Lymphoproliferative disorders in chronic hepatitis C

Journal of Viral Hepatitis, 2004, 11, 302–309 Lymphoproliferative disorders in chronic hepatitis C R. Idilman,1,2 A. Colantoni,1 N. De Maria,1 S. Alkan,3 S. Nand4 and D. H. Van Thiel1 1 Department of Medicine, Division of Gastroenterology and Liver Transplantation, Loyola University Medical Center, Maywood, IL, USA; 2Department of Gastroenterology, Ankara University Medical School, Ankara, Turkey; 3Department of Pathology, and 4Department of Medicine, Division of HematologyOncology, Loyola University Medical Center, Maywood, IL, USA Received in revised form November 2003; accepted for publication November 2003 SUMMARY. Chronic hepatitis C virus (HCV) infection is associated with the development of lymphoproliferative disorders (LPDs). The aim of this investigation was to determine the prevalence and characterization of monoclonal gammopathy and benign and malignant LPDs in individuals with chronic hepatitis C. A total of 233 subjects diagnosed with chronic hepatitis C (male/female ratio: 131/102, median age; 49 years) were studied. Serum and urine were examined for the presence of a monoclonal gammopathy. A bone marrow aspirate and biopsy was obtained in individuals with a monoclonal gammopathy. Thirty-two patients (13.7%, 32 of 233) had a monoclonal gammopathy; 75% of them were benign and were not associated with malignant disorders (24 of 32) while 25% were associated with malignant LPDs or a plasma cell disorder (eight of 32). Two additional subjects without monoclonal gammopathy were diagnosed as having a malignant LPDs. The prevalence of malignant INTRODUCTION Chronic hepatitis C is an insidious form of liver disease that relentlessly but silently progresses to cirrhosis in 20% of cases over a period of 10–30 years [1–3]. Chronic hepatitis C infection has been associated with several extra-hepatic manifestations that include membranoproliferative glomerulonephritis, various autoimmune disorders, idiopathic pulmonary fibrosis, benign and malignant lymphoproliferative disorders (LPDs) [4]. Monoclonal gammopathies (MGs) constitute a group of benign and malignant LPDs characterized by the proliferation of a single clone of plasma cells that produce a Abbreviations: LPDs, Lymphoproliferative disorders; MG, Monoclonal gammopathy; MGUS, Monoclonal gammopathy of undetermined significance. Correspondence: Ramazan Idilman MD, Department of Gastroenterology, Ankara University Medical School, Ibn’i Sina Hospital, Sihhiye, Ankara, 06100 Turkey. E-mail: [email protected]
2004 Blackwell Publishing Ltd LPDs/plasma cell disorder in individuals with HCV-induced chronic liver disease was 4.3%. No difference was found in terms of disease duration, HCV genotype, viral load, alanine aminotransferase level or histopathologic score between the subjects with or without a monoclonal gammopathy. The presence of mixed cryoglobulinaemia was strongly associated with the presence of an underlying malignant disorder. Hence a monoclonal gammopathy is found in 14% of patients with chronic hepatitis C and is associated with malignant B-cell LPD in more than a quarter of such patients. The prevalence of LPDs in individuals with HCV-induced chronic liver disease is greater than that of the normal healthy population. Keywords: cryoglobulinaemia, hepatitis C virus, malignant lymphoproliferative disorders, monoclonal gammopathy, plasma cell disorders. monoclonal protein [5]. The prevalence of MGs in the normal healthy population is approximately 1% [5–7]. The clinical spectrum of MGs ranges from a benign monoclonal gammopathy of undetermined significance (MGUS) to overt multiple myeloma, Waldenstrom’s macroglobulinaemia or malignant lymphoma [5]. Previous studies have reported that about 2–11% of the patients with hepatitis C virus (HCV) infection have a MG [6,7]. Most of these studies have been retrospective and the data reported are frequently in conflict. The HCV may be responsible for the development of benign and malignant LPDs associated with chronic HCV infection [7,8]. The association between mixed cryoglobulinaemia (MC) and chronic HCV infection is extremely strong with more than 95% of patients affected by essential MC having serologic evidence of a current or prior HCV infection [4]. Between 