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FSGS, we frequently have observed inflammatory mononuclear cells in close contact with sclerotic areas of glomeruli,3 which may directly promote FSGS through the release of factors that stimulate cell proliferation and generation of extracellular matrix. In summary, renal damage can develop in patients with the rare NK cell proliferative disease so that their renal function should be carefully monitored. The nature of the involved cells and the pathologic findings suggest a peculiar pathogenetic mechanism and indicate the need for future research into the role of the NK cell system in renal diseases. References
8. Farrant JM, Taylor CG, Beer SF. Nephrotic syndrome in chronic lymphocytic leukaemia. Br J Haematol 1988;69:419. 9. Fer MF, McKinney TD, Richardson RL, Hande KR, Oldham RK, Greco FA. Cancer and the kidney: renal complications of neoplasms. Am J Med 1981;71:704-718. 10. Hara J, Benedict SH, Champagne E, Mak TW, Minden M, Gelfand EW. Comparison of T cell receptor a, b, and g gene rearrangement and expression in T cell acute lymphoblastic leukemia. J Clin Invest 1988;81:989-996. 11. Lanier LL, Philips JH. Evidence for three types of human cytotoxic lymphocyte. Immunol Today 1986;79:132-134. 12. Orman SV, Schechter GP, Whang-Peng J, et al. Nephrotic syndrome associated with a clonal T-cell leukemia of large granular lymphocytes with cytotoxic function. Arch Intern Med 1986,146: 1827-1829. 13. Rambaldi A, Pelicci P, Allavena P, et al. T cell receptor /3 chain gene rearrangements in lymphoproliferative disorders of large granular lymphocytes/natural killer cells. J Exp Med 1985,162: 2156-2162. 14. Rambaldi A, Rossi V, Allavena P, et al. Lymphokine production in Ty lymphoproliferative disorders. Scand J Immunol 1986;23: 183-188. 15. Shaloub RJ. Pathogenesis of lipoid nephrosis: a disorder to T-cell function. Lancet 1974;2:556-560. 16. Seney FD, Federgreen WR, Stein H, Kashgarian M. A review of nephrotic syndrome associated with chronic lymphocytic leukemia. Arch Intern Med 1986;146:137-141. 17. Takihara Y, Champagne E, Griesser H, et al. Sequence and organization of the human T cell delta chain gene. Eur J Immunol 1988;18:283-291. 18. Touchard G, Preud'Homme JL, Aucoutourier P, et al. Nephrotic syndrome associated with chronic lymphocytic leukemia: an immunological and pathological study. Clin Nephrol 1989;31:107116. 19. White CA, Dillman RO, Royston I. Membranous nephropathy associated with an unusual phenotype of chronic lymphocytic leukemia. Cancer 1983;52:2253-2255.
Clonal Immunoglobulin Gene Rearrangement in Nodular Lymphoid Hyperplasia of the Gastrointestinal Tract Associated with Common Variable Immunodeficiency MICHAEL J. LASZEWSKI, M.D., JOHN D. KEMP, M.D., JAMES A. GOEKEN, M.D. FRANK A. MITROS, M.D. CHARLES E. PLATZ, M.D., AND FRED R. DICK, M.D.
The authors report a case of common variable immunodeficiency associated with nodular lymphoid hyperplasia of the gastrointestinal tract in which a clonal population of lymphoid cells was detected by immunophenotypic and genotypic studies on tissue obtained by colonoscopic biopsy. The patient has been followed up for more than 50 months without clinical, radiographic, or pathologic evidence of lymphoma. The significance of clonal rearrangement in the setting of immunodeficiency and the role
Received August 29, 1989; received revised manuscript and accepted for publication February 16, 1990. Address reprint requests to Dr. Dick: Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242.
Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa
of genotypic studies in defining lymphoid malignancy are discussed. (Key words: Common variable immunodeficiency; Nodular lymphoid hyperplasia; Immunophenotype; Gene rearrangement) Am J Clin Pathol 1990;94:338-343
COMMON VARIABLE IMMUNODEFICIENCY (CVID) is a heterogeneous form of primary immunodeficiency associated with hypogammaglobulinemia and a
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1. Bassan R, Buzzetti M, Marini B, Rambaldi A, Allavena P, Barbui T. Investigation of chonic lymphocytosis in adults. Am J Clin Pathol 1988;89:783-787. 2. Bassan R, Pronesti M, Buzzetti M, et al. Autoimmunity and B-cell dysfunction in chronic proliferative disorders of large granular lymphocytes/natural killer cells. Cancer 1989;63:90-95. 3. Bertani T, Zoja C, Abbate M, Rossini M, Remuzzi G. Age-related nephropathy in rats with intact kidneys exposed to diets with different protein contents. Lab Invest 1989;60:197-204. 4. Bottazzi B, Rambaldi A, Introna M, et al. Migratory capacity of large granular lymphocytes from T-gamma-lymphoproliferative disorders. Nat Immun Cell Growth Regul 1986;5:19-27. 5. Chan WC, Link S, Mawle A, Check I, Brynes RK, Winton EF. Heterogeneity of large granular lymphocyte proliferations: delineation of two major subtypes. Blood 1986,68:1142-1153. 6. Chan WC, Winton EF, Waldmann TA. Lymphocytosis of large granular lymphocytes. Arch Intern Med 1986;146:1201-1203. 7. Dabbs DJ, Morel-Maroger Striker L, Mignon F, Striker G. Glomerular lesions in lymphomas and leukemias. Am J Med 1986,80: 63-70.
