|Year : 2013 | Volume
| Issue : 2 | Page : 90-95
B-cell clonality and t(14;18) in Egyptian patients with chronic hepatitis C and its relation to antiviral therapy
Walaa Othman1, Dalia A. Salem1, Sherin M. Abd El-aziz1, Mona M. Arafa2, Raghda E. Farag2, Shahira Eletreby3
1 Department of Clinical Pathology, Faculty of Medicine, Mansoura University, Mansoura, Egypt
2 Department of Tropical Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
3 Department of Internal Medicine, Faculty of Medicine, Mansoura University, Mansoura, Egypt
|Date of Submission||18-Feb-2013|
|Date of Acceptance||18-Feb-2013|
|Date of Web Publication||20-Jun-2014|
Dalia A. Salem
MD, Department of Clinical Pathology, Faculty of Medicine, Mansoura University, 35516 Mansoura
Source of Support: None, Conflict of Interest: None
The mechanism of lymphomagenesis of hepatitis C virus (HCV)-related B-cell lymphoma is unknown. Recently, it has been suggested that HCV may induce B-cell clonal proliferation and t(14;18) translocation in patients chronically infected with the virus.
The aim of this study was to assess the occurrence of immunoglobulin heavy chain (IgH) gene rearrangement and t(14;18) translocation in Egyptian chronic HCV patients and to examine the effect of antiviral treatment on IgH rearrangement and t(14;18) in HCV-infected patients.
Patients and methods
Forty-five Egyptian patients with chronic HCV infection were selected. The level of HCV-RNA in the serum was quantified using the Stratagene Mx3000P Real-Time PCR System at diagnosis and 3, 6, and 12 months from the beginning of the therapy. IgH clonality was detected using multiplex VH-JH (FR2) PCR, whereas t(14;18) was detected using nested PCR before and after antiviral therapy.
After 3 months of antiviral therapy, 24/45 patients (53.3%) showed an early virological response and completed their 12 months of antiviral therapy, after which they showed complete clearance of serum HCV-RNA (responder group). However, 21/45 patients (46.7%) did not show early virological response and hence stopped their therapy (nonresponder group). Clonal IgH rearrangement and t(14;18) were detected in 33.3 and 40% of patients, respectively. The percentage of patients who lose their clonal IgH or t(14;18) varies according to the completion of antiviral therapy. On comparing responder (12 months therapy) versus nonresponder (3 months therapy) groups, the loss of clonal IgH was nonsignificant (100 vs. 66.7%, P=0.134) whereas it was highly significant in terms of regression in t(14;18) (75 vs. 0%, P=0.0006).
In Egyptian patients with chronic HCV infection, the presence of clonal B cell and t(14;18) is a frequent finding. Twelve months of antiviral therapy are efficiently effective for regression of clonal IgH gene rearrangement and t(14;18).
Keywords: chronic hepatitis C, IgH rearrangement, interferon, t(14;18)
|How to cite this article:|
Othman W, Salem DA, Abd El-aziz SM, Arafa MM, Farag RE, Eletreby S. B-cell clonality and t(14;18) in Egyptian patients with chronic hepatitis C and its relation to antiviral therapy. Egypt J Haematol 2013;38:90-5
|How to cite this URL:|
Othman W, Salem DA, Abd El-aziz SM, Arafa MM, Farag RE, Eletreby S. B-cell clonality and t(14;18) in Egyptian patients with chronic hepatitis C and its relation to antiviral therapy. Egypt J Haematol [serial online] 2013 [cited 2020 Feb 20];38:90-5. Available from: http://www.ehj.eg.net/text.asp?2013/38/2/90/134795
| Introduction|| |
Hepatitis C virus (HCV) infection is a major public health problem worldwide; Egypt has one of the world’s highest prevalence of HCV infection. Clinical studies in Egypt showed that 70–90% of patients with chronic hepatitis, cirrhosis, or hepatocellular carcinoma had HCV infections 1,2.
