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Supplementary Data from Hepatocellular Carcinoma in Mongolia Delineates Unique Molecular Traits and a Mutational Signature Associated with Environmental Agents

Purpose:Mongolia has the world's highest incidence of hepatocellular carcinoma (HCC), with ∼100 cases/100,000 inhabitants, although the reasons for this have not been thoroughly delineated.Experimental Design:We performed a molecular characterization of Mongolian (n = 192) compared with Western (n = 187) HCCs by RNA sequencing and whole-exome sequencing to unveil distinct genomic and transcriptomic features associated with environmental factors in this population.Results:Mongolian patients were younger, with higher female prevalence, and with predominantly HBV–HDV coinfection etiology. Mongolian HCCs presented significantly higher rates of protein-coding mutations (121 vs. 70 mutations per tumor in Western), and in specific driver HCC genes (i.e., APOB and TSC2). Four mutational signatures characterized Mongolian samples, one of which was novel (SBS Mongolia) and present in 25% of Mongolian HCC cases. This signature showed a distinct substitution profile with a high proportion of T>G substitutions and was significantly associated with a signature of exposure to the environmental agent dimethyl sulfate (71%), a 2A carcinogenic associated with coal combustion. Transcriptomic-based analysis delineated three molecular clusters, two not present in Western HCC; one with a highly inflamed profile and the other significantly associated with younger female patients.Conclusions:Mongolian HCC has unique molecular traits with a high mutational burden and a novel mutational signature associated with genotoxic environmental factors present in this country.

Supplementary Figure from Hepatocellular Carcinoma in Mongolia Delineates Unique Molecular Traits and a Mutational Signature Associated with Environmental Agents

PDF file, 10.8MB, Supplementary Table 1: Clinical characteristics of human hepatocellular carcinoma samples. Supplementary Table 2: Clinical Characteristics of 5 public HCC datasets. Supplementary Table 3: Wnt-pathway mRNA list: 210 Wnt-related genes from the literature. Supplementary Table 4: Wnt-pathway miRNA list: 49 Wnt-related miRNAs from the literature. Supplementary Table 5: Significantly differentially expressed Wnt-related genes in CTNNB1- and Wnt-TGF_-class samples of the training set. Supplementary Table 6: Summary of dysregulated CTNNB1- and Wnt-TGF_-Wnt-genes according to their cellular localization and their known or suspected function. Supplementary Table 7: Expression of known liver-related Wnt-target-genes. Supplementary Table 8: Correlation of Wnt-related transcription factors to Wnt-target-genes in the training set. Supplementary Table 9: Frequency _catenin IHC positive samples and CTNNB1 mutations Wnt-related and non-Wnt-related subclasses of HCC in the in training (T) and validation (V) sets. Supplementary Table 10: Differentially expressed Wnt-related-miRNAs in 89 HCC samples of the training set. Supplementary Table 11: Enrichment of samples identified by CTNNB1-, Wnt-TGF__ WntGenes- and CTNNB1-mutation-signature in CTNNB1 and Wnt-TGF_ subclasses in 5 independent datasets (Fisher's Exact Test). Supplementary Table 12: 21 Liver Cancer cell lines analyzed by GSEA for gene expression profiles similar to either CTNNB1 or Wnt-TGF_ class. Supplementary Figure 1: Differential Expression of Wnt-pathway mRNAs in the CTNNB1- and Wnt-TGF Beta-classes in the training set. Supplementary Figure 2: Differential Expression of Wnt-pathway-miRNAs in the CTNNB1- and Wnt-TGF Beta-classes in the training set. Supplementary Figure 3: Validation of CTNNB1- and Wnt-TGF Beta-specific Wnt-related mRNA profiles and mutation-status in 5 public HCC datasets. Supplementary Figure 4: Validation of the mutation signature in an independent cohort of HCC. Supplementary Figure 5: Correlation of gene expression data from 21 different liver cancer cell lines with CTNNB1 and Wnt-TGF Beta class signatures by GSEA. Supplementary Figure 6: Baseline luciferase levels in Wnt reporter liver cancer cells stably transduced with TCF/LEF luciferase reporter. Supplementary Figure 7: Sorafenib decreases beta-catenin protein levels in 4 liver cancer cell lines.

