Epstein-Barr virus and Interleukin-28B polymorphism in the prediction of response to interferon therapy in hepatitis C patients

Background and study aims: In chronic hepatitis C virus [HCV], viral and host factors are known to be predictors for anti-viral therapy. IL-28B genotype strongly influences treatment outcome, while Epstein-Barr virus [EBV] co-infection could accelerate the course of chronic HCV infection. This study was conducted to assess whether EBV co-infection adds to the predictive value of IL-28B

Patients and methods: A total of 105 patients with chronic HCV were classified according to their response to treatment into two groups: 38 sustained virological responders [SVRs] and 67 nonresponders [NRs]. Collected sera at baseline and follow-up [FUP] were used for assessing EBV antibodies by enzyme-linked immunosorbent assay [ELISA] and the expression of EBV genes [BNLF-1, BZLF-1, and EBER-2] by polymerase chain reaction [PCR]. Collected peripheral blood was used for detecting IL-28B rs.12979860 single-nucleotide polymorphism

Results: Regarding IL-28B genotype frequencies, a significant difference [p = 0.003] was observed between SVRs [C/C = 51.4%, C/T = 48.6%, T/T = 0%] and NRs [C/C = 25%, C/T = 55%, T/T = 20%]. On assessing EBV infection at baseline and FUP, it was found that 61% and 55% were positive, respectively, with no significant difference between SVRs and NRs. As for anti-viral capsid antigen [VCA] antibodies, the NRs had significantly higher baseline anti-VCA immunoglobulin M [IgM] levels than SVRs [p = 0.01]. While FUP anti-Epstein-Barr nuclear antigen-1 [EBNA-1] IgG reported a significant decline within SVR patients [p = 0.02], neither baseline nor FUP anti-VCA IgG levels showed a statistically significant viral response. Finally, on comparing EBV markers with CC versus CT and TT genotypes, it was found that FUP anti-VCA IgG levels were significantly increased in CC genotype [p = 0.003]

Conclusion: Interleukin-28B polymorphism could be a possible predictor of response to pegylated interferon/ribavirin therapy [PEG-IFN/RBV]. Furthermore, co-infection with EBV did not affect the response to IFN-based therapy in HCV-infected patients

Expression of MMTV-like env gene in Egyptian breast cancer patients

Mouse mammary tumor virus [MMTV] causes breast cancer in mice. DNA sequences related to MMTV-like env gene is detected in human breast cancer [EC] tissue suggesting its etiology in human BC. The objective of our study was to assess the significance of MMTV-like env gene in Egyptian BC women. One hundred and fifty archival formalin-fixed paraffin embedded breast tissues were used and divided into 2 groups; group one included 100 malignant, group two included50 benign tissues. To amplify the MMTV-like env gene, semi-nested PCR was used and to confirm the homology with the MMTV genome direct sequencing was used. MMTV-like env was efficiently detected in36%ofmalignantand 4% of benign breast tissues. Sequence analysis was evident revealed 96% homology with the MMTV genome, but no other significant similarities with the human genome

The presence of the viral sequences was associated significantly with estrogen and progesteron positive cases and insignificantly the other pathological parameters studied. The molecular analysis of breast cancer tissue confirmed the presence of MMTV-like env sequences with significant high percentages in cancerous tissues than in benign one. These data raising the possibility that MMTV viral infection may be involved in the neoplastic process

p53 Mutation in HCV-genotype-4 associated hepatocellular carcinoma in Egyptian patients

HCV-associated hepatocellular carcinoma [HCC] is a common neoplasm in Egypt where genotype-4 is prevalent. In the present study the incidence and pattern of p53 mutations was assessed in relation to HCV-genotype-4 in Egyptian HCC patients. We investigated 25 HCV positive HCCs for p53 mutations over expression in relation to HCV-N53 by immunohistochemistry, SSCP and sequencing. Genotyping was done using LiPA-Il and TRUGENE 5 NC sequencing kit. Results were correlated to standard clinicopathologic prognostic factors for HCC. Thirteen cases showed p53 overexpression, and 10 showed p53 mutation [13 mutations] by sequencing [72% concordance]. The highest mutation rate was in exons 6 and 7 [30%] followed by exons 5 and 8 [20%]. Mutations included 3 transitions, 5 transversions, 3 deletions, and 2 insertions. All exon 7 mutations were at codon 249 specific for AFB1 [AGG-AGT, Arg-Ser] and codon 248 specific for vinyl chloride contamination [CGG-TGG, Arg-Trp]. Other mutations reported are novel. Immunostaining for HCV N53 was detected in 19 cases independent of p53 mutation. p53 aberrations were significantly associated with poor prognostic factors for HCC. However, no specific pattern for p53 mutations was observed in HCV genotype 4-associated HCC and no significant relation between p53 mutations, HCV-N53 expressions or any HCV sub-genotype-4 sequence

