The association between monoclonal gammopathies and chronic liver diseases was reported before the hepatitis C virus (HCV) was identified [4-6]. Recently, HCV infection was seen in 15% of patients who had monoclonal gammopathies without cryoglobulinemia [7]; conversely, the prevalence of monoclonal gammopathies without cryoglobulinemia in patients with chronic HCV infection did not seem to differ from that in the general population [8].

We evaluated the prevalence of monoclonal gammopathies in patients with chronic HCV infection and the possible association of monoclonal gammopathies with particular HCV strains.

Methods

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Patients

From January 1995 to November 1995, all new patients with chronic liver diseases who were attending our outpatient clinic (Semeiotica Medica, Bologna, Italy) were consecutively enrolled and divided into two groups. The first group consisted of 239 patients with HCV infection, and the second consisted of 98 patients with liver diseases with causes other than HCV infection (76 were positive for the hepatitis B surface antigen [HBsAg], 9 had alcoholic liver disease, and 13 had primary biliary cirrhosis). Patients who were currently receiving antiviral or immunosuppressive therapies and patients who had received such treatments in the past were excluded. The study was approved by the ethical committee at the Universita di Bologna, and informed consent was obtained from all patients.

Chronic viral hepatitis was diagnosed on the basis of serum positivity for anti-HCV antibodies (Ortho Diagnostic Systems, Milan, Italy) or HBsAg (AUSRIA II, Abbott Laboratories, Chicago, Illinois), elevated serum alanine aminotransferase level, and liver histologic findings. In the absence of other causes of liver disease, alcoholic liver disease was diagnosed in men who reported a daily alcohol intake of more than 80 g for at least 1 year and in women who reported a daily alcohol intake of more than 60 g for at least 1 year. Primary biliary cirrhosis was diagnosed on the basis of serum positivity for antimitochondrial antibodies and liver histologic findings.

Laboratory Testing for Immunoglobulins

In all serum specimens, we performed electrophoresis of total proteins on acetate gel (Jookoo CTE 5000, Chemetron, Tokyo, Japan); measured immunoglobulin levels by using nephelometry (Behring Diagnostics, Inc., San Jose, California); performed immunoelectrophoresis for monoclonal immunoglobulin component (Immunofixation Electrophoresis, Beckman Instruments, Inc., Fullerton, California); and measured cryoglobulins according to the criteria outlined by Brouet and colleagues [9].

Virologic Tests

In HCV-positive patients who had a monoclonal band, HCV RNA and HCV genotype were determined. Serum HCV RNA was detected by nested polymerase chain reaction and by genotyping, as described elsewhere [10].

Bone Marrow Histology

A bone marrow trephine biopsy was done with a Jamshidi needle on patients who had monoclonal band on immunoelectrophoresis. The tissue samples were fixed and stained according to standard procedures. Immunohistochemistry was performed by using a large panel of antibodies [11]. Histologic examination was done by two experienced pathologists who were blinded to study group assignment.

Statistical Analysis

Categorical variables were compared by chi-square analysis or by the Fisher exact test; continuous variables were analyzed by the Student t-test for unpaired data. A P value less than 0.05 was considered statistically significant. The computations were performed by using SOLO (BMDP Statistical Software, SPS, Inc., Chicago, Illinois).

To evaluate differences in HCV genotype distribution between patients with and patients without monoclonal gammopathies, a group of randomly chosen HCV-positive patients without lymphoproliferative disorders was matched two to one for sex, age, liver histologic findings, and geographic origin with patients who had monoclonal gammopathy.

Results

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The HCV-positive and HCV-negative patient groups did not differ for age, sex, or liver histologic findings (Table 1).

Laboratory Testing for Immunoglobulins

All patients with serum monoclonal band (except one who was HBsAg-positive) belonged to the HCV-positive group (Table 1). The main clinical, laboratory, and histologic findings of HCV-positive patients with monoclonal gammopathy are reported in Table 2 and Table 3. In the HCV-negative patient with monoclonal gammopathy, the monoclonal component was IgM-. Among patients with HCV, those with monoclonal gammopathy were older than those without this condition (59 ± 11 years compared with 51 ± 14 years; P = 0.0041). No differences were found in the patients' sex (15 men and 12 women compared with 126 men and 86 women), liver histologic findings (13 patients with chronic hepatitis and 14 patients with cirrhosis compared with 149 patients with chronic hepatitis and 63 patients with cirrhosis), or prevalence of cryoglobulins (19% [5 of 27] compared with 9% [20 of 212]).

Virologic Findings

Serum HCV RNA was detected in 22 of 27 patients with monoclonal gammopathy (81%); in 11 of these 22 patients (50%), genotype 2a/c was detected as a single infection. This prevalence was significantly higher than the prevalence of genotype 2a/c in matched controls (8 of 44 [18%]; P = 0.009). Conversely, the prevalence of genotype 1b was significantly lower in patients with monoclonal gammopathy (6 of 22 [27%]) than in matched controls (30 of 44 [68%]; P = 0.002). Three patients (14%) with monoclonal gammopathy had mixed infection with genotypes 2a/c and 1b.

