Primers for the detection of single nucleotide polymorphisms (SNPs) of genes IL-18 (-607 C/A and -137 C/G), IFN-γ (+874 A/T) and TNF-α (-238 A/G and -308 A/G).
Hepatocellular carcinoma (HCC) is a primary malignant tumor of the liver which represents a serious public health problem in the world, corresponding to the fifth more frequent malignant neoplasia among men, the eighth among women, and the third cause of cancer death in the world [1,2]. Seventy to ninety percent of HCC cases occur in patients with cirrhosis or with chronic liver disease, with cell injury followed by regeneration mediated by the immune response playing an important role in hepatocarcinogenesis .
In Brazil, a national survey to update HCC epidemiology and clinical profile of patients with HCC (29 centers, with 1,405 patients diagnosed with HCC from 2004 to 2009) showed that the median age at diagnosis was 59 years (1–92 years old; 78% male) and 98% of the patients had cirrhosis (1279/1308), with the hepatitis C virus being the main etiology (54%), followed by hepatitis B virus (16%) and alcohol (14%). In Southeastern and Southern Brazil, hepatitis C virus accounted for over 55% of cases. In the Northeast and North, hepatitis C virus accounted for less than 50%, and hepatitis B virus accounted for 22–25% of cases . In Ribeirão Preto, Southeastern Brazil, the clinical characteristics of 130 patients with HCC attended at the University Hospital of the Faculty of Medicine of Ribeirao Preto, University of São Paulo (HCFMRP-USP) was revised. The mean (± SD) age at the time of HCC diagnosis was 55.6±11.2 years, with 81.5% of them being males. Cirrhosis was present in 89.2% of cases, with 53.4% of the patients being ChildPugh A; chronic hepatitis B or C without cirrhosis was detected in 3.2%, nonalcoholic steatohepatitis (NASH) in 3.8%, and a normal liver in 3.8% .
The human major histocompatibility complex (MHC) represents a set of genes responsible for coding histocompatibility molecules. It is a high density region [3.6 Mb DNA) located on the short arm of chromosome 6, region 6p21.3, which contains more than 200 genes grouped into three classes denoted class I, II and III. Class I genes (classic or class Ia) code for the classic histocompatibility molecules HLA-A, B and C; class II genes code for the histocompatibility molecules HLA-DR, DQ and DP, and class III genes, although included in the MHC, do not code for histocompatibility molecules. Among class I genes, there are also those denoted non-classic or class Ib, which code for the non-classic histocompatibility molecules HLA-E, F and G .
Thus far, 47 alleles have been attributed to the
The expression of
In general, the inflammatory response mediated by the immune system is beneficial to the host; however, when tissue homeostasis is chronically affected the interactions between innate and adaptive immune responses may be deregulated, culminating with chronic inflammation, excessive tissue remodeling, loss of tissue architecture, apoptosis/necrosis and oxidative stress which, under certain circumstances, may increase the risk of tumor development . The cytokines are responsible for the regulation of growth differentiation and activation of immune cells. The ability to produce cytokines by an individual is influenced by genetic components that have been attributed to molecular mechanisms, including variations in the transcription, translation and secretion pathways .
2. Problem statement
Over the last years, regarding HCC, few studies evaluating the role of alleles and genotypes of
Immune response and carcinogenesis
Alleles and genotypes of cytokines polymorphisms and 14bp of HLA-G gene in HCC patients.
3. Method and patients
We evaluated, in a Brazilian cohort, the association of alleles and genotypes of the 14bp insertion/deletion polymorphism of the
This was a retrospective cross-sectional study conducted on 109 patients (89 men) with mean age 55.8±11.4 years for the 14bp insertion/deletion polymorphism of the
The evaluation of the severity of HCC was based on: a) tumor presentation (uninodular, multinodular or diffuse infiltrative); b) nodule size (<5 cm, 5-10 cm or >10 cm); c) Milan criteria (fulfills or does not fulfill); d) metastasis (present or absent); e) histological classification according to Edmondson & Steiner, 1954 (grades I, II, III or IV) .