13 and 54% of individuals with HCV infection can be shown to have MC [4]. Although the causative role of HCV in B-cell LPDs is still unclear, several investigators have reported that the prevalence of HCV infection in patients with B-cell non-Hodgkin’s lymphoma ranges from 1 to 30% Monoclonal gammopathy in HCV infection [7–10]. HCV may exert its oncogenic potential because of an indirect mechanism or directly utilizes other pathways [9]. The aims of the present investigation were to determine the prevalence of MG, and benign and malignant LPDs in individuals with chronic hepatitis C and to characterize the distribution of HCV viral genotypes in individuals with such conditions. METHODS Subjects A total of 233 consecutive HCV positive patients referred for treatment of chronic hepatitis C who were seen at the Loyola University Medical Center from June 1998 to January 2000 and were included in this study. The diagnosis of HCV-induced chronic liver disease in each case was made on the basis of serological and histological data consisting of antiHCV positivity, abnormal serum alanine aminotransferase (ALT) levels, HCV-RNA positivity and a liver biopsy documenting chronic hepatitis. Biochemical tests The presence of a MG was assessed by immunofixation (Hydragel immunofixation system, Sebia Inc., Norcross, GA, USA). Serum immunofixation was performed in all HCV positive subjects. Urine immunofixation was performed in subjects with one phase proteinuria on routine urine analysis, or with malignant LPDs. Serum ALT, aspartate aminotransferase, gamma glutamyl transpeptidase, lactate dehydrogenase, alkaline phosphatase, total bilirubin, white blood cell count (WBC) and serum immunoglobulin levels (immunoglobulin G, A and M levels) were measured using standard reagents and methods by the Clinical Pathology Department of Loyola University in CLIA approved laboratories. Flow cytometry Mononuclear cells from bone marrow or peripheral blood were isolated by Ficoll–Hypaque density gradient centrifugation. Cells were stained with four colour direct immunofluorescence using the antibodies against CD2 (CD, Cluster of Differention), CD3, CD4, CD5, CD8, CD19, CD20, CD22, CD23, CD45 and kappa and lambda immunoglobulin light chains. Plasma cells were gated by selection of the high intensity CD38-positive population and analysed with antibodies against kappa and lambda immunoglobulin light chains. Ten thousand events per sample were analysed on an Epics XL-MCL flow cytometer (Coulter, Miami Lakes, FL USA) utilizing Coulter System II software. The criterion for immunophenotypic marker positivity was labelling of at least 20% of the cells by the marker in question as determined from the histogram scatterplots. 303 Serological tests Hepatitis C virus antibody (Anti-HCV) was assayed using second generation enzyme-linked immunosorbent assays (ELISA; Abbott Laboratories, North Chicago, IL, USA). HCVRNA quantification was performed utilizing a commercially available assay system (Amplicor, Roche Diagnostics, Indianapolis, IN, USA) [11]. Both tests were performed in the Clinical Pathology laboratory of the Department of Pathology at Loyola University Medical Center. The HCV genotyping was performed by ARUP Labs (Salt Lake City, UT, USA) using the reverse transcriptase-polymerase chain reaction (RT-PCR) amplification followed by nucleic acid sequencing. HCV genotypes were assigned based upon the 5¢ UTR sequence analysis and were grouped into six major genotypes. The genotypes were subtyped according to sequence characteristics as described [12]. Cryoglobulin determination Each subject had blood drawn for the detection of cryoglobulins. Specifically, blood was drawn directly into prewarmed (37 C) red-topped tubes. The blood was allowed to clot in a water bath maintained at 37 C and the supernatant plasma was collected at 37 C. The serum has placed in a haematocrit tube sealed with clay and allowed to cool undisturbed at 4 C in a refrigerator for 72 h. The haematocrit tube was then centrifuged for 3 min in a haematocrit centrifuge maintained at 4 C. The presence of the cryocrit was determined [13]. Histology In each case, a liver biopsy was obtained under fluoroscopic guidance (the standard method at our institution) using a 14-gauge Tru-cut needle (Baxter, Valencia, CA, USA). Two cores of tissue were obtained. One core was used for the histopathological assessment; the second core was used for determination of the hepatic content of iron. A bone marrow aspirate and biopsy was obtained in every individual identified as having a MG. A lymph node biopsy was obtained in those with a malignant LPD whenever possible. The tissue samples were fixed, stained and interpreted according to standard procedures utilized by the Hematopathology Section of the Department of Pathology at Loyola University Medical Center. Surface marker studies were performed to identify a monoclonal B-cell population and for immunophenotypic analysis. Lymphomas were classified according to the Revised European American Lymphoma (REAL) classification [14]. Follow-up Blood samples were collected from the subjects for the detection of the presence of a MG using immunofixation every 6 months. If a MG was detected either in serum or
2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 302–309 304 R. Idilman et al. Table 1 Clinical characteristics of all subjects Patient number Age (years) Gender (male/female) Race (African-American/Caucasian) HCV viral load (copies · 105/mL) HCV genotype 1b Initial ALT level (IU/L) Chronic active hepatitis Cirrhosis Cryoglobulinemia RF autoantibody WBC count (N, 2200–8500/mm3) IgG level (N, 694–1618 mg/dL) IgA level (N, 68–378 mg/dL) IgM level (N, 46–266 mg/dL) Chronic hepatitis C with monoclonal gammopathy Chronic hepatitis C without monoclonal gammopathy P-value 32 58 15/17 8/24 82 ± 5 52% 75 ± 5 20 12 22% 38% 5.6 ± 0.5 1996 ± 162 303 ± 35 235 ± 37 201 49 116/85 33/168 74 ± 8 38% 84 ± 6 155 46 12% 27% 6.3 ± 0.6 1681 ± 160 296 ± 35 231 ± 32 ) <0.001 ns ns ns ns ns ns ns ns ns ns <0.05 ns ns HCV, hepatitis C virus; ALT, alanine aminotransferase; RF, rheumatoid factor; WBC, white blood cell; Ig, immunoglobulin; ns, not significant. urine by immunofixation at a follow-up examination, a bone marrow aspirate and biopsy was obtained. Human research approval All subjects involved in this study signed an Institutional Review Board for the Protection of Human Subjects (IRB) approved informed consent before each liver and bone marrow biopsy as well as a separate consent for the collection of blood for research purposes. Moreover, the study conformed to the ethical guidelines of the 1975 Declaration of Helsinki and was approved before its initiation by the IRB at Loyola University Medical Center. Statistical analyses The data were analysed using the Mann–Whitney nonparametric test, Kruskal–Wallis ANOVA one-way and chi-square test. A P-value of <0.05 was considered to be significant. RESULTS All anti-HCV positive patients were HCV-RNA positive in serum as well as in the liver when assessed by PCR. All patients with HCV infection were negative for anti-HIV antibody using ELISA kit. Prevalence of monoclonal gammopathy A MG was found in 32 of the 233 subjects (13.7%) at the first evaluation. The characteristics of the subjects are shown in Table 1. The male/female ratio of the subjects with a MG was 15 of 17. Their average age was 57.9 years (range 34– 86 years) and was significantly greater than that of individuals without a MG (48.9 years, P < 0.001). There was no difference in gender or race between individuals with and without MG (male/female ratio: 15/17 vs 116/85; AfricanAmerican/Caucasian ratio: 8/24 vs 33/168, respectively; P > 0.05). Serum IgG level was significantly higher in subjects with a MG as compared with those without a MG (1996 ± 162 mg/dL vs 1680.7 ± 160 mg/dL, P < 0.05). No differences in serum IgA and IgM levels were found (Serum IgA level, 303 ± 35 mg/dL vs 296 ± 35 mg/dL respectively, serum IgM level 235 ± 37 mg/dL vs 231 ± 32 mg/dL respectively; P > 0.05). There was no significant difference in terms of initial WBC count among the subjects with and without a MG (5600/mm3 vs 6300/mm3) (Table 1). The serum monoclonal gammopathy was characterized in 31 of the 32 subjects as being an IgG type in 14 (eight IgG-j, five IgG-k, one IgG), an IgM type in eight (three IgM-j, four IgM-k, one IgM), an IgA type in three (one IgA-j, two IgA-k), and a lambda light chain type in six. Urine immunofixation was performed in 20 subjects and was found to be positive in five (25%). One subject who had peripheral lymphadenopathy without a detectable MG in the serum tested positive for a monoclonal protein in the urine. Flow cytometric analyses of peripheral blood lymphocytes subpopulation in the subjects with MG were all within the normal range. Among the 32 subjects with a detectable MG, 24 had a benign type (MGUS) which was identified as an IgG type in
