The concordance between plasma DNA and tissue DNA.
Abstract
Hepatocellular carcinoma (HCC) is a major health problem worldwide. The DNA PM of cancer-related genes plays an important role in the development and progression of HCC. The data reported in our studies provide evidence that PM of p73, p14, and O6-MGMT is associated with HCC, whereas PM of the APC gene is more common in chronic hepatitis (CH) cases. Thus, it could be used as a maker for early detection of HCV-induced chronic active hepatitis. A panel of four genes APC, p73, p14, and O6-MGMT independently affected the classification of cases into HCC and CH with accuracy (89.9%), sensitivity (83.9%), and specificity (94.7%). Also, the detection of PM of APC, FHIT, p15, p16, and E-cadherin in peripheral blood of HCV-infected patients is a highly sensitive and specific. Therefore, blood could be used as efficiently as tissue biopsies to assess PM of different genes. This could help in the follow-up of CH patients and early detection of HCC. We did not observe a significant difference in the methylation status according to the virus type HBV versus HCV. So, plasma DNA is a reliable resource for methylation studies in the future, irrespective of the type of hepatitis infection.
Keywords
- hepatitis C virus-genotype 4
- chronic hepatitis
- hepatocellular carcinoma
- promoter methylation
1. Introduction
Hepatocellular carcinoma (HCC) is a major health problem and it is the third most common cause of cancer-related death worldwide [1]. In Egypt HCC ranks the first in males and the second in females after breast cancer. It accounted for 33.6% in males and 13.5% in females [2]. This high incidence of HCC is attributed to the high prevalence of HCV infection, especially genotype 4 in Egypt [3]. HCV infection has an estimated global prevalence of 2.5%, causing chronic liver disease in about 170 million people worldwide [4]. Although it has been estimated that 80% of HCC occurs in cirrhotic livers, the underlying molecular mechanisms of virus associated hepatocarcinogenesis are still unclear [5]. It has been suggested that HCV-encoded proteins may contribute to tumor progression through their direct and indirect interactions with host hepatic cells. Additionally, the generated status of chronic HCV inflammation is accompanied by immune-mediated destruction of infected hepatocytes, oxidative stress, virus-induced apoptosis and DNA damage leading to genomic instability and continuous regeneration that may be incorporated in liver cancer development [6].
Previous studies demonstrated that DNA methylation has a major role in the initiation and progression of various types of human cancers [7, 8]. Aberrant promoter methylation of tumor suppressor genes (TSGs), such as P14 or O6-methylguanine-DNA methyltransferase (O6MGMT) has been reported in relation to HCC development [9].
The term DNA methylation refers to the addition of a methyl group to the cytosine residue in the CpG islands. Normally, CpG islands are not methylated regardless of their transcriptional status, and methylation of the promoter regions of tumor suppressor genes (TSGs) or growth regulatory genes resulted in silencing of those genes, and cancer development. Since it was proven that different types of cancer showed distinct DNA methylation profiles, thus it could be possible to develop specific methylation signatures for those types of cancer [10].
The power of PM as a molecular marker is the ability of detecting its presence in a variety of sample types including fresh specimens, body fluids and archival paraffin-embedded tissues, as well as to the defined localization of the lesion in the CpG islands of the genes. Promoter methylation could be an important early event in the cascade of carcinogenesis and it can also be of important as prognostic and predictive marker [11]. The DNA methylation profiles in HCV-infected patients from Egypt have not been well studied yet, although it has the highest prevalence of HCV infection worldwide with approximately 14% of the population infected [12].
2. Concordance between tissue and plasma DNA methylation in HCC patients
Owing to the crucial effects of DNA promotor methylation in the development and progression of HCC, we investigated the role of DNA methylation events in the tissues of HCC patients for using five tumor suppressor genes: APC, FHIT, p15, p16, and E-cadherin. We also assessed the DNA methylation patterns of these genes in the plasma from the same patients and compared the tissue and plasma patterns [13]. This was done to investigate the concordance between tissue and plasma methylation patterns in Egyptian patients with HCV and/or HBV- associated HCC. Although liver biopsy is the current gold standard for detecting methylation events, imaging techniques are usually sufficient for liver cancer diagnosis and therefore the need of tissue biopsy decreased markedly [14]. So it was essential to search for another tool for detection of promotor methylation in HCC by a simpler, easy and reliable technique.
