Portal System

Briefly, the two key features of NASH, steatosis and inflammation, were categorized as follows: steatosis was determined by analyzing hepatocellular vesicular steatosis, i.e. macrovesicular steatosis and microvesicular steatosis separately, and by hepatocellular hypertrophy as defined below (Fig. 2). Inflammation was scored by analyzing the amount of inflammatory cell aggregates (Fig. 2). The proposed rodent scoring system is shown in Table 4 and options for its use in diagnosis are shown in S1 Fig. The purpose of this scoring system is however not to derive a single score, but to score the individual features.
Macrovesicular steatosis and microvesicular steatosis were both separately scored and the severity was graded, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). The difference between macrovesicular and microvesicular steatosis was defined by whether the vacuoles displaced the nucleus to the side (macrovesicular) or not (microvesicular). Similarly, the level of hepatocellular hypertrophy, defined as cellular enlargement more than 1.5 times the normal hepatocyte diameter, was scored, based on the percentage of the total area affected, into the following categories: 0 (<5%), 1 (5–33%), 2 (34–66%) and 3 (>66%). For hepatocellular hypertrophy the evaluation was merely based on abnormal enlargement of the cells, irrespective of rounding of the cells and/or changes in cytoplasm or the number of vacuoles, and is therefore not a substitute of ballooning. The unweight sum of the scores for steatosis (macrovesicular steatosis, microvesicular steatosis and hypertrophy) thus ranged from 0–9. Both steatosis and hypertrophy were evaluated at a 40 to 100× magnification and only the sheets of hepatocytes were taken into account (terminal hepatic venules and portal tracts etc were excluded).
Inflammation was evaluated by counting the number of inflammatory foci per field using a 100 x magnification (view size of 3.1 mm²). A focus was defined a cluster, not a row, of ≥5 inflammatory cells. Five different fields were counted and the average was subsequently scored into the following categories: normal (<0.5 foci), slight (0.5–1.0 foci), moderate (1.0–2.0 foci), severe (>2.0 foci).
Hepatic fibrosis was identified using Sirius Red stained slides at 40 x magnification and evaluated by scoring whether pathologic collagen staining was absent (only in vessels) or collagen staining observed within the liver slide, the latter further defined as mild, moderate or massive. In addition, the percentage of the total area affected was evaluated using using image analysis of surface area on Sirius red stained slides.

Tissue sections were prepared at a thickness of 4 μm and stained with hematoxylin and eosin according to standard procedures. Two experienced pathologists, who were blinded to the experimental details, assessed liver histology using an Eclipse E800 Microscope (Nikon, Kawasaki, Japan). Ishak scores (a liver fibrosis scoring system) were then determined for each tissue section.

Patients with HCC who underwent combination therapy of ICI (nivolumab, camrelizumab, sintilimab, pembrolizumab, toripalimab, atezolizumab, or tislelizumab) and lenvatinib for unresectable HCC at Zhongshan Hospital between February 2018 and December 2020 were retrospectively enrolled as the combination therapy cohort. Meanwhile, a cohort of patients with HCC receiving neoadjuvant combination therapy and subsequent surgical resection at the same institute from February 2019 to September 2021 was recruited as the neoadjuvant cohort. The inclusion criteria for patients were listed as follows: (1) histopathological or radiological diagnosis of HCC; (2) without a history of other malignancies; (3) availability of complete clinicopathological features and follow-up data; (4) at least one radiological evaluation after the initiation of treatment. Patients without measurable intrahepatic foci were excluded from the analysis.
Clinicopathological information, radiological data, and laboratory parameters at baseline (within 30 days before the start of therapy) were collected. The tumor stage was classified under the Barcelona Clinic Liver Cancer (BCLC) staging system (21 (link)). Liver function was evaluated based on the Child-Pugh score (22 (link)). Approval for this study was granted by the Ethics Committee of Zhongshan Hospital (No. B2020-401) and informed consent was obtained from each patient included in the analysis.

A total of 304 patients treated with lenvatinib monotherapy or lenvatinib plus ICI as first-line treatment for uHCC at Zhongshan Hospital between October 2018 and December 2020 were retrospectively included. The inclusion criteria were: clinically diagnosed as HCC; the Barcelona Clinic Liver Cancer (BCLC) B-C stage; lenvatinib for more than one month; at least one imaginary follow-up; complete baseline information. Tumor differentiation was assessed using the Edmondson grading system and liver function was evaluated using the Child-Pugh scoring system. The BCLC system (14 (link)) and The Guidelines of Primary Liver Cancer in China (15 (link)) were used to determine the tumor stage. To avoid potential bias, the included patients were randomly assigned to the training cohort and validation cohort at a 2:1 ratio. The research was conducted in accordance with the declaration of Helsinki and ethical approvals were obtained from the ethics committee of Zhongshan hospital (B2020-401).