HCV infection has been associated with a series of B-cell lymphoproliferative disorders (LPDs), including essential mixed cryoglobulinemia, B-cell non-Hodgkin’s lymphoma, and monoclonal gammopathies 3. The process by which HCV infection leads to the development of lymphoma is unknown, but growing evidence indicates that HCV may directly stimulate and possibly infect B cells. The chronic stimulation of B cells leads to a potent but restricted antibody response, which, in some cases, undergoes additional oncogenic events and gives rise to a monoclonal B-cell expansion that can be detected by immunoglobulin heavy chain (IgH) gene rearrangement. In addition, an increased prevalence of the t(14;18), which results in deregulated overexpression of the Bcl-2 antiapoptotic protein, occurs in HCV-positive patients that may be a further step for transformation to lymphoma in such patients 4,5.
Because chronic antigenic stimulation by HCV has been shown to play a role in the development of B-cell expansion and malignant transformation, it is possible that eradication of the persistent infection by antiviral treatment may possibly lead to regression of the proliferating clone 6. Antiviral treatment appears to be effective in eliminating the clonal proliferation of B cells in patients with chronic HCV infection and may prevent the subsequent development of lymphoma 7.
The present study aimed to assess the occurrence of IgH gene rearrangement and t(14;18) in Egyptian chronic HCV patients and to examine the effect of antiviral therapy of interferon-α on regression of clonal IgH rearrangement and t(14;18).
| Patients and methods|| |
This was a prospective study in which 45 Egyptian patients with chronic HCV infection (28 men and 17 women, mean age 44.15±3.91 years) were recruited from the Department of Tropical Medicine and Hepatology Unit, Mansoura University Hospitals. The diagnosis of chronic HCV was made on the basis of clinical findings, biochemical profile, and HCV-RNA detection in patients’ sera. Liver biopsy was performed to confirm the diagnosis and rule out other causes of chronic liver diseases according to the consensus recommendations of the Asian Pacific Association for the Study of the Liver 8,9. Written consent was obtained from all patients and the study design was approved by the university ethical committee. All patients received a combination therapy of a fixed weekly dose of 180 μg of the PEG interferon (peginterferon α-2a, Pegasys; Hoffman La Roche, Basel, Switzerland) with ribavirin at a dose adjusted according to body weight. All patients were followed up by PCR for HCV-RNA after 3 months of treatment to identify the early virological response (EVR), that is, a >2 log10 drop in viral load after 3 months compared with the baseline. The patients were divided according to their EVR into ‘responder’ and ‘nonresponder’ groups. The responder group included 24 patients who continued their antiviral therapy for 12 months and were followed up by PCR for HCV-RNA after 6 and 12 months of treatment. End of treatment response (ETR) is defined as normalization of liver enzymes and loss of detectable HCV-RNA by PCR at treatment completion (12 months). The nonresponder group who did not show EVR (showed a ⩽2 log10 drop in viral load after 3 months as compared with baseline) included 21 patients and they had stopped their antiviral treatment.
Assessment of HCV infection
The level of HCV-RNA in the serum was quantified using the Stratagene Mx3000P Real-Time PCR System (Agilent Technologies, California, USA) with a sensitivity of ∼15 IU/ml.
Detection of IgH rearrangement
Genomic DNA was extracted from peripheral blood samples using the Qiagen QIAamp DNA mini prep kit (Cat No: 51304; Qiagen, Santa Clarita, California, USA) according to the manufacturer’s instructions. IgH clonality was detected using multiplex VH-JH (FR2) PCR as described by BIOMED-2 concerted action 10 for all samples in addition to positive (monoclonal) and negative (polyclonal) controls. The primers used are shown in [Table 1]; the reverse primer (JH consensus) was fluorescently labeled to enable post-PCR detection by Gene Mapper. Approximately 100–500 ng DNA was added to a reaction mix containing 10 pmol of each primer, 12.5 μl of 2×PCR master mix (Fermentas, Life Sciences, Vilnius, Lithuania) containing 0.6 U Taq polymerase, and 400 nmol/l of each of dNTPs in PCR buffer containing 4 mmol/l magnesium chloride. The reaction volume was completed to 25 μl with deionized water. The mixture was heated to 94°C for 7 min, and then 35 cycles each of 94°C for 30 s, 60°C for 30 s, and 72°C for 30 s were performed, followed by 10 min at 72°C. All PCR reactions were performed in a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems, California, USA).
