Original Article
Brief Report
Myeloma-Associated Paraprotein Directed against the HIV-1 p24 Antigen in an HIV-1-Seropositive Patient
N Engl J Med 1993; 328:1817-1819June 24, 1993
- Article
The spectrum of hematologic and immunologic abnormalities in human immunodeficiency virus type 1 (HIV-1) infection is broad1,2. Although the incidence of B-cell-associated neoplasms is increased, relatively few cases of multiple myeloma have been described3,4. There are, however, reports of patients in whom serum paraproteins present as single or oligoclonal bands5-11. These paraproteins have previously been thought of as part of the exuberant polyclonal response to HIV-110,11.
In this report, we describe an HIV-1-positive patient with myeloma whose IgG-κ paraprotein specifically recognized the HIV-1 p24 gag antigen. It is possible that an HIV-1 antigen-driven response played an important part in the pathogenesis of this patient's myeloma.
Case Report
A 30-year-old bisexual man presented with acute renal failure, a blood urea nitrogen concentration of 72 mg per deciliter (26 mmol per liter), and a creatinine concentration of 4.7 mg per deciliter (415 μmol per liter). The uric acid concentration was 12.8 mg per deciliter (0.77 mmol per liter). His hemoglobin level was 10.3 g per deciliter, and the hematocrit 31 percent. The formation of rouleaux was noted.
The patient's serum osmolality was 312 mOsm per kilogram, his serum total protein 12.8 g per deciliter, and his serum viscosity 3.1 times that of water (normal, 1.4 to 1.8). Agarose-gel protein electrophoresis and immunofixation of the patient's serum demonstrated an IgG-κ paraprotein at a concentration of 6.4 g per deciliter. Urine protein electrophoresis and immunofixation demonstrated the presence of 0.6 g of IgG-κ and free κ light chains per total volume.
A bone marrow biopsy revealed marked plasmacytosis, with many plasmablasts present. The aspirate contained 34 percent plasma cells, including many immature and binucleate forms. On the basis of the findings of serum and urine paraproteins, bone marrow plasmacytosis, anemia, increased uric acid, and renal failure, a diagnosis of multiple myeloma was considered.
Results
A test for HIV-1 antibody by enzyme immunoassay (Genetic Systems) was strongly positive. Western blot analysis (Bio-Rad) demonstrated multiple reactive bands corresponding to different pol, env, and gag antigens. Western blotting of serial dilutions of the patient's serum showed reactivity with p24 and p55 up to a dilution of 1:10,000, with the reactivity stronger for p24 than for p55.
These results suggested that the serum paraprotein might be directed against the HIV-1 p24 gag antigen. Since affinity chromatography has been used to characterize myeloma paraproteins,12 we tested this hypothesis by passing the patient's serum over a p24 affinity column constructed by coupling recombinant HIV-1 p24 antigen (a gift from Dr. C. DeBouck) to cyanogen bromide-activated Sepharose 4B beads (Pharmacia).
The column was washed with 0.01 M phosphate (pH 7.4) in 0.15 M sodium chloride (phosphate-buffered saline) until the effluent had an optical density at 280 nm comparable to that of phosphate-buffered saline. The effluent was collected and concentrated. Bound antibody was eluted with 4 ml of a glycine buffer (0.1 M, pH 2.0). The fraction containing the eluted antibody was collected, neutralized in TRIS (1.0 M, pH 8.0, 50 microliters per milliliter of solution), and concentrated (1 mg per milliliter in phosphate-buffered saline).