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Recent advances in molecular biology and their application to diagnostic pathology have increased our understanding of lymphocyte differentiation and the development of lymphoid neoplasia.18,21 Detection of immunoglobulin and T-cell receptor beta chain gene rearrangement provides the diagnostic pathologist with a sensitive and relatively specific marker for lymphocyte clonality and the potential for the early detection of neoplasia.4,6,35 We studied fresh-frozen biopsy specimens of gastrointestinal mucosa from a patient with CVID and associated NLH using immunohistochemical and Southern blot hybridization techniques in an effort to characterize the lymphoid component and to determine whether an occult clonal lymphocyte population could be identified. The potential clinical, biologic, and pathologic significances of clonality in the setting of immunodeficiency are discussed. Report of a Case A 15-year-old white girl with insulin-dependent diabetes mellitus was diagnosed with CVID after she had presented with numerous bouts of otitis media, recurrent bronchitis, bacterial pneumonic infections, and a pharyngeal abscess. Her physical examination was remarkable only for the presence of clubbing. Her family history was remarkable in that both her father and a brother had been diagnosed with CVID. Her initial serum immunoglobulins in the autumn of 1984 were IgG 0.33 g/L (normal is 5.50-14.00 g/L), IgA 0.06 g/L (normal is 0.40-3.30 g/L) and IgM 0.22 g/L (normal is 0.55-3.30 g/L). Additional laboratory studies revealed microcytic anemia secondary to iron deficiency. The remaining laboratory studies, including thyroid function tests, were within normal limits. The patient was started on parenteral immunoglobulin therapy. In the spring of 1985 she developed diarrhea and weight loss with subsequent hypoalbuminemia. Rigid sigmoidoscopy revealed multiple sessile and pedunculated polyps. A barium enema showed the colon and terminal ileum to be studded with sessile and pedunculated polyps measuring 0.3-2.0 cm. Multiple colonic biopsies revealed lymphoid and juvenile-type polyps. A small bowel biopsy revealed giardiasis, nodular lymphoid hyperplasia, and a paucity of plasma cells.
Since 1985, she has been followed up as an outpatient, and although there has been no evidence of neoplasia or giardiasis, her lymphoid hyperplasia has not resolved.
Materials and Methods Biopsy specimens were fixed in buffered formalin, sectioned at 5 nm and stained with hematoxylin and eosin for morphologic studies. Immunophenotypic studies were performed on snapfrozen gastrointestinal mucosa obtained from endoscopic biopsies. Reagents used included fluorescein-conjugated polyclonal anti-IgG, anti-IgA, anti-IgD, anti-kappa, and anti-lambda (Kallestad Diagnostics, Austin, TX) and unconjugated monoclonal antibodies directed against the CD-20, CD-5, CD-2, CD-4 and CD-8 determinants (Bl, T4, T8, Tl 1, Coulter, Inc., Hialeah, FL; and T101, Hybritech, Inc., San Diego, CA). The monoclonal reagents were followed with a fluorescein-conjugated polyclonal antimouse G, A, M reagent (Coulter). Direct immunofluorescence microscopy was performed using a Leitz (Rockleigh, NJ) microscope, equipped with a 450-W xenon lamp. Unconjugated polyclonal reagents against immunoglobulins followed by biotinylated secondary antibodies were used in some studies, and then developed by the avidin-biotin complex (ABC) immunoperoxidase technique (BioGenex Laboratories, San Ramon, CA). For immunogenotypic studies, DNA was extracted from tissues with phenol and chloroform, precipitated with ethanol and dissolved. Five /ug of DNA were digested with restriction enzymes (Boehringer Mannheim, Mannheim, West Germany). BamHI and Hindlll restriction enzymes were used for immunoglobulin heavy chain gene (JH) analysis, BamHI for kappa chain gene (CK) analysis, and BamHI and EcoRI for T-cell receptor Beta chain gene (TCR-B) analysis. Placental DNA was used as a control. The digested DNA was electrophoresed on 0.8% agarose gel and transferred onto nitrocellulose paper by the method of Southern.32 The membranes were hybridized with random primed P 32 labeled probes, washed extensively, exposed to x-ray films for 3-72 hours and then developed. A 3.5-kilobase Hindlll fragment TCR-B probe was supplied by Dr. Jeffrey Sklar (Stanford Medical School),39 and 2.5-kilobase EcoRI fragment CK and 2.5kilobase Sau3A fragment JH probes were supplied by Dr. Philip Leder (Harvard Medical School).11,30 Results Pathologic Findings Histologic examination of multiple colonic biopsies revealed pedunculated juvenile-type polyps comprised of normal colonic mucosa lining dilated glands, many of which contained numerous neutrophils and fibrinous de-
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constellation of clinical and pathologic findings, including recurrent sinopulmonary infections, gastrointestinal disorders, and an increased incidence of gastrointestinal and lymphoreticular neoplasms. 512,31 The spectrum of gastrointestinal disease includes giardiasis and nodular lymphoid hyperplasia (NHL) with resultant diarrhea, malabsorption, and protein-losing enteropathy. There is an increased incidence of malignant neoplasms in patients with primary immunodeficiency, and the risk to patients with CVID has been reported to range from 4 to 1 6 % 12,16,33.34 Malignant lymphoma associated with CVID has been reported, with many of the tumors arising in the gastrointestinal tract.12,16,33,34 The association of malignant lymphoma and NLH has been observed both in the setting of CVID 810 ' 20,26 and without apparent hypogammaglobulinemia.14,24,25 The exact mechanism of the immunologic defect in CVID is unknown, and the pathogenic mechanism for the increased incidence of neoplasia is uncertain.
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A.J.C.P. • September 1990
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FIG. 1 (upper, left). Whole mount of the first polypectomy specimen showing large aggregates of submucosal lymphoid tissue (arrow). Hematoxylin and eosin (XI.3). FIG. 2 (upper, right). Reactive germinal centers within the mucosa of a polyp with surrounding perifollicular lymphocytes within the lamina propria. Hematoxylin and eosin (X40). FIGS. 3 and 4 (lower). Focus of immunoblasts within a lymphoid aggregate with less well-defined germinal centers. Hematoxylin and eosin FIG. 3 (left). (X40). FIG. 4 (right). (X160).
