1 Johns Hopkins Bloomberg School of Public Health, Department of Epidemiology
2 Georgetown University, Lombardi Cancer Center
3 Cairo University, Egyptian National Cancer Institute
4 University of Maryland, Department of Epidemiology and Preventive Medicine
5 Minia University, Department of Microbiology
Correspondence: Christopher A Loffredo, Georgetown University, Box 571472, Washington DC 20057-1472, USA. E-mail: CAL9{at}georgetown.edu
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Abstract |
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Methods Cases with B-cell NHL (N = 227) were recruited from the National Cancer Institute of Cairo University, a major referral centre. Controls (N = 227) were patients with fractures being treated at the Kasr El-Aini Orthopaedic Hospital, from the same referral base as the cases, and were frequency-matched by gender, rural versus urban birthplace, and age. Subjects were interviewed about their medical history and possible risk factors, and blood samples were collected for HCV diagnostic tests. Anti-HCV and HCV RNA were determined by enzyme-linked immunoassay and reverse transcription-polymerase chain reaction, respectively. Odds ratios (OR) and 95% CI were calculated from logistic regression models.
Results Overall, 42% of subjects were anti-HCV positive and 33% had HCV RNA. There was a statistically significant unadjusted association of HCV RNA with NHL (OR = 2.3, 95% CI: 1.5, 3.5), which differed slightly by gender (males: OR = 2.1, 95% CI: 1.2, 3.7 versus females: OR = 2.5, 95% CI: 1.3, 4.8). Anti-HCV without HCV RNA was not associated with case status (OR = 0.9, 95% CI: 0.5, 1.6). After adjustment for age, gender, rural versus urban birthplace, and rural versus urban current residence, the association of HCV RNA with the risk of NHL remained statistically significant (OR = 2.9, 95% CI: 1.9, 4.5).
Conclusions These data support the hypothesis that NHL is a malignant outcome of chronic HCV infection.
Accepted 24 February 2004
The World Health Organization (WHO) estimates that 170 million people are infected with hepatitis C virus (HCV).1 An estimated 1215% of Egyptians, or 810 million people, have serological evidence of HCV infection, with higher rates in older age groups and residents of rural areas in Lower and Middle Egypt.2 There is compelling evidence for a large-scale iatrogenic transmission of HCV during a parenteral anti-schistosomal treatment campaign carried out from the 1920s through the 1980s.2 Continued transmission in Egypt has been associated with transfusion of unscreened blood; invasive medical procedures, including Caesarean section and abortion; injections by informal health care providers;3 and haemodialysis.4
Since its identification, HCV has been added to the roster of tumour-associated viruses because of its role in hepatocarcinogenesis. It has also been linked to upwards of 28 extrahepatic disease manifestations, chiefly autoimmune in nature. However, some of these reports are inconclusive, and it remains to be confirmed that HCV is causally associated with all but a few conditions.5
One of the extrahepatic diseases in which HCV has been implicated is B-cell non-Hodgkin's lymphoma (NHL). HCV-associated lymphomas have been observed, but whether they are caused by HCV remains to be shown definitively. There is a suggestion that some B-cell NHL associated with HCV arise from clonal expansion of B-cells with particular immunoglobulin gene rearrangements specific for the E2 protein of the HCV envelope,6,7 which is consistent with the hypothesis that lymphomas develop when B cells proliferate in response to antigen.6 However, no biological mechanism of HCV-associated lymphomagenesis has been definitively elucidated.
Most of the studies reported to date that failed to find an association of HCV with NHL were conducted in areas where the prevalence of HCV was extremely low, leaving open the possibility that such an association actually exists but could not be detected because neither cases nor controls had adequate opportunity for exposure. Working in a population with high prevalence of HCV allowed us to conduct a case-control study with adequate statistical power to assess the question of whether there is an association of chronic HCV infection with NHL.
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Materials and Methods |
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Some participants were new patients who were undergoing tests to establish a diagnosis, and some were returning patients who were about to initiate or were already undergoing treatment; all were recruited within 6 months of initial diagnosis. Provisional cases were those whose lab slips indicated a diagnosis or suspicion of NHL. Their classification as a case was subject to change after confirmation of their diagnoses through pathology data and medical records review. Subjects whose diagnoses were not located or finalized during record review, or who were found to have a diagnosis other than an NHL, were dropped from the study.