We collected paired blood and tissue samples from 28 HCV and/or HBV- associated HCC patients from Egypt. DNA was extracted from those patients (tissue and blood) and the promotor methylation for
We reported a statistically significant concordance between plasma and tissue methylation profiles [13]. The frequency of promoter methylation in tissue and plasma samples for the five tumor suppressor genes was as follows:
Although detection of promoter methylation in the plasma DNA was highly specific, it was not as sensitive for the matching change in tissue DNA, suggesting that DNA promotor methylation in tissues might originate in tumor cells before appearing in the vascular spaces (blood or plasma). The positive predictive value (PPV) was higher than the negative predictive value (NPV) for
3. Methylation profile and viral status
Another interesting finding observed is that, there was no significant correlation between HBV or HCV infection and the incidence of promoter methylation, to suggest whether the viral status could be used to predict methylation and subsequent gene silencing for the five aforementioned tumor suppressor genes [13]. Therefore, plasma DNA could be used as a reliable source for methylation detection in HCC patients irrespective of the type of hepatitis viral infection (Table 2).
HBV | HCV | HBV infection type* | |
---|---|---|---|
APC | 0.107 | 0.634 | 0.508 |
FHIT | 0.545 | 1 | 0.508 |
p15 | 0.481 | 1 | 0.288 |
p16 | 0.295 | 0.639 | 1 |
E-cadherin | 0.273 | 0.629 | 0.66 |
4. Increasing DNA promoter methylation is associated with disease progression from chronic hepatitis C to cirrhosis and hepatocellular carcinoma
As a continuation of our previous studies, which showed a concordance between tissue and plasma DNA methylation, and hence the validity of using plasma DNA methylation profile as a marker for HCC [13], we had assessed the methylation frequency of three tumor suppressor genes (
We found significant differences in the frequency of PM of all studied genes within the different stages of chronic liver disease and HCC (Table 3 and Figure 2). The methylation frequency of
Gene | HCC | HCV with liver cirrhosis | Chronic Hepatitis C | Control | p value |
---|---|---|---|---|---|
n = 208(%) | n = 108(%) | n = 100(%) | n = 100(%) | ||
P14 | M 100 (48.1) | 52 (48.1) | 16 (16) | 8 (8) | 0.008 |
U 108 (51.9) | 56 (51.9) | 84 (84) | 92 (92) | ||
P15 | M 92 (44.2) | 36 (33.3) | 20 (20) | 4 (4) | 0.006 |
U 116 (55.8) | 72 (66.7) | 80 (80) | 96 (96) | ||
O6MGMT | M 84 (40.4) | 60 (55.6) | 20 (20) | 4 (4) | <0.001 |
U 124 (59.6) | 48 (44.4) | 80 (80) | 96 (96) | ||
P73 | M 136 (65.4) | 72 (66.7) | 32 (32) | 4 (4) | <0.001 |
U 72 (34.6) | 36 (33.3) | 68 (68) | 96 (96) |
As for
The methylation frequency of
The methylation frequency of
HCC | Cirrhosis | Chronic hepatitis C | Control | |
---|---|---|---|---|
P14 | HCC | 0.954a | 0.035b | 0.004c |
Cirrhosis | 0.050d | 0.004e | ||
Chronic C | 0.546f | |||
Control | ||||
P15 | HCC | 0.409a | 0.090b | <0.001c |
Cirrhosis | 0.554d | 0.024e | ||
Chronic C | 0.223f | |||
Control | ||||
MGMT | HCC | 0.328a | 0.016b | 0.003c |
Cirrhosis | 0.002d | <0.001e | ||
Chronic C | 0.189f | |||
Control | ||||
TP73 | HCC | 0.858a | 0.037b | <0.001c |
Cirrhosis | 0.058d | <0.001e | ||
Chronic C | 0.026f | |||
Control |
Thus, it could be concluded that, the methylation frequency increases with the progression of liver disease and thus it that could be used to monitor whether a patient with chronic hepatitis C is likely to progress to liver cirrhosis or even HCC or not. Moreover, the process of PM does not represent an early event in hepatocarcinogenesis cascade but it increases and continues with disease progression to cancer.