Figure 1Figure 1
Densitometric Scanning of Agarose-Gel Electrophoretograms of the Patient's Serum, the Affinity-Column Effluent, and Affinity-Purified Antibody. shows densitometric scans of electrophoretograms of the patient's serum, the column effluent, and the eluted antibody. The serum paraprotein bound measurably to the p24 affinity column, as evidenced by a decrease in the concentration of paraprotein in the column effluent. When it was eluted from the column, the immunoadsorbed antibody was present as a single band. Immunofixation demonstrated the affinity-purified antibody to be a single IgG-κ species that comigrated with the serum paraprotein (Figure 2Figure 2
Agarose-Gel Electrophoresis of the Patient's Serum and the Affinity-Purified Paraprotein Eluted from the Recombinant p24 Affinity Column.).HIV-1 Western blot analysis of the eluted antibody to determine its specificity showed strong and weak reactivity, respectively, against the gag antigens p24 and p55. The fainter reactivity for p55 was probably due to cross-reactivity between related gag gene products. Figure 3Figure 3
Western Blot Analysis of the Patient's Serum and the Eluted Antibody. shows that this pattern corresponded to those observed when the serum was serially diluted. In addition, the antibody was tested for reactivity with selected infectious agents other than HIV-1; it did not react with cytomegalovirus, streptolysin O, Treponema pallidum, or Toxoplasma gondii. Together, these findings indicated that the paraprotein in this patient was directed against the p24 antigen.To investigate further the clonal nature of the immune response, Southern blot analysis was performed on cells aspirated from the bone marrow as described elsewhere13. This analysis clearly demonstrated a clonal pattern of immunoglobulin gene rearrangement (Figure 4Figure 4
Southern Blot Analysis of the Patient's Bone Marrow Aspirate.).In addition, cytospin preparations of the aspirate that had been fixed in methanol at 4 °C for 30 minutes were studied by fluorescent immunochemistry. The vast majority of the plasma cells contained IgG-κ (more than 90 percent of those stained with anti-γ as compared with less than 5 percent of those stained with anti-micro; more than 90 percent of those stained with anti-κ as compared with less than 5 percent of those stained with anti-λ). Flow-cytometric analysis of a sample of permeabilized aspirated cells confirmed these results by demonstrating an overall γ:micro ratio of 8:1 and a κ:λ ratio of 7:1.
Discussion
It appears that the paraprotein of this HIV-1-infected patient with myeloma was directed specifically against the HIV-1 p24 antigen. The evidence of specificity stems from the measurable and selective immunoadsorption of the paraprotein from the patient's serum by a recombinant p24 affinity column. The affinity-purified antibody consisted of a single IgG-κ species that comigrated with the serum paraprotein.
Western blot analysis demonstrated antibody reactivity against HIV-1 gag antigens p24 and p55. Southern blot analysis, immunochemical analysis, and flow-cytometric analysis performed on a bone marrow aspirate strongly suggested a monoclonal process, although the possibility that more than one clone existed cannot be excluded without immunoglobulin sequencing.
It is unusual to identify a paraprotein in an HIV-1-positive patient with myeloma that is directed against one specific HIV-1 antigen. There have been previous reports of myeloma paraproteins directed against a wide variety of infectious agents, including bacteria such as streptococcus14-17. These studies suggest the existence of a causal relation between myeloma and antigenic stimulation, reminiscent of an earlier hypothesis that B-cell cancers may also be related to antigenic stimulation18,19.
Free p24 antigen is usually present in the circulation shortly after infection with HIV-1 and before the development of a humoral response20,21. We suggest that the lymphoproliferative disease developed in this patient at least partly because of an antigen-driven response to circulating p24 antigen. The specificity of this myeloma-associated paraprotein for p24 suggests that clonal selection by HIV-1 antigen played a part in the pathogenesis of this plasmacytic disorder.
We are indebted to Ms. Kathy Bayer, Ms. Mattie Gadson, and Ms. Emily Thompson for their expert assistance; to Drs. David Biggs, Marc Kahn, and Robert Rotche for obtaining bone marrow aspirates; and to Drs. Marilyn Senior, Bonnie Mochan, and Lorna Lynn for their assistance.
Source Information
From the Department of Pathology and Laboratory Medicine, University of Pennsylvania School of Medicine, 6 Founders Bldg., 3400 Spruce St., Philadelphia, PA 19104-6082, where reprint requests should be addressed to Dr. Silberstein.
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