Bone Marrow Histology

Of the 27 HCV-positive patients with monoclonal gammopathy, 19 had bone marrow biopsy done at the Institute of Haematology of Policlinico S. Orsola, Bologna, Italy; 6 had biopsies done elsewhere; and 2 refused biopsy. Mature plasma cells that occupied less than 5% of the lacunar spaces were seen in 8 of 19 patients (polytypic plasmacytosis in 4 and monotypic plasmacytosis in 4). In 3 other patients, slightly atypical plasma cells that were monotypic for light-chain immunoglobulins and had focal clustering that occupied 8% to 10% of the lacunar spaces were found; the morphologic diagnosis of low-grade plasmacytoma, stage 1A, was made [12]. In 6 of the 8 remaining biopsy specimens, small lymphocytes were distributed in the interstitium or in small aggregates, and immunophenotyping showed their reactive nature. A plurifocal lymphoid infiltration with diffuse positivity for CD79a led to a diagnosis of B-cell lymphocytic lymphoma in the last 2 patients [13].

Clinical Diagnosis of Gammopathies

According to the Southwest Oncology Group criteria [1], the clinical classifications of monoclonal gammopathies in the 21 HCV-positive patients who were evaluated at our hospital (including the 2 who refused bone marrow biopsy) were monoclonal gammopathy of undetermined significance in 17 patients, smoldering myeloma in 2, and multiple myeloma in 2. In the 6 patients who were evaluated elsewhere, histologic and clinical data were consistent with monoclonal gammopathy of undetermined significance in 1 patient, smoldering myeloma in 1 patient, and multiple myeloma in 4 patients.

Patients with myeloma were significantly older than those with other monoclonal gammopathies (64 ± 3.8 years compared with 57.7 ± 11.7 years; P = 0.045). Monoclonal gammopathy was seen only in patients between 40 and 70 years of age (with the exception of one 19-year-old patient) and peaked in patients who were 60 to 69 years of age (21%). Patients with myeloma tended to have cirrhosis more often than did those with other types of monoclonal gammopathy (Table 2 and Table 3).

Discussion

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Our study shows that monoclonal gammopathies occur more frequently in patients with HCV-related liver diseases (11%) than in patients with other liver diseases (1%), who had a prevalence similar to that reported in the general population [14, 15]. Mangia and coworkers [8], however, found monoclonal gammopathies in 2% of patients with HCV infection. Such variant results can be caused by differences in analytical methods and patient enrollment. Unfortunately, Mangia and coworkers did not report the characteristics of the study sample or the methods used to screen for monoclonal gammopathy. In our study, we screened for serum monoclonal component by using immunoelectrophoresis. This may be important because not all patients with positive results on immunoelectrophoresis showed an appreciable M-peak at protein electrophoresis. Furthermore, we excluded patients who had been treated with interferon because this drug decreases the production of monoclonal immunoglobulin by the myeloma plasma cells [16].

A possible limitation of our study is that in some patients, bone marrow histologic examination was neither done nor evaluated by our pathologists. For all patients, however, we had enough data to apply the Southwest Oncology Group criteria for the final clinical diagnosis [1].

Monoclonal gammopathies are age-related disorders with a prevalence in the general population of about 1% in persons between 25 and 65 years of age, 3% in persons 70 to 80 years of age, and up to 10% in persons older than 80 years of age [2, 14, 15]. This age-related increase was also seen in our patients: Those with monoclonal gammopathies were significantly older, and multiple myeloma was more often seen in older patients. However, the disorder clearly occurred earlier in our study group than in the general population.

The prevalence of cryoglobulinemia in the HCV-positive patients in our study was lower than previously reported [17]. However, this prevalence did not differ in patients with and patients without monoclonal gammopathies. Moreover, the monoclonal component was type IgG in most patients. Thus, monoclonal gammopathies would seem to be B-cell disorders that are distinct from cryoglobulinemia-associated lymphoproliferative disease (which is characterized by an IgM monoclonal component).

An important finding was the association between HCV genotype 2a/c and monoclonal gammopathy. A high prevalence of HCV genotype 2 has also been seen in HCV-positive patients with B-cell non-Hodgkin lymphoma [18] or cryoglobulinemia [10]. The high prevalence of genotype 2a/c is even more striking because genotype 1b is the most prevalent in Italy and is present in 50% to 90% of HCV-infected patients [19]. Therefore, our results reinforce the hypothesis that genotype 2 may be more involved in lymphoproliferative disorders than other genotypes [10]. Genotype 2a/c has been considered a "benign" variant of HCV with respect to severity of liver damage [10, 20]. However, in more than 50% of our patients with monoclonal gammopathy, genotype 2a/c was associated with cirrhosis; this implies a long-standing infection.

In conclusion, our results show that the prevalence of monoclonal gammopathies in patients with HCV-related chronic liver disease is striking and is often associated with genotype 2a/c infection. Monoclonal gammopathy may be seen as an event that occurs late in the course of HCV infection, which supports the hypothesis that prolonged antigenic stimulation may be involved in the development of monoclonal gammopathy. Therefore, screening for monoclonal gammopathy should be indicated in patients with long-standing HCV infection.

Drs. Zignego and Giannini: Istituto di Medicina Interna, Universita di Firenze, viale Morgagni, 85-50134 Firenze, Italy.

Drs. Gherlinzoni and Tura: Istituto di Ematologia ed Oncologia Medica, Universita di Bologna, via Massarenti, 9-40138 Bologna, Italy.

Dr. Boni: Laboratorio Centralizzato, Policlinico S. Orsola, via Massarenti, 9-40138 Bologna, Italy.

Drs. Sabattini and Pileri: Servizio di Anatomia Patologica, Istituto di Ematologia ed Oncologia Medica, Universita di Bologna, via Massarenti, 9-40138 Bologna, Italy.