Deep learning-based phenotyping reclassifies combined hepatocellular-cholangiocarcinoma.

Primary liver cancer arises either from hepatocytic or biliary lineage cells, giving rise to hepatocellular carcinoma (HCC) or intrahepatic cholangiocarcinoma (ICCA). Combined hepatocellular- cholangiocarcinomas (cHCC-CCA) exhibit equivocal or mixed features of both, causing diagnostic uncertainty and difficulty in determining proper management. Here, we perform a comprehensive deep learning-based phenotyping of multiple cohorts of patients. We show that deep learning can reproduce the diagnosis of HCC vs. CCA with a high performance. We analyze a series of 405 cHCC-CCA patients and demonstrate that the model can reclassify the tumors as HCC or ICCA, and that the predictions are consistent with clinical outcomes, genetic alterations and in situ spatial gene expression profiling. This type of approach could improve treatment decisions and ultimately clinical outcome for patients with rare and biphenotypic cancers such as cHCC-CCA.

Contrast-enhanced ultrasound Liver Imaging Reporting and Data System: Lights and shadows in hepatocellular carcinoma and cholangiocellular carcinoma diagnosis.

BACKGROUND: Contrast-enhanced ultrasound (CEUS) is considered a secondary examination compared to computed tomography (CT) and magnetic resonance imaging (MRI) in the diagnosis of hepatocellular carcinoma (HCC), due to the risk of misdiagnosing intrahepatic cholangiocarcinoma (ICC). The introduction of CEUS Liver Imaging Reporting and Data System (CEUS LI-RADS) might overcome this limitation. Even though data from the literature seems promising, its reliability in real-life context has not been well-established yet. AIM: To test the accuracy of CEUS LI-RADS for correctly diagnosing HCC and ICC in cirrhosis. METHODS: CEUS LI-RADS class was retrospectively assigned to 511 nodules identified in 269 patients suffering from liver cirrhosis. The diagnostic standard for all nodules was either biopsy (102 nodules) or CT/MRI (409 nodules). Common diagnostic accuracy indexes such as sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) were assessed for the following associations: CEUS LR-5 and HCC; CEUS LR-4 and 5 merged class and HCC; CEUS LR-M and ICC; and CEUS LR-3 and malignancy. The frequency of malignant lesions in CEUS LR-3 subgroups with different CEUS patterns was also determined. Inter-rater agreement for CEUS LI-RADS class assignment and for major CEUS pattern identification was evaluated. RESULTS: CEUS LR-5 predicted HCC with a 67.6% sensitivity, 97.7% specificity, and 99.3% PPV (P < 0.001). The merging of LR-4 and 5 offered an improved 93.9% sensitivity in HCC diagnosis with a 94.3% specificity and 98.8% PPV (P < 0.001). CEUS LR-M predicted ICC with a 91.3% sensitivity, 96.7% specificity, and 99.6% NPV (P < 0.001). CEUS LR-3 predominantly included benign lesions (only 28.8% of malignancies). In this class, the hypo-hypo pattern showed a much higher rate of malignant lesions (73.3%) than the iso-iso pattern (2.6%). Inter-rater agreement between internal raters for CEUS-LR class assignment was almost perfect (n = 511, k = 0.94, P < 0.001), while the agreement among raters from separate centres was substantial (n = 50, k = 0.67, P < 0.001). Agreement was stronger for arterial phase hyperenhancement (internal k = 0.86, P < 2.7 × 10-214; external k = 0.8, P < 0.001) than washout (internal k = 0.79, P < 1.6 × 10-202; external k = 0.71, P < 0.001). CONCLUSION: CEUS LI-RADS is effective but can be improved by merging LR-4 and 5 to diagnose HCC and by splitting LR-3 into two subgroups to differentiate iso-iso nodules from other patterns.

Nestin as a diagnostic and prognostic marker for combined hepatocellular-cholangiocarcinoma.

