Pathological Findings in Human Autoimmune Lymphoproliferative Syndrome
1998; Elsevier BV; Volume: 153; Issue: 5 Linguagem: Inglês
10.1016/s0002-9440(10)65742-2
ISSN1525-2191
AutoresMegan S. Lim, Stephen E. Straus, Janet K. Dale, Thomas A. Fleisher, Maryalice Stetler‐Stevenson, Warren Strober, Michael C. Sneller, Jennifer M. Puck, Michael J. Lenardo, Kojo S.J. Elenitoba‐Johnson, Albert Y. Lin, Mark Raffeld, Elaine S. Jaffe,
Tópico(s)Lymphoma Diagnosis and Treatment
ResumoThe defects in lymphocyte apoptosis that underlie the autoimmune lymphoproliferative syndrome (ALPS) are usually attributable to inherited mutations of the CD95 (Fas) gene. In this report, we present the histopathological and immunophenotypic features seen in the lymph nodes (n = 16), peripheral blood (n = 10), bone marrow (n = 2), spleen (n = 3), and liver (n = 2) from 10 patients with ALPS. Lymph nodes showed marked paracortical hyperplasia. Interfollicular areas were expanded and populated by T cell receptor-αβ CD3+ CD4−CD8−(double-negative, DN) T cells that were negative for CD45RO. CD45RA+ T cells were increased in all cases studied. The paracortical infiltrate was a result of both reduced apoptosis and increased proliferation, as measured by in situdetection of DNA fragmentation and staining with MIB-1, respectively. The paracortical proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. Many of the paracortical lymphocytes expressed markers associated with cytotoxicity, such as perforin, TIA-1, and CD57. CD25 was negative. In addition, most lymph nodes exhibited florid follicular hyperplasia, often with focal progressive transformation of germinal centers; in some cases, follicular involution was seen. A polyclonal plasmacytosis also was present. The spleens were markedly enlarged, more than 10 times normal size. There was expansion of both white pulp and red pulp, with increased DN T cells. DN T cells also were observed in liver biopsies exhibiting portal triaditis. In the peripheral blood, the T cells showed increased expression of HLA-DR and CD57 but not CD25. CD45RA+ T cells were increased in the four cases studied. Polyclonal B cell lymphocytosis with expansion of CD5+ B cells was a characteristic finding. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS. Of note, two affected family members of one proband developed lymphoma (T-cell-rich B-cell lymphoma and nodular lymphocyte predominance Hodgkin's disease, respectively). The defects in lymphocyte apoptosis that underlie the autoimmune lymphoproliferative syndrome (ALPS) are usually attributable to inherited mutations of the CD95 (Fas) gene. In this report, we present the histopathological and immunophenotypic features seen in the lymph nodes (n = 16), peripheral blood (n = 10), bone marrow (n = 2), spleen (n = 3), and liver (n = 2) from 10 patients with ALPS. Lymph nodes showed marked paracortical hyperplasia. Interfollicular areas were expanded and populated by T cell receptor-αβ CD3+ CD4−CD8−(double-negative, DN) T cells that were negative for CD45RO. CD45RA+ T cells were increased in all cases studied. The paracortical infiltrate was a result of both reduced apoptosis and increased proliferation, as measured by in situdetection of DNA fragmentation and staining with MIB-1, respectively. The paracortical proliferation may be extensive enough to suggest a diagnosis of malignant lymphoma. Many of the paracortical lymphocytes expressed markers associated with cytotoxicity, such as perforin, TIA-1, and CD57. CD25 was negative. In addition, most lymph nodes exhibited florid follicular hyperplasia, often with focal progressive transformation of germinal centers; in some cases, follicular involution was seen. A polyclonal plasmacytosis also was present. The spleens were markedly enlarged, more than 10 times normal size. There was expansion of both white pulp and red pulp, with increased DN T cells. DN T cells also were observed in liver biopsies exhibiting portal triaditis. In the peripheral blood, the T cells showed increased expression of HLA-DR and CD57 but not CD25. CD45RA+ T cells were increased in the four cases studied. Polyclonal B cell lymphocytosis with expansion of CD5+ B cells was a characteristic finding. Taken together, the histopathological and immunophenotypic findings, particularly in lymph nodes and peripheral blood, are sufficiently distinctive to suggest a diagnosis of ALPS. Of note, two affected family members of one proband