Purpose: Hepatocellular carcinoma (HCC) is a heterogeneous cancer with active Wnt signaling. Underlying biologic mechanisms remain unclear and no drug targeting this pathway has been approved to date. We aimed to characterize Wnt-pathway aberrations in HCC patients, and to investigate sorafenib as a potential Wnt modulator in experimental models of liver cancer.Experimental Design: The Wnt-pathway was assessed using mRNA (642 HCCs and 21 liver cancer cell lines) and miRNA expression data (89 HCCs), immunohistochemistry (108 HCCs), and CTNNB1-mutation data (91 HCCs). Effects of sorafenib on Wnt signaling were evaluated in four liver cancer cell lines with active Wnt signaling and a tumor xenograft model.Results: Evidence for Wnt activation was observed for 315 (49.1%) cases, and was further classified as CTNNB1 class (138 cases [21.5%]) or Wnt-TGFβ class (177 cases [27.6%]). CTNNB1 class was characterized by upregulation of liver-specific Wnt-targets, nuclear β-catenin and glutamine-synthetase immunostaining, and enrichment of CTNNB1-mutation-signature, whereas Wnt-TGFβ class was characterized by dysregulation of classical Wnt-targets and the absence of nuclear β-catenin. Sorafenib decreased Wnt signaling and β-catenin protein in HepG2 (CTNNB1 class), SNU387 (Wnt-TGFβ class), SNU398 (CTNNB1-mutation), and Huh7 (lithium-chloride-pathway activation) cell lines. In addition, sorafenib attenuated expression of liver-related Wnt-targets GLUL, LGR5, and TBX3. The suppressive effect on CTNNB1 class–specific Wnt-pathway activation was validated in vivo using HepG2 xenografts in nude mice, accompanied by decreased tumor volume and increased survival of treated animals.Conclusions: Distinct dysregulation of Wnt-pathway constituents characterize two different Wnt-related molecular classes (CTNNB1 and Wnt-TGFβ), accounting for half of all HCC patients. Sorafenib modulates β-catenin/Wnt signaling in experimental models that harbor the CTNNB1 class signature. Clin Cancer Res; 18(18); 4997–5007. ©2012 AACR.

ObjectiveWe previously reported a characterisation of the hepatocellular carcinoma (HCC) immune contexture and described an immune-specific class. We now aim to further delineate the immunogenomic classification of HCC to incorporate features that explain responses/resistance to immunotherapy.DesignWe performed RNA and whole-exome sequencing, T-cell receptor (TCR)-sequencing, multiplex immunofluorescence and immunohistochemistry in a novel cohort of 240 HCC patients and validated our results in other cohorts comprising 660 patients.ResultsOur integrative analysis led to define: (1) the inflamed class of HCC (37%), which includes the previously reported immune subclass (22%) and a new immune-like subclass (15%) with high interferon signalling, cytolytic activity, expression of immune-effector cytokines and a more diverse T-cell repertoire. A 20-gene signature was able to capture ~90% of these tumours and is associated with response to immunotherapy. Proteins identified in liquid biopsies recapitulated the inflamed class with an area under the ROC curve (AUC) of 0.91; (2) The intermediate class, enriched inTP53mutations (49% vs 29%, p=0.035), and chromosomal losses involving immune-related genes and; (3) the excluded class, enriched inCTNNB1mutations (93% vs 27%, pPTK2overexpression due to gene amplification and promoter hypomethylation.CTNNB1mutations outside the excluded class led to weak activation of the Wnt-βcatenin pathway or occurred in HCCs dominated by high interferon signalling and type I antigen presenting genes.ConclusionWe have characterised the immunogenomic contexture of HCC and defined inflamed and non-inflamed tumours. Two distinctCTNNB1patterns associated with a differential role in immune evasion are described. These features may help predict immune response in HCC.

