Stephen C. Ward

MicroRNA-Based Classification of Hepatocellular Carcinoma and Oncogenic Role of miR-517a

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a heterogeneous tumor that develops via activation of multiple pathways and molecular alterations. It has been a challenge to identify molecular classes of HCC and design treatment strategies for each specific subtype. MicroRNAs (miRNAs) are involved in HCC pathogenesis, and their expression profiles have been used to classify cancers. We analyzed miRNA expression in human HCC samples to identify molecular subclasses and oncogenic miRNAs. METHODS We performed miRNA profiling of 89 HCC samples using a ligation-mediated amplification method. Subclasses were identified by unsupervised clustering analysis. We identified molecular features specific for each subclass using expression pattern (Affymetrix U133 2.0; Affymetrix, Santa Clara, CA), DNA change (Affymetrix STY Mapping Array), mutation (CTNNB1), and immunohistochemical (phosphor[p]-protein kinase B, p-insulin growth factor-IR, p-S6, p-epidermal growth factor receptor, β-catenin) analyses. The roles of selected miRNAs were investigated in cell lines and in an orthotopic model of HCC. RESULTS We identified 3 main clusters of HCCs: the wingless-type MMTV integration site (32 of 89; 36%), interferon-related (29 of 89; 33%), and proliferation (28 of 89; 31%) subclasses. A subset of patients with tumors in the proliferation subclass (8 of 89; 9%) overexpressed a family of poorly characterized miRNAs from chr19q13.42. Expression of miR-517a and miR-520c (from ch19q13.42) increased proliferation, migration, and invasion of HCC cells in vitro. MiR-517a promoted tumorigenesis and metastatic dissemination in vivo. CONCLUSIONS We propose miRNA-based classification of 3 subclasses of HCC. Among the proliferation class, miR-517a is an oncogenic miRNA that promotes tumor progression. There is rationale for developing therapies that target miR-517a for patients with HCC.

Wnt-pathway activation in two molecular classes of hepatocellular carcinoma and experimental modulation by sorafenib.

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.

Chronic alcohol-induced liver injury and oxidant stress are decreased in cytochrome P4502E1 knockout mice and restored in humanized cytochrome P4502E1 knock-in mice.

A major pathway for chronic ethanol-induced liver injury is ethanol-induced oxidant stress. Several pathways contribute to mechanisms by which ethanol induces oxidant stress. Although some studies support a role for cytochrome P450 2E1 (CYP2E1), others do not. Most previous studies were conducted in the intragastric infusion model of ethanol administration. There is a need to develop oral models of significant liver injury and to evaluate the possible role of CYP2E1 in ethanol actions in such models. We evaluated chronic ethanol-induced liver injury, steatosis, and oxidant stress in wild-type (WT) mice, CYP2E1 knock out (KO) mice, and humanized CYP2E1 knock-in (KI) mice, in which the human 2E1 was added back to mice deficient in the mouse 2E1. WT mice and the CYP2E1 KO and KI mice (both provided by Dr. F. Gonzalez, National Cancer Institute) were fed a high-fat Lieber-DeCarli ethanol liquid diet for 3weeks; pair-fed controls received dextrose. Ethanol produced fatty liver and oxidant stress in WT mice but liver injury (transaminases, histopathology) was minimal. Ethanol-induced steatosis and oxidant stress were blunted in the KO mice (no liver injury) but restored in the KI mice. Significant liver injury was produced in the ethanol-fed KI mice, with elevated transaminases, necrosis, and increased levels of collagen type 1 and smooth muscle actin. This liver injury in the KI mice was associated with elevated oxidant stress and elevated levels of the human CYP2E1 compared to levels of the mouse 2E1 in WT mice. Activation of JNK and decreased levels of Bcl-2 and Bcl-XL were observed in the ethanol-fed KI mice compared to the other groups. Fatty liver in the WT and the KI mice was associated with lower levels of PPARα and acyl-CoA oxidase. No such changes were found in the ethanol-fed KO mice. These results show that CYP2E1 plays a major role in ethanol-induced fatty liver and oxidant stress. It is the absence of CYP2E1 in the KO mice that is responsible for the blunting of steatosis and oxidant stress because restoring the CYP2E1 restores the fatty liver and oxidant stress. Moreover, it is the human CYP2E1 that restores these effects of ethanol, which suggests that results for fatty liver and oxidant stress from rodent models of ethanol intake and mouse CYP2E1 can be extrapolated to human models of ethanol intake and to human CYP2E1.