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bris. The surface epithelium was focally ulcerated. The stroma was inflamed and in several areas contained dense collagen. Adenomatous, hamartomatous, and/or hyperplastic features were not identified. The polyps contained large foci of lymphoid tissue with germinal centers within the submucosa and a paucity of plasma cells within the lamina propria (Figs. 1 and 2). Some of the lymphoid aggregates contained less well-defined germinal centers and foci of immunoblasts (Figs. 3 and 4). Sessile colonic polyps were comprised of hyperplastic lymphoid aggregates, many with germinal centers. Parasitic forms were not identified. The small bowel biopsies showed normal villi with a normal villous-to-crypt ratio. Parasitic forms consistent with Giardia were identified. The lamina propria and submucosa contained lymphoid nodules and a paucity of plasma cells.
lit C
Discussion Common variable immunodeficiency is a primary immunodeficient state manifested by hypogammaglobulinemia that usually presents later in life with recurrent sinopulmonary infections. There is a high but variable incidence of associated gastrointestinal disorders, including giardiasis, nodular lymphoid hyperplasia, a sprue-like
TCR-p Chain
Bam HI
Bam HI
Hind I I I
III 111 S*
So
Sj
ment of the JH and CK bands, whereas the second specimen with the polyclonal phenotype showed only germline bands by Southern blot hybridization (Figs. 5 and 6). The TCR-B blots showed no clonal rearrangement in either specimen (Fig. 7). The apparent absence of JH rearrangement with Hindlll probably is due to comigration of the rearranged band with the germline band. 6 The CK rearrangement initially appears inconsistent with the presence of a lambda immunophenotype; however, about 25% of lambda-producing tumors will show a clonal CK rearrangement, probably due to a nonproductive rearrangement of an allele that does not go on to produce lambda light chains.1
JH
C
S,
FlG. 6. Southern blot analysis of the immunoglobulin kappa light chain gene (CK) using BamHl restriction endonuclease digestion. Germline bands are present in both the control (C) and patient specimen lanes (SI and S2). An additional clonal rearranged band (arrow) is present in the first patient specimen (SI).
C
S-j
So
FlG. 5. Southern blot analysis of the immunoglobulin heavy chain (JH)gene using BamHl and Hindlll restriction endonuclease digestions. Germline bands are present in the control (C) and the patient specimen lanes (SI and S2). An additional clonal rearranged band (arrow) is present on the BamHl digested DNA from the first patient specimen (SI).
f 0
Hind III
i
8 , 8 2
-|?—§—i— c
S1
S2
FlG. 7. Southern blot analysis of the T-cell receptor beta chain gene (TCR-B) using BamHl and EcoRI restriction endonuclease digestions. Only germline bands are present in both the control (C) and patient specimen lanes (SI and S2).
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The immunophenotypic analyses were performed on two consecutive polypectomy specimens submitted fresh and snap-frozen in the spring of 1985 and the spring of 1986. The first, a large rectal lymphoid polyp, showed predominantly B-cells with most of the cells IgM and CD20 positive. Within the germinal centers, kappa and lambda light chains were present in normal numbers; however in an area with less well-defined germinal centers, a focus of immunoblasts appeared to be IgM-lambda restricted, suggesting the possibility of a small clonal population. IgG, IgA, and IgD were not identified within the germinal center cell population. Plasma cells were present in decreased numbers, and IgA staining plasma cells were not identified. Scattered within the lamina propria were T-cells with a normal mature peripheral T-cell phenotype. The second specimen, examined approximately one year later, showed polyclonal staining of hyperplastic lymphoid tissue with a paucity of plasma cells. No clonal proliferation was identified. Immunogenotypic studies were performed on both specimens. The first specimen showed clonal rearrange-
BamHl
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The immunophenotypic data in the present case are in keeping with that previously reported in patients with CVID and NLH. 27 The lymphoid follicles within the Peyer's patches were populated with lymphocytes bearing surface IgM-kappa and IgM-lambda, and there were decreased numbers of IgG and IgA containing cells. The perifollicular regions were populated by normal numbers of T-cells. In CVID associated with NLH, the number of plasma cells within the lamina propria is decreased, although the number of circulating B-lymphocytes is normal.5,35'36'37 Our case is unusual in that an apparent focal monoclonal expansion of B-cells was identified immunophenotypically and was then verified by gene rearrangement studies performed on the first polypectomy specimen. Malignant lymphomas of the gastrointestinal tract in adults have been reported to be associated with NLH, both with 810 ' 20 ' 26 and without14,24,25 immunodeficiency. Several authors postulate that this association may be more than just fortuitous and have further suggested that a similar relationship may exist between the benign lymphoepithelial lesion of the salivary gland seen with Sjogren's syndrome and the development of lymphoma. 14 Advances in the application of molecular biologic techniques have provided the diagnostic pathologist with
new tools for recognizing early clonal lymphoid proliferations but also have raised questions concerning oncogenesis and immunoregulation of lymphocyte proliferation. In the majority of situations, clonal immunoglobulin and antigen receptor gene rearrangement appear to be sensitive and relatively specific indicators of malignancy.4'6,35 In immunodeficient states, however, whether primary or secondary, the diagnostic interpretation of gene rearrangement may be difficult, especially in the setting of histopathology which is not diagnostic of malignancy.6 This is most evident in solid organ and bone marrow transplantation, where the Epstein-Barr Virus (EBV) has been shown to lead to lymphoproliferative disorders (LPD) with polyclonal, oligoclonal, or monoclonal B-cell proliferations. It is thought that in these situations there is a lack of T-cell suppression of B-cells containing the EBV genome, and that this can lead to a fatal lymphoproliferative disorder {i.e., lymphoma). 7,17,29 The cases of malignant lymphoma associated with CVID and NLH have not been studied immunogenotypically or for the presence of the EBV genome. Clonal gene rearrangement with "benign" histology has been reported in lymphoepithelial lesions of the salivary gland in patient's with Sjogren's syndrome, 9 angioimmunoblastic lymphadenopathy with dysproteinemia,6'22 AIDS associated lymphadenopathy, 628 and lymphoid hyperplasia in the Wiskott-Aldrich syndrome.4'6 All of these disorders, as well as the present case, have in common an immunologic defect with a tendency for exaggerated Bcell proliferation and a predisposition for the development of lymphoma. In the present case, the finding of an occult clonal expansion at the molecular level without obvious histologic evidence of neoplasia may simply reflect the best response available (in terms of affinity and/or avidity) from a deficient immune system to an unknown antigenic stimulus. It also seems conceivable that other such clonal expansions may develop and then regress without necessarily proceeding to malignant transformation. Perhaps the most appropriate terminology in this setting would be "monoclonal gene rearrangement of uncertain significance"15 analogous to that put forward by Kyle,19 in reference to monoclonal gammopathies. Although in our patient there has been no evidence of subsequent neoplastic transformation, we cannot entirely exclude the possibility that an isolated incipient lymphoma was completely excised during the polypectomy. Our findings are further evidence that the detection of antigen receptor gene rearrangement, hence clonality, should not necessarily be equated with neoplasia, especially in the setting of immunodeficiency. Molecular biologic techniques are providing us with new insights into the nature of lymphocyte differentiation and neoplasia and challenge the pathologist to carefully define criteria for the diagnosis of lymphoid malignancy.
