Limpieza láser

Targeted genome sequencing was used in order to detect cancer-related somatic mutations in human samples. 1000 Genomes Project (The Genomes Project, 2015) database was used to evaluate the SNPs and non-harmful mutations. Due to the fact that we only worked with pathological tissue, there was no possibility to eliminate person-specific SNPs from analysis, thus we had to rely on 1000 Genome Project. The final data set from Project contains data for 2,504 presumably healthy individuals from 26 populations, which allows to estimate the level of harmfulness of one mutation or another. However, the presence of certain mutation in presumably healthy individual does not necessarily mean that this mutation is not harmful, and that the individual will not develop a disease later. A disease could be on early developing stage, thus not affecting the picture of blood tests of subjective feeling of well-being. Another issue with presumably harmless SNPs that the Project does not consider the combination of those SNPs. Possibly the combination of several ‘harmless’ SNPs can cause the disease. Another limitation of this work was the application of panel sequencing. The panel sequencing is restricted to the known set of mutations, unlike the whole genome sequencing. In our cohort were patients with no mutations detected, which does not mean that individuals had no mutations. Nonetheless, various mutations were detected, identified in confirmed in the cohort.

CTNNB1

Several CTNNB1 mutations are specific for tumor samples and were detected in 15% of HCC. 23% of Group B samples contained CTNNB1 mutations: c.A121G, p.T41A; c.T133C, p.S45P; c.C134T, p.S45F; c.C110T, p.S37F; c.C98T, p.S33F, all of which were located in exon 3 (Gamallo, et al., 1999).

CTNNB1 (c.C134T, p.S45F) mutation has been shown in patients with desmoid tumors (this type of tumors originates from fibroblasts) (Colombo, et al., 2013; Mullen, et al., 2013; Nishida, et al., 2016). Nishida et al. and Mullen at al. also found CTNNB1 (c.A121G, p.T41A) mutation in desmoid tumors. Doyen et al. show the CTNNB1 (c.A121G, p.T41A) and CTNNB1 (c.T133C, p.S45P) mutations and suggest that a certain CTNNB1 mutations are necessary to provide the tumor with

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the growth advantage (Doyen, et al., 2016). Kolble et al. describes the CTNNB1 (c.T133C, p.S45P) mutation being present in colorectal cancer and suggests that this specific mutation if restricted to a hepatic metastasis (Kolble, et al., 2000). CTNNB1 (c.C98T, p.S33F) mutation can be involved in the development of colorectal cancer (Alomar, et al., 2016).

Studies of Gamallo et al., Kobayashi et al. and Moreno-Bueno et al. report the CTNNB1 (c.A121G, p.T41A) and CTNNB1 (c.C110T, p.S37F) mutations existence in ovarian and endometrial carcinomas (Gamallo, et al., 1999; Kobayashi, Sagae, Nishioka, Tokino, & Kudo, 1999; Moreno- Bueno, et al., 2001). Kobayashi et al. suggest, that each mutation of CTNNB1 with the serine/threonine substitution results in the downregulation of beta-catenin through phosphorylation by GSK-3 beta kinase. The work of Palacios and Gamallo also evaluated the role of mutation of CTNNB1 on exon 3, confirming the (c.A121G, p.T41A) and (c.C110T, p.S37F) mutations, implicating CTNNB1 mutations in ovarian malignant transformation (Palacios & Gamallo, 1998).

None of the above mentioned mutations were previously described in HCC, however the suggested by several authors mechanism of malignisation of ovarian and endometric tissues could be applicable to the liver cancer development. Alternatively, considering the desmoid tumors publications (Colombo, et al., 2013; Mullen, et al., 2013; Nishida, et al., 2016), the origin of the tumors, diagnosed as hepatocellular, can be questionable. Here we excluded the ovarian cells origin because 5 out of 6 patients with CTNNB1 mutations are males (information about 1 patient is not available).

However, several CTNNB1 mutations are specific for tumor samples and were detected in 15% of HCC. This observation can indicate that CTNNB1 mutation is not a necessary but sufficient factor for the CCM changes, and, when present, affects metabolism in a drastic way, which has an impact on major liver functions. Thus, the above mentioned CTNNB1 mutations should further be validated on larger cohorts, for potential use as a prognostic biomarker of survival or metastatic status.

Overall, CTNNB1 mutations present in current cohort correlated with more drastic changes in CCM, which were be detected by proteome analysis. CTNNB1 mutations should further be validated on larger cohorts for potential use as a prognostic biomarker of survival or metastatic status. Additionally, the mechanistic studies of the influence of CTNNB1 mutations on the changes of malignancy potential and invasiveness can be performed on liver and HCC cell lines.

109 TP53

The TP53 (c.216delC, p.V73fs) mutation was frequently met in the full cohort. This specific mutation is described by Mouradov et al. in the KM12 colon cancer cell line (Mouradov, et al., 2014). This paper also present the TP53 (c.902delC, p.P301fs), which was met in the cohort twice, in another colon cancer cell line – HCA-7. In addition, TP53 (c902delC, P301fs) was confirmed to be present in a colon cancer patient (Giannakis, et al., 2014).

PIK3CA

Janku et al. reported PIK3CA (c.A3140G, p.H1047R) mutation to be found in colorectal cancer patients (Janku, et al., 2012). Reiner et al. studied the cohort of 50 patient with HCC and found PIK3CA (c.A3140G, p.H1047R) mutation if 2% of them. Their results suggest that somatic PIK3CA mutations play role in the frequent activation of the PI3K/AKT pathway in carcinomas of the biliary tract and liver (Riener, Bawohl, Clavien, & Jochum, 2008).

TP53, CTNNB1, and PIK3CA mutations are known in the literature to be associated with HCC in HBV and HCV contaminated patients (Tornesello, et al., 2013). However, in our cohort these mutations are present not exclusively in patients with HBV or HCV.

The other mutations represented in Table 2 show no solid evidence in the literature to be involved in HCC development or functioning processes. The mutations were detected in the samples with fatty liver, hepatitis B and C, fibrotic liver, cirrhosis, and HCC. However, no specific mutations, apart from CTNNB1, were confirmed to be present only in HCC. No clear distinction between cancer and precancerous conditions could be achieved exclusively by the mutation analysis.

Overall my analysis unravels that the mutations in HCC are more wide spread than expected.