggest that among antiapoptotic Bcl-2 proteins, targeting Bcl-xL may break resistance to radiation in HNSCC, synovial sarcoma and NSCLC in vitro. In NSCLC, Mcl-1 might be a promising target that needs further investigation.

Gene expression-based prediction of pazopanib efficacy in sarcoma.

BACKGROUND: The multi-receptor tyrosine kinase inhibitor pazopanib is approved for the treatment of advanced soft-tissue sarcoma and has also shown activity in other sarcoma subtypes. However, its clinical efficacy is highly variable, and no reliable predictors exist to select patients who are likely to benefit from this drug. PATIENTS AND METHODS: We analysed the molecular profiles and clinical outcomes of patients with pazopanib-treated sarcoma enrolled in a prospective observational study by the German Cancer Consortium, DKTK MASTER, that employs whole-genome/exome sequencing and transcriptome sequencing to inform the care of young adults with advanced cancer across histology and patients with rare cancers. RESULTS: Among 109 patients with available whole-genome/exome sequencing data, there was no correlation between clinical parameters, specific genetic alterations or mutational signatures and clinical outcome. In contrast, the analysis of a subcohort of 62 patients who underwent molecular analysis before pazopanib treatment and had transcriptome sequencing data available showed that mRNA levels of NTRK3 (hazard ratio [HR] = 0.53, p = 0.021), IGF1R (HR = 1.82, p = 0.027) and KDR (HR = 0.50, p = 0.011) were independently associated with progression-free survival (PFS). Based on the expression of these receptor tyrosine kinase genes, i.e. the features NTRK3-high, IGF1R-low and KDR-high, we developed a pazopanib efficacy predictor that stratified patients into three groups with significantly different PFS (p < 0.0001). Application of the pazopanib efficacy predictor to an independent cohort of patients with pazopanib-treated sarcoma from DKTK MASTER (n = 43) confirmed its potential to separate patient groups with significantly different PFS (p = 0.02), whereas no such association was observed in patients with sarcoma from DKTK MASTER (n = 97) or The Cancer Genome Atlas sarcoma cohort (n = 256) who were not treated with pazopanib. CONCLUSION: A score based on the combined expression of NTRK3, IGF1R and KDR allows the identification of patients with sarcoma and with good, intermediate and poor outcome following pazopanib therapy and warrants prospective investigation as a predictive tool to optimise the use of this drug in the clinic.

MGMT inactivation as a new biomarker in patients with advanced biliary tract cancers.

Biliary tract cancers (BTCs) have poor prognosis and limited therapeutic options. The impact of O6 -methylguanine-DNA methyltransferase (MGMT) inactivation in advanced BTC patients is not established. We investigated the prevalence, prognostic, and predictive impact of MGMT inactivation in two multicenter cohorts. MGMT inactivation was assessed through PCR and immunohistochemistry (IHC) in an Italian cohort; the results were then externally validated using RNA sequencing (RNA-seq) data from the BTC subcohort of the Molecularly Aided Stratification for Tumor Eradication Research (MASTER) precision oncology program of the National Center for Tumor Diseases Heidelberg and the German Cancer Consortium. Among 164 Italian cases, 18% presented MGMT promoter hypermethylation (> 14%) and 73% had negative MGMT protein expression. Both were associated with worse overall survival (OS; HR 2.31; P < 0.001 and HR 1.99, P = 0.012, respectively). In the MASTER cohort, patients with lower MGMT mRNA expression showed significantly poorer OS (median OS [mOS] 20.4 vs 31.7 months, unadjusted HR 1.89; P = 0.043). Our results suggest that MGMT inactivation is a frequent epigenetic alteration in BTC, with a significant prognostic impact, and provide the rationale to explore DNA-damaging agents in MGMT-inactivated BTCs.

Caspase-8 in endothelial cells maintains gut homeostasis and prevents small bowel inflammation in mice.

The gut has a specific vascular barrier that controls trafficking of antigens and microbiota into the bloodstream. However, the molecular mechanisms regulating the maintenance of this vascular barrier remain elusive. Here, we identified Caspase-8 as a pro-survival factor in mature intestinal endothelial cells that is required to actively maintain vascular homeostasis in the small intestine in an organ-specific manner. In particular, we find that deletion of Caspase-8 in endothelial cells results in small intestinal hemorrhages and bowel inflammation, while all other organs remained unaffected. We also show that Caspase-8 seems to be particularly needed in lymphatic endothelial cells to maintain gut homeostasis. Our work demonstrates that endothelial cell dysfunction, leading to the breakdown of the gut-vascular barrier, is an active driver of chronic small intestinal inflammation, highlighting the role of the intestinal vasculature as a safeguard of organ function.

