CD4/CD8 Ratio could be predictor of burden hepatocellular carcinoma in Egyptian chronic hepatitis C after combined sofosbuvir and daclatasvir therapy

Background During the first years of the use of direct acting Hepatitis C antiviral drugs (DAAS), several studies reported a possible correlation between this new era of treatment and an increased risk of Hepatocellular carcinoma (HCC). Its development could possibly be favored by the changes in the immunological milieu and the different cellular behavior after eradication of HCV infection with them. For this reason, this study aimed to address the immunological effect of DAAS Subject & methods Prospective paired -sample design, carried out on 90 naïve chronically infected HCV patients before and after receiving a combination therapy of sofosbuvir; at a dose of 400 mg once daily and daclatasvir; at a dose of 60 mg once daily for 12 weeks and follow up for one year. immunological tests including: total T cell count, T helper cell count, T cytotoxic cell count and natural killer cell count in peripheral blood through (CD3, CD3/CD4, CD3/CD8 and CD56 respectively) by Fluorochrome monoclonal antibodies labelled with specific dyes through Multiparameter, FACSCanto ™ II flow cytometer (Becton Dickinson, USA). Result Concerning the immunological changes, total T cells (CD3+), Natural killer cells showed non-significant decrease at end of therapy while significant decrease in T helper cells (CD3+CD4+) T cytotoxic cells (CD3+CD8+) compared to pre-treatment value. Long follow up revealed 26.6% developed focal HCC, in more addition, multivariate analysis show CD4/CD8 ratio could be predictor as well as sex for early development of HCC after combined DAAS therapy. Conclusion HCV treatment by DAAS produces significant decrease in T helper, T cytotoxic cells in CHC patients at the end of therapy. 26.6% developed focal HCC with independent CD4/CD8 predictor for burden malignancy. Further large extended population study is needed for clarify this concern.


Introduction
HCV is a global health burden affecting approximately 71 million people worldwide with Egypt having the highest prevalence 1 . It causes annual deaths of approximately 399,000 which result mostly from complications 2 . HCV is a blood-borne virus transmitted mainly through infected blood and blood products 3 . It is a major cause of liver fibrosis, cirrhosis and hepatocellular carcinoma (in a rate of 1-7% each year). Hepatocellular carcinoma (HCC) is the third common cause of cancer death worldwide predisposing to one million deaths annually [4][5] . Innate immunity plays the primary defense mechanism against HCV infection through Type I Interferon (IFN) production that makes the cells ready & promotes them to fight infection, checks viral replication, induces adaptive immune response in addition to stimulating certain cells as natural killer cells, dendritic cells & Kupffer cells 6 . NK cells represent an important arm of innate immunity that plays a critical role in HCV eradication. The stimulated NK cells recruit virus specific T cells, promote the antiviral immune response in the liver & eliminate virus infected hepatocytes directly through cytolytic mecha-nisms & indirectly by releasing cytokines such as INF-γ & TNF-α 7 . HCV antigens are presented on MHC CLASS I & II molecules on the outer surface of the cell where they are identified by CD8+ & CD4+ T cells respectively. In more, HCV peptides liberated from damaged virus-infected hepatocytes are picked up by myeloid DCs that migrate to the draining lymph nodes (LNs) where they present HCV peptides on HLA class II particles with higher expression of CD80 & CD86 costimulatory particles that react with & stimulate HCV specific T helper cells 8  Interferon-based therapy was the core therapy of chronic HCV infection with cure rate of about 50% & with many side effects. This therapy was recently replaced by new drugs called direct acting antivirals (DAAS) with high cure rate indicated by achieving sustained virological response (SVR) in more than 90% of patients with shorter duration of treatment & little adverse effects than interferon-based therapy. These DAAS target viral replication through affecting viral proteins essential for it such as ns3/4a inhibitors, ns5a inhibitors & ns5b inhibitors [10][11] . This high SVR achieved by DAAS was expected to reduce the risk of HCC. However, the effect of treatment by DAAS on recurrence & occurrence of new HCC in patients with cirrhosis after successful treatment is conflicting & controversial 12 . Several studies reported increased incidence of HCC recurrence, de novo HCC & aggressive relapse of HCC during & after receiving DAAS treatment. High recurrence of 27% raised the debate that DAAS may be incriminated in predisposition of HCC recurrence or new occurrence [13][14] . Affection of immune system after viral eradication by DAAS may be a contributing factor to HCC recurrence or de novo occurrence 15 . For this reason, the current study aimed to evaluate certain immune markers (total T cells, T helper cells, T cytotoxic cells and natural killer cells) in chronic HCV patients treated with sofosbuvir and daclatasvir before therapy and after 12 weeks and 48 weeks of therapy to detect the effect of this combination therapy of direct acting antiviral therapy on the immune status of these patients.

