SEPT9 Expression in Hepatic Nodules: An Immunohistochemical Study of Hepatocellular Neoplasm and Metastasis.

The methylated SEPT9 DNA ( mSEPT9 ) in plasma is a US Food and Drug Administration (FDA)-approved screening biomarker in colorectal cancer and is emerging as a promising diagnostic and prognostic biomarker in hepatocellular carcinoma (HCC). We evaluated the SEPT9 protein expression by immunohistochemistry (IHC) in various hepatic tumors from 164 hepatectomies and explants. Cases diagnosed as HCC (n=68), hepatocellular adenoma (n=31), dysplastic nodule (n=24), and metastasis (n=41) were retrieved. SEPT9 stain was performed on representative tissue blocks showing tumor/liver interface. For HCC, archived IHC (SATB2, CK19, CDX2, CK20, and CDH17) slides were also reviewed. The findings were correlated with demographics, risk factors, tumor size, alpha fetoprotein levels at diagnosis, T stage and oncologic outcomes, with significance defined as P <0.05. Percentage of SEPT9 positivity differed significantly among hepatocellular adenoma (3%), dysplastic nodule (0%), HCC (32%), and metastasis (83%, P <0.001). Compared with patients with SEPT9- HCC, those with SEPT9+ HCC were older (70 vs. 63 y, P =0.01). The extent of SEPT9 staining correlated with age ( rs =0.31, P =0.01), tumor grade ( rs =0.30, P =0.01), and extent of SATB2 staining ( rs =0.28, P =0.02). No associations were found between SEPT9 staining and tumor size, T stage, risk factors, CK19, CDX2, CK20, or CDH17 expression, alpha fetoprotein levels at diagnosis, METAVIR fibrosis stage, and oncologic outcome in the HCC cohort. SEPT9 is likely implicated in liver carcinogenesis in a HCC subset. Similar to mSEPT9 DNA measurement in liquid biopsies, SEPT9 staining by IHC may prove helpful as an adjunct diagnostic biomarker with potential prognostic ramifications.

Inflammatory Hepatocellular Adenoma with Elevated Serum Protein Induced by Vitamin K Absence/Antagonist-II in Adult Males.

Two men (24 and 34 years of age) with a single hypervascular liver tumor were admitted to our hospital. The tumors were diagnosed as hepatocellular adenoma (HCA) by an ultrasound-guided biopsy and classified as inflammatory type by immunohistochemical staining. Considering the risk of malignant transformation, they underwent surgical resection. Although the serum levels of protein induced by vitamin K absence/antagonist-II (PIVKA-II) were slightly elevated, they normalized after the resection. The diagnosis of HCA including malignant transformation is often difficult by image findings alone. Careful immunohistochemical examinations are very useful for the diagnosis and classification of subgroups, including malignant transformation. In addition, we proved that HCA without malignant transformation expresses PIVKA-II.

Chronic dietary toxicity and carcinogenicity study with potassium perfluorooctanesulfonate in Sprague Dawley rats.

To investigate toxicity and neoplastic potential from chronic exposure to perfluorooctanesulfonate (PFOS), a two-year toxicity and cancer bioassay was conducted with potassium PFOS (K⁺ PFOS) in male and female Sprague Dawley rats via dietary exposure at nominal K⁺ PFOS concentrations of 0, 0.5, 2, 5, and 20 µg/g (ppm) diet for up to 104 weeks. Additional groups were fed 20 ppm for the first 52 weeks, after which they were fed control diet through study termination (20 ppm Recovery groups). Scheduled interim sacrifices occurred on Weeks 4, 14, and 53, with terminal sacrifice between Weeks 103 and 106. K⁺ PFOS appeared to be well-tolerated, with some reductions in body weight occurring in treated rats relative to controls over certain study periods. Male rats experienced a statistically significant decreased trend in mortality with significantly increased survival to term at the two highest treatment levels. Decreased serum total cholesterol, especially in males, and increased serum urea nitrogen were consistent clinical chemistry observations that were clearly related to treatment. The principal non-neoplastic effect associated with K⁺ PFOS exposure was in livers of males and females and included hepatocellular hypertrophy, with proliferation of endoplasmic reticulum, vacuolation, and increased eosinophilic granulation of the cytoplasm. Statistically significant increases in hepatocellular adenoma were observed in males (p=0.046) and females (p=0.039) of the 20 ppm treatment group, and all of these tumors were observed in rats surviving to terminal sacrifice. The only hepatocellular carcinoma observed was in a 20 ppm dose group female. There were no treatment-related findings for thyroid tissue in rats fed K⁺ PFOS through study termination; however, male rats in the 20 ppm Recovery group had statistically significantly increased thyroid follicular cell adenoma, which was considered spurious. There was no evidence of kidney or bladder effects. In rats, the dietary dose estimated as the lower 95% confidence limit of the benchmark dose for a 10% increase in hepatic tumors was 8 ppm for both sexes. Recent mechanistic studies suggest a PPARα/CAR/PXR-mediated mode of action for the liver tumors observed in the present two-year study.

