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Schwertheim et al. Hepatoma Res 2020;6:41 I http://dx.doi.org/10.20517/2394-5079.2020.23 Page 3 of 14

Table 1. Clinical and pathological parameters of the study group with 72 HCC cases
All (n = 72)
Mean age (years) at diagnosis (range) 62 (17-99)
Gender (Male/Female) 55/17
Liver morphology
Non-cirrhotic 44
Cirrhotic 22
Fibrotic 6
Background
Underlying disease unknown 43
Alcohol abuse 2
Hepatitis B 12
Hepatitis C 14
Hepatitis B + C 0
Alpha-1-antitrypsin deficiency 1
Primary biliary cirrhosis 0
Autoimmunhepatitis 0
Tumor staging
pT1a/b 35
pT2 24
pT3 8
pT4 5
Grading
G1 9
G2 40
G3/G4 23
Nodal status
pN0 68
pN1 4
Lymph vessel infiltration
L0 72
Blood vessel infiltration
V0 41
V1 31
Resection status
R0 61
R1 10
R2 1
Observation period (in days) post surgery
Minimum 55
Maximum 3009

HCC: Hepatocellular carcinoma

Tissue microarray construction and immunohistochemistry
We investigated the expression of selected candidate proteins immunohistochemically using tissue
microarrays (TMAs). Regions of tumors were selected with matching H&E stained slides and marked on the
donor block. Construction of the TMAs was performed by using a manual tissue-array instrument (Beecher
[6]
Instruments, Silver Spring, MD, USA) as described before . Briefly, we took three 1-mm-thick tissue cores
from each specimen. Each TMA contained three corresponding tumor-free liver tissue cores as controls
and cores with myocardial tissue for TMA orientation and 10 sections of about 3 µm each were cut from
each TMA. Immunohistochemistry (IHC) of the paraffin sections for the antibodies β-catenin, KDM2A,
glutamine synthethase, ubiquitin, p62, LC3B, cathepsin B and cathepsin D was conducted as described
[6]
before by using an automated staining device (Dako Autostainer, Dako, Glostrup, Denmark). Further, we
stained one section from each TMA with H&E; Supplementary Table 1 provides detailed information on the
antibodies used and staining protocols. Additionally, we included negative controls in every run: slides were