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syndrome, achlorhydria and pernicious anemia. 512 ' 31 Malignant neoplasms have been reported to occur with increased frequency, with primary gastrointestinal lymphoma and gastric carcinoma being the most common tumors. 1216,33 The disorder may be familial; however, a mode of inheritance has not been identified.31 In general, follicular lymphoid hyperplasia is the rule with normal to enlarged tonsillar tissue, NLH, and splenomegaly.512 The immunologic defect appears to involve the final stage of B-cell maturation. The numbers of circulating B- and T-cells usually are normal, but there is an apparent decrease to absence of mature plasma cells and impairment of immunoglobulin synthesis.5,36'37 In some patients, however, deficient T-helper function or excessive T-suppressor activity have been implicated as the cause of deficient immunoglobulin production.31,37 Nodular lymphoid hyperplasia is a well-recognized entity that most often is encountered as a normal variant in the pediatric population, usually involving the terminal ileum and/or colon,23 and in immunodeficiency states. 213 In recent years, cases of NLH in immunocompetent adults have been reported24'25'38 and of interest is the presence of Giardia in these patients as well as those with known CVID. Whether this may represent some form of localized gastrointestinal immunodeficiency is uncertain. The causal relationship of the parasitosis to the development of NLH has been a point of debate in the literature.3'37,38 In the majority of the cases reported, as in our case, the NLH did not regress after eradication of the infection.3,25
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Acknowledgments. The authors wish to thank Linda Schneekloth and Marcia Wood for typing the manuscript and Joel Carl for his photographic assistance.
References
20. Lamers CBHW, Wagener DJT, Assmann KJM, Van Tongeren JHM. Jejunal lymphoma in a patient with primary adult-onset hypogammaglobulinemia and nodular lymphoid hyperplasia of the small intestine. Dig Dis Sci 1980;25:553-557. 21. Leder P. The genetics of antibody diversity. Sci Am 1982;246(5): 102-115. 22. Lipford EH, Smith HR, Pittaluga S, et al. Clonality of angio-immunoblastic lymphadenopathy and implications for its evolution to malignant lymphoma. J Clin Invest 1987;79:637-642. 23. Louw JH. Polypoid lesions of the large bowel in children, with particular reference to benign polyposis. J Pediatr Surg 1968,3:195209. 24. Matuchansky C, Morichau-Beauchant M, Touchard G, et al. Nodular lymphoid hyperplasia of the small bowel associated with primary jejunal malignant lymphoma. Gastroenterology 1980;78:15871592. 25. Matuchansky C, Touchard G, Lemaire M, et al. Malignant lymphoma of the small bowel associated with diffuse nodular hyperplasia. N Engl J Med 1985;313(3): 166-171. 26. Maurer HS, Gotoff SP, Allen L, Bolan J. Malignant lymphoma of the small intestine in multiple family members—association with an immunologic deficiency. Cancer 1976;37:2224-2231. 27. Nagura H, Kohler PF, Brown WR. Immunocytochemical characterization of the lymphocytes in nodular lymphoid hyperplasia of the bowel. Lab Invest 1979;40(l):66-73. 28. Pelicci P-G, Knowles DM, Arlin ZA, et al. Multiple monoclonal B cell expansions and c-myc oncogene rearrangements in acquired immune deficiency syndrome-related lymphoproliferative disorders. J Exp Med 1986;164:2049-2076. 29. Purtillo DT, Klein G. Introduction to Epstein-Barr virus and lymphoproliferative diseases in immunodeficient individuals. Cancer Res 1981:41:4209. 30. Ravetch JV, Siebenlist U, Korsmeyer S et al. Structure of the human immunoglobulin locus: characterization of embryonic and rearranged J and D genes. Cell 1981;27:583-591. 31. Rosen FS, Cooper MD, Wedgwood RJP. The primary immunodeficiencies (part II). N Engl J Med 1984;311(5):300-310. 32. Southern EM. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol 1975;98:503-517. 33. Spector BD, Perry GS III, Kersey JH. Genetically determined immunodeficiency diseases (GDID) and malignancy: report from the immunodeficiency—cancer registry. Clin Immunol Immunopathol 1978;11:12-29. 34. Waldmann TA, Strober W, Blaese RM. Immunodeficiency disease and malignancy—various immunologic deficiencies of man and the role of immune processes in the control of malignant disease. Ann Intern Med 1972;77:605-628. 35. Waldmann TA, Davis MM, Bongiovanni KF, Korsmeyer SJ. Rearrangements of genes for the antigen receptor on T cells as markers of lineage and clonality in human lymphoid neoplasms. N Engl J Med 1985;313:776-783. 36. Waldmann TA, Blaese RM, Broder S, Krakauer RS. Disorders of suppressor immunoregulatory cells in the pathogenesis of immunodeficiency and autoimmunity. Ann Intern Med 1978;88: 226-238. 37. Waldmann TA. Immunodeficiency: immunoregulation and immunogenetics. Clin Immunol Immunopathol 1986;40:25-36. 38. Ward H, Jalan KN, Maitra TK, Agarwal SK, Mahalanabis D. Small intestinal nodular lymphoid hyperplasia in patients with giardiasis and normal serum immunoglobulins. Gut 1983;24:120-126. 39. Weiss LM, Hy E, Wood GS, et al. Clonal rearrangements of T-cell receptor genes in Mycosis fungoides and dermatopathic lymphadenopathy. N Engl J Med 1985;313:539-544.