Objectives The 8th edition American Joint Committee on Cancer (AJCC) staging system for hepatocellular carcinoma (HCC) has been criticized for failing to stratify patients. We aimed to reassess and modify the tumor staging criteria for HCC. Methods Three independent study cohorts were collected and analyzed. Results The initial cohort consists of 103 patients with HCC. By Kaplan-Meier survival analysis, the 8th edition failed to distinguish between T1b and T2. Only tumor size and large vessel invasion, but not small vessel invasion or other histopathologic parameters, predicted HCC survival. We modified the T staging criteria by eliminating small vessel invasion while emphasizing tumor size in the middle categories (T2 and T3), which achieved more even distribution of cases and significantly improved risk stratifications (P Conclusions Our study showed that tumor size, but not small vessel invasion, predicts survival in patients with HCC. We suggest incorporating our modified T staging criteria in future AJCC revisions.

Portal hypertension (PH) is associated with complications such as ascites and esophageal varices and is typically diagnosed through invasive hepatic venous pressure gradient (HVPG) measurement, which is not widely available. In this study, we aim to assess the diagnostic performance of 2D/3D MR elastography (MRE) and shear wave elastography (SWE) measures of liver and spleen stiffness (LS and SS) and spleen volume, to noninvasively diagnose clinically significant portal hypertension (CSPH) using HVPG measurement as the reference.In this prospective study, patients with liver disease underwent 2D/3D MRE and SWE of the liver and spleen, as well as HVPG measurement. The correlation between MRE/SWE measures of LS/SS and spleen volume with HVPG was assessed. ROC analysis was used to determine the utility of MRE, SWE, and spleen volume for diagnosing CSPH.Thirty-six patients (M/F 22/14, mean age 55 ± 14 years) were included. Of the evaluated parameters, 3D MRE SS had the strongest correlation with HVPG (r = 0.686, p0.001), followed by 2D MRE SS (r = 0.476, p = 0.004). 3D MRE SS displayed the best performance for diagnosis of CSPH (AUC = 0.911) followed by 2D MRE SS (AUC = 0.845) and 3D MRE LS (AUC = 0.804). SWE SS showed poor performance for diagnosis of CSPH (AUC = 0.583) while spleen volume was a fair predictor (AUC = 0.738). 3D MRE SS was significantly superior to SWE LS/SS (p ≤ 0.021) for the diagnosis of CSPH.SS measured with 3D MRE outperforms SWE for the diagnosis of CSPH. SS appears to be a useful biomarker for assessing PH severity. These results need further validation.• Spleen stiffness measured with 2D and 3D MR elastography correlates significantly with hepatic venous pressure gradient measurement. • Spleen stiffness measured with 3D MR elastography demonstrates excellent performance for the diagnosis of clinically significant portal hypertension (AUC 0.911). • Spleen stiffness measured with 3D MR elastography outperforms liver and spleen stiffness measured with shear wave elastography for diagnosis of clinically significant portal hypertension.

