We report an unusual case of Epstein-Barr virus (EBV)associated mucosal-associated lymphoid tissue (MALT) lymphoma involving the lungs, kidneys, and axillary lymph nodes in a child with congenital hypoadrenalism and panhypopituitarism. The patient presented with an aggressive clinical course and histologic evolution. Initial biopsies (1994) of the lung and kidney revealed histologic features of low-grade B-cell MALT lymphoma with lymphoepithelial lesions within the renal tubules and bronchial epithelium. Subsequent biopsies (1996, 1997, and 1999) revealed progressively greater cytologic atypia, polymorphism, and necrosis; an increased mitotic rate; and a preponderance of large cells, indicative of progression from a low-grade to a high-grade MALT lymphoma. Immunophenotyping of the lung and lymph node lesions revealed identical surface marker profiles: cells were CD19^sup +^, CD20^sup +^, immunoglobulin (Ig) G^sup +^, kappa^sup +^, lambda^sup -^, CD5^sup -^, CD10^sup -^, CD23^sup -^, and Ig^sup -^, and also negative for T cell markers. Genotypic analysis demonstrated the presence of immunoglobulin heavy chain rearrangement and monoclonality of EBV in the lung lesion by Southern blot hybridization and polymerase chain reaction (PCR). The clinicopathologic features suggest that these lesions might represent an immunosupression-related continuum of low-grade to high-grade MALT lymphomas. Infection with EBV may have contributed to this tumor's aggressive clinical and histologic evolution.
(Arch Pathol Lab Med. 2000;124:1520-1524)
Mucosal-associated lymphoid tissue (MALT) lymphomas are a distinctive subset of non-Hodgkin's lymphomas. They are low grade, arise from extranodal lymphoid tissue, and tend to remain localized for prolonged periods.1 These lymphomas often occur in adults and are associated with a history of autoimmune disease such as Sjogren's syndrome or Hashimoto's thyroiditis or with chronic gastritis and Helicobacter pylori infection.2,3 Histologically, MALT lymphomas are characterized by extranodal (mucosal)-based proliferations of lymphoid cells of intermediate size with moderately irregular nuclei; pale cytoplasm; and well-defined cell membranes.2 These cells have been called "centrocyte-like cells" and often infiltrate normal epithelial structures, forming so-called lymphoepithelial lesions.2,4 Clinical and histologic progression to higher-grade lesions can occur.3,5
Epstein-Barr virus (EBV) has been implicated as a causative agent in a number of lymphoproliferative disorders, including infectious mononucleosis, Burkitt's lymphoma, nasal-type natural killer-cell lymphoma, and lymphoproliferative disorders related to immunodeficiency.6 Even though the specific molecular changes accompanying EBV associated lymphoproliferative disorders are not well defined, structural alterations of 1 or more proto-oncogenes such as c-myc and bcl-6 probably play a role in these disorders.6 Epstein-Barr virus is also more commonly associated with high-grade lymphomas than with low-grade lymphomas.7 However, to the best of our knowledge, no evidence exists indicating that EBV plays a role in the development of low-grade MALT lymphomas. This report documents a pediatric patient with B-cell MALT lymphoma with histologic progression to a polymorphic largecell lymphoma. The latter lymphoma exhibited histologic features reminiscent of the end spectrum of posttransplant lymphoproliferative disorders (PTLD). Using Southern blot hybridization, monoclonal EBV genome was detected, indicating a possible role for EBV in the transformation of the low-grade MALT lymphoma to high-grade large-cell lymphoma.
REPORT OF A CASE
A 9-year-old boy with a history of dysmorphic features and hypoadrenalism presented with right shoulder pain and fever in July 1994. Laboratory data included a hematocrit of 40%; a white blood cell count of 11.5 x 10^sup 9^/L with 69% neutrophils,14% lymphocytes, 11% monocytes, 2% eosinophils, 4% bands; a platelet count of 39.7 x 10^sup 9^/L; a blood urea nitrogen of 15 mg/dL; a creatinine of 0.3 mg/dL; and a lactate dehydrogenase (LDH) of 373 IU/L. Physical examination revealed a small child (less than third percentile in height and weight) with small ears and micrognathia without nasal structural abnormality. A chest radiograph and computerized tomographic (CT) scan revealed a nodular density in the right lung base and a right renal mass. Histologically, both lesions were diagnosed as low-grade MALT type B-cell lymphoma. The patient was treated with high-dose chemotherapy including adriamycin and prednisone for 1 year, to a maximal adriamycin dose of 360 mg/kg. He remained clinically stable.
