CTNNB1 polymorphism (rs121913407) in circulating tumor DNA (ctDNA) in Egyptian hepatocellular carcinoma patients

Abstract

Background

Hepatocellular carcinoma (HCC) represents the sixth most common cancer worldwide and the fourth in Egypt. Persistent inflammation and specific somatic mutations in driving genes play a major role in the development of HCC. One of these somatic mutations is CTNNB1 mutations with subsequent activation of β-catenin in HCC, associated with a risk of malignant transformation. In this study, we investigate the clinical utility of peripheral blood circulating tumor DNA (ctDNA) CTNNB1 (rs121913407) in HCC patients compared to pathological chronic hepatitis C virus (HCV) patients and healthy controls.

Methods

Our study is a case-control study at the Ain Shams Centre for Organ Transplantation, Ain Shams University Hospitals, enrolling twenty-eight adult HCC patients (twelve early HCC patients and sixteen advanced HCC patients), ten patients with chronic hepatitis C as a disease control group, and ten healthy controls. We collected plasma and stored at −80 °C. We detected mutations in the gene locus CTNNB1 rs121913407 by real-time PCR.

Results

All of our studied cases (early and advanced HCC) in addition to HCV and healthy control groups were CTNNB1 wild (TT) genotype. There was statistical significant difference between early and late cases of HCC as regards AFP and AST.

Conclusions

None of our recruited subjects showed CTNNB1 rs121913407 gene mutation. Further studies on larger number of patients are needed to clarify and confirm the clinical utility of CTNNB1 single-nucleotide polymorphism in the pathogenesis of HCC related to HCV in Egyptian population.

Background

Hepatocellular carcinoma is a primary tumor of the liver, which arises from liver cells and constitutes about 90% of all primary liver cancer types that usually develops in the setting of chronic liver disease, particularly in patients with chronic hepatitis B and C. HCC has a rising incidence in Egypt mostly due to high prevalence of viral hepatitis and its complications [1, 2].

Over the past decade, advances in genomic research have increased our knowledge of HCC molecular pathogenesis. However, the exact molecular mechanisms underlying the development of HCC are still unclear [3].

According to the current European Association for Study of the Liver (EASL) guidelines, one of the unmet needs in HCC research is to develop new tools for early detection including the assessment of liquid biopsy [4]. In addition, without a liver biopsy, assessment of the genomic profile becomes a challenge. This can be addressed by a noninvasive liquid biopsy which provides actionable genomic information without the risk of complications. Recently, circulating tumor DNA (ctDNA) has attracted extensive attention as a promising component of liquid biopsy. Circulating tumor DNAs are mutant DNAs released into the circulation by tumor cells and can be assessed through analysis of plasma from a blood sample of HCC patient. A liquid biopsy, represented in a blood sample, can be used to assess ctDNA in a quest to comprehensively profile the tumor genome better than conventional sampling methods. This qualifies it as a better vehicle to provide information about abnormalities in genes for guiding targeted therapy, unveiling drug resistance, and monitoring treatment response [5, 6].

Recent studies have identified specific somatic mutations in driving genes that appear to contribute to tumor initiation and progression. One of these somatic mutations is CTNNB1 mutations with subsequent activation of β-catenin in HCC, associated with a risk of malignant transformation. Notably, hepatitis B and C infections have different impacts on other driver genes frequently mutated in HCC and belong to key signaling pathways of oncogenesis as the WNT/β-catenin pathway and the P53 cell cycle pathway [3].

According to ClinGen Allele Registry, the variant CTNNB1 rs121913407:c.133T>C (p.Ser45Pro) lies within coding transcript of the gene CTNNB1, with substitution of cytosine for thymine at nucleotide position 133. This is a missense mutation causing the associated protein reference sequence amino acid number 45 to be changed from serine to proline. CTNNB1 maps to the short arm of chromosome 3 (3p22.1). The variant is present on chromosome 3 nucleotide number 41224645 (chr3: g.41224645T>C on assembly GRCh38). This SNP NM_001904.4 (CTNNB1): c.133T>C (p.Ser45Pro) is included in ClinVar and recorded on ensembl as rs121913407 SNP, with details about different synonyms for the same variant on OMIM, ClinGen, UniProtKB, and dbSNP. It is included in ClinVar as a somatic pathogenic variant in HCC and as a missense mutation that is pathogenic or likely pathogenic [7, 8].