STAT1 and STAT3 Exhibit a Crosstalk and Are Associated with Increased Inflammation in Hepatocellular Carcinoma.

Liver cancers, which are mostly hepatocellular carcinoma (HCC) and cholangiocarcinoma (CCA), are very aggressive tumors with poor prognosis. Therapeutic options with curative intent are largely limited to surgery and available systemic therapies show limited benefit. Signal transducer and activator of transcription 1 (STAT1) and 3 (STAT3) are key transcription factors activated by pro-inflammatory cytokines such as interferon-γ (IFN-γ) and interleukin-6 (IL-6). In this study, we combined in vitro cell culture experiments and immunohistochemical analyses of human HCC (N = 124) and CCA (N = 138) specimens. We observed that in the absence of STAT3, IL-6 induced the activation of STAT1 and its target genes suggesting that IL-6 derived from the tumor microenvironment may activate both STAT1 and STAT3 target genes in HCC tumor cells. In addition, STAT1 and STAT3 were highly activated in a subset of HCC, which exhibited a high degree of infiltrating CD8- and FOXP3-positive immune cells and PD-L1 expression. Our results demonstrate that STAT1 and STAT3 are expressed and activated in HCC and tumor infiltrating immune cells. In addition, HCC cases with high STAT1 and STAT3 expression also exhibited a high degree of immune cell infiltration, suggesting increased immunological tolerance.

Transforming Growth Factor-ß Activated Kinase 1 (Tak1) Is Activated in Hepatocellular Carcinoma, Mediates Tumor Progression, and Predicts Unfavorable Outcome.

Although knowledge on inflammatory signaling pathways driving cancer initiation and progression has been increasing, molecular mechanisms in hepatocarcinogenesis are still far from being completely understood. Hepatocyte-specific deletion of the MAPKKK Tak1 in mice recapitulates important steps of hepatocellular carcinoma (HCC) development, including the occurrence of cell death, steatohepatitis, dysplastic nodules, and HCCs. However, overactivation of Tak1 in mice upon deletion of its deubiquitinase Cyld also results in steatohepatitis and HCC development. To investigate Tak1 and Cyld in human HCCs, we created a tissue microarray to analyze their expression by immunohistochemistry in a large and well-characterized cohort of 871 HCCs of 561 patients. In the human liver and HCC, Tak1 is predominantly present as its isoform Tak1A and predominantly localizes to cell nuclei. Tak1 is upregulated in diethylnitrosamine-induced mouse HCCs as well as in human HCCs independent of etiology and is further induced in distant metastases. A high nuclear Tak1 expression is associated with short survival and vascular invasion. When we overexpressed Tak1A in Huh7 cells, we observed increased tumor cell migration, whereas overexpression of full-length Tak1 had no significant effect. A combined score of low Cyld and high Tak1 expression was an independent prognostic marker in a multivariate Cox regression model.

Integrative analysis reveals early and distinct genetic and epigenetic changes in intraductal papillary and tubulopapillary cholangiocarcinogenesis.

OBJECTIVE: A detailed understanding of the molecular alterations in different forms of cholangiocarcinogenesis is crucial for a better understanding of cholangiocarcinoma (CCA) and may pave the way to early diagnosis and better treatment options. DESIGN: We analysed a clinicopathologically well-characterised patient cohort (n=54) with high-grade intraductal papillary (IPNB) or tubulopapillary (ITPN) neoplastic precursor lesions of the biliary tract and correlated the results with an independent non-IPNB/ITPN associated CCA cohort (n=294). The triplet sample set of non-neoplastic biliary epithelium, precursor and invasive CCA was analysed by next generation sequencing, DNA copy number and genome-wide methylation profiling. RESULTS: Patients with invasive CCA arising from IPNB/ITPN had better prognosis than patients with CCA not associated with IPNB/ITPN. ITPN was localised mostly intrahepatic, whereas IPNB was mostly of extrahepatic origin. IPNB/ITPN were equally associated with small-duct and large-duct type intrahepatic CCA. IPNB exhibited mutational profiles of extrahepatic CCA, while ITPN had significantly fewer mutations. Most mutations were shared between precursor lesions and corresponding invasive CCA but ROBO2 mutations occurred exclusively in invasive CCA and CTNNB1 mutations were mainly present in precursor lesions. In addition, IPNB and ITPN differed in their DNA methylation profiles and analyses of latent methylation components suggested that IPNB and ITPN may have different cells-of-origin. CONCLUSION: Integrative analysis revealed that IPNB and ITPN harbour distinct early genetic alterations, IPNB are enriched in mutations typical for extrahepatic CCA, whereas ITPN exhibited few genetic alterations and showed distinct epigenetic profiles. In conclusion, IPNB/ITPN may represent a distinctive, intermediate form of intrahepatic and extrahepatic cholangiocarcinogenesis.