In March 1996, a routine follow-up CT scan revealed left axillary lymphadenopathy as well as a density in the left kidney. A diagnosis of MALT type B-cell lymphoma with large-cell transformation was rendered from the lymph node biopsy. The patient received 2 courses of intensive chemotherapy including ot-interferon. In January 1997, the patient presented with recurrent pneumonia. The admission laboratory data showed a white blood cell count of 17 x 10^sup 9^/L, with 94% neutrophils, 2.7% lymphocytes, and 2.7% monocytes. The hematocrit was 33%, the platelet count was 26.3 x 10^sup 9^/L, and the LDH was 997 IU/L. A chest radiograph showed a right upper lobe infiltrate and an open biopsy of this lesion was diagnosed as a high-grade MALT type B-cell lymphoma. The patient continued to receive alpha-interferon and also received 4 weekly doses of Rituxan anti-CD20 monoclonal antibody. The patient had multiple recurrent pneumonias and remained in poor condition. In March 1999, the patient was admitted for worsening shortness of breath, fever, and hepatosplenomegaly. Laboratory studies showed a hematocrit of 40%, a white blood cell count of 1.0 x 10^sup 9^/L, a platelet count of 9.8 x 10^sup 9^/L, and an LDH of 2785 IU/L. Measurement of serum immunoglobulin levels gave the following values: IgA, 32 (normal, 65-349 mg/dL); IgG, 413 (normal, 638-1700 mg/dL); and IgM, 5 (normal, 200-440 mg/dL). A CT scan showed diffuse nodular infiltrates of both lungs. The infiltrates had increased in number and size when compared with the prior 1997 study. An open lung biopsy was performed and the lesion was diagnosed as a high-grade, diffuse, large B-cell lymphoma. The patient expired of multiple organ failure 20 days after admission. A postmorterm examination was not performed. The parents of the patient were unrelated and in good health; his 2 siblings and other relatives have no similar clinical findings.
MATERIALS AND METHODS
Immunofluorescence and Immunohistochemical Analyses
The Becton-Dickinson fluorescence-activated cell-sorter (Becton-Dickinson, San Jose, Calif) was employed for flow cytometric analysis. Cell suspensions from lung tissue and lymph node were prepared in RPMI (Roswell Park Memorial Institute) buffer and stained with fluorescein- or phycoerythrin-conjugated monoclonal antibodies for surface immunophenotyping (Pharmacia Biotech, Uppsala, Sweden). A broad panel of fluorochrome-conjugated monoclonal antibodies were used for the following cell surface markers: CD2, CD3, CD4, CDS, CD7, CDB, CD10, CD14, CD15, CD19, CD20, CD30, CD33, IgG, IgM, lambda, and kappa (Becton Dickinson). Controls were set up by replacing the monoclonal antibodies with a mouse immunoglobulin of the same isotype. After gating lymphoid cells on forward versus 90 deg light scatter, a minimum of 5000 cells were evaluated and multiparameter analysis of gated cell populations was performed to provide immunophenotypic information.
Immunostains were performed on formalin-fixed, paraffin-embedded tissue sections by using a peroxidase-labeled detection system with antibodies to CD3, CD20, IgG, IgM, kappa, lambda, CD45R0, and CD45RA (Dako Corporation, Carpinteria, Calif) according to a protocol (Ventana Medical System, Tucson, Ariz) described previously.8
Deoxyribonucleic Acid Extraction, Southern Blot Analysis, and Polymerase Chain Reaction
Genomic deoxyribonucleic and (DNA) was extracted from cryopreserved tissue using the salting-out procedure and immunoglobulin gene rearrangement was probed by Southern blot or polymerase chain reaction (PCR) as described previously.8 Briefly DNA was isolated with saturated sodium chloride solution after sodium dodecyl sulfate (SDS)-proteinase K lysis of the tissue. The resulting samples of DNA were digested with the restriction endonucleases HindIII, and BamHI, electrophoresed in agarose gels, denatured with alkali, neutralized, and transferred to nitrocellulose filters. The filters were hybridized with ^sup 32^P labeled DNA probes containing the immunoglobulin heavy-chain-- joining determinant region. The filter was then washed in standard sodium citrate and SDS at 60 deg C for 2 hours and autoradiographed at -70 deg C for 16 hours to 72 hours. Human tonsil tissue served as a germline control.
The EBV genome and clonality were determined by Southern blot hybridization and PCR with a probe specific for the EBV repeated genomic terminal repeat, as previously described.8,9 An EBV-containing Burkitt lymphoma cell line (Daudi) was used as an EBV positive control.