2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 302–309 Monoclonal gammopathy in HCV infection 305 Table 2 Characteristics of the 24 subjects with a MGUS Patient Age Sex Race HCV genotype Histology Serum monoclonal Urine monoclonal Cryo RF BM findings 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 70 73 54 50 86 44 55 60 46 60 58 67 42 59 63 70 57 43 47 34 61 61 51 47 F M M F M F M F M F M M M M F F F M M F F F M F C C C C C C C B C C B C C B C C B B C C C C C B 1b 1b NA 2b 2a NA 1b 1a NA 2b NA NA 1a 1b 1b 1a 1b 1a NA 1a NA 1b 1a 1b Cirrhosis CAH Cirrhosis Cirrhosis Cirrhosis CAH Cirrhosis CAH CAH CAH CAH CAH CAH CAH Cirrhosis CAH CAH CAH CAH Cirrhosis Cirrhosis CAH CAH CAH IgG-k IgM-k IgG-k IgG-j k k IgG-j IgA-k IgM k IgG-j IgG-k IgM-j IgA-k IgG-j IgG IgM-k IgG-j IgM-k IgG-j k IgG-k IgM-j IgG-j NA Proteinuria NA NA Negative Negative Negative Negative Negative Proteinuria Proteinuria Negative Negative Proteinuria Proteinuria NA Present NA Present NA Proteinuria NA NA NA ) ) ) ) ) ) ) ) + ) + ) ) ) ) ) ) ) ) ) ) ) ) ) 35 <20 <20 <20 <20 <20 <20 29 216 <20 <20 <20 573 <20 128 <20 146 <20 <20 <20 <20 225 260 <20 pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl pl cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell cell 7% 6% 3% 3% 3% 4% 5% 8% 3% 7% 5% 3% 4% 5% 3% 3% 4% 2% 3% 3% 3% 2% 3% 4% MGUS, monoclonal gammopathy of undetermined significance; M, male; F, female; HCV, hepatitis C virus; RF, rheumatoid factor; NA, not available; CAH, chronic active hepatitis; Ig, immunoglobulin; C, Caucasian; B, African-Americans. 12 (seven IgG-j, four IgG-k, one IgG), an IgM type in six (two IgM-j, three IgM-k, one IgM), an IgA-k type in two, and a lambda light chain type in four (Table 2). The bone marrow biopsy in each case demonstrated a normal trilineage haematopoiesis with normal or slightly increased numbers of plasma cells. The plasma cells were small and mature appearing and showed an interstitial plasma cell distribution without cluster formation. Flow cytometric analysis demonstrated plasma cell clonality. No aberrant B or T cell population was detected and no significant changes in the Tcells subpopulation analyses were identified. No additional new MG or loss of the presence MG was observed during a 2-year follow-up. (Tables 2 and 3). On the contrary, 23% (seven of 31) of the HCV positive individuals with cryoglobulinaemia had a MG positive. The cryoglobulinaemia was asymptomatic in the vast majority of cases with only six of the 31 cases (19.3%) having clinical purpura and/or vasculitic lesion, which occurred exclusively on the lower legs. The prevalence of a clinical peripheral neuropathy was only slightly greater (10 of 31, 32.3%). Low grade proteinuria was present in eight of 31 (25.8%). A detectable RF titre (>20 IU/mL) was found in 33.3% of subjects with a MGUS (eight of 24) and 50% of subjects with MG associated with a malignant LPDs/plasma cells disorder (four of eight) (P ¼ P > 0.05 ns) (Tables 2 and 3). Prevalence of cryoglobulins No difference was found in the prevalence of cryoglobulin and rheumatoid factor (RF) positivity (>20 IU/mL) between subjects with and without a MG (7/32, 22% vs 24/201, 12% and 12/32, 38% vs 54/201, 27% respectively, P > 0.05). Cryoglobulinaemia was detected in only two of the 24 subjects with a MGUS while it was present in five of the eight subjects with MG associated with a malignant LPDs/plasma cells disorder (2/24, 8.3% vs 5/8, 62.5%, P < 0.01) Prevalence of malignant LPDs/plasma cell disorder The remaining eight of the 32 subjects with a detectable MG had a malignant LPDs or plasma cell disorder assessed by the presence of abnormal appearing plasma cell occurring in clusters. Seven of these eight had a detectable serum MG which consisted of an IgG type in two (one IgG-j, one IgG-k), an IgM type in two (one IgM-j, one IgM-k), an IgA type in one (one IgA-j), a lambda light chain type in two (Table 3).
