ASS1 Overexpression: A Hallmark of Sonic Hedgehog Hepatocellular Adenomas; Recommendations for Clinical Practice.

Until recently, 10% of hepatocellular adenomas (HCAs) remained unclassified (UHCA). Among the UHCAs, the sonic hedgehog HCA (shHCA) was defined by focal deletions that fuse the promoter of Inhibin beta E chain with GLI1. Prostaglandin D2 synthase was proposed as immunomarker. In parallel, our previous work using proteomic analysis showed that most UHCAs constitute a homogeneous subtype associated with overexpression of argininosuccinate synthase (ASS1). To clarify the use of ASS1 in the HCA classification and avoid misinterpretations of the immunohistochemical staining, the aims of this work were to study (1) the link between shHCA and ASS1 overexpression and (2) the clinical relevance of ASS1 overexpression for diagnosis. Molecular, proteomic, and immunohistochemical analyses were performed in UHCA cases of the Bordeaux series. The clinico-pathological features, including ASS1 immunohistochemical labeling, were analyzed on a large international series of 67 cases. ASS1 overexpression and the shHCA subgroup were superimposed in 15 cases studied by molecular analysis, establishing ASS1 overexpression as a hallmark of shHCA. Moreover, the ASS1 immunomarker was better than prostaglandin D2 synthase and only found positive in 7 of 22 shHCAs. Of the 67 UHCA cases, 58 (85.3%) overexpressed ASS1, four cases were ASS1 negative, and in five cases ASS1 was noncontributory. Proteomic analysis performed in the case of doubtful interpretation of ASS1 overexpression, especially on biopsies, can be a support to interpret such cases. ASS1 overexpression is a specific hallmark of shHCA known to be at high risk of bleeding. Therefore, ASS1 is an additional tool for HCA classification and clinical diagnosis.

Adeno-associated virus in the liver: natural history and consequences in tumour development.

OBJECTIVE: Adeno-associated virus (AAV) is a defective mono-stranded DNA virus, endemic in human population (35%-80%). Recurrent clonal AAV2 insertions are associated with the pathogenesis of rare human hepatocellular carcinoma (HCC) developed on normal liver. This study aimed to characterise the natural history of AAV infection in the liver and its consequence in tumour development. DESIGN: Viral DNA was quantified in tumour and non-tumour liver tissues of 1461 patients. Presence of episomal form and viral mRNA expression were analysed using a DNAse/TaqMan-based assay and quantitative RT-PCR. In silico analyses using viral capture data explored viral variants and new clonal insertions. RESULTS: AAV DNA was detected in 21% of the patients, including 8% of the tumour tissues, equally distributed in two major viral subtypes: one similar to AAV2, the other hybrid between AAV2 and AAV13 sequences. Episomal viral forms were found in 4% of the non-tumour tissues, frequently associated with viral RNA expression and human herpesvirus type 6, the candidate natural AAV helper virus. In 30 HCC, clonal AAV insertions were recurrently identified in CCNA2, CCNE1, TERT, TNFSF10, KMT2B and GLI1/INHBE. AAV insertion triggered oncogenic overexpression through multiple mechanisms that differ according to the localisation of the integration site. CONCLUSION: We provided an integrated analysis of the wild-type AAV infection in the liver with the identification of viral genotypes, molecular forms, helper virus relationship and viral integrations. Clonal AAV insertions were positive selected during HCC development on non-cirrhotic liver challenging the notion of AAV as a non-pathogenic virus.

Repeat surgery in HNF1alpha-inactivated adenomatosis.

BACKGROUND AND AIMS: Stopping oral contraceptives following nodule detection usually prevents further hepatocellular growth (HCA); rare cases of growth have been reported after surgery. The aim of the study was to review our resected HCA cases and their outcomes and more specifically, growth. METHODS: We retrieved all HCA cases that required a second intervention and HCA growth cases of none resected HCA after resection of one or several HCAs. RESULTS: Out of the 210 resected classified HCA cases, a second resection was performed in 5 cases, 4 of which were in women with HNF1alpha-inactivated adenomatosis (H-adenomatosis) and had a favorable outcome. The fifth case was the occurrence of an inflammatory HCA, 3 years after resection of a previous one. Of the 65 resected HNF1-inactivated HCAs (H-HCAs), the nodules that remained continued to increase very slowly in 3 adenomatosis cases. After surgery, the liver became dysmorphic years later in one case, and the nodules grew but not significantly in another case. After the diagnosis of adenomatosis, progressive growth leads to surgery 12 years later in the last case. CONCLUSION: These results confirm that, in rare H-adenomatosis, size of nodules may increase very slowly, probably in part through coalescence of micro H-HCAs and leading occasionally to a second resection.