Selection of controls
Control subjects free from cancer were sampled by gender and birthplace from among all patients of the Kasr El Aini Faculty of Medicine Orthopaedic Hospital in Cairo, Egypt. They were frequency-matched to the case group by rural versus urban birthplace, gender, and 5-year age category. The rationale for selecting orthopaedic patients, all of whom were being treated for fractures, was: (1) they would be representative of the source population of the cases by region, since both hospitals draw patients from the same area and (2) they would be representative of the general population with respect to HCV infection, since HCV positivity and fracture are exceedingly likely to be independent. Potential controls had to be 18 years old, and physically and mentally capable of participating.
Interview procedures
Ethical review boards at all involved institutions approved the study. Written or witnessed oral informed consent was obtained from all participants before enrolment. Trained research assistants administered a standardized Arabic-language questionnaire in a face-to-face interview that lasted 30 minutes. The questionnaire asked about residence, employment, and smoking histories; exposures to pesticides and other industrial or agricultural substances; social and educational factors; and medical history. On completion of the interview, a blood specimen was collected. There was no further follow-up with the study subjects.
Laboratory assays
Within 6 hours of collection, the blood was separated and the serum divided into aliquots and stored at 80°C. Samples were later thawed and tested for anti-HCV antibody by Abbott HCV enzyme-linked immunoassay (EIA) 3.0 (Abbott Park, IL, USA) according to the manufacturer's instructions. Samples were initially tested for HCV RNA by direct nested reverse transcription-polymerase chain reaction (RT-PCR) as described previously.8 A positive result by the direct RT-PCR method was considered truly positive, and no further investigation was done. A sample that was negative by both direct RT-PCR and EIA was considered negative. However, all samples that tested negative by direct nested RT-PCR and positive by EIA were re-tested by conventional RT-PCR, which differed from direct RT-PCR in that it included an RNA purification step.
In all cases where formalin-fixed tissue from NHL cases was available at the Egyptian National Cancer Institute, immunophenotyping for B- and T-cell markers was performed at the Department of Pathology. Identification of B- and T-cell surface markers was carried out using pan-B (CD-20) and pan-T (CD-45) monoclonal antibodies with the DAKO EnVision System (Code No. K4006, DAKO, Carpinteria, CA, USA). This is a two-step indirect staining technique similar to the avidinbiotin complex method. The test was performed according to the manufacturer's instructions in the package insert. Cases that tested positive for the B-cell marker were retained, while those positive for the T-cell marker were dropped from analysis, regardless of previous classification based on histological examination of haematoxylin and eosin-stained slides.
Statistical analyses
Environmental exposures from the questionnaire were examined as dichotomous, ever/never variables, and age (in 2000) was included as a continuous variable. Region of current residence was categorized as Upper Egypt and Deserts (hereafter referred to as Upper Egypt) and Lower Egypt. This geographical distinction should not be confused with the distinction between rural and urban, which is based on participants' self-reported birth in a rural or urban area and was used as a matching criterion. Rural and urban areas might be located in either Upper or Lower Egypt.
Characteristics of cases and controls in the entire sample and in males and females were compared either by Pearson's 2 test (for categorical variables) or by a t-test (for continuous variables). Point estimates and exact 95% CI were calculated for unadjusted unmatched odds ratios (OR) using PCR-negative participants as the reference group in the first case, regardless of EIA results, and those negative for HCV by both PCR and EIA as the reference group for the joint analysis of EIA and PCR. Because both HCV infection and NHL occur more frequently in males than in females, analyses were stratified to determine if any observed HCV-NHL association differed by gender.
Adjusted OR and 95% CI were obtained by standard logistic regression. Potential confounders were identified as variables whose inclusion in a logistic regression model resulted in a change of 5% in the baseline OR.9 Final multivariate logistic regression models included HCV RNA, age, gender, urban versus rural birthplace, and the variables identified as confounders from the preliminary analyses. Reported P-values were based on two-tailed tests. All statistical analyses were performed using SAS, version 9.0 (Cary, NC).
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Results |
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Table 1 shows the distribution of demographic and other selected characteristics in cases and controls. Cases and controls were well matched by place of birth (rural versus urban), age, and gender, but controls were more likely overall to reside currently in a rural area (P < 0.01). Controls were more likely to be born and to reside in Upper Egypt, regardless of gender (P < 0.01 for entire sample and for males; P = 0.05 for females). Agricultural use of pesticides (P = 0.86) and total deliveries (P = 0.70) were not statistically different between cases and controls. Cases and controls did not differ in their reports of blood transfusions (P = 0.10). Cases were more likely to have had injection treatments for schistosomiasis (P < 0.01).