Genetic distance and heterogenecity between quasispecies is a critical predictor to IFN response in Egyptian patients with HCV genotype-4

HCV is one of the major health problem in Egypt, where it is highly prevalent. Genotype 4 is the most common genotype of HCV and its response to treatment is still a controversy. HCV genotype 4 quasispecies diversity within the 5' untranslated region [5'UTR] was studied in a series of 22 native Egyptian patients with chronic hepatitis C virus with no previous treatment who satisfied all NIH criteria for combined treatment of pegylated IFN and ribavirine and was correlated with the outcome of treatment. The study also included 7 control patients with no antiviral treatment. HCV sequencing was done using the TRUGENE HCV 5-NC genotyping kit. At the 48[th] week of treatment, 15 patients [68%] showed virological response. Whereas HCV-RNA was still detected in 7 patients [32%] in this period; of those, 6 experienced a partial virological response followed by viral breakthrough during treatment. Only one patient did not show any virological or chemical response. The four females included in this study were all responders. There was a significant correlation between the response rate and lower fibrosis [p=0.026] as well as the total number of mutation spots [including all the insertions, deletions, transitions and transversions] [p=0.007, p= 0.035]. Patients who responded to interferon treatment had statistically significant less number in both transitions [p=0.007] and the genetic distances between the quasispecies [p= 0.035]. So, viral genetic complexity and variability may play a role in the response to IFN treatment. The consensus alignment of all three groups revealed no characteristic pattern among the three groups. However, the G to A transitions at 160 was observed among non responders who need further study to confirm this observation

Role of mismatch repair genes [h MLHI, h PMS1, h PMS2, GTBP/h MSH6, h MSH2] in the pathogenesis of hepatocellular carcinoma

DNA mismatch repair [MMR] is an important mechanism involved in maintaining fidelity of genomic DNA. Abnormalities in at least one of five MMR genes are implicated in the development of many cancers and the associated micro satellite instability [MSI]. By using a newly developed multiplex reverse transcription -PCR assay, the expression of the five known MMR [hMLH1, hPMS1, hPMS2, GTBP/hMSH6, hMSH2] were evaluated in 33 human HCC cases as well as 16 cases from the normal distant hepatic tissue samples [NDHT] were also evaluated. Twenty- five of them were associated with HCV infection. This was done in an attempt to determine the role of MMR genes in the development of HCC. The beta actin gene was used as an internal control for RNA degradation and DNA contamination and as well as a reference for quantifying the levels of their transcription. Out of the 33 studied HCC cases, 30 cases [90.9%] showed reduction in the expression of one or more of the 5 studied MMR genes. Reduced expression of hMSH2 was found in [71.9%], hMLH1 [53.3%], GTBP [51.1%], hPMS2 [33.3%] and hPMSI [6%]. Correlation analysis showed a strong significant correlation [P= 0.0069] between reduced expression of hPMS2 and GTBP [P=0.0034] as well as hPMS2 and non-cirrhosis [P=0.0197]. Chi-square analysis showed a significant correlation between reduced expression of hMLHl and grade II. On the other hand, 57.1%, 50%, 20%, 18.8% and 6% of the NDHT showed reduced expression of hMSH2, hMLHI, GTBP, hPMS2 and hPMSI respectively. Multivariate analysis showed significant correlation between HCC and hMSH2 [P= 0.008], hMLH1 [P=0.001] and GTBP [P=0.032], also between hPMS2, GTBP and HCC infected with HCV cases [P< 0.001, 0.002]. It could finally be concluded that reduced expression of hPMS2 is likely associated with growth advantage and stimulates proliferation changes that have encouraged malignant development in non- cirrhotic HCV infected patients via acquisition of more genetic damage and the MMR defects that occur at an early stage of hepatocarcinogenesis

Comparative analysis of different PCR techniques for detection of HCV in hepatocellular carcinoma patients

Three different isolation procedures and a primer set from the 5-TUR were compared using 3 variable PCR techniques [direct PCR, semi-nested PCR and single tube RT-PCR]. This was applied for detection of HCV- RNA in sera from 30 hepatocellular carcinoma patients positive for HCV antibodies by both EIA and immunoblotting techniques. It was found that RNA extraction with silica [Boom et al., 1990] and semi-nested PCR technique were the most sensitive procedures. By this combined method the study was able to detect 20 copies of the in vitro transcribed RNA compared to 50 copies and 100 copies using 1 tube RTPCR and direct PCR techniques, respectively