developed lymphoma (T-cell-rich B-cell lymphoma and nodular lymphocyte predominance Hodgkin's disease, respectively). Autoimmune lymphoproliferative syndrome (ALPS) is a disorder characterized by generalized, nonmalignant lymphadenopathy, hypergammaglobulinemia, lymphocytosis, splenomegaly, and autoimmune phenomena. A distinct feature of ALPS, and an early clue to its nature, is the occurrence of markedly increased numbers and percentage of T cell receptor (TCR)-αβ CD4−CD8−, double-negative (DN) T cells in the circulation and lymphoid tissues. Sneller et al1Sneller MC Straus SE Jaffe ES Jaffe JS Fleisher TA Stetler-Stevenson M Strober W A novel lymphoproliferative/autoimmune syndrome resembling murine lpr/gld disease.J Clin Invest. 1992; 90: 334-341Crossref PubMed Scopus (267) Google Scholar first reported a detailed clinical and immunological study of two patients, recognizing similarities to the MRL and C3H/Hej strains of mice possessing the lpr and gld mutations, respectively. Genetic elucidation of the lpr andgld loci as representing recessive mutations in the genes encoding CD95 (Fas/Apo-1) and CD95L (FasL), respectively,2Watanabe-Fukunaga R Brannan PE Copeland NG Jenkins N Nagata S Lymphoproliferation disorder in mice explained by defects in Fas antigen that mediates apoptosis.Nature. 1992; 356: 314-317Crossref PubMed Scopus (2819) Google Scholar, 3Takahashi T Tanaka M Rannan CI Jenkins NA Copeland NG Suda T Nagata S Generalized lymphoproliferative disease in mice, caused by a point mutation in the Fas ligand.Cell. 1994; 76: 969-977Abstract Full Text PDF PubMed Scopus (1495) Google Scholar, 4Lynch DH Watson ML Alderson MR Baum PR Miller RE Tough T Gibson M Davis-Smith T Smith CA Hunter K Bhat D Din W Goodwin RG Seldin MF The mouse Fas-ligand gene is mutated in gld mice and is part of a TNF family gene cluster.Immunity. 1994; 1: 131-140Abstract Full Text PDF PubMed Scopus (328) Google Scholar led to the discovery of functional CD95 mutations in ALPS patients.5Fisher GH Rosenberg FJ Straus SE Dale JK Middleton LA Lin AY Strober W Lenardo MJ Puck JM Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1319) Google Scholar, 6Drappa J Vaishnaw A Sullivan KE Chu J Elkon KB Fas gene mutations in the Canale-Smith syndrome, an inherited lymphoproliferative disorder associated with autoimmunity.N Engl J Med. 1996; 335: 1643-1649Crossref PubMed Scopus (428) Google Scholar, 7Rieux-Laucat F Le Diest F Hivroz C Roberts IA Debatin KM Fischer A de Villartay JP Mutations in Fas associated with human lymphoproliferative syndrome and autoimmunity.Science. 1995; 268: 1347-1349Crossref PubMed Scopus (1200) Google Scholar, 8Le Deist F Emile J-F Rieux-Laucat F Benkerrou M Roberts I Brousse N Fischer A Clinical, immunological and pathological consequences of fas-deficient conditions.Lancet. 1996; 348: 719-723Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar With the ability to diagnose specific CD95 mutations in ALPS cases, it was appreciated that patients affected with this disorder had been included in published series by a number of investigators of undefined chronic lymphoproliferation or splenomegaly with associated autoimmune phenomena.9Canale VC Smith CH Chronic lymphadenopathy simulating malignant lymphoma.J Pediatr. 1967; 70: 891-899Abstract Full Text PDF PubMed Scopus (181) Google Scholar, 10Rao LM Shahidi NT Opitz JM Hereditary splenomegaly with hypersplenism.Clin Genet. 1974; 5: 379-386Crossref PubMed Scopus (10) Google Scholar CD95 is a 48-kd type I transmembrane protein belonging to the tumor necrosis factor receptor family. Interaction of CD95 and its ligand plays a critical role in controlling the homeostasis of peripheral lymphocytes by inducing signals leading to cellular apoptosis; thus, aberrations in this interaction would be expected to impair the apoptosis of normal and autoreactive lymphocytes. Indeed, mice bearing the lpr or gld mutations, who thus have defective expression of CD95 or its ligand, respectively, develop massive lymphoid hyperplasia and autoimmunity.11Gillette-Ferguson I Sidman CL A specific intercellular pathway of apoptotic cell death is defective in the mature peripheral T cells of autoimmune lpr and gld mice.Eur J Immunol. 1994; 24: 1181-1185Crossref PubMed Scopus (79) Google Scholar, 12Adachi M Suematsu S Kondo T Ogasawara J Tanaka T Yoshida N Nagata S Targeted mutation in the Fas gene causes hyperplasia in peripheral lymphoid organs and liver.Nature Genet. 