The aim of this work was to study the clinical utility of peripheral blood ctDNA CTNNB1 rs121913407 c.133T>C in HCC patients compared to pathological chronic hepatitis C virus (HCV) patients and healthy controls.

Methods

Sample size

This is an exploratory study which was approved by the Faculty of Medicine Ain Shams University Research Ethics Committee (FMASU R92/2020).

Patient selection

In our study, we included Egyptian patients with HCV-related HCC attending the HCC clinic and Ain Shams Centre for Organ Transplantation (ASCOT), Ain Shams University Hospitals, between October 2020 and April 2021. The study included twenty-eight adult HCC patients, ten patients with chronic hepatitis C as a disease control group, and ten healthy controls.

Diagnosis of the studied cases

Based on EASL guidelines 2018, we diagnosed cirrhotic patients depending on noninvasive criteria and/or pathology. We based diagnosis on the identification of the typical hallmarks of HCC, which is the combination of hypervascularity in late arterial phase (defined as arterial phase hyperenhancement [APHE] according to LI-RADS [Liver Imaging Reporting and Data System] classification and washout on portal venous and/or delayed phases, which reflects the vascular derangement occurring during hepatocarcinogenesis [9]. We determined clinical staging of HCC according to Barcelona Clinic Liver Cancer (BCLC) staging classification and Child-Turcotte-Pugh staging (CTP) [10, 11].

We divided patients into two groups according to eligibility for liver transplantation into two groups: early HCC (EHCC) and advanced HCC patients (AHCC). Group 1: early HCC (EHCC) patients (n = 12) who fulfilled either Milan criteria, single lesion ≤ 5 cm or up to 3 lesions ≤ 3 cm each in the absence of tumor vascular invasion or evidence of extrahepatic metastases [12], or University of California, San Francisco (UCSF) criteria, which considered a single lesion ≤ 6.5 cm or 2–3 lesions ≤ 4.5 cm each, with total tumor diameter ≤ 8 cm are accepted [13]. Additional inclusion criteria were alpha-fetoprotein < 200 ng/mL and the absence of macrovascular invasion or distant metastatic spread as documented by triphasic pelviabdominal CT, CT chest, bone scan, and/or PET scan if needed [12, 13]. Group 2: advanced HCC patients (AHCC) (n = 16) and fulfilling HCC are beyond the abovementioned criteria (Milan or UCSF) and/or BCLC stage C, the presence of macrovascular invasion by duplex ultrasound, and/or triphasic CT. The presence of metastasis including lymph node invasion as documented by triphasic pelvi-abdominal CT, CT chest, bone scan, and/or PET scan if needed and alpha-fetoprotein > 1000 ng/mL are additional criteria for inclusion in this group.

Exclusion criteria were patients having other degenerative conditions affecting cfDNA concentrations like autoimmune diseases or other malignancies, non-HCV-related HCC, cystic liver focal lesions (hepatic abscesses, hydatid cysts), metastatic liver focal lesions (cancer colon, cancer breast), and refusal to sign informed consent.

All subjects included in this study were subjected to the following: full history taking and thorough clinical examination and radiological workup including the following: abdominal ultrasound/duplex and spiral triphasic abdominal CT and/or MRI. In addition, we performed laboratory investigations, including complete blood count, international normalized ratio (INR), alpha-fetoprotein (AFP), renal function tests (BUN, creatinine), liver function tests including aspartate aminotransferase (AST), alanine aminotransferase (ALT), albumin, and total bilirubin.

For the purpose of genotyping, we collected 10 mL of blood on EDTA vacutainer tubes for ctDNA extraction. Circulating tumor DNA (ctDNA) was extracted from the plasma of at least 10 mL peripheral venous blood samples in EDTA. Blood samples were processed within 2 h after venipuncture by a two-step centrifugation method: the first spin at 1600 g for 10 min to remove the majority of blood cells and a second spin at 16,000 g for another 10 min to remove the cellular debris. The plasma was subpackaged in RNase DNase-free tubes and stored at −80 °C until use. The ctDNA was extracted using the GeneJET Whole Blood Genomic DNA Purification Mini Kit according to the manufacturer’s instructions (Thermo Fisher Scientific, USA).