Histologic and Immunopathologic Examination
Biopsies of the right lower lobe of the lung and right kidney were performed in 1994. The pulmonary parenchyma was infiltrated by a dense lymphoplasmacytic infiltrate composed of centrocyte-like lymphoid cells, as well as numerous plasmacytoid cells and plasma cells (Figure 1, A). The centrocyte-like cells were small to intermediate in size with a moderate amount of clear cytoplasm. The nuclei appeared angulated, with inconspicuous nucleoli. Some of the neoplastic cells had infiltrated respiratory epithelium, producing lymphoepithelial lesions (Figure 1, A). In some foci, large numbers of large cells with a plasmacytoid appearance, dispersed nuclear chromatin, and distinct nucleoli were identified in clusters or intermixed with the centrocyte-like cells (Figure 1, A). Mitotic figures were rare and there was no evidence of necrosis. The diagnosis of low-grade MALT B-cell lymphoma with an increased number of large cells was rendered. Examination of the renal mass showed extensive lymphoplasmacytic infiltration of the renal cortex similar to that observed in the lung. The centrocyte-like cell population was more prominent in this lesion. Numerous plasmacytoid cells, plasma cells, and immunoblasts were admixed with the centrocytic cells. Lymphoepithelial lesions involving the proximal tubules were present (Figure 1, B), a typical histologic feature of low-grade MALT B-cell lymphoma.
Immunohistochemical staining revealed that both lesions were CD20^sup +^ and CD3^sup -^. Immunostaining for kappa and lambda light chains showed predominant staining of kappa light chain in both lesions, indicating a monotypic kappa population. Multiparameter analysis of the gated cell populations by flow cytometry revealed that cells were CD19^sup +^, CD20^sup +^, IgG^sup +^, kappa^sup +^, CD5^sup -^, CD10^sup -^, CD23^sup -^, IgM^sup -^, and lambda^sup -^, and also negative for T-cell markers. These results represent a phenotype of MALT lymphoma.3
The left axillary lymph node biopsy of 1996 revealed a similar diffuse lymphoproliferative process, but with a "starry-sky" pattern (Figure 2). Compared with the previous biopsies, this lesion had a greater preponderance of large cells, a much higher mitotic rate, and greater cytologic atypia. The cell population appeared more polymorphic and included numerous atypical immunoblasts and Reed-Steinberg-like cells (Figure 2), a pattern reminiscent of the polymorphic B-cell lymphoma observed in the setting of immunosuppression following organ transplant (posttransplant lymphoproliferative disorders [PTLD]).8,10,11
Immunophenotypic analysis of the lymph node by flow cytometry and immunohistochemistry revealed the same phenotypic features noted in the previous lung biopsy. The histologic similarity and identical phenotype when compared with the prior lung biopsy suggested that the 2 lesions were a continuum of the same process. Therefore, a diagnosis of MALT type B-cell lymphoma with large-cell transformation was rendered.
The open-lung biopsy of the right upper lobe of the lung in 1997 revealed a diffuse lymphoid infiltrate that resembled the previous lung biopsy, but had greater architectural distortion, focal necrosis, and greater mitotic activity and cytologic atypia. Scattered lymphoepithelial lesions were identified in residual bronchial structures (Figure 3, insert). The lymphoid cells included polymorphous small, intermediate, and large lymphoid cells; lymphoplasmacytoid cells; mature plasma cells; and immunoblasts. Some of the large transformed immunoblasts showed atypical nuclei and resembled Reed-Sternberg cells and ReedSternberg cell variants (Figure 3). These histologic features also resembled the continuous spectrum of PTLD. Immunostains confirmed that the phenotype was identical to that of the previous lung biopsy. The lesion was diagnosed as high-grade MALT type B-cell lymphoma.
The biopsies of both lungs in 1999 showed complete architectural distortion; extensive necrosis; an increased number of large bizarre cells; an increased number of Reed-Steinberg cells and their variants; an increased number of histiocytes; and depletion of lymphoid cells (Figure 4). Immunostains revealed only a few scattered B-positive, large bizarre cells and abundant reactive histiocytes. The lesion was diagnosed as recurrent, high-grade, diffuse large B-cell lymphoma; this diagnosis was supported by ancillary molecular studies using PCR.