Use of RNA interference to modulate liver adenoma development in a murine model transgenic for hepatitis B virus.

Chronic hepatitis B virus (HBV) infection is closely related to the development of severe liver complications, including hepatocellular carcinoma. In previous studies, we reported that in vivo long-term HBV suppression in transgenic mice can be achieved without apparent toxicity by short hairpin RNA sequentially delivered using adeno-associated viral (AAV) vectors of different serotypes. Our goal herein was to address the clinical utility of this delivery system and, in particular, to determine whether RNA interference (RNAi) and its ability to induce long-term HBV suppression will modulate the development of HBV-associated liver pathology. As a model system, we used a unique HBV transgenic mouse model, containing a 1.3 times over length of the HBV genome, on the ICR mouse background. These transgenic mice produce high serum HBV titers comparable with human chronic HBV patients, and, importantly, manifest characteristic HBV-associated pathology, including progressive hepatocellular injury and the development of hepatocellular adenoma. Using this system, we injected animals with AAV vectors expressing either HBV-specific or a control luciferase-specific short hairpin RNA and followed animals for a total of 18 months. We report herein that AAV-mediated RNAi therapy profoundly inhibits HBV replication and gene expression, with a significant reduction in hepatic regeneration, liver enzymes and, importantly, the appearance of liver adenomas. Indeed, the therapeutic effect of RNAi correlated with the reduction in HBV titers. Our data demonstrate that appropriately designed RNAi therapy has the potential to prevent formation of HBV-associated hepatocellular adenoma.

Insulin resistance, steatohepatitis, and hepatocellular carcinoma in a new congenic strain of Fatty Liver Shionogi (FLS) mice with the Lep(ob) gene.

In order to examine the influence of obesity on metabolic disorder and liver pathogenesis of the Fatty Liver Shionogi (FLS) mouse, which develops hereditary fatty liver and spontaneous liver tumors, we established a new congenic strain named FLS-Lep(ob). The Lep(ob) gene of the C57BL/6JWakShi (B6)-Lep(ob)/Lep(ob) mouse was transferred into the genome of the FLS mouse, by backcross mating. FLS-Lep(ob)/Lep(ob) mice were maintained by intercrossing between Lep(ob)-heterozygous littermates. The FLS-Lep(ob)/Lep(ob) mice of both sexes developed remarkable hyperphagia, obesity and type 2 diabetes mellitus. At 12 weeks of age, glucosuria was detected in all male and female FLS-Lep(ob)/Lep(ob) mice. Biochemical examination demonstrated that the FLS-Lep(ob)/Lep(ob) mice have severe hyperlipidemia and hyperinsulinemia. The livers of FLS-Lep(ob)/Lep(ob) mice showed microvesicular steatosis and deposition of large lipid droplets in hepatocytes throughout the lobules. The steatohepatitis-like lesions including the multifocal mononuclear cell infiltration and clusters of foamy cells were observed earlier in FLS-Lep(ob)/ Lep(ob) mice than in FLS mice. B6-Lep(ob)/Lep(ob) mice did not show hepatic inflammatory change. Furthermore, FLS-Lep(ob)/Lep(ob) mice developed multiple hepatic tumors including hepatocellular adenomas and carcinomas following steatohepatitis. In conclusion, the FLS-Lep(ob)/Lep(ob) mice developed steatohepatitis and hepatic tumors following hepatic steatosis. The FLS-Lep(ob)/Lep(ob) mouse with obesity and type 2 diabetes mellitus might be a useful animal model for human non-alcoholic steatohepatitis (NASH).