We assessed mutation in the gene locus CTNNB1 c.133T>C (p.Ser45Pro) by real-time PCR (RT-PCR). The sequence information of the primers and probes primers and probes is illustrated in Table 1. Real-time PCR reactions were done in a final volume of 20 μL, using 20 ng of extracted DNA. We used TaqMan Genotyping PCR Master Mix and SNP Genotyping Assay (Thermo Fisher Scientific, USA). We used volumes for PCR reaction mix according to manufacturer’s instructions (Table 2). Cycling conditions were as follows: initial activation at 95 °C for 10 min and then denaturation; 40 cycles of 95 °C for 15 s, annealing/extension; 57 °C for 15 s, and 72 °C for 1 min in a DT-Lite real-time PCR system (DNA technology, Russia).

Table 1 Sequence information of the primers and probes for the RT-PCR assay

Table 2 PCR reaction mix in each sample

Statistical analysis

For this purpose, we used a GraphPad Prism statistical software version (5.01). We expressed the values for the biochemical markers as mean and standard deviation in the case of parametric data and as median and interquartile ranges (IQR) in case of skewed data, while we summarized categorical variables using frequency measures. For comparative analysis, we used Wilcoxon’s rank-sum test (Mann-Whitney U), chi-square test, and Kruskal-Wallis test. In all statistical analyses, p < 0.05 was considered significant.

Results

The demographic characteristics of all studied subjects and statistical comparison between the various studied parameters are included in Table 3. Regarding Child-Turcotte-Pugh (CTP), 39.3% of HCC patients were stage A, 39.3% stage B, and 21.4% of them at stage C. Also, regarding BCLC staging, 7.1% of HCC patients were at stage 0, 25% stage A, 21.4% stage B, 25% stage C, and 21.4% of them at stage D as demonstrated in Table 3.

Table 3 Tumor-related characteristics in HCC group (n = 28)

The descriptive and comparative statistics of the demographic data and various studied parameters in healthy control, HCV, and HCC patients are shown in Table 4. Serum levels of AST, ALT, INR, and total bilirubin were highly significantly increased in the HCC group as compared to the healthy control group and HCV group (p < 0.05), while serum albumin was significantly decreased in the HCC group as compared to both healthy control group and HCV (p < 0.05).

Table 4 Demographic characteristics of all studied subjects and statistical comparison between the various studied parameters in HCC patients versus healthy ccontrol and HCV patients^ab

The descriptive and comparative statistics of demographic data and various studied parameters in the EHCC patients and the AHCC patients are shown in Table 5. Serum levels of AFP were highly significantly increased in the AHCC group as compared to the EHCC group (p < 0.01). Also, serum AST, INR, BUN, and creatinine were statistically significantly increased in the AHCC group as compared to the EHCC group (p < 0.05, respectively). On the contrary, serum albumin was significantly decreased in AHCC group (p < 0.05). Regarding CTNNB1 c.133T>C genotyping, all of our studied cases (early and advanced HCC) in addition to HCV and healthy control groups were CTNNB1 wild (TT) genotype (Table 6). We demonstrate a representative of results of the cycle threshold (Ct) for a run on 5 EHCC, and 6 AHCC samples in the study in Table 7, and the fluorescence curves showing the Ct in Fig. 1.

Table 5 Demographic characteristics of all studied subjects and statistical comparison between EHCC and AHCC^a using Wilcoxon’s rank-sum test and between all the studied groups using Kruskal-Wallis test^b

Table 6 CTNNB1 c.133T>C genotyping in HCC, HCV, and healthy control groups

Table 7 Cycle thresholds for EHCC and AHCC patients for wild genotype (VIC probe) on DT-Lite PCR system

Fig. 1

Showing the fluorescence curves for the studied samples on DT-Lite PCR system graphical display

Discussion

Hepatocellular carcinoma frequently arises in the context of chronic cellular injury with consequent DNA damage and genetic alterations [14].

The fundamental pathogenic event in the development of HCC is genetic mutation resulting in aberrant activation of signal transduction Wnt/β-catenin pathway, which plays a critical role in initiating and sustaining hepatic carcinogenesis [15, 16].

β-catenin, encoded by CTNNB1 gene, is the main effector signaling molecule in the canonical Wnt pathway [17]. In malignant hepatocytes, β-catenin loses its physiological function as a cell-adhesion molecule accumulates resulting in cellular proliferation and metastasis [18]. CTNNB1 mutations were identified in about 20–40% of liver cancers [19].