Background & aims: single-agent anti-PD1 checkpoint inhibitors convey outstanding clinical benefits in a small fraction (∼20%) of patients with advanced hepatocellular carcinoma (aHCC) but the molecular mechanisms determining response are unknown. To fill this gap, we herein analyze the molecular and immune traits of aHCC in patients treated with anti-PD1. Methods: overall, 111 tumor samples from patients with aHCC were obtained from 13 centers before systemic therapies. We performed molecular analysis and immune deconvolution using whole-genome expression data (n = 83), mutational analysis (n = 72), and histologic evaluation with an endpoint of objective response. Results: among 83 patients with transcriptomic data, 28 were treated in frontline, whereas 55 patients were treated after tyrosine kinase inhibitors (TKI) either in second or third line. Responders treated in frontline showed upregulated interferon-γ signaling and major histocompatibility complex II-related antigen presentation. We generated an 11-gene signature (IFNAP), capturing these molecular features, which predicts response and survival in patients treated with anti-PD1 in frontline. The signature was validated in a separate cohort of aHCC and >240 patients with other solid cancer types where it also predicted response and survival. Of note, the same signature was unable to predict response in archival tissue of patients treated with frontline TKIs, highlighting the need for fresh biopsies before immunotherapy. Conclusion: interferon signaling and major histocompatibility complex-related genes are key molecular features of HCCs responding to anti-PD1. A novel 11-gene signature predicts response in frontline aHCC, but not in patients pretreated with TKIs. These results must be confirmed in prospective studies and highlights the need for biopsies before immunotherapy to identify biomarkers of response.

Patients receiving immunosuppressive therapy for inflammatory bowel disease may be susceptible to non-cirrhotic portal hypertension, now referred to as porto-sinusoidal vascular disease. Here we describe a patient treated with long-term azathioprine for Crohn’s disease who developed porto-sinusoidal vascular disease with obliterative portal venopathy without nodular regenerative hyperplasia on histology. Specific signs of portal hypertension were present, including porto-systemic collaterals on imaging. Histopathologic findings of porto-sinusoidal vascular disease support the hypothesis of endothelial cell injury induced by 6-thioguanine secondary to azathioprine use.

AIMS Lymphoepithelioma-like carcinomas (LELCs) are uncommon epithelial cancers characteristically showing two distinct components consisting of malignant epithelial cells and prominent dense lymphoid infiltrate. Hepatic LELCs consist of two types, the lymphoepithelioma-like hepatocellular carcinoma and lymphoepithelioma-like cholangiocarcinoma (LEL-CCA), with the latter being strongly associated with Epstein-Barr virus (EBV). METHODS AND RESULTS We present a series of three cases of intrahepatic biliary EBV-associated LEL tumours in which the biliary epithelial component showed a distinctly benign appearance, instead of the usual malignant epithelial features of a typical CCA or EBV-associated LEL-CCA. In the lesions, the biliary epithelium showed interconnecting glands or cords of cells. All had a very low proliferation (Ki-67) index. Immunohistochemistry for IDH1 and TP53 performed on two cases was negative and molecular tests for EGFR and KRAS gene mutations performed on one were negative. Prognosis was very good in all three cases, with patients alive with no evidence of disease 24-62 months after surgery. Intriguingly, all three cases had co-infection of HBV and EBV. These cases are also discussed in the context of the 63 cases of LEL-CCA available in the literature, with a focus on epidemiology, clinicopathological features and potential research interests. CONCLUSIONS Based on the distinct clinicopathological features and unique survival benefits, we believe these tumours represent the benign end of the spectrum of EBV-associated lymphoepithelial biliary carcinomas. Whether these tumours require a revision of the current nomenclature to 'lymphoepithelioma-like neoplasm of the biliary tract with probable low malignant potential' will require more detailed analysis with larger case-series.