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1. Aisenberg AC, Wilkes BM, Jacobson JO, Harris NL. Immunoglobulin gene rearrangement in adult non-Hodgkin's lymphoma. Am J Med 1987;82:738-744. 2. Ajdukiewicz AB, Youngs GR, Bouchier IAD. Nodular lymphoid hyperplasia with hypogammaglobulinemia. Gut 1972;13:589-595. 3. Ament ME, Rubin CE. Relation of giardiasis to abnormal intestinal structure and function in gastrointestinal immunodeficiency syndromes. Gastroenterology 1972;62(2):216-226. 4. Arnold A, Cossman J, Bakhshi A, Jaffe ES, Waldmann TA, Korsmeyer SJ. Immunoglobulin-gene rearrangement as unique clonal markers in human lymphoid neoplasms. N Engl J Med 1983,309(26): 1593-1599. 5. Buckley RH. Humoral immunodeficiency. Clin Immunol Immunopathol 1986;40:13-24. 6. Cossman J, Uppenkamp M, Sundeen J, Coupland R, Raffeld M. Molecular genetics and the diagnosis of lymphoma. Arch Pathol Lab Med 1988;112:117-127. 7. Davey DD, Kamat D, Laszewski MJ, et al. Epstein-Barr virus-related lymphoproliferative disorders following bone marrow transplantation: an immunologic and genotypic analysis. Mod Pathol 1989;2(l):27-34. 8. Durham JC, Stephens DS, Rimland D, Nassar VH, Spira TJ. Common variable hypogammaglobulinemia complicated by an unusual T-suppressor/cytotoxic cell lymphoma. Cancer 1987;59: 271-276. 9. Fishleder A, Tubbs R, Hesse B, Levine H. Uniform detection of immunoglobulin-gene rearrangement in benign lymphoepithelial lesions. N Engl J Med 1987;316(18):1118-1121. 10. Gonzalez-Vitale JC, Gomez LG, Goldblum RM, Goldman AS, Patterson M. Immunoblastic lymphoma of small intestine complicating late-onset immunodeficiency. Cancer 1982;49:445-449. 11. Heiter PA, Maizel JV, Leder P. Evolution of human immunoglobulin KJ region genes. J Biol Chem 1982;257:1516. 12. Hermans PE, Diaz-buxo J A, Stobo ID. Idiopathic late-onset immunoglobulin deficiency. Am J Med 1976;61:221-237. 13. Jacobsen KW, deShazo RD. Selective immunoglobulin A deficiency associated with nodular lymphoid hyperplasia. J Allergy Clin Immunol 1979;64(6):516-521. 14. Kahn LB, Novis BH. Nodular lymphoid hyperplasia of the small bowel associated with primary small bowel reticulum cell lymphoma. Cancer 1974;33:837-844. 15. Kamat D, Laszewski MJ, Kemp JD, et al. The diagnostic utility of immunophenotyping and immunogenotyping in the pathologic evaluation of lymphoid proliferations. Med Pathol 1990;3(2): 105— 112. 16. Kersey JH, Spector BD, Good RA. Primary immunodeficiency diseases and cancer: the immunodeficiency-cancer registry. Int J Cancer 1973;12:333-347. 17. Klein G, Purtillo DT. Summary: symposium on Epstein-Barr virusinduced lymphoproliferative diseases in immunodeficient patients. Cancer Res 1981;41:4302. 18. Korsmeyer SJ, Hieter PA, Sharrow SO, Goldman CK, Leder P, Waldmann TA. Normal human B cells display ordered light chain gene rearrangements and deletions. J Exp Med 1982; 156:975985. 19. Kyle RA. Monoclonal gammapathy of undetermined significance: natural history in 241 cases. Am J Med 1978;64:814.
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FSGS, we frequently have observed inflammatory mononuclear cells in close contact with sclerotic areas of glomeruli,3 which may directly promote FSGS through the release of factors that stimulate cell proliferation and generation of extracellular matrix. In summary, renal damage can develop in patients with the rare NK cell proliferative disease so that their renal function should be carefully monitored. The nature of the involved cells and the pathologic findings suggest a peculiar pathogenetic mechanism and indicate the need for future research into the role of the NK cell system in renal diseases. References
8. Farrant JM, Taylor CG, Beer SF. Nephrotic syndrome in chronic lymphocytic leukaemia. Br J Haematol 1988;69:419. 9. Fer MF, McKinney TD, Richardson RL, Hande KR, Oldham RK, Greco FA. Cancer and the kidney: renal complications of neoplasms. Am J Med 1981;71:704-718. 10. Hara J, Benedict SH, Champagne E, Mak TW, Minden M, Gelfand EW. Comparison of T cell receptor a, b, and g gene rearrangement and expression in T cell acute lymphoblastic leukemia. J Clin Invest 1988;81:989-996. 11. Lanier LL, Philips JH. Evidence for three types of human cytotoxic lymphocyte. Immunol Today 1986;79:132-134. 12. Orman SV, Schechter GP, Whang-Peng J, et al. Nephrotic syndrome associated with a clonal T-cell leukemia of large granular lymphocytes with cytotoxic function. Arch Intern Med 1986,146: 1827-1829. 13. Rambaldi A, Pelicci P, Allavena P, et al. T cell receptor /3 chain gene rearrangements in lymphoproliferative disorders of large granular lymphocytes/natural killer cells. J Exp Med 1985,162: 2156-2162. 14. Rambaldi A, Rossi V, Allavena P, et al. Lymphokine production in Ty lymphoproliferative disorders. Scand J Immunol 1986;23: 183-188. 15. Shaloub RJ. Pathogenesis of lipoid nephrosis: a disorder to T-cell function. Lancet 1974;2:556-560. 16. Seney FD, Federgreen WR, Stein H, Kashgarian M. A review of nephrotic syndrome associated with chronic lymphocytic leukemia. Arch Intern Med 1986;146:137-141. 17. Takihara Y, Champagne E, Griesser H, et al. Sequence and organization of the human T cell delta chain gene. Eur J Immunol 1988;18:283-291. 18. Touchard G, Preud'Homme JL, Aucoutourier P, et al. Nephrotic syndrome associated with chronic lymphocytic leukemia: an immunological and pathological study. Clin Nephrol 1989;31:107116. 19. White CA, Dillman RO, Royston I. Membranous nephropathy associated with an unusual phenotype of chronic lymphocytic leukemia. Cancer 1983;52:2253-2255.
