Whipple disease is rarely associated with the subsequent development of lymphoma, although the two may be related. We report a case in which a BCL2 rearrangement indicative of a clonal B-cell population was detected in a patient with active Whipple disease. After antibiotic therapy, the BCL2-rearranged population disappeared.

Case Report

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A 44-year-old man was referred because of possible abdominal lymphoma. He presented with a 2-year history of weight loss, lethargy, and seronegativity for rheumatoid factor. Six months before hospitalization, he had had intermittent episodes of diarrhea with fluid retention. A physical examination showed gross ascites. Although the liver and spleen were not enlarged, a computed tomographic scan showed massive involvement of mesenteric lymph nodes with hypodense infiltrates, cystic formation, and concentric thickening of the jejunal wall. Results of a neurologic examination were normal. The patient had a hemoglobin level of 102 g/L, a platelet count of 1120 x 10⁹/L, a leukocyte count of 15.5 x 10⁹/L, and a lymphocyte count of 0.75 x 10⁹/L. Duodenal and mesenteric lymph node biopsy specimens were infiltrated by periodic acid-Schiff-positive macrophages with microcyst formation containing lipid material. Microorganisms could not be cultured. Tropheryma whippelii was suspected, and Whipple disease was diagnosed.

The patient initially received intravenous penicillin G (12 000 000 U/d) and tobramycin (2 g/d) for 15 days and then received trimethoprim-sulfamethoxazole therapy (trimethoprim, 166 mg/d, and sulfamethoxazole, 320 mg/d) for 1 year. Improvement was noted after the first month of treatment, and the size of the mesenteric lymph nodes as measured by echography was normal 1 year after treatment was begun.

Methods

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Samples

We extracted DNA samples from the patient's bone marrow, mesenteric lymph nodes (at the time of diagnosis), and blood, which we also tested 3 and 12 months after antibiotic therapy. Samples of DNA from two patients with Whipple disease served as positive controls for T. whippelii. Placental and human spleen DNA samples were used as negative controls.

Polymerase Chain Reaction Assay

We detected T. whippelii sequences using a procedure previously described by Relman and colleagues [2]. Briefly, 500 ng of genomic DNA was amplified using primers pW3FE and pW2RB. The amplified product, obtained from the initial lymph node specimen, was cloned into pUC18 and sequenced for confirmation. One clone, which was completely homologous to the sequence of the 16S recombinant DNA of T. whippelii (GenBank data base, accession number M87484), was designated pWN1 and used after being labeled as a probe for confirmation of the specificity of the PCR products obtained from the other samples. We tested each sample at least twice in different experiments.

Southern Blot Analysis

The configurations of the immunoglobulin heavy chain, immunoglobulin light chain, T-cell receptor, BCL2 gene, and myc gene were analyzed by Southern blotting as previously described [3]. We successively hybridized the prepared blots with probes to the immunoglobulin (JH, J, C); a probe to the ß chain of the T-cell receptor; three probes to the BCL2 gene, including a 2.8-kb EcoRI-HindIII fragment containing the major breakpoint region (BCL2 mbr), a 4-kb EcoRI fragment containing the minor breakpoint cluster, and a 1.6-kb EcoRI complementary DNA fragment used to screen for rearrangements of the 5' BCL2 region [4]; and a probe to the myc oncogene (1.4-kb ClaI/EcoRI containing the third exon) [5].

Results

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Duodenal and lymph node biopsy specimens showed massive infiltration by periodic acid-Schiff-positive macrophages (Figure 1, top left and top right). Bone marrow examination also showed small gram-positive rod-like organisms compatible with T. whippelii within histiocytic cells.