1995; 11: 294-300Crossref PubMed Scopus (339) Google Scholar, 13Adachi M Suematsu S Suda T Watanabe D Fukuyama H Ogasawara J Tanaka T Yoshida N Shigekazu N Enhanced and accelerated lymphoproliferation in Fas-null mice.Biochem. 1996; 93: 2131-2136Google Scholar In vitro studies of B and T cells from ALPS patients and their family members verify that in humans, as well as in mice, CD95 mutations lead to defective lymphocyte apoptosis. The precise role of CD95 in lymphocyte homeostasis in these individuals is not completely understood. This fact is illustrated by the wide spectrum of clinical and immunological findings in ALPS patients and the occurrence of family members who possess identical mutations and defective in vitro apoptosis but few or no clinical signs of ALPS.14Infante AJ, Britton HA, DeNapoli T, Middelton LA, Lenardo MJ, Jackson CE, Wang J, Fleisher T, Straus SE, Puck JM: The clinical spectrum in a large kindred with autoimmune lymphoproliferative syndrome (ALPS) due to a fas mutation that impairs lymphocyte apoptosis. J Pediatr (in press)Google Scholar In this study, we report the spectrum of histological and immunophenotypic features of 10 patients with ALPS. The potential functional properties of expanded DN T cells characteristic of this syndrome are also discussed. Ten patients referred to the National Institutes of Health for evaluation of generalized lymphadenopathy, splenomegaly, peripheral lymphocytosis, and autoimmune phenomena, along with available relatives, were evaluated. The studies were performed under approved research protocols of the National Institute of Allergy and Infectious Diseases and the National Human Genome Research Institute and with the patients' written consent. Peripheral blood, lymph nodes, spleen, bone marrow, and liver were available for examination. ALPS was defined as the presence of chronic, nonmalignant lymphoproliferation, defective lymphocyte apoptosis in vitro, and greater than or equal to 1% TCR-αβ CD3+CD4−CD8− T cells.15Sneller MC Wang J Dale JK Strober W Middleton LA Choi Y Fleisher TA Lim MS Jaffe ES Puck JM Lenardo MJ Straus SE Clinical, immunologic and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis.Blood. 1997; 89: 1341-1348Crossref PubMed Google Scholar Two family members of one proband (patient 3) had lymphoma; these tissues also were obtained for histological analysis. Anticoagulated peripheral blood specimens were stained for flow cytometry using a whole-blood lysis method and analyzed with a FACScan and Cell Quest software (Becton Dickinson, San Jose, CA). In selected cases (three lymph nodes and one spleen), cell suspensions were prepared from freshly resected tissues and stained with fluorescein-isothiocyanate-conjugated or phycoerythrin-conjugated murine monoclonal antibodies (listed in Table 1) and analyzed by flow cytometry using a FACScan and Cell Quest software.Table 1Monoclonal Antibodies Used in Immunophenotypic AnalysisAntigenNameSourceCD1aOKT6Ortho Diagnostics, Raritan, NDCD2Leu5bBecton Dickinson, Mountain View, CACD3Leu-4Becton DickinsonCD4Leu-3a, 1F6*Used in paraffin sections only.Becton Dickinson; Novocastra Labs,*Used in paraffin sections only. Newcastle-on-Tyne, UKCD5Leu-1Becton DickinsonCD7Leu-9Becton DickinsonCD8Leu-2a, 144*Used in paraffin sections only.Becton Dickinson; K. Gatter,*Used in paraffin sections only. Oxford University, Oxford, UKCD11cLeu-M5Becton Dickinson,CD16Leu-11Becton Dickinson,CD25Anti-TacT. Waldmann, National Cancer InstituteCD56Leu-19Becton DickinsonCD57Leu-7Becton DickinsonCD20Leu-16, L26*Used in paraffin sections only.Becton Dickinson; Dako,*Used in paraffin sections only. Carpinteria, CACD19Leu-12Becton Dickinsonκ light chainBethesda Research Laboratories, Bethesda, MDλ light chainBethesda Research LaboratoriesCD30BerH2DakoCD38Leu-17DakoHLA-DRMIB-1*Used in paraffin sections only.Ki-67DakoTCR γδTCRδ1Becton Dickinson; T-Cell Sciences, Cambridge, MATCR αββF1Becton Dickinson; T-Cell SciencesPerforin*Used in paraffin sections only.P1-8H. Yagita, Juntendo University, Tokyo, JapanCGP*Used in paraffin sections only.TIA-1Coulter, Hialeah, FLPolyclonal CD3*Used in paraffin sections only.DakoCD43*Used in paraffin sections only.Leu-22Becton DickinsonCD45RA†Used in flow cytometry only.Leu 18Becton DickinsonCD45RO*Used in paraffin sections only.A6Immunotech, Westbrook, MECGP, cytotoxic granule-associated protein.* Used in paraffin sections only.