Clonal Immunoglobulin Gene Rearrangement in Nodular Lymphoid Hyperplasia of the Gastrointestinal Tract Associated with Common Variable Immunodeficiency MICHAEL J. LASZEWSKI, M.D., JOHN D. KEMP, M.D., JAMES A. GOEKEN, M.D. FRANK A. MITROS, M.D. CHARLES E. PLATZ, M.D., AND FRED R. DICK, M.D.
The authors report a case of common variable immunodeficiency associated with nodular lymphoid hyperplasia of the gastrointestinal tract in which a clonal population of lymphoid cells was detected by immunophenotypic and genotypic studies on tissue obtained by colonoscopic biopsy. The patient has been followed up for more than 50 months without clinical, radiographic, or pathologic evidence of lymphoma. The significance of clonal rearrangement in the setting of immunodeficiency and the role
Received August 29, 1989; received revised manuscript and accepted for publication February 16, 1990. Address reprint requests to Dr. Dick: Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa 52242.
Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa
of genotypic studies in defining lymphoid malignancy are discussed. (Key words: Common variable immunodeficiency; Nodular lymphoid hyperplasia; Immunophenotype; Gene rearrangement) Am J Clin Pathol 1990;94:338-343
COMMON VARIABLE IMMUNODEFICIENCY (CVID) is a heterogeneous form of primary immunodeficiency associated with hypogammaglobulinemia and a
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1. Bassan R, Buzzetti M, Marini B, Rambaldi A, Allavena P, Barbui T. Investigation of chonic lymphocytosis in adults. Am J Clin Pathol 1988;89:783-787. 2. Bassan R, Pronesti M, Buzzetti M, et al. Autoimmunity and B-cell dysfunction in chronic proliferative disorders of large granular lymphocytes/natural killer cells. Cancer 1989;63:90-95. 3. Bertani T, Zoja C, Abbate M, Rossini M, Remuzzi G. Age-related nephropathy in rats with intact kidneys exposed to diets with different protein contents. Lab Invest 1989;60:197-204. 4. Bottazzi B, Rambaldi A, Introna M, et al. Migratory capacity of large granular lymphocytes from T-gamma-lymphoproliferative disorders. Nat Immun Cell Growth Regul 1986;5:19-27. 5. Chan WC, Link S, Mawle A, Check I, Brynes RK, Winton EF. Heterogeneity of large granular lymphocyte proliferations: delineation of two major subtypes. Blood 1986,68:1142-1153. 6. Chan WC, Winton EF, Waldmann TA. Lymphocytosis of large granular lymphocytes. Arch Intern Med 1986;146:1201-1203. 7. Dabbs DJ, Morel-Maroger Striker L, Mignon F, Striker G. Glomerular lesions in lymphomas and leukemias. Am J Med 1986,80: 63-70.
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Recent advances in molecular biology and their application to diagnostic pathology have increased our understanding of lymphocyte differentiation and the development of lymphoid neoplasia.18,21 Detection of immunoglobulin and T-cell receptor beta chain gene rearrangement provides the diagnostic pathologist with a sensitive and relatively specific marker for lymphocyte clonality and the potential for the early detection of neoplasia.4,6,35 We studied fresh-frozen biopsy specimens of gastrointestinal mucosa from a patient with CVID and associated NLH using immunohistochemical and Southern blot hybridization techniques in an effort to characterize the lymphoid component and to determine whether an occult clonal lymphocyte population could be identified. The potential clinical, biologic, and pathologic significances of clonality in the setting of immunodeficiency are discussed. Report of a Case A 15-year-old white girl with insulin-dependent diabetes mellitus was diagnosed with CVID after she had presented with numerous bouts of otitis media, recurrent bronchitis, bacterial pneumonic infections, and a pharyngeal abscess. Her physical examination was remarkable only for the presence of clubbing. Her family history was remarkable in that both her father and a brother had been diagnosed with CVID. Her initial serum immunoglobulins in the autumn of 1984 were IgG 0.33 g/L (normal is 5.50-14.00 g/L), IgA 0.06 g/L (normal is 0.40-3.30 g/L) and IgM 0.22 g/L (normal is 0.55-3.30 g/L). Additional laboratory studies revealed microcytic anemia secondary to iron deficiency. The remaining laboratory studies, including thyroid function tests, were within normal limits. The patient was started on parenteral immunoglobulin therapy. In the spring of 1985 she developed diarrhea and weight loss with subsequent hypoalbuminemia. Rigid sigmoidoscopy revealed multiple sessile and pedunculated polyps. A barium enema showed the colon and terminal ileum to be studded with sessile and pedunculated polyps measuring 0.3-2.0 cm. Multiple colonic biopsies revealed lymphoid and juvenile-type polyps. A small bowel biopsy revealed giardiasis, nodular lymphoid hyperplasia, and a paucity of plasma cells.
Since 1985, she has been followed up as an outpatient, and although there has been no evidence of neoplasia or giardiasis, her lymphoid hyperplasia has not resolved.