BACKGROUND & AIMS: Combined hepatocellular-cholangiocarcinoma (cHCC-CCA) is a rare primary liver cancer (PLC) associated with a poor prognosis. Given the challenges in its identification and its clinical implications, biomarkers are critically needed. We aimed to investigate the diagnostic and prognostic value of the immunohistochemical expression of Nestin, a progenitor cell marker, in a large multicentric series of PLCs. METHODS: We collected 461 cHCC-CCA samples from 32 different clinical centers. Control cases included 368 hepatocellular carcinomas (HCCs) and 221 intrahepatic cholangiocarcinomas (iCCAs). Nestin immunohistochemistry was performed on whole tumor sections. Diagnostic and prognostic performances of Nestin expression were determined using receiver-operating characteristic curves and Cox regression modeling. RESULTS: Nestin was able to distinguish cHCC-CCA from HCC with AUCs of 0.85 and 0.86 on surgical and biopsy samples, respectively. Performance was lower for the distinction of cHCC-CCA from iCCA (AUCs of 0.59 and 0.60). Nestin, however, showed a high prognostic value, allowing identification of the subset of cHCC-CCA ("Nestin High", >30% neoplastic cells with positive staining) associated with the worst clinical outcome (shorter disease-free and overall survival) after surgical resection and liver transplantation, as well as when assessment was performed on biopsies. CONCLUSION: We show in different clinical settings that Nestin has diagnostic value and that it is a useful biomarker to identify the subset of cHCC-CCA associated with the worst clinical outcome. Nestin immunohistochemistry may be used to refine risk stratification and improve treatment allocation for patients with this highly aggressive malignancy. LAY SUMMARY: There are different types of primary liver cancers (i.e. cancers that originate in the liver). Accurately identifying a specific subtype of primary liver cancer (and determining its associated prognosis) is important as it can have a major impact on treatment allocation. Herein, we show that a protein called Nestin could be used to refine risk stratification and improve treatment allocation for patients with combined hepatocellular carcinoma, a rare but highly aggressive subtype of primary liver cancer.

Deletion of Lactate Dehydrogenase-A Impairs Oncogene-Induced Mouse Hepatocellular Carcinoma Development.

BACKGROUND AND AIMS: Hepatocellular carcinoma (HCC) is a multistep process whereby abnormally proliferating cancer cells undergo extensive metabolic reprogramming. Metabolic alterations in hepatocarcinogenesis depend on the activation of specific oncogenes, thus partially explaining HCC heterogeneity. c-Myc oncogene overexpression, frequently observed in human HCCs, leads to a metabolic rewiring toward a Warburg phenotype and production of lactate, resulting in the acidification of the extracellular space, favoring the emergence of an immune-permissive tumor microenvironment. Here, we investigated whether Ldha genetic ablation interferes with metabolic reprogramming and HCC development in the mouse. METHODS: We characterized the metabolic reprogramming in tumors induced in C57BL/6J mice hydrodynamically cotransfected with c-Myc and h-Ras. Using the same experimental model, we investigated the effect of Ldha inhibition-gained through the inducible and hepatocyte-specific Ldha knockout-on cancer cell metabolic reprogramming, number and size of HCC lesions, and tumor microenvironment alterations. RESULTS: c-Myc/h-Ras-driven tumors display a striking glycolytic metabolism, suggesting a switch to a Warburg phenotype. The tumors also exhibited enhanced pentose phosphate pathway activity, the switch of glutamine to sustain glutathione synthesis instead of the tricarboxylic acid cycle, and the impairment of oxidative phosphorylation. In addition, Ldha abrogation significantly hampered tumor number and size together with an evident inhibition of the Warburg-like metabolic feature and a remarkable increase of CD4+ lymphocytes compared with Ldha wild-type livers. CONCLUSIONS: These results demonstrate that Ldha deletion significantly impairs mouse HCC development and suggest lactate dehydrogenase as a potential target to enhance the efficacy of the current therapeutic options.

The Warburg Effect 97 Years after Its Discovery.

The deregulation of the oxidative metabolism in cancer, as shown by the increased aerobic glycolysis and impaired oxidative phosphorylation (Warburg effect), is coordinated by genetic changes leading to the activation of oncogenes and the loss of oncosuppressor genes. The understanding of the metabolic deregulation of cancer cells is necessary to prevent and cure cancer. In this review, we illustrate and comment the principal metabolic and molecular variations of cancer cells, involved in their anomalous behavior, that include modifications of oxidative metabolism, the activation of oncogenes that promote glycolysis and a decrease of oxygen consumption in cancer cells, the genetic susceptibility to cancer, the molecular correlations involved in the metabolic deregulation in cancer, the defective cancer mitochondria, the relationships between the Warburg effect and tumor therapy, and recent studies that reevaluate the Warburg effect. Taken together, these observations indicate that the Warburg effect is an epiphenomenon of the transformation process essential for the development of malignancy.

Mitochondrial toxicity and body shape changes during nucleos(t)ide analogues administration in patients with chronic hepatitis B.