To assess the performance of gadoxetate dynamic contrast-enhanced (DCE) MRI of the liver and spleen for noninvasive diagnosis of portal hypertension (PH).Thirty-five patients (M/F 22/13, mean age 55 years) with chronic liver disease who underwent hepatic venous pressure gradient (HVPG) measurements were prospectively enrolled in this IRB-approved study. All patients underwent multiparametric MRI including gadoxetate DCE-MRI acquisition. Model-based and model-free DCE-MRI analyses were performed. The correlation between DCE-MRI parameters and HVPG was assessed. ROC analysis was employed to determine the diagnostic performance of DCE-MRI parameters alone and in combination for prediction of PH and clinically significant (CS)PH (HVPG5 and ≥ 10 mmHg, respectively).Mean HVPG was 7.0 ± 5.0 mmHg (range 0-18 mmHg). Twenty-one (60%) patients had PH, of whom 9 had CSPH. Modeled liver uptake fraction fOur results demonstrate the potential utility of hepatocyte uptake parameters and spleen interstitial fraction obtained with gadoxetate DCE-MRI for the diagnosis of PH and CSPH.• Liver uptake and spleen interstitial fraction estimates from gadoxetate DCE-MRI are significantly correlated with portal pressure measurements. • Liver uptake rate shows good diagnostic performance for the diagnosis of portal hypertension. • The combination of liver uptake rate with spleen interstitial fraction exhibits excellent diagnostic performance for the diagnosis of clinically significant portal hypertension.

Hepatocellular adenoma (HCA) is a rare benign tumor of the liver with low risk of malignant transformation. It is associated with oral contraceptives/anabolic steroid use, metabolic disease, and rarely, vascular abnormalities. We report an interesting case of HCA arising in a background of diffuse hepatic nodular regenerative hyperplasia (NRH) in a 40-year-old female patient with systemic lupus erythematosus (SLE). She presented with sudden-onset refractory ascites, elevated liver enzymes, diffuse hepatic nodularity and mass lesions on imaging concerning for malignancy. Targeted biopsies of the mass lesion were performed with inconclusive diagnoses. The patient ultimately underwent resection of the mass, which was confirmed as HCA, inflammatory type, arising in a background of NRH. It is not uncommon for SLE patients to have liver manifestations such as NRH, but HCA arising in NRH has not been previously reported. Our case reveals an unusual relationship between HCA and hepatic vasculopathy in the clinical context of a systemic inflammatory condition, the mechanism by which is not fully understood.

Multiparametric magnetic resonance imaging (mpMRI) combining different techniques such as MR elastography (MRE) has emerged as a noninvasive approach to diagnose and stage liver fibrosis with high accuracy allowing for anatomical and functional information.To assess the diagnostic performance of mpMRI including qualitative and quantitative assessment of MRE, liver surface nodularity (LSN) measurement, hepatic enhancement ratios postgadoxetic acid, and serum markers (APRI, FIB-4) for the detection of liver fibrosis.IRB-approved retrospective.Eighty-three adult patients.1.5T and 3.0T MR systems. MRE and TTwo independent observers analyzed qualitative color-coded MRE maps on a scale of 0-3. Regions of interest were drawn to measure liver stiffness on MRE stiffness maps and on pre- and postcontrast TA multivariable logistic analysis was performed to identify independent predictors of liver fibrosis. Receiver operating characteristic (ROC) analysis evaluated the performance of each imaging technique for detection of fibrosis, in comparison with serum markers.Liver stiffness measured with MRE provided the strongest correlation with histopathologic fibrosis stage (r = 0.74, P0.001), and the highest diagnostic performance for detection of stages F2-F4, F3-F4, and F4 (areas under the curve [AUCs] of 0.87, 0.91, and 0.89, respectively, P0.001) compared to other methods. Qualitative assessment of MRE maps showed fair to good accuracy for detection of fibrosis (AUC range 0.76-0.84). Multivariable logistic analysis identified liver stiffness and FIB-4 as independent predictors of fibrosis with AUCs of 0.90 (F2-F4), 0.93 (F3-F4) and 0.92 (F4) when combined.Liver stiffness measured with MRE showed the best performance for detection of liver fibrosis compared to LSN and gadoxetic acid uptake, with slight improvement when combined with FIB-4.3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2018;47:1552-1561.

