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

Abstract

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.

Fig. 1

ASS1 T versus NT liver expression ratio in HCA subtypes quantified by mass spectrometry. The red line indicates T/NT ratio = 1.

Fig. 2

Comparative analysis of PTGDS and ASS1 IHC of shHCA. (A) Comparative IHC of three different cases (cases 66, 242, and 178) with anti‐PTGDS (left) and ASS1 (right) antibodies. PTGDS staining is interpreted as diffuse/moderate (a), mild intensity (c) or negative (e); ASS1 is clearly overexpressed in the three corresponding cases (b,d,f), contrasting with NT liver. (B) Summary of comparative IHC results on the 22 shHCA cases of the Bordeaux cohort (24 tumors). PTGDS labeling intensity: mild (1), moderate (2), and focal (f). ASS1 labeling: homogeneous (H) and heterogeneous (h).

Fig. 3

Histology and IHC of shHCA (surgical specimens). (A) H&E is compatible/typical with the diagnosis of shHCA; tumor (T) is a well‐differentiated, nonencapsulated hepatocellular proliferation with bland aspect, contrasting with steatotic (a) or nonsteatotic (b) NT liver; T consists of clear cells (c) often associated with hypereosinophilic and packed cells related to ischemia (d), blood cavities at different stages of degradation (e‐h), damaged vascular wall with detachment from hepatocytes row (i), red blood cells (asterisk) and fibrous bands (arrows) related to remodeling of the tumor (j). (B) Typical ASS1 staining on NT liver (“honey comb”) (a) and on T liver: diffuse (b) or heterogeneous (c,d). Scale bars represent original magnification; when the bar is missing, magnification is ×1.

Fig. 4

IHC results with anti‐ASS1 antibody on the whole cohort of 67 patients: the three different series (A), in comparison with H&E classification (B), and global result in 67 UHCAs (C).

Fig. 5

Biopsies of shHCA. (A) Biopsies with typical ASS1 IHC. ASS1 is clearly overexpressed in T compared with NT in two different fragments (case BinP1) or in the two parts (line) of the same biopsy (Case BCo1). (B) Biopsies analyzed by mass spectrometry–based proteomic. ASS1 is clearly overexpressed in T compared with NT fragments in case B162 with a high T:NT ASS1 ratio; ASS1 is quite difficult to interpret in biopsy BBDX3, but ASS1 (and other biomarkers) T:NT ratios quantified by mass spectrometry allowed us to assess the diagnosis of shHCA. Representative T and NT zones have been magnified. Abbreviation: NI, nonidentified.

Fig. 6

Algorithm for shHCA diagnosis.