Our study purpose was to evaluate mitochondrial (mt)DNA and RNA in peripheral blood mononuclear cells (PBMCs) and body shape changes (BSC) in HBV-infected patients. mtDNA and mtRNA were measured in PBMCs. The presence of BSC was evaluated through a questionnaire and clinical evaluation. A total of 157 subjects were enrolled, of these 107 were HBV-infected patients, 54 receiving nucleoside analogues (NAs, Group A), 53 naive to antivirals (Group B) and 50 age-sex matched controls (Group C). All HBV-treated patients had negative HBV-DNA. Twenty (37,0%) received lamivudine + adefovir, 20 (37.0%) tenofovir, 2 (3.7%) lamivudine and 12 (22.2%) entecavir. Therapy median duration was 38 months (IQR 20-60) in NA-treated patients. Group A showed significantly higher mtDNA/nuclear (n) DNA ratio (p = 0.000008) compared to Group C and Group B (p = 0.002). Group B showed significantly higher mtDNA/nDNA ratio compared to Group C (p = 0.017). Group A and B had significantly lower mtRNA/nRNA ratio compared to Group C (p = 0.00003 and p = 0.00006, respectively). Tenofovir and entecavir showed less impact compared to lamivudine + adefovir. mtDNA/nDNA ratio positively (Rho = 0.34, p < 0.05) and mtRNA/nRNA ratio negatively (Rho = -0.34, p < 0.05) correlated with therapy duration. BSC were significantly more frequent in Group A [10/54 (18.5%)] compared to Group B [3/53 (5.6%, p = 0.04)] and Group C [0/50, (p = 0.0009)]. In conclusion, long-term NA therapy was associated both to mitochondrial toxicity and BSC, showing significant differences in mtDNA and mtRNA levels. Tenofovir and entecavir showed lower impact on alterations, compared to 1st generation NA.

Inhibition of MELK Protooncogene as an Innovative Treatment for Intrahepatic Cholangiocarcinoma.

Background and Objectives: Intrahepatic cholangiocarcinoma (iCCA) is a pernicious tumor characterized by a dismal outcome and scarce therapeutic options. To substantially improve the prognosis of iCCA patients, a better understanding of the molecular mechanisms responsible for development and progression of this disease is imperative. In the present study, we aimed at elucidating the role of the maternal embryonic leucine zipper kinase (MELK) protooncogene in iCCA. Materials and Methods: We analyzed the expression of MELK and two putative targets, Forkhead Box M1 (FOXM1) and Enhancer of Zeste Homolog 2 (EZH2), in a collection of human iCCA by real-time RT-PCR and immunohistochemistry (IHC). The effects on iCCA growth of both the multi-kinase inhibitor OTSSP167 and specific small-interfering RNA (siRNA) against MELK were investigated in iCCA cell lines. Results: Expression of MELK was significantly higher in tumors than in corresponding non-neoplastic liver counterparts, with highest levels of MELK being associated with patients' shorter survival length. In vitro, OTSSP167 suppressed the growth of iCCA cell lines in a dose-dependent manner by reducing proliferation and inducing apoptosis. These effects were amplified when OTSSP167 administration was coupled to the DNA-damaging agent doxorubicin. Similar results, but less remarkable, were obtained when MELK was silenced by specific siRNA in the same cells. At the molecular level, siRNA against MELK triggered downregulation of MELK and its targets. Finally, we found that MELK is a downstream target of the E2F1 transcription factor. Conclusion: Our results indicate that MELK is ubiquitously overexpressed in iCCA, where it may represent a prognostic indicator and a therapeutic target. In particular, the combination of OTSSP167 (or other, more specific MELK inhibitors) with DNA-damaging agents might be a potentially effective therapy for human iCCA.

"Verteporfin exhibits anti-proliferative activity in embryonal and alveolar rhabdomyosarcoma cell lines".

Rhabdomyosarcoma (RMS) is a pediatric tumor, which arises from muscle precursor cells. Recently, it has been demonstrated that Hippo Pathway (Hpo), a pathway that regulates several physiological and biological features, is involved in RMS tumorigenesis. For instance, an upregulation of the Hpo downstream effector Yes-Associated Protein 1 (YAP) leads to the development of embryonal rhabdomyosarcoma (eRMS) in murine activated muscle satellite cells. On the other hand, the YAP paralog transcriptional co-activator with PDZ-binding motif (TAZ) is overexpressed in alveolar rhabdomyosarcoma (aRMS) patients with poor survival. YAP and TAZ exhibit both cytoplasmic and nuclear functions. In the nucleus, YAP binds TEADs (TEA domain family members) factors and together they constitute a complex that is able either to activate the transcription of several genes such as MYC, Tbx5 and PAX8 or to maintain the stability of others like p73. Due to the key role of YAP and TAZ in cancer, the identification and/or development of new compounds able to block their activity might be an effective antineoplastic strategy. Verteporfin (VP) is a molecule able to stop the formation of YAP/TEAD complex in the nucleus. The aim of this study is to evaluate the action of VP on RMS cell lines. This work shows that VP has an anti-proliferative activity on all RMS cell lines analyzed. Depending on RMS cell lines, VP affects cell cycle differently. Moreover, VP is able to decrease YAP protein levels, and to induce the activation of apoptosis mechanism through the cleavage of PARP-1. In addition, Annexin V assay showed the activation of apoptosis and necrosis after VP treatment. In summary, the ability of VP to disrupt RMS cell proliferation could be a novel and valuable strategy to improve the therapeutic approaches in treating rhabdomyosarcoma.

