Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

Background & Aims: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.Methods: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n = 164) and intrahepatic cholangiocarcinoma (iCCA) (n = 149).Results: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliaryderived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p < 0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p = 0.008), showed significant upregulation of transforming growth factor (TGF)-beta signaling and enrichment of inflammation-related and immune response signatures (p < 0.001). Stem-cell tumors were characterized by spaltlike transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p < 0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.Conclusions: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-b signaling.Lay summary: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Intratumoral heterogeneity and clonal evolution in liver cancer

Clonal evolution of a tumor ecosystem depends on different selection pressures that are principally immune and treatment mediated. We integrate RNA-seq, DNA sequencing, TCR-seq and SNP array data across multiple regions of liver cancer specimens to map spatio-temporal interactions between cancer and immune cells. We investigate how these interactions reflect intra-tumor heterogeneity (ITH) by correlating regional neo-epitope and viral antigen burden with the regional adaptive immune response. Regional expression of passenger mutations dominantly recruits adaptive responses as opposed to hepatitis B virus and cancer-testis antigens. We detect different clonal expansion of the adaptive immune system in distant regions of the same tumor. An ITH-based gene signature improves single-biopsy patient survival predictions and an expression survey of 38,553 single cells across 7 regions of 2 patients further reveals heterogeneity in liver cancer. These data quantify transcriptomic ITH and how the different components of the HCC ecosystem interact during cancer evolution

Feasibility and reproducibility of liver surface nodularity quantification for the assessment of liver cirrhosis using CT and MRI

Purpose: To assess intra-observer, inter-observer and inter-modality (CT vs. MRI) reproducibility of liver surface nodularity (LSN) scores measured with software used for detection of liver fibrosis. Methods: This IRB-approved retrospective study included patients with both abdominal CT and MRI within 6 months of histopathologic sampling. Two independent observers used post-processing software to quantify LSN scores on axial non-contrast CT (NCT), axial contrast-enhanced CT (CECT), axial T2-weighted (T2W) HASTE, and axial and coronal post-gadoxetic acid T1-weighted (T1W) images obtained during the hepatobiliary phase (HBP). Ten slices were used to acquire the LSN scores. Intra-observer, inter-observer, and inter-modality (CT vs. MRI) reproducibility were assessed with intraclass correlation coefficient (ICC) and coefficients of variability (CV). Accuracy for detection of cirrhosis was evaluated for each technique. Results: 26 patients (M/F 19/7, mean age 57 years), including 7 with cirrhosis (26.9%), were assessed. Technical failure occurred with NCT (1/23, 4.3%) and T2 HASTE (8/28, 28.6%). Intra-observer reproducibility was excellent for NCT, CECT, axial and coronal T1W HBP [ICC â¥Â 0.92, CV â¤Â 8%]. Inter-observer reproducibility was also excellent for NCT and CECT (ICC â¥Â 0.95, CV â¤Â 7.3%) and for coronal T1W HBP (ICC = 0.84, CV = 5.6%). There was fair to moderate agreement between CT and MRI (ICC 0.20â0.44). There were significant differences in mean LSN scores between non-cirrhotic and cirrhotic patients with NCT (2.6 vs. 4.2, p = 0.04) and T1W HBP (3.7 vs. 4.6; p = 0.01) images, with AUCs of 0.81 and 0.82, respectively. Conclusions: LSN measurement is highly reproducible with NCT and post-contrast T1W HBP on MRI, with different results obtained between CT and MRI. Keywords: Liver surface nodularity, Fibrosis, Cirrhosis, CT, MR

Detection of liver fibrosis using qualitative and quantitative MR elastography compared to liver surface nodularity measurement, gadoxetic acid uptake, and serum markers

BACKGROUND: 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. PURPOSE: 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. STUDY TYPE: IRB-approved retrospective. SUBJECTS: Eighty-three adult patients. FIELD STRENGTH/SEQUENCE: 1.5T and 3.0T MR systems. MRE and T1 -weighted postgadoxetic acid sequences. ASSESSMENT: Two 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 T1 -weighted images to measure hepatic enhancement ratios. Software was used to generate LSN measurements. Histopathology was used as the reference standard for diagnosis of liver fibrosis in all patients. STATISTICAL TESTS: A 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. RESULTS: Liver stiffness measured with MRE provided the strongest correlation with histopathologic fibrosis stage (r = 0.74, P < 0.001), 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, P < 0.001) 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. DATA CONCLUSION: 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. LEVEL OF EVIDENCE: 3 Technical Efficacy: Stage 2 J. Magn. Reson. Imaging 2017

Evaluation of HCC response to locoregional therapy: validation of MRI-based response criteria versus explant pathology