Based on our data regarding the high methylation frequency of
5. Analysis of DNA methylation events of the 11 tested genes among the studied groups
A high methylation frequency was reported for all studied genes (except for p15) in the PBL and tissues with increasing methylation index as the disease progresses (Figure 3). The PM of the 11 tested genes assessed in 13 NHT samples showed no methylation events in
Genes | Normal liver N = 13 (%) | Chronic hepatitis (CH) | Hepatocellular carcinoma HCC | p-Value* | |||
---|---|---|---|---|---|---|---|
(Tissue) (38) (%) | (PBL) (20) (%) | (HCC) (31) (%) | (ANT) (31) (%) | (CH and HCC) | (CH and ANT) | ||
APC | 4 (30.8) | 33 (86.8) | 16 (80) | 13 (41.9) | 14 (45.2) | <0.001 | <0.001 |
FHIT | 2 (15.4) | 20 (52.6) | 6 (30) | 21 (67.7) | 20 (64.5) | 0.204 | 0.005 |
P15 | 0 (0) | 0 (0) | 0 (0) | 5 (16.1) | 5 (16.1) | 0.010 | # |
P73 | 0 (0) | 8 (21.1) | 1 (5.0) | 26 (83.9) | 23 (74.2) | <0.001 | <0.001 |
P14 | 6 (46.2) | 17 (44.7) | 10 (50) | 28 (90.3) | 28 (90.3) | <0.001 | <0.001 |
P16 | 3 (23.1) | 15 (39.5) | 9 (45) | 14 (45.2) | 19 (61.3) | 0.634 | 0.390 |
DAPK | 3 (23.1) | 22 (57.9) | 12 (60) | 21 (67.7) | 22 (71) | 0.401 | 0.023 |
RARb | 0 (0) | 0 (00) | 0 (0) | 5 (16.1) | 3 (9.7) | 0.015 | # |
RASSF | 0 (0) | 26 (68.4) | 20 (100) | 31 (100) | 31 (100) | 0.001 | <0.001 |
O6O6-MGMT | 0 (0) | 10 (26.3) | 10 (50.0) | 21 (67.7) | 20 (64.5) | <0.001 | <0.001 |
CDH1 | 3 (23.1) | 7 (18.4) | 8 (40.0) | 17 (54.8) | 14 (45.2) | 0.002 | 0.004 |
Our data regarding the
6. Genes methylation could be used as a biomarker for diagnosis of HCC and CH
The Coordination of methylation at the 11 tested genes was analyzed in our study using the Mann–Whitney U test through comparing the status of each gene (M or U) with the MI calculated with the remaining genes (Table 6). The combined effect of the studied methylated genes as biomarkers for diagnosis of HCC and CH has been determined (assessed) using the stepwise logistic regression, and accordingly only
Parameter | Regression estimate | P value | Odds ratio | 95% CI | for OR |
---|---|---|---|---|---|
APC | −3.606 | 0.003 | 0.027 | 0.003 | 0.287 |
p73 | 3.671 | 0.001 | 39.302 | 4.752 | 325.017 |
P14 | 3.638 | 0.009 | 38.014 | 2.492 | 579.829 |
O6-MGMT | 2.589 | 0.014 | 13.311 | 1.685 | 105.132 |
Within the identified genes panel which independently affected the classification of cases into HCC and CH in this study, p14 only showed a high MF in HCC cases. Our data in this context confirmed those of Anzola et al. [30] and Yang et al. [20] who reported that
Similar to
A significant difference in the MFs of
7. Concordance between PM and protein expression of APC and O6MGMT
We assessed the protein expression of
8. Conclusion
We conclude that DNA PM of multiple cancer-related genes plays an important role in the development and progression of HCC and therefore, it could be detected in different stages of disease progression from hepatitis to HCC. The data reported in our study provide evidence that PM of
Moreover, a panel of four genes
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