A meta-analysis including 2334 liver cancer cases from twenty-two studies showed that accumulation of β-catenin in the cytoplasm and/or nucleus significantly correlated with poor prognosis [18]. Hence, we examined the polymorphism of CTNNB1 c.133T>C in early and advanced HCC patients compared to pathological chronic HCV patients and healthy controls.

All of our studied cases (early and advanced HCC) in addition to HCV and healthy control groups were CTNNB1 wild (TT) genotype. None of our recruited HCC cases showed CTNNB1 gene mutation. The reason for these negative results may be due to the fact that all cases analyzed in our study were cirrhotic HCC patients, and CTNNB1 mutations were described to be particularly prevalent in non-cirrhotic HCC patients [20].

Our results are in agreement with Lombardo et al. [3] who revealed the absence of CTNNB1 mutation in exon 3 of all frozen tumor liver specimens from 67 HCC Italian patients using Sanger sequencing. Forty (59.7%) of the 67 patients with HCC had HCV. They mentioned that although somatic mutations are expected to be found in CTNNB1 gene which is considered a driver gene for HCC development, these mutations show variable frequencies in different geographic areas, possibly depending on liver disease etiology and environmental factors. They added that in the absence of CTNNB1 somatic mutations, possibly in these patients, activation of Wnt/β-catenin could be induced independently as in previous studies on adrenal aldosterone-producing adenomas [3].

Our results are in disagreement with Tornesello et al. [21] who found that mutations in exon 3 of CTNNB1 gene occurred in HCV-related HCCs (17.5%) from Italians in Naples. Tornesello et al. [21] performed the analysis of CTNNB1 exon 3 using the direct sequencing analysis. A similar lower mutation frequency was previously observed among French HCC cases using whole-exome DNA sequencing [22]. This discrepancy between our results and earlier studies may be due to other researchers using a more sensitive next-generation sequencing technique by the researchers on a different sample represented in HCC tissue from a dissimilar population of European patients. Moreover, it is well known that genetic origin and heterogeneity affect the mutation rates. Previous study has indicated that mutation pattern and frequencies could be variable according to heterogeneity in host genetic, etiology, and geographical regions (23). This is the first study on Egyptian patients for SNPs in gene locus CTNNB1 c.133T>C in a liquid biopsy, a different population from those done in previous studies [23].

Another relevant observation by Tornesello et al. [21] is the presence of CTNNB1 gene mutations in HCC patients in a comparable frequency in Asia and Europe. They observed that these HCC patients with mutated gene were significantly younger relative to those with wild type. In North Africa, only 4 out of 42 HCC patients revealed CTNNB1 mutations. The mean age in our studied patients was 60 years old which could contribute in addition to the different geographic regions of our population to the negative results.

In discordance with our results, Lee et al. [24] reported the presence of CTNNB1 mutations in HCC patients which were not associated with any clinicopathologic factors. Lee et al. [24] performed the analysis of CTNNB1 mutations using PCR amplification and Sanger sequencing analysis. Moreover, CTNNB1 mutations were present in 13.2% (10/76) of HCC related to HBV and in 14.3% (1/7) HCC related to HCV. The small number of cases in our study, although greater than HCC-HCV patients in Lee et al. (2016), in addition to their use of a more sensitive NGS technology on a tissue sample, may explain the absence of concordance between our results [24].

As a justification to our results which failed to identify any mutation, Park et al. [25] described that CTNNB1 mutations were not found in precursor lesions of HCC and were not uniformly present in all tumors, indicating that these mutations are late events in hepatocarcinogenesis. Furthermore, intratumoral genetic heterogeneity is a practical challenge, and it is known that HCC shows morphological and immunophenotypical heterogeneity, which most probably presents different genetic alterations among HCC patients [26].

The limitation of this study is its nature as a small pilot study without previous genetic information on the frequency of CTNNB1 mutation in the Egyptian population. Such a situation makes it mandatory to enroll a larger number of patients to explore the frequency and the role of such mutation in HCC.

Conclusion

Our study fails to prove evidence for the clinical utility of CTNNB1 rs121913407 c.133T>C (p.Ser45Pro) in Egyptian HCC patients. We attribute our observed negative results to the small number of cases in our study and the need for the study of more patients to reach a definitive conclusion. Furthermore, we need to support our speculations by sequencing either ctDNA from blood samples or DNA from tissue samples from the same patients to confirm the absence of mutations or their presence with low frequencies and using spatial sequencing which studies single cells to reveal cancer genome obtained from separate multiple cells in HCC tumor tissue.