2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 302–309 306 R. Idilman et al. Table 3 Characteristics of 10 subjects with a malignant LPDs and plasma cell disorder HCV Serum Urine Patient Age Sex Race genotype Histology monoclonal monoclonal Cryo RF BM findings Histological diagnosis Site Diffuse Renal large B cell CLL Bone marrow MALToma stomach 1 65 M C 1b Cirrhosis Negative Positive + <20 Negative 2 50 M C 1a CAH IgA-j NA + 46 LPD 3 54 F C 2b CAH k Positive ) <20 4 58 M C 2a Cirrhosis k Negative + <20 Plasma cells 13% Negative 5 66 F B 1b CAH Negative ) 6 70 F C 1b Cirrhosis IgG-j Proteinuria + 7 49 M C 1b CAH Negative Negative ) 8 71 F B 1b CAH IgM-j NA ) 9 67 F C 1b Cirrhosis IgM-k Positive + 10 47 M B NA CAH NA ) IgG-k Negative Diffuse Stomach large B cell <20 plasma MM Bone cells >17% marrow 356 Plasma MM bone cells >20% marrow 85 Negative Follicular Cervical Grade II 1130 LPD CLL Bone marrow 254 Negative Diffuse Mediastinal large B cell <20 Negative Diffuse Axillary large B cell HCV, hepatitis C virus; RF, rheumatoid factor; CAH, chronic active hepatitis; C, Caucasian; B, African-Americans, MM; multiple myeloma, LPD; lymphoproliferative disorder, CLL; chronic lymphocytic leukaemia; Ig, immunoglobulin; NA, not available. Patient number Age (years) Gender (male/female) Race (African-American/Caucasian) HCV viral load (copies · 105/mL) HCV genotype 1b others (1a, 2a/b) Initial ALT level (IU/L) Chronic active hepatitis Knodell’s score Cirrhosis Cryoglobulinemia RF autoantibody RF levels (if>20 IU/mL) MGUS Malignant LPD’s and PCD 24 56.6 ± 2.4 12/12 6/18 74 ± 7 47% 53% 80 ± 6 16 9.2 ± 0.7 8 8% 33% 202 ± 61 10 60.3 ± 3.4 5/5 3/7 58 ± 14 67% 33% 65 ± 12 6 9.4 ± 1.3 4 50% 50% 374 ± 197 P-value – ns ns ns ns ns Table 4 The clinical status of HCV infection in individuals with monoclonal gammopathy associated with and without malignant LPDs and plasma cell disorder ns ns ns ns <0.01 ns ns HCV, hepatitis C virus; MGUS, monoclonal gammopathy of undetermined significance; ALT, alanine aminotransferase; RF, rheumatoid factor; LPDs, lymphoproliferative disorders; PCD, plasma cell disorder; ns, not significant. The remaining one had a MG detectable in the urine. There was no significant difference in terms of age, gender, race between subjects with benign MG and those who had a malignant LPDs/plasma cell disorder (Table 4). Two additional individuals without MG in either serum or urine had peripheral lymphoadenopathy and were diagnosed as having a malignant LPD. Thus, the total number of individuals with malignant LPDs/plasma cells disorder in the
2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 302–309 Monoclonal gammopathy in HCV infection study population was 10 with a prevalence was 4.3% (10 of 233). Among these 10 subjects, six had a B-cell NHL lymphoma, two had chronic lymphocytic leukaemia (CLL) and two had multiple myeloma (Table 3). Three of the lymphomas were nodal and three presented as extra-nodal lesion. One of the extra-nodal lymphoma was a low grade mucosa-associated lymphoid tissue (MALT) lymphoma of stomach and showed a mixed lymphocytic infiltration of centrocyte-like cells, lymphoplasmacytoid cells and mature plasma cells. There were occasional lymphoepitheloid lesions present. Helicobacter-like organisms were not detected by histologic examination. One patient had a grade II follicular lymphoma involving cervical lymph nodes. Two cases involving the bone marrow were diagnosed as being CLL with typical immunophenotype expression (CD19, CD20, CD5 and CD23) for this diagnosis. One case had a prominent lymphoplasmacytoid differentiation by histology. Four subjects had a diffuse large cell lymphoma