Combination therapy for hepatocellular carcinoma: Additive preclinical efficacy of the HDAC inhibitor panobinostat with sorafenib

BACKGROUND & AIMS Hepatocellular carcinoma (HCC) is a heterogeneous cancer in which sorafenib is the only approved systemic therapy. Histone deacetylases (HDAC) are commonly dysregulated in cancer and therefore represent promising targets for therapies, however their role in HCC pathogenesis is still unknown. We analyzed the expression of 11 HDACs in human HCCs and assessed the efficacy of the pan-HDAC inhibitor panobinostat alone and in combination with sorafenib in preclinical models of liver cancer. METHODS Gene expression and copy number changes were analyzed in a cohort of 334 human HCCs, while the effects of panobinostat and sorafenib were evaluated in three liver cancer cell lines and a murine xenograft model. RESULTS Aberrant HDAC expression was identified and validated in 91 and 243 HCCs, respectively. Upregulation of HDAC3 and HDAC5 mRNAs was significantly correlated with DNA copy number gains. Inhibiting HDACs with panobinostat led to strong anti-tumoral effects in vitro and vivo, enhanced by the addition of sorafenib. Cell viability and proliferation declined, while apoptosis and autophagy increased. Panobinostat increased histone H3 and HSP90 acetylation, downregulated BIRC5 (survivin) and upregulated CDH1. Combination therapy with panobinostat and sorafenib significantly decreased vessel density, and most significantly decreased tumor volume and increased survival in HCC xenografts. CONCLUSIONS Aberrant expression of several HDACs and copy number gains of HDAC3 and HDAC5 occur in HCC. Treatment with panobinostat combined with sorafenib demonstrated the highest preclinical efficacy in HCC models, providing the rationale for clinical studies with this novel combination.

Intrahepatic Cholangiocarcinoma: New Insights in Pathology

Cholangiocarcinomas are malignant tumors that derive from cholangiocytes of small intrahepatic bile ducts or bile ductules (intrahepatic cholangiocarcinoma; ICC), or of large hilar or extrahepatic bile ducts (extrahepatic cholangiocarcinoma; ECC). ICC and ECC differ in morphology, pathogenesis, risk factors, treatment, and prognosis. This review focuses on ICC, which is rising in incidence with the emergence of hepatitis C virus (HCV) infection as a risk factor. The authors examined 73 ICC, which were resected at The Mount Sinai Medical Center in New York City, and reviewed the literature. The tumors were categorized into classical and nonclassical ICCs based on histopathology. Classical ICCs (54.8%) were characterized by a tubular, glandular, or nested pattern of growth, were significantly associated with tumor size of more than 5 cm and the absence of underlying liver disease and/or advanced fibrosis. Nonclassical ICCs (45.2%) consisted of tumors with trabecular architecture, tumors that exhibited features of extrahepatic carcinomas, and carcinomas considered to be derived from hepatic progenitor cells, i.e., combined hepatocellular/cholangiocarcinomas and cholangiolocellular carcinomas (ductular type of ICC). They were smaller and often arose in chronic liver disease, mostly HCV infection, and/or with significant fibrosis. The role of immunohistochemistry in the diagnosis of ICC and the importance of the new American Joint Committee on Cancer Staging System for ICC are also discussed.