Patients and Methods
This study was paired -sample design, one arm prospective carried out on 90 naïve chronically infected HCV patients before and after receiving a combination therapy of sofosbuvir; at a dose of 400 mg once daily and daclatasvir; at a dose of 60 mg once daily for 12  carcinoma (HCC) were excluded. All patients were subjected to the following: Full history taking, complete clinical examination with the following investigations done before receiving any treatment and after 12-week therapy with sofosbuvir and daclatasvir and follow up for one year after therapy.
Blood was distributed in three tubes (about 200 µl in each tube calculated according to the equation 1000/ WBC count). One tube contained only blood (auto) to adjust auto fluorscent background before analysis, sec-ond tube contained blood with 5 µl anti CD3, CD4 and CD8 mAbs labelled with FITC, APC and PE respectively and the third tube contained blood with 5 µl PE-labelled anti CD56 mAb. Blood was then incubated with fluorochrome-conjugated monoclonal antibodies for 15 minutes in the dark at room temperature. Then 2ml of ammonium chloride were added to each tube to induce lysis of red blood cells. The samples were then vortexed and incubated in the dark for 15 minutes followed also by vortex. Centrifugation at a speed of 3000 RPM was then done for 5 minutes and supernatant was discarded, then the pellet was formed. This was followed by wash with 0.5 ml or 1 ml of phosphate buffered saline for two times or more till clearance of red blood cells. Vortex was then done followed by centrifugation followed by wash until all blood around the pellet was cleared to get rid of debris and 500 µl PPS were left on the specimen. Eight-colour flow cytometry was then done using FACSCanto (Becton Dickinson and company, BD Bioscience, San Jose, CA 95131 USA) and data were analysed using FACS ADIV-IA software to detect surface marker expression through flourochrome-labeled monoclonal antibodies. Gating strategy was done using bright CD45 mononuclear cells with counting total CD3, absolute count of CD4 and CD8 from total CD3 with calculating CD4/CD8 ratio in addition to counting natural killer cells which express CD56. By plotting CD3 versus CD4, we can get total T cell (CD3 + ) count and T helper cell count (CD3 + CD4 + ) and by plotting CD3 versus CD8, we can get also total T cell count and T cytotoxic cell count (CD3 + CD8 + ). Also, by plotting CD4 versus CD8, we can obtain CD4/CD8 ratio. the count of natural killer cells through (CD56 + ).

Sample size
Sample size was calculated using PASS software (version 2008). Estimation relied upon a previous study by17.

Statistical analysis
Data were entered and analyzed using IBM-SPSS software (version 25). Qualitative data were expressed as frequency and percentage. Quantitative data were initially tested for normality using Shapiro-Wilk's test with data being normally distributed if p>0.050. Quantitative data were expressed as mean ± standard deviation (SD) if normally distributed or median and interquartile range (IQR) if no Paired-Samples t-test was used if data were normally distributed in both readings. The non-paramet-ric alternative Wilcoxon signed ranks test was used if not. Repeated-measures ANOVA test was used if data were normally distributed in all readings. results were considered as statistically significant if p value ≤ 0.050.