† Used in flow cytometry only. Open table in a new tab CGP, cytotoxic granule-associated protein. Lymph nodes (n = 16), spleens (n = 3), livers (n = 2), and bone marrows (n = 2) were examined in formalin-fixed, paraffin-embedded hematoxylin and eosin (H&E)-stained sections. Immunophenotypic studies were performed on paraffin sections using the ABC immunoperoxidase technique as previously described.16Elenitoba-Johnson KSJ Kumar S Lim MS Kingma DW Raffeld M Jaffe E Marginal zone B-cell lymphoma with monocytoid B-cell lymphocytes in pediatric patients without immunodeficiency: a report of two cases.Am J Clin Pathol. 1997; 107: 92-98PubMed Google Scholar In seven cases yielding five lymph nodes and two spleens, snap-frozen material was studied using cryoimmunohistochemical techniques. The antibodies used for paraffin-section and frozen-section immunohistochemical studies are listed in Table 1. In one patient (17), no paraffin or frozen sections were available for immunohistochemical studies. Similarly, unstained sections were not available for the spleen from patient 3. In situ detection of cells undergoing apoptosis was determined in histological sections using the terminal deoxynucleotide transferase (Tdt)-mediated dUTP-biotin nick-end labeling (TUNEL) method according to the Apoptag kit (Oncor, Gaithersburg, MD). TCR γ-chain gene and immunoglobulin heavy chain (IgH) gene rearrangement analyses were performed using polymerase chain reaction (PCR) amplification techniques on high molecular weight DNA extracted from peripheral blood mononuclear cells or paraffin-embedded tissue sections as previously described.16Elenitoba-Johnson KSJ Kumar S Lim MS Kingma DW Raffeld M Jaffe E Marginal zone B-cell lymphoma with monocytoid B-cell lymphocytes in pediatric patients without immunodeficiency: a report of two cases.Am J Clin Pathol. 1997; 107: 92-98PubMed Google Scholar, 17McCarthy KP Sloane JP Kabarowski JH Matutes E Wiedermann LM A simplified method of detection of clonal rearrangement of the TCR-γ chain gene.Diagn Mol Pathol. 1992; 1: 173-179PubMed Google Scholar Because of early suspicions that chronic Epstein-Barr virus infection might play a role in the chronic lymphadenopathy of these patients,1Sneller MC Straus SE Jaffe ES Jaffe JS Fleisher TA Stetler-Stevenson M Strober W A novel lymphoproliferative/autoimmune syndrome resembling murine lpr/gld disease.J Clin Invest. 1992; 90: 334-341Crossref PubMed Scopus (267) Google Scholar lymph node sections from five patients were examined for the expression of the Epstein-Barr viral RNA by in situ hybridization using the EBER-1 probe.18Natkunam Y Elenitoba-Johnson KS Kingma DW Kamel O Epstein-Barr virus strain type and latent membrane protein-1 gene deletions in lymphoma in patients with rheumatic disease.Arthritis Rheum. 1997; 40: 1152-1156Crossref PubMed Scopus (10) Google Scholar Fas (CD95) gene mutations were identified, as previously described.5Fisher GH Rosenberg FJ Straus SE Dale JK Middleton LA Lin AY Strober W Lenardo MJ Puck JM Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1319) Google Scholar Ten patients originally came to medical attention 2 months to 5 years after birth because of persistent lymph node and spleen enlargement. Detailed clinical data for 9 of these 10 patients have been presented previously by Sneller et al.15Sneller MC Wang J Dale JK Strober W Middleton LA Choi Y Fleisher TA Lim MS Jaffe ES Puck JM Lenardo MJ Straus SE Clinical, immunologic and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis.Blood. 1997; 89: 1341-1348Crossref PubMed Google Scholar Other clinical manifestations included recurrent bouts of hemolytic anemia, neutropenia, idiopathic thrombocytopenia, and glomerulonephritis.15Sneller MC Wang J Dale JK Strober W Middleton LA Choi Y Fleisher TA Lim MS Jaffe ES Puck JM Lenardo MJ Straus SE Clinical, immunologic and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis.Blood. 