BACKGROUND AND AIMS: This study evaluates the performance of various magnetic resonance imaging (MRI) response criteria for the prediction of complete pathologic necrosis (CPN) of hepatocellular carcinoma (HCC) post locoregional therapy (LRT) using explant pathology as a reference. METHODS: We included 61 patients (male/female 46/15; mean age 60years) who underwent liver transplantation after LRT with transarterial chemoembolization plus radiofrequency or microwave ablation (n=56), or (90)Yttrium radioembolization (n=5). MRI was performed <90days before liver transplantation. Three independent readers assessed the following criteria: RECIST, EASL, modified RECIST (mRECIST), percentage of necrosis on subtraction images, and diffusion-weighted imaging (DWI), both qualitative (signal intensity) and quantitative (apparent diffusion coefficient [ADC]). The degree of necrosis was retrospectively assessed at histopathology. Intraclass correlation coefficient (ICC) and Cohen's kappa were used to assess inter-reader agreement. Logistic regression and receiver operating characteristic analyses were used to determine imaging predictors of CPN. Pearson correlation was performed between imaging criteria and pathologic degree of tumor necrosis. RESULTS: A total of 97HCCs (mean size 2.3±1.3cm) including 28 with CPN were evaluated. There was excellent inter-reader agreement (ICC 0.77-0.86, all methods). EASL, mRECIST, percentage of necrosis and qualitative DWI were all significant (p<0.001) predictors of CPN, while RECIST and ADC were not. EASL, mRECIST and percentage of necrosis performed similarly (area under the curves [AUCs] 0.810-0.815) while the performance of qualitative DWI was lower (AUC 0.622). Image subtraction demonstrated the strongest correlation (r=0.71-0.72, p<0.0001) with pathologic degree of tumor necrosis. CONCLUSIONS: EASL/mRECIST criteria and image subtraction have excellent diagnostic performance for predicting CPN in HCC treated with LRT, with image subtraction correlating best with pathologic degree of tumor necrosis. Thus, MR image subtraction is recommended for assessing HCC response to LRT. LAY SUMMARY: The assessment of hepatocellular carcinoma (HCC) tumor necrosis after locoregional therapy is essential for additional treatment planning and estimation of outcome. In this study, we assessed the performance of various magnetic resonance imaging (MRI) response criteria (RECIST, mRECIST, EASL, percentage of necrosis on subtraction images, and diffusion-weighted imaging) for the prediction of complete pathologic necrosis of HCC post locoregional therapy on liver explant. Patients who underwent liver transplantation after locoregional therapy were included in this retrospective study. All patients underwent routine liver MRI within 90days of liver transplantation. EASL/mRECIST criteria and image subtraction had excellent diagnostic performance for predicting complete pathologic necrosis in treated HCC, with image subtraction correlating best with pathologic degree of tumor necrosis

PAXgene Fixation for Pancreatic Cancer: Implications for Molecular and Surgical Pathology

Genomic profiling of pancreatic cancer using small core biopsies has taken an increasingly prominent role in precision medicine. However, if not appropriately preserved, nucleic acids (NA) from pancreatic tissues are known to be susceptible to degradation due to high intrinsic levels of nucleases. PAXgene fixation (PreAnalytix, Switzerland) represents a novel formalin-free tissue preservation method. We sought to compare the NA and histomorphological preservation of pancreatic cancer tissues preserved with PAXgene-fixed paraffin-embedding (PFPE) and formalin-fixed paraffin-embedding (FFPE). Tissues from 19 patients were obtained prospectively from pancreaticoduodenectomy specimens and evaluated by four gastrointestinal pathologists. The extracted NA were quantified by Nanodrop and Qubit and assessed for quality by qPCR, targeted next-generation sequencing (NGS) assay, and RNA-sequencing. Our results demonstrated that, when assessed blindly for morphological quality, the four pathologists deemed the PFPE slides adequate for diagnostic purposes. PFPE tissues enable greater yields of less fragmented and more amplifiable DNA. PFPE tissues demonstrated significantly improved quality control (QC) metrics in a targeted NGS assay including Median Absolute Pair-wise Difference (MAPD) scores. Our results support the use of PAXgene fixative for the processing of specimens from pancreatic cancers with the potential benefits of improved yields for more amplifiable DNA in low-yield biopsy specimens and its ideal use for amplicon-based NGS assays

Hepatocellular carcinoma in patients cured of chronic hepatitis C: Minimal steatosis

Abstract Background Successful treatment of hepatitis C reduces liver inflammation and fibrosis; however, patients remain at risk of developing hepatocellular carcinoma (HCC). Aims To identify risk factors for new‐onset HCC in patients cured of hepatitis C. Methods Imaging, histological, and clinical data on patients whose first HCC was diagnosed >12 months of post‐SVR were analyzed. Histology of 20 nontumor tissues was analyzed in a blinded manner using the Knodel/Ishak/HAI system for necroinflammation and fibrosis/cirrhosis stage and the Brunt system for steatosis/steatohepatitis. Factors associated with post‐SVR HCC were identified by comparison with HALT‐C participants who did not develop post‐SVR HCC. Results Hepatocellular carcinoma was diagnosed in 54 patients (45 M/9F), a median of 6 years of post‐SVR [interquartile range (IQR) =1.4‐10y] at a median age of 61 years (IQR, 59–67). Approximately one‐third lacked cirrhosis, and only 11% had steatosis on imaging. The majority (60%) had no steatosis/steatohepatitis in histopathology. The median HAI score was 3 (1.25–4), indicating mild necroinflammation. In a multivariable logistic regression model, post‐SVR HCC was positively associated with non‐Caucasian race (p = 0.03), smoking (p = 0.03), age > 60 years at HCC diagnosis (p = 0.03), albumin1 (p = 0.05), and platelets <100 × 103 cells/μL (p < 0.001). Alpha fetoprotein ≥4.75 ng/mL had 90% specificity and 71% sensitivity for HCC occurrence. Noncirrhotic patients had larger tumors (p = 0.002) and a higher prevalence of vascular invasion (p = 0.016) than cirrhotic patients. Conclusions One‐third of patients with post‐SVR HCC did not have liver cirrhosis; most had no steatosis/steatohepatitis. Hepatocellular carcinomas were more advanced in noncirrhotic patients. Results support AFP as a promising marker of post‐SVR HCC risk