Results
The demographic, clinical characterization and basic laboratory data are shown in tables (1)(2)(3).  Impact of Drug on patients' Immunological criteria of the studied cases: At 3 months no significant changes total CD3; CD4/CD8 ratio; NK while significant reduction was observed in T-helper cells CD3/CD4 and T-cytotoxic cells CD3/CD8 in study cases (       HCV therapy has been revolutionized in the few recent years by the appearance of direct-acting antiviral agents (DAAS). These greatly effective, very well-tolerated DAAS of several classes have largely substituted PEG-IFN and RBV. The advantage of these new regimens is that; they produce greater sustained virologic response (SVR) rates with more possibility of adherence to therapy in addition to much fewer side effects and much less diminished health-related quality of life during therapy 22 .
The elevated rates of SVR attained in Chronic hepatitis C(CHC) patients treated with DAAS were expected to raise the hope of a significant reduction in HCC occurrence and recurrence. Unexpectedly, patients who cleared HCV with DAAS have been found to encounter increased aggressiveness and elevated rates of HCC recurrence (28% and 29% respectively) after a complete response to resection or local ablation within only 6 months of ther-apy13-14. On the other hand, increased risk of HCC recurrence after DAAS treatment in CHC patients after receiving curative cancer treatments couldn't be revealed by three independent prospective French cohorts 23 . These contradictory results have increased commentaries and criticism about this controversial issu 24 . Despite the unclear effects of DAAS therapy on the rate of HCC occurrence or recurrence, it would be important to study the immunological alterations in CHC patients treated with DAAS [25][26] .
Sofosbuvir is the principal DAA in all published reports of DAAS related HCC. In spite of that, the supposed relation between DAAS in general, sofosbuvir or sofosbuvir linked metabolites and carcinogenesis requires more analysis. Although, multiple theories were hypothesized to clarify this supposed linkage, none of them had a strong proof of concept 13 . This study aimed to detect the effect of sofosbuvir and daclatasvir combination therapy of DAAS on the immune status in chronic hepatitis c patients follow up of patients for one year and could be predictor of burden Hepatocellular carcinoma. This Egyptian study was carried out on 90 naive chronically infected hepatitis C patients, treated by combined DAAS antiviral therapy for 12 weeks then follow up for one year. The present study revealed significant reduction of both ALT, AST at the end of completion therapy, this finding similar to same observation on similar population with both combined therapy [27][28] . As HCV is believed to be non-cytopathic, prompt depression, either quantitatively or qualitatively, of intrahepatic cytotoxic inflammatory cells might be the cause of prompt AST/ALT normalization 29 . No significant change was observed in serum albumin level between the end of therapy and pre-treatment period. This was similar to the results conducted by [27][28][29][30] . While disagree with study by Fayed et al. showed an increase in albumin level 31 . The difference between our study and this study may be due to the possibility that longer duration may be needed for the improvement in serum albumin level and consequently the synthetic function of the liver as reported by Maruoka et al. who noted that over the first two years after combined interferon treatment, serum albumin level increased gradually then plateaued 32 . This improvement reinforces the belief that intrahepatic inflammation directly, participate in reduced synthetic ability of the liver and that ameliorating inflammation can reconstruct liver function to some degree 33 .
Total bilirubin level at the termination of therapy was not found at statistically significant change from pre-treatment level. This was also in agreement with the results obtained [34][35] .
As regarding renal function assessment by Serum creatinine had non-significantly different level at end of therapy compared to pre-treatment level. This was also in agreement with [27][28][29][30][31][32][33][34] . while in agreement with study by Sulkowski et al. that assessed sofosbuvir and daclatasvir in untreated patients and previously treated patients who failed telaprevir and bocebrevir treatment showed increase of creatinine at the end of therapy 36 . This difference in observation may be related to the effect load of drug therapy used in protocol regiment on renal function. Considering the hematological parameters, White blood cell count (WBC) wasn't significantly changed at end of therapy compared to pre-treatment. This finding similar to [31][32][33][34][35][36][37] . In contrast, the study by Elsharkawy et al. showed significant reduction of WBCs at the end of treatment 30 .
Hemoglobin level showed non-significant change at the end of therapy, similar to observation of [34][35][36][37][38] In contrast, there was significant decrease of hemoglobin at the end of therapy compared to pre-treatment 36 . Platelet count decreased significantly at 2 months of therapy compared to pre-treatment level. The level increased at end of treatment but was still lower than the pre-treatment level. Platelet count decreased at the termination of therapy when compared to healthy subjects in the study of 38 while there was non-significant decrease in platelet count 3 months post-therapy compared to pre-treatment level in the study of 37 and at the end of therapy compared to baseline in the study of 31  The emergence of HCC was linked, to baseline risk fac-tors including advanced fibrosis stage, HBV co-infection or age. Also, it was suggested by another hypothesis that DAAS promote dysregulation of immune surveillance mechanisms after the very rapid viral eradication; this hypothesis has been promoted by several studies. The reconstitution of innate immunity with the downregulation of type II and III IFNs receptors and IFN-stimulated genes. A decreased stimulation of IFN may subsequently permit the growth of neoplastic cells as the INF has anti-angiogenic and anti-proliferative characteristics that DAAs lack. Also, the reduction in the cytotoxic activity of NK cells in the liver is one of the immune system changes that have been described after HCV eradication, which supports a more rapid progression of HCC foci 29 . This finding was confirmed by an interesting study carried out by Monto and his colleagues who observed that NK cell inhibitory KIR/HLA types were found in all 11 patients who developed HCC after second generation DAA indicating the genetic based impaired immune-surveillance capacity in those patients 47 . MicroRNA (miRNA) 122 that is the major miRNA in hepatocytes could be related to another potential mechanism. As re it acts as a tumor suppressor gene in HCC 48 . Interestingly, down regulation of miRNA 122 after achieving SVR through DAA therapy was observed; this may contribute to an elevated risk of HCC recurrence 49 . Eventually, another significant study by Villani et al. revealed that the level of vascular endothelial growth factor becomes significantly elevated during treatment with DAA and remains elevated for 3 months after treatment cessation that may finally lead to the occurrence/recurrence of HCC 50 .Unique identification of T-cell population in HCC could help in targeted therapy or patient may benefit from immunotherapy later on, CAR-T cell in future could destroy malignant cells 51 .
Limitations of the present study include limited scope of research population. Also, we didn't assess the hepatic immune changes and the phenotypic changes after DAAS therapy. So, further studies with large sample size, prolonged follow up for 2 years or more and wide scope of research with different genotypes are needed to confirm the results of the present work and to investigate different changes in peripheral immunity in correlation with hepatic immunity and phenotypic immune changes.

Conclusion
HCV treatment by DAAS produces non-significant decrease in the count of total T cells, natural cells, while significant reduction in T helper cells, T cytotoxic cells, CD4/CD8 ratio in peripheral blood of CHC patients at the end of therapy. longer periods of follow up re-vealed26.6% developed HCC. Regression analysis revealed significant CD4/C8 ratio as early predictor of HCC after DAAs therapy.