Immunoglobulin Gene Rearrangements and EBV Detection from Lung Biopsies
The clonality and cell lineage of the lung lesions were analyzed by Southern blot hybridization (1994) and PCR (1999). Clonal immunoglobulin gene rearrangement of the heavy chain gene was identified using the restriction enzymes BamHI, HindIII or BglII, and Xbal digestion. This result was consistent with the immunophenotypic studies and suggested clonal B-cell expansion. The presence of EBV infection and clonality of the EBV genome were determined by evaluating the number and size of EBV terminal fragments by Southern blot hybridization (1994) and PCR (1999). The lung tissues from the 1994 and 1999 biopsies were hybridized to a probe containing the terminal tandem repeats of the EBV genome. A single strongly hybridized fragment was detected, indicating EBV monoclonal expansion and presence of the virus within the lesion.
This case documents the occurrence of pulmonary and renal low-grade MALT B-cell lymphoma with evidence of progressive large-cell transformation early in the clinical course of a 9-year-old boy. To the best of our knowledge, this is the youngest patient reported with a high-grade MALT type lymphoma.10 In contrast to the majority of MALT lymphomas, this lymphoma disseminated early to the lungs, lymph nodes, and kidneys; the kidney is a rare site for MALT lymphoma. Monoclonal EBV genome was demonstrated, suggesting a role for EBV in the clinical and histologic evolution of this lesion. This is the first case that demonstrates the histologic evolution from a lowgrade to a high-grade MALT lymphoma with polymorphic transformation complicated by EBV infection. The patient's immunodeficient status is believed to have contributed to the process.
The tumor initially (1994) had features of a low-grade MALT lymphoma with marked plasmacytoid differentiation and an increased number of large cells. This histologic appearance was reminiscent of plasmacytic hyperplasia in PTLD. The demonstration of monoclonality by both phenotypic and genotypic studies proved a malignant B-cell process. The phenotypic features determined by flow cytometry supported the diagnosis of MALT lymphoma rather than another low-grade lymphomas, such as small lymphocytic lymphoma, mantle-cell lymphoma, or follicular center-cell lymphoma.3
As the disease process progressed, the patient presented with worsening respiratory symptoms, more severe cytopenia, and increasing LDH levels, and also developed hepatosplenomegaly. Subsequent biopsies during this period (1996, 1997, and 1999) from the axillary lymph nodes and lung revealed a similar but progressively higher grade of diffuse lymphoproliferative process, representing a histologic progression from low-grade to high-grade MALT lymphoma with polymorphic transformation. The highgrade tumor was characterized by greater architectural distortion, necrosis, greater mitotic activity and cytologic atypia, increased polymorphism, and a preponderance of large cells. Some of the large atypical cells resembled Reed-Sternberg cells. These histologic features resembled those seen in immunodeficiency-related lymphoproliferative disorders such as PTLD.11
We recently reported a case of polymorphic B-cell lymphoma in a child infected with the human immunodeficiency virus.8 The patient had an EBV associated, highgrade lymphoma with similar polymorphous cell infiltrates and geographic necrosis; the lymphoma had an aggressive clinical course. The findings in the present case support a role for EBV in the pathogenesis of high-grade, "polymorphic" lymphomas that arise in immunosuppressed patients. A monoclonal EBV genome was demonstrated in the lung biopsy, a feature also usually assodated with immunodeficiency-related lymphoproliferative disorders. These findings suggested that the patient might indeed have had an underlying immunodeficiency state. The clinical history of recurrent sinusitis and pneumonia and low serum levels of IgA, IgG, and IgM in his last admission support this concept.
The presence of the EBV genome indicates that the virus may have played a role in the large-cell transformation and dissemination. Although the mechanisms that cause large-cell transformation are unknown, other studies support this hypothesis. For example, EBV has been detected more frequently in high-grade lymphomas than in lowgrade lymphomas.10 Recent in situ hybridization studies have demonstrated that EBV can be recognized only in the large-cell component but not in the low-grade component of MALT lymphoma.12 The proposed role of EBV in the development of EBV associated lymphoproliferative disorders involves the activation of oncogenes or inactivation of tumor suppressor genes. Although translocation and subsequent activation of the c-myc oncogene does not occur as frequently in immunocompromised lymphoproliferative disorders as in Burkitt's lymphoma,13 c-myc translocation has been associated with high-grade progression of lymphoma in transplant patients.14 Further investigation is required to determine whether c-myc or other oncogenes such as bcl-6 are involved in the histologic progression of MALT lymphoma.