Mixed hepatocellular cholangiocarcinoma tumors: Cholangiolocellular carcinoma is a distinct molecular entity

Background & Aims: Mixed hepatocellular cholangiocarcinoma (HCC-CCA) is a rare and poorly understood type of primary liver cancer. We aimed to perform a comprehensive molecular characterization of this malignancy.Methods: Gene expression profiling, DNA copy number detection, and exome sequencing using formalin-fixed samples from 18 patients with mixed HCC-CCA were performed, encompassing the whole histological spectrum of the disease. Comparative genomic analysis was carried out, using independent datasets of HCC (n = 164) and intrahepatic cholangiocarcinoma (iCCA) (n = 149).Results: Integrative genomic analysis of HCC-CCAs revealed that cholangiolocellular carcinoma (CLC) represents a distinct biliaryderived entity compared with the stem-cell and classical types. CLC tumors were neural cell adhesion molecule (NCAM) positive (6/6 vs. 1/12, p < 0.001), chromosomally stable (mean chromosomal aberrations 5.7 vs. 14.1, p = 0.008), showed significant upregulation of transforming growth factor (TGF)-beta signaling and enrichment of inflammation-related and immune response signatures (p < 0.001). Stem-cell tumors were characterized by spaltlike transcription factor 4 (SALL4) positivity (6/8 vs. 0/10, p < 0.001), enrichment of progenitor-like signatures, activation of specific oncogenic pathways (i.e., MYC and insulin-like growth factor [IGF]), and signatures related to poor clinical outcome. In the classical type, there was a significant correlation in the copy number variation of the iCCA and HCC components, suggesting a clonal origin. Exome sequencing revealed an average of 63 non-synonymous mutations per tumor (2 mean driver mutations per tumor). Among those, TP53 was the most frequently mutated gene (6/21, 29%) in HCC-CCAs.Conclusions: Mixed HCC-CCA represents a heterogeneous group of tumors, with the stem-cell type characterized by features of poor prognosis, and the classical type with common lineage for HCC and iCCA components. CLC stands alone as a distinct biliary-derived entity associated with chromosomal stability and active TGF-b signaling.Lay summary: Molecular analysis of mixed hepatocellular cholangiocarcinoma (HCC-CCA) showed that cholangiolocellular carcinoma (CLC) is distinct and biliary in origin. It has none of the traits of hepatocellular carcinoma (HCC). However, within mixed HCC-CCA, stem-cell type tumors shared an aggressive nature and poor outcome, whereas the classic type showed a common cell lineage for both the HCC and the intrahepatic CCA component. The pathological classification of mixed HCC-CCA should be redefined because of the new molecular data provided. (C) 2017 European Association for the Study of the Liver. Published by Elsevier B.V. All rights reserved

Single Topic Conference on Autoimmune Liver Disease from the Canadian Association for the Study of the Liver

Autoimmune liver disease (AILD) spans a spectrum of chronic disorders affecting the liver parenchyma and biliary system. Three main categories of AILD are autoimmune hepatitis (AIH), primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC). This review condenses the presentation and discussions of the Single Topic Conference (STC) on AILD that was held in Ottawa, Ontario, in November 2019. We cover generalities regarding disease presentation and clinical diagnosis; mechanistic themes; treatment paradigms; clinical trials, including approaches and challenges to new therapies; and looking beyond traditional disease boundaries. Although these diseases are considered autoimmune, the etiology and role of environmental triggers are poorly understood. AILDs are progressive and chronic conditions that affect survival and quality of life. Advances have been made in PBC treatment because second-line treatments are now available (obeticholic acid, bezafibrate); however, a significant proportion still present suboptimal response. AIH treatment has remained unchanged for several decades, and data suggest that fewer than 50% of patients achieve a complete response and as many as 80% develop treatment-related side effects. B-cell depletion therapy to treat AIH is in an early stage of development and has shown promising results. An effective treatment for PSC is urgently needed. Liver transplant remains the best option for patients who develop decompensated cirrhosis or hepatocellular carcinoma within specific criteria, but recurrent AILD might occur. Continued efforts are warranted to develop networks for AILD aimed at assessing geo-epidemiological, clinical, and biochemical differences to capture the new treatment era in Canada