Successful Split Liver-Kidney Transplant for Factor H Associated Hemolytic Uremic Syndrome

BACKGROUND AND OBJECTIVES A male infant with a family history of thrombotic microangiopathy developed atypical hemolytic uremic syndrome (aHUS). DESIGN, SETTING, PARTICIPANTS, & MEASUREMENTS Case report. RESULTS Genetic analysis demonstrated a heterozygous mutation (S1191L) of CFH, the gene coding complement factor H (CFH). The child suffered many episodes of HUS, each treated with plasma exchange. In time, despite initiation of a prophylactic regimen of plasma exchange, his renal function declined significantly. At the age of 4 yr he received a (split liver) combined liver-kidney transplant (LKT) with preoperative plasma exchange and enoxaparin anticoagulation. Initial function of both grafts was excellent and is maintained for nearly 2 yr. CONCLUSIONS This report adds to the small but growing number of individuals in whom LKT has provided a favorable outcome for aHUS associated with CFH mutation, expands the technique of using a split liver graft, and describes the unique histologic features of subclinical liver disease in HUS.

Fibrolamellar carcinoma: a review with focus on genetics and comparison to other malignant primary liver tumors.

Fibrolamellar carcinoma is a rare primary malignant liver neoplasm that usually affects adolescents and young adults with no underlying liver disease. Morphologically, the tumor cells resemble oncocytic hepatocytes arranged in cords with a stroma of lamellated collagen fibers. Immunohistochemical studies have found that fibrolamellar carcinomas express markers associated with both biliary (CK7 and epithelial membrane antigen) and hepatocytic (heppar-1and glypican-3) differentiation, as well as markers associated with hepatic progenitor cells (CK19 and EpCAM) and stem cells (CD133 and CD44). Genetic studies show fewer alterations compared with classic hepatocellular carcinoma. Pooled data from comparative genomic hybridization studies show that fibrolamellar carcinomas have fewer and less frequent genomic alterations when compared with classic hepatocellular carcinoma, cholangiocarcinoma, and hepatoblastoma. Of the alterations seen in fibrolamellar carcinoma, the most frequent are gains in 1q and 8q (also frequently seen in other hepatic tumors) and loss of 18q. Fibrolamellar carcinoma also has less frequent methylation of tumor suppressor promoters compared with hepatocellular carcinoma and minimal alterations in mitochondrial DNA. Fibrolamellar carcinoma is associated with better survival than hepatocellular carcinoma and cholangiocarcinoma, presumably due to the young age of the patients and the lack of cirrhosis. These features make more aggressive surgical therapy possible. There is currently very little information on the effectiveness of chemotherapy for fibrolamellar carcinoma.

Unique genomic profile of fibrolamellar hepatocellular carcinoma.

BACKGROUND & AIMS Fibrolamellar hepatocellular carcinoma (FLC) is a rare primary hepatic cancer that develops in children and young adults without cirrhosis. Little is known about its pathogenesis, and it can be treated only with surgery. We performed an integrative genomic analysis of a large series of patients with FLC to identify associated genetic factors. METHODS By using 78 clinically annotated FLC samples, we performed whole-transcriptome (n = 58), single-nucleotide polymorphism array (n = 41), and next-generation sequencing (n = 48) analyses; we also assessed the prevalence of the DNAJB1-PRKACA fusion transcript associated with this cancer (n = 73). We performed class discovery using non-negative matrix factorization, and functional annotation using gene-set enrichment analyses, nearest template prediction, ingenuity pathway analyses, and immunohistochemistry. The genomic identification of significant targets in a cancer algorithm was used to identify chromosomal aberrations, MuTect and VarScan2 were used to identify somatic mutations, and the random survival forest was used to determine patient prognoses. Findings were validated in an independent cohort. RESULTS Unsupervised gene expression clustering showed 3 robust molecular classes of tumors: the proliferation class (51% of samples) had altered expression of genes that regulate proliferation and mammalian target of rapamycin signaling activation; the inflammation class (26% of samples) had altered expression of genes that regulate inflammation and cytokine enriched production; and the unannotated class (23% of samples) had a gene expression signature that was not associated previously with liver tumors. Expression of genes that regulate neuroendocrine function, as well as histologic markers of cholangiocytes and hepatocytes, were detected in all 3 classes. FLCs had few copy number variations; the most frequent were focal amplification at 8q24.3 (in 12.5% of samples), and deletions at 19p13 (in 28% of samples) and 22q13.32 (in 25% of samples). The DNAJB1-PRKACA fusion transcript was detected in 79% of samples. FLC samples also contained mutations in cancer-related genes such as BRCA2 (in 4.2% of samples), which are uncommon in liver neoplasms. However, FLCs did not contain mutations most commonly detected in liver cancers. We identified an 8-gene signature that predicted survival of patients with FLC. CONCLUSIONS In a genomic analysis of 78 FLC samples, we identified 3 classes based on gene expression profiles. FLCs contain mutations and chromosomal aberrations not previously associated with liver cancer, and almost 80% contain the DNAJB1-PRKACA fusion transcript. By using this information, we identified a gene signature that is associated with patient survival time.