A total of 202 healthy individuals (56 females and 146 males) with a mean age (±SD) of 33.3±8.3 years and from the same geographic region as the patients studied were used as controls for the evaluation of the frequency of 14bp,
For the determination of the 14bp insertion/deletion, IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) genotypes, we first extracted genomic DNA from peripheral leukocytes by the salting out technique . The HLA-G 14bp insertion/deletion genotypes at exon 8 of the HLA-G locus was analyzed as described: 200ng of genomic DNA were amplified in a 25mL reaction mixture containing 0.20mM dNTP (Invitrogen, Carlsbad, CA), 0.2mM of each primer, 0.5U Taq DNA polymerase (Invitrogen, Carlsbad, CA), 1.5mM MgCl2 and a 1x PCR buffer (0.2M Tris–HCl, pH 8.5; 0.5M KCl). After an initial denaturation step at 94°C for 5 minutes, samples were submitted to 30 additional cycles at 94°C for 45 seconds, 56°C for 45 seconds and 72°C for 1 minute, with a final extension cycle at 72°C for 7 minutes with 5´-TGTGAAACAGCTGCCCTGTG-3´ as the forward primer and 5´-AAGGAATGCAGTTCAGCATGA-3´ as the reverse primer [26,27]. After DNA amplification by PCR, the reaction products were submitted to 10% polyacrylamide gel electrophoresis under non-denaturing conditions followed by silver impregnation. The presence of 345 bp fragments corresponded to the deletion allele, while the 359 bp fragment corresponded to the 14bp insertion allele.
|TNF 238 UP||AGGCAATAGGTTTTGAGGGCCAT|
The alleles and genotypes of
For statistical analysis, the allele and genotype frequencies were calculated by the direct count method in all groups. Adherence of genotypic proportions to Hardy-Weinberg expectations was determined by the exact test of Guo and Thompson  using the GENEPOP software v. 4.0.10. The presence of a significant association between polymorphisms of the same gene was evaluated by a likelihood ratio test of probability of linkage disequilibrium using the ARLEQUIN software, v. 3.1 . If a positive association was detected, but the gameteic phase was unknown, the PHASE (v. 2 package)  and EM algorithms  were used to reconstruct the TNF or IL haplotypes. Allele, genotype and haplotype frequencies were compared by the two-tailed Fisher exact test using the GraphPad InStat 3.06 software, which was also used to estimate the odds ratio (OR) and its 95% confidence interval (95%CI). The level of significance was set at P<0.05.
Cirrhosis was observed in 89% of the patients, and major underlying causes included: hepatitis C (35%), alcohol plus hepatitis C (25%), alcohol (18%), hepatitis B (9%), alcohol plus hepatitis B (4%), alcohol plus hepatitis C plus hepatitis B (1%), hereditary hemochromatosis (1%), non-alcoholic steatohepatitis (1%), autoimmune hepatitis (1%) and cryptogenic cirrhosis (5%). Four percent of the patients had no underlying liver disease, 4% non-alcoholic steatohepatitis and 3% chronic hepatitis without cirrhosis (two hepatitis B and one hepatitis C). Fifty-four percent (59/109) of the patients met the Milan criteria for liver transplantation . Metastasis search was performed in 88% of cases (17% had metastasis and 93% did not). Tumors <5cm, 5-10cm and >10 cm were found in 57%, 18% and 8% respectively. Diffuse infiltrative HCC totaled 15% of the cases. Histological evaluation of HCC was performed in 39% (42/109) of subjects with 62% presenting Edmondson-Steiner I or II and 38% III or IV.