involving the mediastinum, stomach, kidney and axillary lymph nodes. The two patients with MM had weakness-fatigue, bone pain and anaemia. One of these two had chronic hepatitis. The other had cirrhosis. Both had HCV genotype 1b infection. Bone marrow biopsy showed increased plasma cell numbers being 17 and 20% respectively in these two cases. Chronic HCV infection No significant difference in terms of source of infection, initial ALT levels (75 ± 5 IU/L vs 84 ± 6 IU/L respectively; P > 0.05) and HCV viral load (82 ± 5 · 105 copies/mL vs 74 ± 8 · 105 copies/mL respectively; P > 0.05) was observed among subjects with and without a MG (Table 1). Among the 32 HCV positive subjects with MG, 20 (male/ female ratio 10/10, age 56.3 ± 2.2 years) had chronic hepatitis without cirrhosis, while 12 had histologically confirmed cirrhosis (male/female ratio 5/7, age 61.1 ± 3.6 years). There was no significant difference in terms of the histopathologic diagnosis between subjects with and without a MG [20 chronic active hepatitis (CAH), 12 cirrhosis vs 155 CAH, 46 cirrhosis; P > 0.05] (Table 1). No correlation in terms of the presence of MG with the elevation of IgG among individuals with/without cirrhosis was observed (75%, 9/12 vs 60%, 12/20 P > 0.05). The HCV genotype was identified in 25 HCV positive subjects with a MG; seven (28%) had genotype 1a, 13 (52%) had genotype 1b and five (20%) had genotype 2a/2b. Among individuals without a MG, 38% were positive for genotype 1b. No significant difference was found in genotype distribution between subjects with and without a MG (HCV genotype 1b; 52% vs 38% respectively, P > 0.05) (Table 1). No differences in the severity of the primary liver disease (CAH/Cirrhosis ratio, 16/8; P > 0.05), initial ALT levels (80 ± 6 IU/L vs 65 ± 12 IU/L respectively; P > 0.05), frequency and size of the lymphoid follicles in the liver, HCV 307 viral load (74 ± 7 vs 58 ± 14 respectively, P > 0.05) or distribution of HCV genotype (HCV genotype 1b; 47% vs 67% respectively, P > 0.05) was detected between subjects with a MGUS and those with a MG associated with malignant LPDs/plasma cell disorder. DISCUSSION Previous studies have shown that HCV is a lymphotropic virus [6–8]. In fact, HCV antigens have been localized by immunofluorescence in peripheral blood monocytes/macrophages, bone marrow and B and T lymphocytes of chronically HCV infected individuals [9,10,15,16]. The biological relevance of such a finding is under intense investigation, particularly because chronic HCV infection has been proposed as being a cause for the development of a LPD. In the present study, the detection rate in HCV positive subjects for a MG using immunofixation was 14%, a value much greater than that reported for the normal population in which the prevalence of a MG is 1% [5–7,17]. This result is comparable with studies by Andreone et al. [6], who observed the presence of a monoclonal band in the serum of 11% of HCV positive individuals using immunoelectrophoresis. The age of the HCV positive subjects with a MG was significantly greater than that of chronic hepatitis C subjects without a MG (58 years vs 49 years, P < 0.001). In fact, the frequency of MGUS in a normal population is age-dependent being about 1% in individuals >50 years and 3% in those >70 years [17]. The results of the present study suggest that MG appears in subjects with chronic hepatitis C at an earlier age. Forty-four per cent of the HCV positive subjects (14 of 32) with a MG had an IgG monoclonal protein. Moreover, the average IgG level was significantly greater in those with a MG than those without such a protein (1996 ± 162 vs 1680.7 ± 160, P < 0.05). Thus, as