Geographic necrosis, karyorrhexis, and polymorphous atypical cell infiltrates were found in the present case and was reminiscent of findings in EBV positive lymphoproliferative disorders such as infectious mononucleosis, posttransplant disorders, and iatrogenic lymphoproliferative disorders.11 Therefore, EBV appears to play a role not only in lymphomagenesis, but also in nonneoplastic cell prolif eration. EBV may induce the expression of cytokines and trigger an exuberant reactive process. The Epstein-Barr virus infects neoplastic and nonneoplastic cells and expresses a range of latent-cycle viral proteins including latent membrane protein-1.15 This protein has been shown to resemble proteins of the superfamily of tumor necrosis factor receptors and to interact with the family of tumor necrosis factor-receptor-associated factors,15 thereby producing cytokine and chemokine through activation of the nuclear factor-kappa (kappa)B pathway.16
In summary, the clinicopathologic features of this case suggest that the lesions might represent an immunosupression-related continuum of low-grade to high-grade MALT lymphomas. Epstein-Barr virus infection may have contributed to this lymphoma's aggressive clinical and histologic evolution.
1. Isaacson PG, Wright DH. Malignant lymphoma of mucosa-associated lymphoid tissue. A distinctive type of B-cell lymphoma. Cancer 1983;52:1410-1416.
2. Isaacson PG, Spencer ). Malignant lymphoma of mucosa-associated lymphoid tissue. Histopathology. 1987;11:445-462.
3. Harris N. Low-grade B-cell lymphoma of mucosa-associated lymphoid tissue and monocytoid B-cell lymphoma. Arch Pathol Lab Med. 1993;117:771-775. 4. Harris N. Extranodal lymphomas and mucosa-associated lymphoid tissue (MALT): a unifying concept. Am J Surg Pathol. 1991;15:879-884.
S. Isaacson PG. Gastrointestinal lymphoma. Hum Pathol.1994;25:1020-1029. 6. Weiss LM, Chang KL. Association of the Epstein-Barr virus with hematolymphoid neoplasia. Adv Anat Pathol. 1996;3:1-15.
7. Hummel M, Anagnostopoulos I, Korbjun P. EBV in B-cell non-Hodgkin's lymphomas: unexpected infection pattern and different incidence in low- and high-grade type. J Pathol. 1995;173:263-271.
8. Tao J, Valderrama E. Lymphoproliferative disorders in the lungs of children with acquired immunodeficiency syndrome: a report of two cases. Am / Surg Pathol. 1999;23:560-566.
9. Tao J, Savargaonkar P, Vallejo C, Cesarman E, Fuchs A. Aggressive natural killer (NK) cell lymphoma presenting as anterior mediastinal mass in a patient with acquired immune deficiency syndrome (AIDS). Arch Pathol Lab Med. 2000; 124:304-309.
10. Liu Q, Ohshima K, Masuda Y, Kikuchi M. Detection of the EBV in primary gastric lymphoma by in situ hybridization. Pathol Int. 1995;45:131-136.
11. Swerdlow SH. Classification of the posttransplant lymphoproliferative disorders: from the past to the present. Semin Diagn Pathol. 1997;14:2-7.
12. Vasef MA, Weiss LM, Chen YY, Medeiros LJ. Gastric lymphoepitheliomalike carcinoma and jejunal B-cell MALT lymphoma with large cell transformation. Am / Clin Pathol. 1996;105:560-566.
13. Brusamolino E, Pagnucco G, Bernasconi C. Secondary lymphomas: a review on lymphoproliferative diseases in immunocompromised hosts. Prevalence, clinical features and pathogenetic mechanisms. Haematologica. 1989;74:605622.
14. Locker J, Nalesnik M. Molecular genetic analysis of lymphoid tumors arising after organ transplantation. Am J Pathol. 1989;135:977-987.
15. Liebowitz D. EBV and a cellular signaling pathway in lymphomas from immunosuppressed patients. N Engl J Med. 1998;338:1413-1421.
16. Barnes PJ, Karin M. Nuclear factor-KB-a pivotal transcription factor in chronic inflammatory diseases. N Eng! J Med. 1997;336:1066-1071.
Jianguo Tao, MD, PhD; Leonard Kahn, MD
Accepted for publication March 8, 2000.
From the Department of Pathology, Long Island Jewish Medical Center, the Long Island Campus for the Albert Einstein College of Medicine, New Hyde Park, NY.
Reprints: Jianguo Tao, MD, PhD, Department of Pathology, Long Island Jewish Medical Center, 270-05 76th Ave, New Hyde Park, NY 11040.
Copyright College of American Pathologists Oct 2000
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