4.1. 14bp insertion/deletion polymorphism
Genotype frequencies of patient and control groups were in accordance to Hardy-Weinberg Equilibrium. The 14bp*D allele was more frequent in cases of HCC than in controls (0.6514 vs. 0.5619;
|Control||177 [0.4381]||227 [0.5619]||45 [0.2228]||87 [0.4307]||70 [0.3465]|
|HCC||76 [0.3486]||142 [0.6514]||16 [0.1468]||44 [0.4037]||49 [0.4495]|
|Diffuse||7 [0.2500]||21 [0.7500]||1 [0.0714]||5 [0.3571]||8 [0.5714]|
|Multinodular||14 [0.3684]||24 [0.6316]||13 [0.1579]||9 [0.4211]||6 [0.4211]|
|Uninodular||55 [0.3667]||95 [0.6333]||12 [0.1600]||31 [0.4133]||32 [0.4767]|
|>10cm||6 [0.3333]||12 [0.6667]||1 [0.1111]||4 [0.4444]||4 [0.4444]|
|5-10cm||11 [0.2750]||29 [0.7250]||1 [0.0500]||9 [0.4500]||10 [0.5000]|
|<5cm||51 [0.4113]||73 [0.5887]||13 [0.2097]||25 [0.4032]||24 [0.3871]|
|Present||4 [0.2857]||10 [0.7143]||0 [0.0000]||4 [0.5714]||3 [0.4286]|
|Absent||63 [0.3539]||115 [0.6461]||13 [0.1461]||37 [0.4157]||39 [0.4382]|
|Yes||46 [0.3898]||72 [0.6102]||11 [0.1864]||24 [0.4068]||24 [0.4068]|
|No||30 [0.3000]||70 [0.7000]||5 [0.1000]||20 [0.4000]||25 [0.5000]|
|I-II||24 [0.4615]||28 [0.5385]||5 [0.1923]||14 [0.5385]||7 [0.2692]|
|III-IV||16 [0.5000]||16 [0.5000]||4 [0.2500]||8 [0.5000]||4 [0.2500]|
|>10 cm ||0.7577||0.7577||0.5320||1.0000||1.0000|
|>10 cm ||0.6132||0.6132||0.6769||1.0000||0.7318|
|5-10 cm ||0.1372||0.1372||0.1698||0.7963||0.4382|
4.2. IL-18 (-607 C/A; rs1946518 and -137 C/G; rs187238), IFN-γ (+874 A/T; rs62559044) and TNF-α (-238 A/G; rs361525 and -308 A/G; rs1800629) polymorphism
The genotypification of the polymorphisms of the genes
|C||62 [0.2768]||120 [0.2970]||0.6464||0.90||0.63-1.30|
|G||162 [0.7232]||284 [0.7030]||0.6464||1.10||0.77-1.59|
|A||92 [0.4107]||129 [0.3193]||0.0235||1.48||1.06-2.08|
|C||132 [0.5893]||275 [0.6807]||0.0235||0.67||0.48-0.94|
|A||130 [0.5856]||240 [0.5941]||0.8652||0.96||0.69-1.35|
|T||92 [0.4144]||164 [0.4059]||0.8652||1.03||0.74-1.44|
|A||41 [0.1847]||39 [0.0965]||0.0025||2.12||1.32-3.40|
|G||181 [0.8153]||365 [0.9035]||0.0025||0.47||0.29-0.76|
|A||30 [0.1351]||32 [0.0792]||0.0351||1.82||1.07-3.08|
|G||192 [0.8649]||372 [0.9208]||0.0351||0.55||0.32-0.93|
When the -607C/A SNP of
|CG||48 [0.4286]||84 [0.4185]||0.9051||1.05||0.66-1.68|
|CC||7 [0.0625]||18 [0.0891]||0.5157||0.68||0.27-0.68|
|GG||57 [0.5089]||100 [0.4950]||0.9063||1.06||0.67-1.68|
|CA||56 [0.5000]||105 [0.5198]||0.8138||0.92||0.58-1.47|
|AA||18 [0.1607]||12 [0.0594]||0.0048||3.03||1.40-6.55|
|CC||38 [0.3393]||85 [0.4208]||0.1845||0.71||0.44-1.14|
|AT||50 [0.4505]||82 [0.4059]||0.4741||1.20||0.75-1.91|
|AA||40 [0.3604]||79 [0.3911]||0.6276||0.88||0.54-1.42|
|TT||21 [0.1892]||41 [0.2030]||0.8823||0.92||0.91-1.65|
|GA||41 [0.3694]||39 [0.1931]||0.0011||2.44||1.45-4.12|
|AA||0 [0.0000]||0 [0.0000]||1.0000||1.81||0.03-92.15|
|GG||70 [0.6306]||163 [0.8069]||0.0011||0.40||0.24-0.69|
|GA||30 [0.2703]||26 [0.1287]||0.0031||2.51||1.39-4.51|
|AA||0 [0.0000]||3 [0.0149]||0.5548||0.25||0.01-4.99|
|GG||81 [0.7297]||173 [0.8564]||0.0098||0.45||0.25-0.80|
No significant differences in the allele or genotype frequencies of SNPs of
Evaluation of SNP -607C/A of
The 14bp*D allele in gene
Chen et al., in a study of 150 individuals of the Chinese Han population, showed that genotypes of the 14bp insertion/deletion polymorphism of
Many mechanisms of tumor escape have been proposed in the literature, some of them local and others systemic. Particularly important among them is the expression of immunomodulatory molecules in the tumoral microbiota, as well as the expression of soluble suppressive factors by the tumoral cells. HLA-G represents one of these immunomodulatory molecules, playing an important role in the mechanisms of immunotolerance by the inhibition of the activity of NK cells, cytotoxic T lymphocytes and antigen-presenting cells [9,36].