is the case for the normal population, a MG of the IgG-type is the most common monoclonal immunoglobulin found in chronic HCV subject. In previous studies a high prevalence of anti-HCV antibodies has been reported in individuals with malignant LPD [8,18–22]. Hausfater et al. [20] in a French population, reported a prevalence of a malignant LPD of 2.5%, this finding is much lower than in North American and Southern European populations. On the contrary, Ohsawa et al. [21] in a Japanese population and Mele et al. [22] in Italian population, reported a moderate association between HCV infection and NHL [21, 22]. In the present study found that 4.3% of the individuals with chronic hepatitis C had malignant LPDs/plasma cell disorder. Clonal expansion of CD19+ B lymphocytes has been demonstrated in hepatic tissue in cases of chronic HCV infection. Moreover, with treatment and eradication of HCV, a reduction in the rate of B-cell clonal proliferation and expansion has been reported [15,16,18,23,24].
2004 Blackwell Publishing Ltd, Journal of Viral Hepatitis, 11, 302–309 308 R. Idilman et al. Several investigators have reported a strong association between the presence of MC and HCV infection [4,23–28]. In the present study, the overall prevalence of cryoglobulinaemia was 22% in individuals with chronic hepatitis C with a MG. The prevalence of cryoglobulinaemia in HCV positive subjects with a MG associated with malignant LPD’s and plasma cell disorder was significantly higher than that present in individuals who are HCV positive with a MGUS as well as in HCV positive individuals without MG (50, 8, 12% respectively; P < 0.01). However no significant difference in the prevalence of cryoglobulinaemia among individuals with chronic hepatitis C with and without a MG was found (22% vs 12%, P ¼ ns). On the contrary, 23% of the HCV positive individuals with cryoglobulinaemia were MG positivity. Noncryoglobulinaemic HCV positive individuals were found also to be MG positive, a finding similar to that reported by Schott et al. [28,29]. Based upon those results, we suggest that HCV positive individuals with cryoglobulinaemia should be investigated for the presence of a MG and the possibility of a malignant LPDs. The prevalence of RF activity in individuals with chronic HCV infection ranges from 24 to 76% [23,30]. RF production in individuals with chronic hepatitis C can be associated with either an oligoclonal or monoclonal intrahepatic B-cell expansion [23]. In the present study, no differences in the prevalence of RF reactivity among HCV positive subjects with a MG associated with or without a malignant LPDs/ plasma cell disorder as well as in individuals with chronic hepatitis C without a MG. No significant difference in gender, race, initial ALT levels, disease duration, HCV viral load or the severity of the primary liver disease between HCV positive subjects with and without a MG was observed. There are limited data available on the distribution of HCV genotype among individuals with B-cell clonal LPD [31,32]. A high prevalence of HCV genotypes 2a and 2c has been reported recently by Italian investigators [6,31,33] despite the fact that genotype 1b is the most common HCV genotype found in Italy. Based upon this disparity, these investigators have suggested that HCV genotype 2a/c might be uniquely involved in the pathogenesis of HCV-associated LPDs [6,31]. In contrast to these studies, in the present series, no significant difference in HCV genotype distribution was seen between individuals with and without a MG, although HCV genotype 1b was more common in patients with a MG. 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