Materials and Methods Biopsy specimens were fixed in buffered formalin, sectioned at 5 nm and stained with hematoxylin and eosin for morphologic studies. Immunophenotypic studies were performed on snapfrozen gastrointestinal mucosa obtained from endoscopic biopsies. Reagents used included fluorescein-conjugated polyclonal anti-IgG, anti-IgA, anti-IgD, anti-kappa, and anti-lambda (Kallestad Diagnostics, Austin, TX) and unconjugated monoclonal antibodies directed against the CD-20, CD-5, CD-2, CD-4 and CD-8 determinants (Bl, T4, T8, Tl 1, Coulter, Inc., Hialeah, FL; and T101, Hybritech, Inc., San Diego, CA). The monoclonal reagents were followed with a fluorescein-conjugated polyclonal antimouse G, A, M reagent (Coulter). Direct immunofluorescence microscopy was performed using a Leitz (Rockleigh, NJ) microscope, equipped with a 450-W xenon lamp. Unconjugated polyclonal reagents against immunoglobulins followed by biotinylated secondary antibodies were used in some studies, and then developed by the avidin-biotin complex (ABC) immunoperoxidase technique (BioGenex Laboratories, San Ramon, CA). For immunogenotypic studies, DNA was extracted from tissues with phenol and chloroform, precipitated with ethanol and dissolved. Five /ug of DNA were digested with restriction enzymes (Boehringer Mannheim, Mannheim, West Germany). BamHI and Hindlll restriction enzymes were used for immunoglobulin heavy chain gene (JH) analysis, BamHI for kappa chain gene (CK) analysis, and BamHI and EcoRI for T-cell receptor Beta chain gene (TCR-B) analysis. Placental DNA was used as a control. The digested DNA was electrophoresed on 0.8% agarose gel and transferred onto nitrocellulose paper by the method of Southern.32 The membranes were hybridized with random primed P 32 labeled probes, washed extensively, exposed to x-ray films for 3-72 hours and then developed. A 3.5-kilobase Hindlll fragment TCR-B probe was supplied by Dr. Jeffrey Sklar (Stanford Medical School),39 and 2.5-kilobase EcoRI fragment CK and 2.5kilobase Sau3A fragment JH probes were supplied by Dr. Philip Leder (Harvard Medical School).11,30 Results Pathologic Findings Histologic examination of multiple colonic biopsies revealed pedunculated juvenile-type polyps comprised of normal colonic mucosa lining dilated glands, many of which contained numerous neutrophils and fibrinous de-
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constellation of clinical and pathologic findings, including recurrent sinopulmonary infections, gastrointestinal disorders, and an increased incidence of gastrointestinal and lymphoreticular neoplasms. 512,31 The spectrum of gastrointestinal disease includes giardiasis and nodular lymphoid hyperplasia (NHL) with resultant diarrhea, malabsorption, and protein-losing enteropathy. There is an increased incidence of malignant neoplasms in patients with primary immunodeficiency, and the risk to patients with CVID has been reported to range from 4 to 1 6 % 12,16,33.34 Malignant lymphoma associated with CVID has been reported, with many of the tumors arising in the gastrointestinal tract.12,16,33,34 The association of malignant lymphoma and NLH has been observed both in the setting of CVID 810 ' 20,26 and without apparent hypogammaglobulinemia.14,24,25 The exact mechanism of the immunologic defect in CVID is unknown, and the pathogenic mechanism for the increased incidence of neoplasia is uncertain.
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A.J.C.P. • September 1990
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FIG. 1 (upper, left). Whole mount of the first polypectomy specimen showing large aggregates of submucosal lymphoid tissue (arrow). Hematoxylin and eosin (XI.3). FIG. 2 (upper, right). Reactive germinal centers within the mucosa of a polyp with surrounding perifollicular lymphocytes within the lamina propria. Hematoxylin and eosin (X40). FIGS. 3 and 4 (lower). Focus of immunoblasts within a lymphoid aggregate with less well-defined germinal centers. Hematoxylin and eosin FIG. 3 (left). (X40). FIG. 4 (right). (X160).
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bris. The surface epithelium was focally ulcerated. The stroma was inflamed and in several areas contained dense collagen. Adenomatous, hamartomatous, and/or hyperplastic features were not identified. The polyps contained large foci of lymphoid tissue with germinal centers within the submucosa and a paucity of plasma cells within the lamina propria (Figs. 1 and 2). Some of the lymphoid aggregates contained less well-defined germinal centers and foci of immunoblasts (Figs. 3 and 4). Sessile colonic polyps were comprised of hyperplastic lymphoid aggregates, many with germinal centers. Parasitic forms were not identified. The small bowel biopsies showed normal villi with a normal villous-to-crypt ratio. Parasitic forms consistent with Giardia were identified. The lamina propria and submucosa contained lymphoid nodules and a paucity of plasma cells.
lit C
Discussion Common variable immunodeficiency is a primary immunodeficient state manifested by hypogammaglobulinemia that usually presents later in life with recurrent sinopulmonary infections. There is a high but variable incidence of associated gastrointestinal disorders, including giardiasis, nodular lymphoid hyperplasia, a sprue-like
TCR-p Chain
Bam HI
Bam HI
Hind I I I
III 111 S*
So
Sj
ment of the JH and CK bands, whereas the second specimen with the polyclonal phenotype showed only germline bands by Southern blot hybridization (Figs. 5 and 6). The TCR-B blots showed no clonal rearrangement in either specimen (Fig. 7). The apparent absence of JH rearrangement with Hindlll probably is due to comigration of the rearranged band with the germline band. 6 The CK rearrangement initially appears inconsistent with the presence of a lambda immunophenotype; however, about 25% of lambda-producing tumors will show a clonal CK rearrangement, probably due to a nonproductive rearrangement of an allele that does not go on to produce lambda light chains.1
JH
C
S,
FlG. 6. Southern blot analysis of the immunoglobulin kappa light chain gene (CK) using BamHl restriction endonuclease digestion. Germline bands are present in both the control (C) and patient specimen lanes (SI and S2). An additional clonal rearranged band (arrow) is present in the first patient specimen (SI).
C
S-j
So
FlG. 5. Southern blot analysis of the immunoglobulin heavy chain (JH)gene using BamHl and Hindlll restriction endonuclease digestions. Germline bands are present in the control (C) and the patient specimen lanes (SI and S2). An additional clonal rearranged band (arrow) is present on the BamHl digested DNA from the first patient specimen (SI).
f 0
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i
8 , 8 2
-|?—§—i— c
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S2
FlG. 7. Southern blot analysis of the T-cell receptor beta chain gene (TCR-B) using BamHl and EcoRI restriction endonuclease digestions. Only germline bands are present in both the control (C) and patient specimen lanes (SI and S2).