To detect the PCR product by Gene Mapper, 1 μl of fluorescently labeled PCR product was mixed with 10 μl of deionised HiDiformamide (Applied Biosystems) and 1 μl of genescan internal lane 500 ROX size standard (Applied Biosystems). The mixture was electrophoresed through an acrylamide containing polymer, POP4 (Applied Biosystems), which was then analyzed using an ABI 310 Genetic Analyzer (Applied Biosystems) using the associated gene mapper software (Applied Biosystems). Clonal rearrangements were identified as single peaks of valid size range (250–295 bp). The peak height should be twice that of adjacent peaks when it is present within a polyclonal background 11.
Detection of t(14;18)
All DNA samples were tested for amplificability by PCR using primers for the globin gene [Table 2]. Each sample was tested for t(14;18) by nested PCR according to Gribben et al. 12. This method has a sensitivity limit of about 10−5–10−6. PCR was performed in a 25 μl final volume using 100–500 ng of DNA, 20 pmol of oligonucleotide primers, and 12.5 μl of 2×PCR master mix (Fermentas, Life Sciences) containing 0.6 U Taq polymerase and 400 nmol/l of each of dNTPs in PCR buffer containing 4 mmol/l magnesium chloride. The initial amplification was performed using 5′ MBR-out and 3′ JH-out primers [Table 2]. The mixtures were initially heated at 95°C for 4 min, followed by 35 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 30 s, with a 10-min final extension at 72°C. The second PCR amplification was performed under identical conditions using 1 μl of the first PCR product and internal 5′ MBR-in and 3′ JH-in primers [Table 2]. Amplified products were analyzed on 2% agarose gels stained by ethidium bromide. With each experiment, both positive [t(14;18) positive cell line; DOHH2] and negative control samples were used with the other samples.
|Table 2: Primers used for the detection of bcl-2 translocation using nested PCR|
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Statistical analysis was carried out using SPSS (SPSS Inc., Chicago, Illinois, USA), version 17.0, for Windows. Comparison between groups was carried out using Student’s t-test for continuous variables and the χ2-test for categorical variables. All values were presented as mean±SD or number and percent. Correlations between t(14;18) and other variables were established using Pearson’s or Spearman’s rank correlation. The Mann–Whitney U-test was used for the continuous ordinal data between two qualitative variables. Variables that achieved statistical significance with the univariate analysis were included in multiple regression analysis to evaluate the independent factors associated with t(14;18) or IgH rearrangement. P values less than 0.05 were considered statistically significant.
| Results|| |
We studied 45 patients with chronic HCV infection who had received PEG-interferon-ribavirin antiviral therapy for 3 months. Twenty-four cases showed EVR and completed their antiviral therapy (responder group), whereas 21 cases did not show EVR and hence stopped their antiviral therapy (nonresponder group). The baseline mean viral load detected in both patients’ groups sera before antiviral treatment was 5.1×105±0.5×105 IU/ml. All patients in the responder group showed ETR and achieved a negative PCR test for the detection of HCV-RNA after 12 months of antiviral therapy.
[Table 3] summarizes the baseline clinical, biochemical, and molecular data of both chronic HCV patient groups. There were no statistical differences between both groups in these parameters.
|Table 3: Baseline characteristics of HCV patients in responder and nonresponder groups|
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Regression analysis [Table 4] shows that clonal IgH is only associated with the high fibrosis stage (P=0.05), whereas t(14;18) is correlated with high levels of alanine transaminase and aspartate aminotransferase, high viral load, high level of alpha feto protein (AFP), and the presence of steatosis (P=0.008, 0.02, 0.002, 0.001, and 0.031, respectively).
|Table 4: Correlation between t(14;18) and clonal IgH rearrangement with other parameters (Spearman’s rank correlation)|
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[Table 5] and [Figure 1] show that 6/6 patients (100%) within the responder group who were positive for clonal IgH rearrangement became negative for this rearrangement after they had completed their antiviral therapy (12 months), whereas only 6/9 patients (66.7%) of the nonresponder group who had this clonal rearrangement became negative after 3 months of therapy. However, the difference between both groups in loss monoclonal IgH rearrangement is not statistically significant.