Immune cell-derived C3a and C5a costimulate human T cell alloimmunity.

Emerging evidence indicates that complement provides costimulatory signals for murine T cells but whether complement impacts human T cells remains unclear. We observed production of complement activation products C3a and C5a during in vitro cultures of human T cells responding to allogeneic dendritic cells (DC). Both partners expressed the receptors for C3a (C3aR) and C5a (C5aR) and C3aR‐ and C5aR‐antagonists inhibited T cell proliferation. Recombinant C3a/C5a promoted CD4+ T cell expansion, bypassed the inhibitory effects of CTLA4‐Ig, and induced AKT phosphorylation, the latter biochemically linking C3aR/C5aR to known T cell signaling pathways. Lowering DC C3a/C5a production by siRNA knockdown of DC C3 reduced T cell alloresponses. Conversely downregulating DC expression of the complement regulatory protein decay–accelerating factor increased immune cell C3a/C5a and augmented T cell proliferation, identifying antigen presenting cells as the dominant complement source. Pharmacological C5aR blockade reduced graft versus host disease (GVHD) scores, prolonged survival, and inhibited T cell responses in NOD scid γcnull mouse recipients of human peripheral blood mononuclear cells, verifying that the mechanisms apply in vivo. Together our findings unequivocally document that immune cell–derived complement impacts human T cell immunity and provide the foundation for future studies targeting C3aR/C5aR as treatments of GVHD and organ transplant rejection in humans.

Plasma cell hepatitis in hepatitis C virus patients post–liver transplantation: Case‐control study showing poor outcome and predictive features in the liver explant

Plasma cell hepatitis (PCH) is characterized by plasma cell infiltration seen in allografts of patients who underwent liver transplantation (LT) for conditions other than autoimmune hepatitis. We identified 40 PCH patients who underwent LT for hepatitis C virus (HCV) by searching our pathology database (1994–2006) for the keywords liver allograft, lymphoplasmacytic, and plasma cell(s). We selected 2 control patients who received LT for HCV for each PCH case. The control patients were matched according to date of LT and availability of biopsy material at the time interval to development of PCH in PCH patients. Explant and post‐LT biopsy slides were blindly reviewed by 2 liver pathologists and the severity of the plasma cell infiltrate was scored. A score of 3 (plasma cells composing >30% of the infiltrate) defined PCH in allograft biopsies. Five random areas of dense inflammation were also examined in explant livers and the highest score was used. Poor outcome was defined as death or advanced fibrosis (stage ≥ 4 of 6). We found that PCH patients were more likely to have worse outcomes than control patients (65% versus 40%, P < 0.01), including increased mortality (50% versus 30%, P < 0.05). Kaplan‐Meier survival analysis showed significantly worse survival for PCH patients from 4 to 10 years post‐LT (P < 0.05). Explants from 40% of PCH patients had a score of 3 compared to 18% of control patients (P < 0.01). We found that the development of PCH is associated with poor outcome in patients undergoing LT for HCV. The association of significant plasma cell infiltrates in native livers of HCV patients developing PCH suggests that some patients may have a predisposition to developing PCH. Liver Transpl 15:1826–1833, 2009.