Background and Aims: Cholangiocarcinoma (CCA) is the second most common primary hepatic malignancy. Based on its anatomical location, CCA can be divided into intrahepatic (iCCA) or extrahepatic (eCCA), with differences in etiology, pathogenesis and clinical management. Few studies have focused on the molecular profiling of eCCA as a single entity, even though it accounts for the most prevalent subtype. Thus, integrative genomic analysis of eCCA would provide critical understanding for the biological traits of this tumor. Methods: 189 FFPE primary eCCA treated by resection were collected at seven international centers from 1995 to 2015. Median survival of the cohort was of 48.5mo. Whole gene-expression profiles were submitted to unsupervised clustering by NMF consensus. Clusters were characterized by Gene Set Enrichment Analysis and Ingenuity Pathway Analysis. Activation of signaling pathways (mTOR/pRPS6 and HER2) was assessed by immunohistochemistry (IHC). Molecular features were correlated with clinico-pathological data. Screening of most prevalent somatic mutations and copy number aberrations is ongoing. Results: We have identified four distinct molecular subtypes of eCCA (cophenetic coefficient=0.995). Tumors classified within the metabolic class (18.7%) were enriched by gene signatures defining bile and fatty acid metabolism (p Conclusions: Transcriptome-based subtyping of eCCA identifies four distinct molecular classes (metabolic, proliferation, mesenchymal and immune) that correlate with clinical-pathological characteristics. These findings enhance the opportunities for therapeutic development in this tumor with dismal prognosis and without approved molecular treatments. Citation Format: Robert Montal, Wei Qiang Leow, Carla Montironi, Laia Bassaganyas, Agrin Moeini, Daniela Sia, Roser Pinyol, Laia Cabellos, Judit Peix, Miho Maeda, Carlos Villacorta, Parissa Tabrizian, Christine Sempoux, Beatriz Minguez, Tim Pawlik, Ismail Labgaa, Lewis Roberts, Manel Sole, Maria Isabel Fiel, Swan Thung, Sasan Roayaie, Augusto Villanueva, Myron Schwartz, Josep Maria Llovet. Integrative molecular classification of extrahepatic cholangiocarcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 4618.

The up-to-seven (Up-to-7) criteria [with 7 being the sum of the size and number of tumors for any given hepatocellular carcinoma (HCC)] have been recently proposed to identify potential candidates for liver transplantation (LT) among patients exceeding the Milan criteria. The aim of this study was to compare the ability of the available pathologic staging systems (the Milan, University of California San Francisco, and Up-to-7 criteria) to predict recurrence. A study population of 479 HCC transplanted patients was identified from prospectively collected databases at Mount Sinai Medical Center (New York, NY) and the University of Padua (Padua, Italy). The best pathologic staging system was identified with log rank, proportion separation index (PSEP), and Cox analyses. Pathologic tumor characteristics (tumor number, tumor size, sum of diameters, macroscopic and microscopic vascular invasion, and grading) were then tested by univariate and multivariate Cox analyses in the prognostic subgroups within and beyond the calculated criteria. The Up-to-7 criteria performed as the best pathologic staging system, the calculated 1-, 3-, and 5-year recurrence probabilities being 4%, 8%, and 14% within the criteria (n = 355) and 22%, 45%, 51% beyond the criteria (n = 124; P0.0001) and the calculated PSEP being 0.27 (95% confidence interval = 0.23-0.31). In multivariate analysis, only biological variables (vascular invasion and tumor grade) significantly predicted recurrence beyond the Up-to-7 criteria. A 3-stage pathologic staging system with a potential to be applied in the preoperative setting was thus created: within the Up-to-7 criteria (recurrence rate = 8%), beyond the Up-to-7 criteria without macrovascular invasion and poorly differentiated grade (recurrence rate = 24%), and beyond the Up-to-7 criteria with macrovascular invasion and/or poorly differentiated grade (recurrence rate = 45%). In conclusion, HCC patients within the pathologic Up-to-7 criteria were associated with a low risk of recurrence after LT. Beyond these criteria, however, a significant proportion of patients with a good HCC biological profile had an acceptable risk of recurrence.