The 14bp insertion/deletion polymorphism in exon 8 of
The tumoral microbiota or even the cells that underwent mutation per se can induce the expression of HLA-G. Studies have demonstrated the expression of HLA-G in various malignant tumors, although with variations in the percentage of lesions expressing the molecule. In studies involving renal cell carcinoma , endometrial adenocarcinoma  and gastric cancer , at least 30% of the tumors exhibited HLA-G expression.
Regarding hepatic diseases, Souto et al., in a study of 74 liver biopsies of individuals with chronic HBV infection and 10 specimens obtained from previously healthy cadaver liver donors, demonstrated that 77% of the samples of chronic HBV hepatitis presented HLA-G expression in the hepatocytes, as opposed to none of the controls . These authors detected a case of HCC, in which HLA-G expression was not detected in the tumor cells but was detected in adjacent non-tumoral hepatic tissue.
Lin et al. evaluated by immunohistochemistry the expression of HLA-G in 219 HCC and adjacent nontumoral tissue samples. The expression of HLA-G was observed in 50.2% of HCC samples vs. 0% of normal corresponding adjacent tissue. Evaluation of HCC stages showed that HLA-G expression was detected in 37.8%, 41.9% and 71.4% of cases in stages I, II and III, respectively. The data reported by these authors revealed that the expression of HLA-G was strongly correlated with advanced HCC stage and that soluble HLA-G was significantly more elevated in the plasma of patients with HCC compared to healthy controls . Similarly, Cai et al. studied the expression of HLA-G by immunohistochemistry in 173 HCC specimens and observed that HLA-G expression was associated with the prognosis of HCC, especially in the early stages of the disease, with higher HLA-G expression being independently associated with shorter overall survival and greater tumor recurrence after surgical resection .
Recently, a study involving 267 patients divided as anti-HBs positive healthy individuals (n=50), chronic HBV carriers (n=45), active hepatitis B (n=46), liver cirrhosis (n=46) and early-stage HCC patients (n=80) showed that serum concentrations of soluble human leukocyte antigen-G (sHLA-G) were significantly higher in the active hepatitis B and HCC groups compared to the other groups (P<0.05). Moreover, the concentrations of sHLA-G were higher in the patients with HCC than in those with liver cirrhosis or active hepatitis B, suggesting that serum sHLA-G concentrations may be associated with the different phases of hepatitis B infection. They did not find any association between sHLA-G concentrations and HCC stage, number of tumors, pathologic grade and presence of vascular invasion . Another study, examining fifteen SNPs in the non-classical class I alleles, found that the SNPs rs17875380, rs41557518, rs114465251, and rs115492845 were associated with susceptibility to chronic hepatitis B infection or HCC, and HLA-F*01:04, HLA-G*01:05N, and HLA-E*01:01 were associated with hepatitis B or hepatitis B with HCC, concluding that these polymorphisms may play an important role in immune surveillance of hepatitis B and HCC, possibly leading to immune responses to virus or cancer cells .
It has been demonstrated that, in addition to genetic factors, the microbiota, for example, strees inducers, hypoxia and cytokines (interferons, IL-10, TNF-α), influences the expression of HLA-G, so that more studies are needed for a better understanding of the interaction of molecules derived from the tumor and from host factors .
In summary, the present findings show that the deletion allele of the
A higher frequency of the
We observed that alleles
The expression of
Studies evaluating the association of polymorphisms of
Literature evidence has demonstrated that IL-18 is a pleiotropic cytokine that enhances the Th1 or Th2 immune response according to the medium and to genetics. In the presence of IL-12, IL-18 induces IFN-γ secretion by NK and T cells, activating the Th1 response, important for defense against tumor cells. On the other hand, IL-18 can increase tumor growth via increased stimulation of VEGF and of the immune response, and also stimulate solid tumor metastasis . A possible explanation for this fact is the increased Th1 response in the early stages of the cancer which, however, is replaced with Th2 as tumor malignancy worsens with tumor development. Thus, as the tumor develops,
Regarding the +874 A/T polymorphism of the
Our results suggest that the 14bp-deletion allele in