SNAI1 Promotes the Cholangiocellular Phenotype, but not Epithelial-Mesenchymal Transition, in a Murine Hepatocellular Carcinoma Model.

Hepatocellular carcinoma (HCC) is the most common type of liver cancer and has limited treatment options. Snail family transcriptional repressor 1 (SNAI1) is a master regulator of epithelial-mesenchymal transition (EMT) and has been implicated in HCC initiation and progression. However, the precise role of SNAI1 and the way it contributes to hepatocarcinogenesis have not been investigated in depth, especially in vivo. Here, we analyzed the functional relevance of SNAI1 in promoting hepatocarcinogenesis in the context of the AKT/c-Met-driven mouse liver tumor model (AKT/c-Met/SNAI1). Overexpression of SNAI1 did not accelerate AKT/c-Met-induced HCC development or induce metastasis in mice. Elevated SNAI1 expression rather led to the formation of cholangiocellular (CCA) lesions in the mouse liver, a phenotype that was paralleled by increased activation of Yap and Notch. Ablation of Yap strongly inhibited AKT/c-Met/SNAI-induced HCC and CCA development, whereas inhibition of the Notch pathway specifically blocked the CCA-like phenotype in mice. Intriguingly, overexpression of SNAI1 failed to induce EMT, indicated by strong E-cadherin expression and lack of vimentin expression by AKT/c-Met/SNAI tumor cells. SNAI1 mRNA levels strongly correlated with the expression of CCA markers, including SOX9, CK19, and EPCAM, but not with EMT markers such as E-CADHERIN and ZO-1, in human HCC samples. Overall, our findings suggest SNAI1 regulates the CCA-like phenotype in hepatocarcinogenesis via regulation of Yap and Notch. SIGNIFICANCE: These findings report a new function of SNAI1 to promote cholangiocellular transdifferentiation instead of epithelial-mesenchymal transition in hepatocellular carcinoma.

PI3K/AKT/mTOR-dependent stabilization of oncogenic far-upstream element binding proteins in hepatocellular carcinoma cells.

UNLABELLED: Transcription factors of the far-upstream element-binding protein (FBP) family represent cellular pathway hubs, and their overexpression in liver cancer (hepatocellular carcinoma [HCC]) stimulates tumor cell proliferation and correlates with poor prognosis. Here we determine the mode of oncogenic FBP overexpression in HCC cells. Using perturbation approaches (kinase inhibitors, small interfering RNAs) and a novel system for rapalog-dependent activation of AKT isoforms, we demonstrate that activity of the phosphatidylinositol-4,5-biphosphate 3-kinase/AKT pathway is involved in the enrichment of nuclear FBP1 and FBP2 in liver cancer cells. In human HCC tissues, phospho-AKT significantly correlates with nuclear FBP1/2 accumulation and expression of the proliferation marker KI67. Mechanistic target of rapamycin (mTOR) inhibition or blockade of its downstream effector eukaryotic translation initiation factor 4E activity equally reduced FBP1/2 concentrations. The mTORC1 inhibitor rapamycin diminishes FBP enrichment in liver tumors after hydrodynamic gene delivery of AKT plasmids. In addition, the multikinase inhibitor sorafenib significantly reduces FBP levels in HCC cells and in multidrug resistance 2-deficient mice that develop HCC due to severe inflammation. Both FBP1/2 messenger RNAs are highly stable, with FBP2 being more stable than FBP1. Importantly, inhibition of phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR signaling significantly diminishes FBP1/2 protein stability in a caspase-3/-7-dependent manner. CONCLUSION: These data provide insight into a transcription-independent mechanism of FBP protein enrichment in liver cancer; further studies will have to show whether this previously unknown interaction between phosphatidylinositol-4,5-biphosphate 3-kinase/AKT/mTOR pathway activity and caspase-mediated FBP stabilization allows the establishment of interventional strategies in FBP-positive HCCs.

Overexpression of far upstream element (FUSE) binding protein (FBP)-interacting repressor (FIR) supports growth of hepatocellular carcinoma.