View larger version (161K): [in this window] [in a new window]   Figure 1. Studies in a patient with Whipple disease. Top left. Whipple disease in the mesenteric lymph nodes. Normal underlying architecture is destroyed and replaced by histiocytic cells with foamy cytoplasm and fatty cysts (original magnification x 250). Top right. Duodenum involvement with periodic acid-Schiff-positive foamy histiocytes in the lamina propria (original magnification x 250). Bottom left. Southern blot hybridization of polymerase chain reaction products with the 284 base-pair digoxigenin-labeled pWN1 probe. Lane 1 has a 1-kb DNA ladder serving as a size marker, lane 2 shows lymph node DNA, lane 3 shows blank water control, lane 4 shows bone marrow DNA, lane 5 represents a negative control extract from human spleen, and lane 6 represents a positive control with the clone pWN1 itself. Bottom right. Rearrangement of the BCL2 gene in different tested samples. Hybridization was done with a 32P-labeled 2.8-kb BCL2 mbr probe. Enzymes used are indicated below each lane. Lanes 1 to 3 represent the lymph node DNA; lanes 4 and 5 represent the bone marrow extract; and lanes 6 and 7 represent whole blood extract at the time of diagnosis and 3 months after treatment. The rearranged band from lane 6 has disappeared in lane 7. The normal germline bands are strong, and the rearranged bands are indicated by arrowheads.

Polymerase chain reaction assays that were done using the primers specific to Whipple disease generated a product of the predicted size (284 base pairs) when DNA samples extracted from the patient's lymph nodes, bone marrow, and blood were used. The DNA samples that had been extracted from the patient's initial blood sample generated a weak band that failed to amplify after 3 and 12 months of treatment. As a specific probe for subsequent Southern blot analysis, we used the nucleotide sequence of the pWN1 clone that was identical to the domain that spans nucleotides 965 through 1231 of the T. whippelii 16S recombinant DNA [2] (Figure 1, bottom left).

Southern blot analysis showed an isolated BCL2-mbr rearrangement in the DNA samples that we extracted from the patient's lymph nodes, bone marrow, and whole blood. This rearranged band was found with three different enzymes (BamHI, BglII, and EcoRI), excluding a possible restriction-site polymorphism. The rearranged band did not appear in blood samples taken 3 and 12 months after treatment (Figure 1, bottom right). No other probes showed an abnormality.

Discussion

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We report finding a clonal BCL2 gene rearrangement in a patient with Whipple disease. BCL2 gene rearrangements only occur in B cells and are associated with follicular lymphomas. We found no evidence of lymphoma in the results of clinical and histologic examinations of the patient. These examinations, however, showed characteristic Whipple disease, which was confirmed by a PCR assay for T. whippelii and by the patient's response to antibiotic therapy.

Although the detection of a BCL2 major break-point rearrangement suggests the presence of clonal B cells, we could not find a rearrangement in the immunoglobulin gene locus, despite using several probes. This may be because of the higher sensitivity of the BCL2 rearrangement detection by Southern blotting or because the BCL2 rearrangement might exist in a non-B-cell population. Because such a genetic abnormality has never been described in non-B cells, we believe it is more likely that the clonal population is of B-cell origin. Previous studies have identified functional abnormalities of B cells in patients with Whipple disease, but genetic abnormalities have not been described in patients without associated lymphoma [6, 7].

The clonal population was detected in the patient's blood samples only during the active phase of disease and not when the patient was in remission, suggesting that there may be a relation between the clonal population and Whipple disease. It is interesting to speculate that this clone was antigenically driven by T. whippelii and expanded to levels that could be detected by Southern blotting and PCR analysis. After successful treatment and eradication of the antigenic stimulus, the clone regressed.

Lymphomas are associated with rare cases of Whipple disease [8, 9], and these may be cases in which oncogenic transformation occurs in the antigenically driven clone. Precedent for such a model exists in Helicobacter pylori infection, which can be associated with mucosal-related lymphomas [10]. Antibiotic treatment may be effective against these lymphomas, supporting a model of antigenically driven clonal proliferations [11].

Drs. Pron and Pierre: Service de Microbiologie, Hopital Necker-Enfants Malades, 149 rue de Sevres, 75743 Paris Cedex 15, France.

Drs. Lefranc and Soua: Laboratoire d'ImmunoGenetique Moleculaire, Institut de Genetique Moleculaire de Montpellier, UMR CNRS 9942, BP505I, 34033 Montpellier Cedex 1, France.

Dr. Angonin: Service d'Anatomo-Pathologie, Hopital Universitaire Jean Minjoz, 25030 Besancon Cedex, France.

Drs. de Wazieres and Dupond: Service de Medecine Interne, Hopital Universitaire Jean Minjoz, 25030 Besancon Cedex, France.