|Table 5: Frequency of IgH rearrangement before and after treatment in relation to virological response (&khgr;2-test)|
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|Figure 1: The effect of antiviral treatment on IgH gene rearrangement in patients with chronic HCV infection. Detection of IgH rearrangement was performed by FR2 Biomed-2 multiplex primers with a subsequent gene scan. Upper electropherogram shows monoclonal peak in a patient with HCV before treatment; lower electropherogram shows polyclonal IgH rearrangement after treatment. HCV, hepatitis C virus; IgH, immunoglobulin heavy chain.|
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As shown in [Table 6] and [Figure 2], t(14;18) was detected in 12/24 of the patients in the responder patient group (50%); nine of these (75%) patients lost this translocation after they completed their antiviral therapy (12 months). In the nonresponder group, 6/21 patients (28.6%) had t(14;18) and none of them lost this translocation after incomplete therapy (3 months). There was a statistically significant difference between both groups with respect to loss of t(14;18) after antiviral therapy (P=0.006).
|Table 6: Frequency of t(14;18) before and after treatment in relation to virological response (&khgr;2-test)|
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|Figure 2: The effect of antiviral treatment on t(14;18) in patients with chronic HCV infection. Detection of t(14;18) by MBR bcl-2/JH nested PCR in PBMCs from patients with chronic HCV infection before and after antiviral treatment. Lane 1: molecular weight marker; lanes 2 and 3: presence of t(14;18) in both pre-treatment and post-treatment samples; lanes 4 and 5: absence of t(14;18) in both pre-treatment and post-treatment samples; lanes 6 and 7: loss of pretreatment t(14;18) in the post-treatment sample; lanes 8 and 9: presence of t(14;18)i in both pre-treatment and post-treatment samples; lane 10: positive control cell line (DOHH2); lane 11: negative control. HCV, hepatitis C virus; PBMCs, peripheral blood mononuclear cells.|
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| Discussion|| |
Clinical studies in Egypt showed that 70–90% of patients with chronic hepatitis, cirrhosis, or hepatocellular carcinoma had HCV infections. Extrahepatic disease manifestations may cause slight complications of chronic infection or in turn dominate its course. These extrahepatic manifestations of HCV infection include autoimmune phenomena as well as an association with B-cell lymphoproliferation and non-Hodgkin’s lymphoma 13.
HCV-related LPD pathogenesis is a multifactorial and multistep process. Current data suggest that the starting points of this process are represented by the cooperation between a sustained and persistent stimulation (leading to lymphoproliferation) and antiapoptotic mechanisms [as caused by t(14;18) and bcl-2 overexpression] acting on the B-cell compartment. A predisposing genetic background and the progressive addition of genetic aberrations may be responsible for the final evolution to a particular LPD 14. Also, the clonal proliferation of B cells in patients with chronic HCV infection appears to be responsive to antiviral treatment that may prevent or treat HCV-related LPDs. This phenomenon may be related to a direct effect of interferon on the proliferating clone or to an indirect effect by eradicating the antigenic stimulus 7.
In this study, HCV-RNA sequences were detected in the sera of 45 chronic HCV patients with mean viral loads of 5.1×105±0.5×105 IU/ml as a baseline load. Twenty-four patients (53%) achieved EVR after 3 months of antiviral therapy; consequently, they completed their antiviral therapy and showed ETR after 12 months of treatment (responder group). However, 21 patients (47%) did not achieve EVR and discontinued their antiviral treatment (nonresponder group). The rates of EVR detected in our study (53%) was lower than most published studies that showed EVR rates from 69 to 78% after 3 months of antiviral therapy 15–17, and this may be because of the small number of patients in our study. However, Legrand-Abravanel et al. 18 reported only a 50% EVR rate after 3 months of therapy in their study. All patients who achieved EVR in this study could achieve ETR, which also varies from most of the studies in which the ETR rate is usually less than the EVR rate by 1–7.5% 15, 16, 19. Again, this may be because of fewer patients in our study or minor changes in the treatment modalities between the studies.
Clonal IgH rearrangement was detected in 15/45 (33.3%) of our HCV patients, with no statistical differences between the responder and the nonresponder groups (25 and 42.9%, respectively). The frequency of clonal IgH gene rearrangement in chronically HCV-infected patients in previously published studies varies widely from 10 to 58.6% 7,20–22. The discrepancy in these results may be because of differences in the number of patients, ethnic background, and more importantly the different methods used for detection of IgH clonality with different sensitivities and specificities.