Simultaneous resection of liver cell adenomas and an intrahepatic portosystemic venous shunt with elevation of serum PIVKA-II level.

A 27-year-old woman with no history of liver disease or oral contraceptive use presented with sudden abdominal pain. Laboratory data showed mild liver dysfunction with jaundice. Computed tomography and angiography revealed centrally located large liver cell adenomas (LCAs) and an intrahepatic portosystemic venous shunt (IHPSS) in the left lobe. The serum des-gamma-carboxy prothrombin (known as "protein induced by a lack of vitamin K or antagonist II," PIVKA-II) level was extremely high (6,647 mAU/ml), indicating malignant transformation of the tumors. Under the diagnosis of LCAs and IHPSS, the patient underwent simultaneous resection of the four liver tumors and portovenous shunt, and the hepatic vascular abnormality was resolved. The pathological diagnosis was LCAs without hepatocellular carcinoma. Immunohistochemical analysis with an anti-PIVKA-II monoclonal antibody showed positive staining of the adenoma cells. This case shows that LCA without malignant transformation can produce PIVKA-II, leading to high serum levels of PIVKA-II. Simultaneous resection of multiple tumors and closure of the portosystemic shunt are strongly recommended in a patient with LCA associated with IHPSS.

Quantitative comparison of alpha-fetoprotein and albumin mRNA levels in hepatocellular carcinoma/adenoma, non-tumor liver and blood: implications in cancer detection and monitoring.

Early detection of recurrence is valuable for monitoring hepatocellular carcinoma (HCC) progression. By quantitative reverse transcriptase polymerase chain reaction (RT-PCR), we derived calibration curves for alpha-fetoprotein (afp) and albumin (alb) mRNAs using 40 matched tumors and non-tumor liver tissues from HCC/adenoma patients. We prospectively quantified tumor cells and non-tumor liver cells in 62 patients' blood samples before, during and after surgery. Expression of both mRNAs was heterogeneous (1-10(5)-fold) between tumors and HepG2 cell line. The alb-mRNA levels in non-tumor liver cells were 2-10-fold higher than in tumor cells. The afp-mRNA levels in HCC cells were 30-1000-fold higher than in non-tumor cells. The alb-mRNA level in blood may reflect the number of liver cells, whereas the afp-mRNA level may represent mostly the number of HCC cells. We found different ratios of circulating HCC cells to non-tumor liver cells during the clinical course of patients, in association with the subsequent development of recurrence/metastasis. This approach may prove useful for detecting and monitoring HCC progression.

Hepatocellular adenomas in glycogen storage disease type I and III: a series of 43 patients and review of the literature.

BACKGROUND: Hepatocellular adenomas may develop in patients with glycogen storage disease types I and III, and the malignant degeneration of adenomas in hepatocellular carcinoma has been reported in ten cases. The aim of this work was to study the characteristics of hepatic adenomas in a large series of 43 patients with glycogen storage disease types I and III and to determine the optimal means of follow-up. METHODS: The charts of 43 patients with glycogen storage disease type I and III were studied. In all these patients, abdominal ultrasonography and the determination of serum alpha-fetoprotein had been performed yearly and serum concentrations of several proteins were determined once. RESULTS: 51.8% of patients with type I and 25% of patients with type III glycogen storage disease had hepatic adenomas at the time of the study. The male to female ratio was 2 to 1 in type I, and no female had adenomas in type III. No evidence of malignant transformation was observed during the follow-up period. Serum concentrations of several proteins were significantly higher in patients with hepatic adenomas than in patients without such lesions. CONCLUSIONS: In patients with glycogen storage disease type I and III, the determination of alpha-fetoprotein serum concentration has to be combined with yearly hepatic ultrasound examinations. Other investigations such as CT scan should be considered when the size of any adenoma increases. The malignant transformation of hepatocellular adenoma into hepatocellular carcinoma remains a rare event.