Unexpected discovery of small HNF1α-inactivated hepatocellular adenoma in pathological specimens from patients resected for liver tumours.

BACKGROUND AND AIMS: It is rare but not uncommon to discover micro/small HNF1α-inactivated hepatocellular adenoma (H-HCA) outside the context of resected H-HCA. We aimed to review our cases of micro/small H-HCA discovered by chance on different kinds of liver resected specimens. METHODS: We retrieved cases of micro/small H-HCA discovered by chance on resected specimens outside the context of H-HCA. All these nodules were liver fatty acid binding protein (LFABP)-negative contrasting with normal positivity in the surrounding non-tumoural liver, ruling out the possibility of focal steatosis or other subtypes of micro-HCAs. RESULTS: We identified 19 micro/small H-HCA cases. In 16 cases they were discovered in patients who underwent surgery for benign nodules including one haemangioma, six focal nodular hyperplasia, seven inflammatory HCA (including one with b-catenin activation), one HCA, whose subtype could not be identified because of massive necrosis/hemorrhage, and one hepatocellular carcinoma. In two additional cases, patients followed up for a melanoma underwent liver surgery to remove micro nodules possibly related to a metastatic process. Finally in one case a micro nodule was seen and resected during a cholecystectomy. CONCLUSION: Taken together, H-HCAs are more frequent than we initially supposed as micro and small HCAs cannot all be detected by routine ultrasound. Despite no information on the potential growth of these micro/small H-HCAs, there is no argument to stop oral contraceptives or to ask for a specific regular surveillance. The association of different subtypes of HCAs with focal nodular hyperplasia suggests they share or have common etiological factors.

Radiofrequency ablation versus surgical resection for hepatocellular carcinoma within the Milan criteria: A study of 281 Western patients.

OBJECTIVES: The aim of this study was to compare survival between radiofrequency ablation (RFA) and surgical resection (SR) in patients with hepatocellular carcinoma (HCC) within Milan criteria. METHODS: From January 2004 to December 2013 we consecutively and retrospectively included all patients with first occurrence of HCC within Milan criteria receiving SR or RFA as first-line treatment. The cumulative overall survival (OS) and disease-free survival (DFS) were compared after inverse probability weighting (including confounding factor). RESULTS: A total of 281 patients (RFA 178, SR 103) were enrolled. In multivariate Cox regression RFA and SR were not independent predictors of survival or recurrence. The respective weighted 5 years OS and DFS for patients with propensity scores between 0.1-0.9 in the SR and RFA groups were 54-33% and 60-16.9%, P = 0.695 and P = 0.426, respectively. Local tumour progression rate did not differ according to treatment (P = 0.523). Major complication rate was higher in the SR group, P = 0.001. Hospitalisation duration was lower in the RFA group (mean 2.19 days, range 2-7) than in the SR group (mean 10.2 days, range 3-30), P < 0.001. CONCLUSION: This large Western study has shown that OS and DFS did not differ after RFA (using mainly multipolar devices) and SR, for HCC within the Milan criteria in a European population, with a shorter hospitalisation time and a lower complication rate for RFA.

Pathological Diagnosis of Hepatocellular Cellular Adenoma according to the Clinical Context.

In Europe and North America, hepatocellular adenomas (HCA) occur, classically, in middle-aged woman taking oral contraceptives. Twenty percent of women, however, are not exposed to oral contraceptives; HCA can more rarely occur in men, children, and women over 65 years. HCA have been observed in many pathological conditions such as glycogenosis, familial adenomatous polyposis, MODY3, after male hormone administration, and in vascular diseases. Obesity is frequent particularly in inflammatory HCA. The background liver is often normal, but steatosis is a frequent finding particularly in inflammatory HCA. The diagnosis of HCA is more difficult when the background liver is fibrotic, notably in vascular diseases. HCA can be solitary, or multiple or in great number (adenomatosis). When nodules are multiple, they are usually of the same subtype. HNF1 α -inactivated HCA occur almost exclusively in woman. The most important point of the classification is the identification of ß -catenin mutated HCA, a strong argument to identify patients at risk of malignant transformation. Some HCA already present criteria indicating malignant transformation. When the whole nodule is a hepatocellular carcinoma, it is extremely difficult to prove that it is the consequence of a former HCA. It is occasionally difficult to identify HCA remodeled by necrosis or hemorrhage.