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Table 2 shows HCV results by case and control status and unadjusted OR by HCV infection status. The first OR column compares cases and controls with evidence of HCV RNA by RT-PCR to those having no HCV RNA. In the entire sample, the OR was 2.3 and was statistically significant (95% CI: 1.5, 3.5). Stratifying by gender shows this association was quite similar in females (OR = 2.5) and in males (OR = 2.1). The second OR column in Table 2 incorporates anti-HCV antibody data to stratify the OR by infection status, with the reference group being those who were negative for all HCV markers. These results show that the associations in males and females were limited to those with evidence of current infection with HCV (i.e. HCV RNA positive), and was not present in those with evidence only of previous infection (HCV RNA negative but anti-HCV positive).
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Among those cases whose tumour samples were analysed by immuno-histochemistry (N = 58), the crude OR compared with all 227 controls for HCV was 1.7 (95% CI: 0.9, 3.2).
Table 3 shows the per cent change in the age-, gender-, and birthplace-adjusted HCV-NHL OR with the addition of each of the variables that differed significantly between cases and controls in Table 1. For the entire sample and especially for males alone, adjustment for current residence in Upper Egypt resulted in a slight increase in the OR for HCV, in the order of 7% to 17%. For females adjustment for residence in Upper Egypt resulted in only a 3% change in the OR. Adjustment for total number of deliveries (for females) resulted in no fluctuations of the HCV-NHL OR.
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Discussion |
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The authors of one large, well-designed study concluded that their results did not support an association of HCV with NHL, although the data they report show otherwise. Pioltelli and others33 matched 300 NHL cases to 600 non-cancer age- and sex-matched controls. Calculations from data reported in that article indicate an HCV-NHL OR of 2.1 (95% CI: 1.4, 3.1), but the authors misreported the CI as (95% CI: 0.7, 27.0) and concluded that they had a non-significant result. On this basis they called for a re-evaluation of the HCVNHL association they had previously reported. In fact, the results of their study are very similar to ours and provide strong support for that association.
The principal finding of the current study is a positive association of current HCV infection with NHL. We found no increased risk of NHL among participants who had cleared infection with HCV or who showed no sign of ever having been infected.
Finding the association only among those currently infected with the virus is consistent with hypotheses about how HCV might increase NHL risk over time, with a minimum latency of several years between HCV infection and NHL development. Conversely, because NHL is a sign of a disordered immune system, it could represent a lower rate of successful viral clearance by NHL patients.
The only possible routes of HCV transmission for which we knew the time of exposure were blood transfusions and anti-schistosomal injections, but when we excluded subjects who reported these exposures after 1994 we did not find major changes in the OR for HCV. Other exposures that we did not assesse.g. injections, surgeries, or tattoosmight have infected participants in the 5 years prior to the study. Still, it is reassuring that removing the influence of such major potential sources of HCV transmission as blood transfusion and anti-schistosomal injection did not affect the estimate of association.
Misclassification of cases and controls may have occurred in this study as diagnoses could not always be independently confirmed due to lack of pathology material. Likewise, immunohistochemical testing to differentiate B-cell from T-cell NHL was done on only a small subset. Immunohistochemistry and histology review were planned as tools with which to establish and confirm diagnoses for all cases, but actually served as quality controls for the diagnoses obtained from the pathology database and medical charts since they could be done in only a few cases.
Some reports have indicated that the relationship of HCV with NHL is confined to particular NHL subtypes, specifically to lymphoplasmacytoid lymphoma/immunocytoma associated with mixed cryoglobulinaemia type II, or, in the absence of mixed cryoglobulinaemia, with follicular centre, marginal zone, and diffuse large NHL.14,34 While immunohistochemical subtyping of NHL was not performed in this study, histopathological classification identified only three lymphoplasmacytic lymphomas in our sample, all of which were negative for HCV RNA. Further immunohistochemistry studies on these materials are in progress in this study population.
The results presented here describe a strong association of chronic HCV infection with the risk of developing NHL, which persisted after adjustment in multivariate models and after several sensitivity analyses. Much remains unknown about the natural history of HCV infection and its possible contribution to carcinogenesis; however, our data suggest that NHL may be among the malignant complications of chronic HCV infection.
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Acknowledgments |
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References |
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