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The immunophenotypic analyses were performed on two consecutive polypectomy specimens submitted fresh and snap-frozen in the spring of 1985 and the spring of 1986. The first, a large rectal lymphoid polyp, showed predominantly B-cells with most of the cells IgM and CD20 positive. Within the germinal centers, kappa and lambda light chains were present in normal numbers; however in an area with less well-defined germinal centers, a focus of immunoblasts appeared to be IgM-lambda restricted, suggesting the possibility of a small clonal population. IgG, IgA, and IgD were not identified within the germinal center cell population. Plasma cells were present in decreased numbers, and IgA staining plasma cells were not identified. Scattered within the lamina propria were T-cells with a normal mature peripheral T-cell phenotype. The second specimen, examined approximately one year later, showed polyclonal staining of hyperplastic lymphoid tissue with a paucity of plasma cells. No clonal proliferation was identified. Immunogenotypic studies were performed on both specimens. The first specimen showed clonal rearrange-
BamHl
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The immunophenotypic data in the present case are in keeping with that previously reported in patients with CVID and NLH. 27 The lymphoid follicles within the Peyer's patches were populated with lymphocytes bearing surface IgM-kappa and IgM-lambda, and there were decreased numbers of IgG and IgA containing cells. The perifollicular regions were populated by normal numbers of T-cells. In CVID associated with NLH, the number of plasma cells within the lamina propria is decreased, although the number of circulating B-lymphocytes is normal.5,35'36'37 Our case is unusual in that an apparent focal monoclonal expansion of B-cells was identified immunophenotypically and was then verified by gene rearrangement studies performed on the first polypectomy specimen. Malignant lymphomas of the gastrointestinal tract in adults have been reported to be associated with NLH, both with 810 ' 20 ' 26 and without14,24,25 immunodeficiency. Several authors postulate that this association may be more than just fortuitous and have further suggested that a similar relationship may exist between the benign lymphoepithelial lesion of the salivary gland seen with Sjogren's syndrome and the development of lymphoma. 14 Advances in the application of molecular biologic techniques have provided the diagnostic pathologist with
new tools for recognizing early clonal lymphoid proliferations but also have raised questions concerning oncogenesis and immunoregulation of lymphocyte proliferation. In the majority of situations, clonal immunoglobulin and antigen receptor gene rearrangement appear to be sensitive and relatively specific indicators of malignancy.4'6,35 In immunodeficient states, however, whether primary or secondary, the diagnostic interpretation of gene rearrangement may be difficult, especially in the setting of histopathology which is not diagnostic of malignancy.6 This is most evident in solid organ and bone marrow transplantation, where the Epstein-Barr Virus (EBV) has been shown to lead to lymphoproliferative disorders (LPD) with polyclonal, oligoclonal, or monoclonal B-cell proliferations. It is thought that in these situations there is a lack of T-cell suppression of B-cells containing the EBV genome, and that this can lead to a fatal lymphoproliferative disorder {i.e., lymphoma). 7,17,29 The cases of malignant lymphoma associated with CVID and NLH have not been studied immunogenotypically or for the presence of the EBV genome. Clonal gene rearrangement with "benign" histology has been reported in lymphoepithelial lesions of the salivary gland in patient's with Sjogren's syndrome, 9 angioimmunoblastic lymphadenopathy with dysproteinemia,6'22 AIDS associated lymphadenopathy, 628 and lymphoid hyperplasia in the Wiskott-Aldrich syndrome.4'6 All of these disorders, as well as the present case, have in common an immunologic defect with a tendency for exaggerated Bcell proliferation and a predisposition for the development of lymphoma. In the present case, the finding of an occult clonal expansion at the molecular level without obvious histologic evidence of neoplasia may simply reflect the best response available (in terms of affinity and/or avidity) from a deficient immune system to an unknown antigenic stimulus. It also seems conceivable that other such clonal expansions may develop and then regress without necessarily proceeding to malignant transformation. Perhaps the most appropriate terminology in this setting would be "monoclonal gene rearrangement of uncertain significance"15 analogous to that put forward by Kyle,19 in reference to monoclonal gammopathies. Although in our patient there has been no evidence of subsequent neoplastic transformation, we cannot entirely exclude the possibility that an isolated incipient lymphoma was completely excised during the polypectomy. Our findings are further evidence that the detection of antigen receptor gene rearrangement, hence clonality, should not necessarily be equated with neoplasia, especially in the setting of immunodeficiency. Molecular biologic techniques are providing us with new insights into the nature of lymphocyte differentiation and neoplasia and challenge the pathologist to carefully define criteria for the diagnosis of lymphoid malignancy.
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syndrome, achlorhydria and pernicious anemia. 512 ' 31 Malignant neoplasms have been reported to occur with increased frequency, with primary gastrointestinal lymphoma and gastric carcinoma being the most common tumors. 1216,33 The disorder may be familial; however, a mode of inheritance has not been identified.31 In general, follicular lymphoid hyperplasia is the rule with normal to enlarged tonsillar tissue, NLH, and splenomegaly.512 The immunologic defect appears to involve the final stage of B-cell maturation. The numbers of circulating B- and T-cells usually are normal, but there is an apparent decrease to absence of mature plasma cells and impairment of immunoglobulin synthesis.5,36'37 In some patients, however, deficient T-helper function or excessive T-suppressor activity have been implicated as the cause of deficient immunoglobulin production.31,37 Nodular lymphoid hyperplasia is a well-recognized entity that most often is encountered as a normal variant in the pediatric population, usually involving the terminal ileum and/or colon,23 and in immunodeficiency states. 213 In recent years, cases of NLH in immunocompetent adults have been reported24'25'38 and of interest is the presence of Giardia in these patients as well as those with known CVID. Whether this may represent some form of localized gastrointestinal immunodeficiency is uncertain. The causal relationship of the parasitosis to the development of NLH has been a point of debate in the literature.3'37,38 In the majority of the cases reported, as in our case, the NLH did not regress after eradication of the infection.3,25
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Acknowledgments. The authors wish to thank Linda Schneekloth and Marcia Wood for typing the manuscript and Joel Carl for his photographic assistance.
References
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