Value and limits of routine histology alone or combined with glutamine synthetase immunostaining in the diagnosis of hepatocellular adenoma subtypes on surgical specimens.

Immunohistochemistry is a valid method to classify hepatocellular adenoma (HCA). The aim was to test the performance of routine histology combined to glutamine synthetase (GS) staining to identify the 2 major HCA subtypes: HNF1 α inactivated (H-HCA) and inflammatory HCA (IHCA). 114 surgical cases, previously classified by immunohistochemistry, were analysed. Group A comprised 45 H-HCAs, 44 IHCAs, and 9 ß -catenin-activated IHCAs (b-IHCA), and group B, 16 b-HCA and unclassified HCA (UHCA). Steatosis was the hallmark of H-HCA. IHCA and b-IHCA were mainly characterized by inflammation, thick arteries, and sinusoidal dilatation; b-IHCA could not be differentiated from IHCA by routine histology. Group B was identified by default. A control set (91 cases) was analyzed using routine and GS stainings (without knowing immunohistochemical results). Among the 45 H-HCAs and 27 IHCAs, 40 and 24 were correctly classified, respectively. Among the 10 b-IHCAs, 4 were identified as such using additional GS. Eight of the 9 HCAs that were neither H-HCA nor IHCA were correctly classified. Conclusion. Routine histology allows to diagnose >85% of the 2 major HCA subtypes. GS is essential to identify b-HCA. This study demonstrates that a "palliative" diagnostic approach can be proposed, when the panel of specific antibodies is not available.

Role of contrast-enhanced sonography in differentiation of subtypes of hepatocellular adenoma: correlation with MRI findings.

OBJECTIVE: Hepatocellular adenomas (HCAs) are divided into three subtypes according to genotype and phenotype. The two main subgroups are hepatocyte nuclear factor 1α (HNF1α)-inactivated HCA and inflammatory HCA. Specific imaging features of these subgroups of adenoma have been delineated with MRI. The aim of this study was to document the contrast-enhanced sonographic (CEUS) features specific for adenoma subtypes and to correlate them with MRI findings. MATERIALS AND METHODS: We retrospectively analyzed data on 38 patients with HCA confirmed at pathologic examination in all cases. All cases were classified with MRI. RESULTS: HNF1α-inactivated HCA (n = 16) was found to have a homogeneous hyperechoic aspect at baseline gray-scale sonography, isovascularity or moderate hypervascularity with mixed filling in the arterial phase, and isoechogenicity in the portal and late portal venous phases. Homogeneous hyperechogenicity during B-mode sonography was the most specific pattern (sensitivity, 88%; specificity, 91%) and correlated with diffuse fat repartition observed on MR images obtained with chemical-shift sequences. In inflammatory HCA (n = 17) the association of arterial hypervascularity with centripetal filling, linear vascularities, peripheral rim of sustained enhancement, and central washout in the late venous phase was specific (sensitivity, 64%; specificity, 100%). Discrepancy between delayed washout during CEUS and sustained enhancement during MRI could be related to differences between gadolinium and microbubbles in diffusing in the interstitial spaces. In the five other HCA cases (four unclassified, one ß-catenin activated) CEUS showed characteristics of benign hepatocellular tumors with no specific features. CONCLUSION: HNF1α-inactivated HCA and inflammatory HCA had characteristic CEUS patterns. Delayed washout, an unusual finding in benign hepatic lesions, is of particular interest and was a characteristic of inflammatory HCA subtype.

Hepatocellular adenoma subtypes: the impact of overweight and obesity.

BACKGROUND: Hepatocellular adenomas (HCA) are rare benign tumours occurring mainly in women using oral contraceptives. With the appearance of a generation of new oral contraceptives, the number of HCA cases was anticipated to decrease, but in fact this was not observed in our practice. The influence of obesity was therefore suspected as a new co-factor. AIMS: The aim of this study was to analyze the impact of overweight/obesity in our cohort of HCA patients classified according to their subtypes. RESULTS: The number of HCA noticeably increased faster in the 2001-2011 period compared to the 1990-2000 period. This phenomenon concurred with an increasing number of patients overweight or obese. Females still represented the great majority of overweight/obese patients presenting HCA, however, overweight/obese male patients constituted a new entity in the inflammatory HCA and ß-catenin activated- inflammatory HCA subgroups. CONCLUSION: We propose that overweight/obesity may soon represent a major risk of malignant transformation of HCA, possibly via the IL-6 pathway.