Multimarker Panels for Detection of Early Stage Hepatocellular Carcinoma: A Prospective, Multicenter, Case-Control Study.

Hepatocellular carcinoma (HCC), the sixth most common cancer worldwide, has an incidence rate equal to mortality. Over 80% of HCC cases occur within a high-risk population, mainly patients with both cirrhosis and chronic hepatitis B or C. With a 5-year survival rate ranging from <16% for advanced HCC to >90% for early stage HCC, there is a high medical need for the early detection of HCC. In this study, we systematically evaluated biomarkers mentioned in international guidelines and peer-reviewed literature for HCC surveillance and diagnosis with the aim of identifying combinations that display high sensitivity and specificity for early stage HCC. Fifty biomarkers were measured in the first sample panel, panel A (n = 110), and subjected to univariate analysis. Of these, 35 biomarkers (38 assays) from panel A and an additional 13 biomarkers from the literature were prioritized for subsequent multivariate evaluation with lasso regression and exhaustive search of two- to four-biomarker combinations (panel B). Panel B included 1,081 samples from patients with HCC (n = 308) or with chronic liver diseases (n = 740). Among all patients, 61.0% had hepatitis B, 32.9% had hepatitis C, and 60.5% had cirrhosis; 40.6% of patients with HCC had early stage cancer. Protein induced by vitamin K absence-II (PIVKA-II; also known as des-gamma-carboxy prothrombin [DCP]) and alpha-fetoprotein (AFP) demonstrated the best clinical performance, both individually and in combination, and the addition of a third biomarker (Lens culinaris agglutinin-reactive fraction of AFP [AFP-L3], cartilage oligomeric matrix protein [COMP], insulin-like growth factor-binding protein 3 [IGFBP3], or matrix metalloproteinase 3 [MMP3]) further increased sensitivity for the detection of both early stage and all-stage HCC. The addition of age and sex to the three-biomarker panel resulted in an improved diagnostic performance. Conclusion: The combination of AFP and PIVKA-II, with either IGFBP3, COMP or MMP3, plus age and sex, demonstrated the best performance for the detection of early- and all-stage HCC. These novel panels performed similar to that of the GALAD score (sex [gender], age, plus serum levels of AFP, AFP-L3 and DCP [PIVKA-II]), a promising screening tool developed for HCC detection.

The Transmembrane Receptor TIRC7 Identifies a Distinct Subset of Immune Cells with Prognostic Implications in Cholangiocarcinoma.

Cholangiocarcinoma (CCA) is a heterogeneous malignancy with a dismal prognosis. Therapeutic options are largely limited to surgery and conventional chemotherapy offers limited benefit. As immunotherapy has proven highly effective in various cancer types, we have undertaken a quantitative immunohistopathological assessment of immune cells expressing the immunoinhibitory T cell immune response cDNA 7 receptor (TIRC7), an emerging immunoinhibitory receptor, in a cohort of 135 CCA patients. TIRC7+ immune cells were present in both the tumor epithelia and stroma in the majority of CCA cases with the highest levels found in intrahepatic CCA. While intraepithelial density of TIRC7+ immune cells was decreased compared to matched non-neoplastic bile ducts, stromal quantity was higher in the tumor samples. Tumors exhibiting signet ring cell or adenosquamous morphology were exclusively associated with an intraepithelial TIRC7+ phenotype. Survival analysis showed intraepithelial TIRC7+ immune cell density to be a highly significant favorable prognosticator in intrahepatic but not proximal or distal CCA. Furthermore, intraepithelial TIRC7+ immune cell density correlated with the number of intraepithelial CD8+ immune cells and with the total number of CD4+ immune cells. Our results suggest the presence and prognostic relevance of TIRC7+ immune cells in CCA and warrant further functional studies on its pharmacological modulation.