Our study showed a considerable prevalence of t(14;18) in chronic HCV patients that reached 40%, with a higher percentage in the responder group in comparison with the nonresponder group (50 and 28.6%, respectively). In other studies, the occurrence of t(14;18) in chronic HCV patients varies from 24 to 48% 2, 6, 23. However, in contrast to our findings, Sansonno et al. 24 failed to find specific bcl-2/IgH amplicons either in liver tissue or in peripheral blood mononuclear cells in HCV-infected patients. Their findings supported the concept that the production of IgH gene rearrangements was not associated with bcl-2/IgH chromosomal translocation.
Univariant analysis has been carried out to study the correlation between the occurrence of clonal IgH or t(14;18) with many clinical and laboratory variables. No significant correlation was found between the occurrence of clonal IgH and other clinical or laboratory data except the fibrosis stage (P=0.05). However, high level of liver enzymes alanine transaminase (P=0.008), aspartate aminotransferase (P=0.02), high viral load (P=0.002), high level of AFP (P=0.001), and the presence of steatosis (P=0.031) may be considered as predictor factors for the presence of t(14;18). No previous publications have been found to support or deny our findings. However, all these variables may be signs associated with the severity of HCV infection, which in turn may induce more B-cell stimulation.
The results of the current study support that antiviral treatment may help to prevent or treat HCV-related LPDs 25 as the completion of this treatment in our study leads to the disappearance of clonal IgH and t(14;18) with different degrees. All patients (6/6, 100%) with clonal IgH rearrangement within the responder group lost this clonality after the completion of their 12-month antiviral therapy. However, only 6/9 patients (66.7%) of the nonresponder group with this clonal rearrangement became negative after incomplete therapy (3 months). However, the difference between both groups in the loss of monoclonal IgH rearrangement did not reach statistical significance (P=0.134). The results of Zuckerman et al. 7 showed significant difference (P<0.02) in loss of IgH clonality after treatment between treated and nontreated patients with HCV (77 and 12.5%, respectively). Nevertheless, his nontreated group did not receive any antiviral treatment unlike our ‘nonresponder’ group, who received antiviral treatment for at least 3 months. However, their results were in agreement with ours in the context of virological response as they stated that loss of IgH in the patients who received antiviral treatment was strongly associated with virological response. In more detail, 86% of his treated patients in whom IgH rearrangement became negative had cleared the virus at the end of therapy. In contrast, Saadoun et al. 26 observed that monoclonal IgH gene rearrangement persisted in splenic lymphoma with villous lymphocytes patients with HCV infection after antiviral treatment irrespective of the hematologic and virological response, although the majority of their patients achieved a sustained complete hematologic response after clearance of HCV-RNA. This may be because of the more overt malignant nature of their patients’ disease in contrast to our patients, who did not have any evident malignant hematologic disease.
In terms of the effect of antiviral treatment on t(14;18), it was found that there was a statistically significant difference (P=0.006) in the number of patients who lost their t(14;18) after the completion of 12-month antiviral therapy in the responder group (9/12; 75%) when compared with the nonresponder group and hence did not complete their treatment (3/12; 25%). These results are in agreement with Zuckerman et al. 7, who reported the disappearance of t(14;18) in 6/7 (86%) treated patients and that was strongly associated with virologic response to treatment. In addition, Giannelli et al. 25 reported the disappearance of t(14;18) in 9/14 (64.3%) patients who achieved a complete virological response (complete clearance of HCV-RNA from their blood), in 6/10 (60%) of patients with a partial virological response (persistently detectable HCV viremia, but with more than one-half reduction of initial load), and in none/6 (0%) who did not show any virological response. Ibrahim et al. 2 concluded that the antiviral therapy for 6 months did not significantly decrease the frequency of t(14;18) compared with nontreated HCV patients despite biochemical and virological response to therapy. Other studies showed that the close association between the virological response and the loss of B-cell monoclonality and t(14;18) indicated that the more effective the antiviral therapy, the more likely the loss of B-cell clonality. In addition, translocated B cells have been shown to reappear after virological relapse at the end of treatment 27.
| Conclusion|| |
In Egyptian patients with chronic HCV infection, the presence of B-cell clonality by analyzing the IgH gene rearrangement and t(14;18) are frequent findings. Twelve months of antiviral therapy are efficiently effective for regression of clonal IgH gene rearrangement and t(14;18). The loss of clonal IgH rearrangement and t(14;18) is closely associated with virological response to antiviral therapy.
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[Figure 1], [Figure 2]
[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6]