UNLABELLED: The far upstream element binding protein (FBP) and the FBP-interacting repressor (FIR) represent molecular tools for transcriptional fine tuning of target genes. Strong overexpression of FBP in human hepatocellular carcinoma (HCC) supports tumor growth and correlates with poor patient prognosis. However, the role of the transcriptional repressor FIR in hepatocarcinogenesis remains poorly delineated. We show that overexpression of FIR correlates with tumor dedifferentiation and tumor cell proliferation in about 60% of primary HCCs. Elevated FIR levels are associated with genomic gains of the FIR gene locus at chromosome 8q24.3 in human HCC specimens. In vitro, nuclear enrichment of FIR supports HCC cell proliferation and migration. Expression profiling of HCC cells after small interfering RNA (siRNA)-mediated silencing of FIR identified the transcription factor DP-1 (TFDP1) as a transcriptional target of FIR. Surprisingly, FIR stimulates the expression of FBP in a TFDP1/E2F1-dependent manner. FIR splice variants lacking or containing exon 2 and/or exon 5 are expressed in the majority of HCCs but not in normal hepatocytes. Specific inhibition of FIR isoforms with and without exon 2 revealed that both groups of FIR splice variants facilitate tumor-supporting effects. This finding was confirmed in xenograft transplantation experiments with lentiviral-infected short hairpin RNA (shRNA) targeting all FIR variants as well as FIR with and without exon 2. CONCLUSION: High-level nuclear FIR does not facilitate repressor properties but supports tumor growth in HCC cells. Thus, the pharmacological inhibition of FIR might represent a promising therapeutic strategy for HCC patients with elevated FIR expression.

Yes-associated protein up-regulates Jagged-1 and activates the Notch pathway in human hepatocellular carcinoma.

BACKGROUND & AIMS: Cancer cells often lose contact inhibition to undergo anchorage-independent proliferation and become resistant to apoptosis by inactivating the Hippo signaling pathway, resulting in activation of the transcriptional co-activator yes-associated protein (YAP). However, the oncogenic mechanisms of YAP activity are unclear. METHODS: By using cross-species analysis of expression data, the Notch ligand Jagged-1 (Jag-1) was identified as a downstream target of YAP in hepatocytes and hepatocellular carcinoma (HCC) cells. We analyzed the functions of YAP in HCC cells via overexpression and RNA silencing experiments. We used transgenic mice that overexpressed a constitutively activated form of YAP (YAP(S127A)), and measured protein levels in HCC, colorectal and pancreatic tumor samples from patients. RESULTS: Human HCC cell lines and mouse hepatocytes that overexpress YAP(S127A) up-regulated Jag-1, leading to activation of the Notch pathway and increased proliferation. Induction of Jag-1, activation of Notch, and cell proliferation required binding of YAP to its transcriptional partner TEA domain family member 4 (TEAD4); TEAD4 binding required the Mst1/2 but not ß-catenin signaling. Levels of YAP correlated with Jag-1 expression and Notch signaling in human tumor samples and correlated with shorter survival times of patients with HCC or colorectal cancer. CONCLUSIONS: The transcriptional regulator YAP up-regulates Jag-1 to activate Notch signaling in HCC cells and mouse hepatocytes. YAP-dependent activity of Jag-1 and Notch correlate in human HCC and colorectal tumor samples with patient survival times, suggesting the use of YAP and Notch inhibitors as therapeutics for gastrointestinal cancer. Transcript profiling: microarray information was deposited at the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jxepvsumwosqkve&acc=GSE35004).

Hepatocarcinogenesis following pancreatic islet transplantation in streptozotocin- and autoimmune-diabetic rats.

Epidemiological studies assign a role to insulin signalling deregulation and diabetes mellitus in human hepatocarcinogenesis. The underlying mechanisms, however, remain largely unknown. To unravel the molecular pathogenesis of insulin-induced hepatocarcinogenesis, we generated an experimental animal model: after transplantation of only a low number of isologous pancreatic islets into the livers of diabetic rats, mild diabetes persists and the beta cells are maximally stimulated to permanently secrete insulin. As a consequence, liver acini, draining the hyperinsulinemic blood from islet grafts, show insulin-induced adaptive alterations simultaneously resembling preneoplastic foci of chemically-induced hepatocarcinogenesis models. These lesions progress to hepatocellular tumours within 6 and 24 months. Hepatocarcinogenesis is accompanied by alterations in hepatocytes metabolisms and changes in signal transduction pathways that, in the beginning, can be attributed solely to insulin action. In this review, we summarize our findings that may help understanding the oncogenic potential of diabetes mellitus in the human liver.