The cohort comprised 160 tumours (131 men, 29 women). 93 tumours developed in liver cirrhosis (Ishak stage 5-6), 67 tumours arose in non-cirrhotic liver tissue (Ishak stage 0-4). In women, the proportion of HCC in non-cirrhotic liver tissue was significantly higher than in men (p < 0.04). Most patients were older than 60 years at first diagnosis (mean age of men 63.9 years and of women 68.8 years). Patients with HCC in cirrhosis were slightly younger (mean age 64.0 years) than patients with HCC in non-cirrhotic parenchyma (mean age 70.0 years). Hotspot hTERT promotor mutations were detected in 101 HCC (63.1 %) with equal sex distribution. In 100 cases a TERTC228T mutation was identified, 1 HCC harboured a TERTC250T mutation. Few cases had a background of hemochromatosis, PSC, autoimmune hepatitis and Fanconi anemia. Clinico-pathological characteristics are summarised in table 2.
Molecular analysis
hTERT promotor mutation was significantly more frequent in HCC arising in cirrhosis than in HCC in non-cirrhosis and additionally independent from sex distribution (p < 0.01) (Table 1, Table 3). hTERT promotor mutation was significantly more frequent in patients over 60 years than in patients under 60 years (p < 0.01). Generally, 78.9% of HCCs in liver cirrhosis with underlying chronic illness harboured a hTERT promotor mutation, compared to 68.2 % without underlying chronic liver disease (p = 0.3) (Table 2). HCC revealed a higher frequency of hTERT promotor mutation in clinically documented cases of chronic alcoholic liver disease (ALD), HBV and HCV infection (p = 0.04; p = 0.81; p = 0.25) compared to cases without underlying liver disease (Table 2). In liver cirrhosis, the frequency of hTERT promotor mutation increased from HCC G1 (50%) compared to HCC G3 (94%) (p = 0.02). In contrast, in non-cirrhotic liver tissue the frequency of hTERT promotor mutation decreased from HCC G1 (50%) to HCC G3 (36,4%).
hTERT promotor mutation was detected in nearly all histological subtypes of HCC (Fig. 1). The highest mutation rates were observed in solid and steatohepatitic HCC subtype in cirrhosis (100% and 88.3%, respectively). Only 62.5% of solid and 33.3% of steatohepatitic carcinomas harbored a hTERT promotor mutation in the group of 67 HCC in non-cirrhosis. HCC in liver cirrhosis presented with a significantly smaller mean diameter (3.9 cm in mutated and in wildtype HCC) than tumours in non-cirrhosis (6.2 cm resp. 7.7 cm in mutated resp. wildtype HCC) (p < 0.05).
The analysis of HCC arising in non-cirrhotic liver with minor histopathological damage and the frequency of hTERT promotor mutation in these tumours were one main interest of this study. Our collective did not include HCC on liver cell adenoma. 29 cases had eighter a history of ALD or HBV or HCV infection or a combination of these illnesses. In strong contrast to HCC in liver cirrhosis, only 41.4% of these tumours harbored hTERT promotor mutations (compared to 78.9 % of hTERT promotor mutations in HCC in liver cirrhosis, p < 0.01). Furthermore, 47.4% of 38 tumours lacking any clinical risk factors for HCC had a hTERT promotor mutation; and even 8 of 18 tumours which arose in non-cirrhotic liver tissue without histopathological changes were mutated.
50% of all tumours in a non-cirrhotic background with moderate fibrosis (stage 2-4), moderate inflammation (mHAI > 3) and steatosis (> 10%) harboured hTERT promotor mutations (Table 3). Frequency of hTERT promotor mutations amounted 30% in tumours which developed in virtually non-affected liver tissue without fibrosis (stage 0), without or with only sparse inflammation (mHAI 0 / 1) and without steatosis. This difference, although not significant, reveals an increasing hTERT promotor mutation rate associated with chronically affected liver parenchyma even in non-cirrhotic liver tissue. Remarkably, a small subgroup of poorly differentiated HCC (grade 3) in non-cirrhotic liver parenchyma without any histopathological changes was identified (n=7), without a hTERT promotor mutation.
For survival analysis age-matched multivariate Cox regression analysis (patients < 60 years vs ≥ 60 years) was performed (Fig. 2). In a period over 5 years, patients with hTERT promotor mutated HCC generally had a similar outcome as patients with HCC, wildtype (p = 0.32).
In a second step the HCC collective was divided into four subgroups (Fig. 2): Subgroup 0: non-cirrhosis, hTERT wildtype; subgroup 1: cirrhosis, hTERT wildtype; subgroup 2: non-cirrhosis, hTERT mutated; subgroup 3: cirrhosis, hTERT mutated. The difference in age-correlated cumulative overall survival over 3, 5 and 16 years was insignificant (3 years p = 0.2, 5 years p = 0.13, 16 years p = 0.22).
Immunohistochemical hTERT expression in HCC and tumour-bearing liver tissue
Furthermore, we analyzed nuclear hTERT antibody expression in HCC with and without hTERT hotspot promotor mutations, as well as in tumour-bearing normal liver tissue. Either a homogenous or a heterogenous immunohistochemical reaction pattern was observed. In most cases only few tumour cells revealed a strong nuclear staining, whereas the majority had a moderate or weak nuclear positivity in the same tissue fragment (Fig. 3). Both antibodies showed a similar labelling pattern in HCCs and in tumour-bearing liver tissue, with a significantly stronger expression and higher nuclear staining intensity in tumour tissue (LS-B95: Mean H score in tumor 106.4, mean H score in non-tumorous liver 75.9, p < 0.01; 2D8: Mean H score in tumor 103, mean H score in non-tumorous liver 79.5, p < 0.01, t statistic for independent samples). Comparison of hTERT promotor mutated HCC and HCC wildtype revealed a mildly stronger immunoreaction in the first group, but these differences were not significant (LS-B95: p = 0.28, 2D8: p = 0.16). hTERT expression was similar in cirrhotic and non-cirrhotic liver tissue, independent from the presence of a molecular proved hTERT promotor mutation.