1997; 89: 1341-1348Crossref PubMed Google Scholar Nearly all patients exhibited pathological autoantibodies, most of which were directed at platelets or red blood cells. Important early clinical clues as to the difference between these patients and patients with defined lymphoproliferative malignancies or viral infections were the prolonged stable clinical course and the lack of opportunistic infections. In the course of their diagnostic evaluations, the patients had undergone lymph node, bone marrow, and liver biopsies, often more than once. Splenectomy was performed on 7 of the 10 patients. Indications for splenectomy were hypersplenism, refractory hemolytic anemia or idiopathic thrombocytopenia, or suspicion for lymphoma. Subsequently, all 10 patients (4 males and 6 females) and their relatives provided informed consent and were evaluated at the National Institutes of Health. All patients showed defective lymphocyte apoptosis in vitro, and all were analyzed for the presence of CD95 gene mutation using either peripheral blood mononuclear cells or paraffin-embedded tissue.5Fisher GH Rosenberg FJ Straus SE Dale JK Middleton LA Lin AY Strober W Lenardo MJ Puck JM Dominant interfering Fas gene mutations impair apoptosis in a human autoimmune lymphoproliferative syndrome.Cell. 1995; 81: 935-946Abstract Full Text PDF PubMed Scopus (1319) Google Scholar Eight of ten patients demonstrated a specific mutation in various parts of the CD95 gene. Investigation of family members demonstrated that all mutations were inherited with one exception; patient 6 had a confirmed de novo mutation. The two remaining patients (patients 11 and 14) did not have an identifiable mutation in the CD95 gene but were diagnosed as ALPS based on the clinical and laboratory findings as well as demonstrable apoptotic defects, as defined by Sneller et al.15Sneller MC Wang J Dale JK Strober W Middleton LA Choi Y Fleisher TA Lim MS Jaffe ES Puck JM Lenardo MJ Straus SE Clinical, immunologic and genetic features of an autoimmune lymphoproliferative syndrome associated with abnormal lymphocyte apoptosis.Blood. 1997; 89: 1341-1348Crossref PubMed Google Scholar, 19Dianzani U Bragardo M DiFranco D Alliaudi C Scagni P Buonfiglio D Redoglia V Bonissoni S Correra A Dianzani I Ramenghi U Deficiency of the Fas apoptosis pathway without Fas gene mutations in pediatric patients with autoimmunity/lymphoproliferation.Blood. 1997; 89: 2871-2879PubMed Google Scholar As detailed in Table 2, 16 lymph nodes from 10 patients with ALPS were subjected to histological analysis. The lymph node architecture was intact. The most prominent and consistent finding was the marked paracortical expansion by lymphocytes in varying stages of immunoblastic transformation. The immunoblasts were intermingled with mature lymphocytes and polyclonal plasma cells (Figure 1). The paracortical lymphocytes were intermediate to large in size and round to oval in shape and contained moderate amounts of clear to more deeply staining eosinophilic cytoplasm. The paracortical lymphoid cells demonstrated a high proliferative index as determined by MIB-1 positivity and frequent mitoses. In several cases, the cellular proliferation was so marked that a diagnosis of malignant lymphoma was suspected by the referring pathologist.Table 2Lymph Node Findings in ALPS PatientsPatientAge at PresentationGenderCD95 MutationParacortical expansionFollicular hyperplasiaFollicular involutionPTGC1*Percentage of DN T cells that also express CD45RA.4 months†Percentage of CD20 cells that also express CD5.M+++−+218 monthsF++++−35 yearsM+++−+42 yearsM+++−−52 monthsM+++−+64 monthsF+++++119 monthsF−++++149 monthsF−++−+174 monthsF+++−−203 yearsF++++−M, male; F, female. Open table in a new tab M, male; F, female. There was a conspicuous absence of histiocytes containing apoptotic bodies within the paracortex, in contrast to what is characteristically seen in other reactive conditions. The status of apoptosis was determined by TUNEL assay, which demonstrated few cells undergoing programmed cell death in the interfollicular areas but readily identified numerous positive cells within the germinal centers (data not shown). An additional histological finding of note was the presence of a spectrum of reactive germinal center changes, including florid follicular hyperplasia. Progressive transformation of germinal centers (PTGC) was observed focally in involved lymph nodes (6/10 cases). A minority of lymph nodes showed areas of atrophic follicles with regressive changes as seen in Castleman's disease (4/10). Prominent postcapillary venules were seen in the interfollicular region of some cases. Immunophenotypically, the majority of the paracortical cells were CD3+ T cells (Figure 2A), of which only a small proportion showed staining with CD4 (Figure 2B) or CD8 (Figure 2C). An increase in DN T cells was also documented by flow cytometry. DN T cells ranged from 27% to 54% of mononuclear cells, representing 51% to 78% of αβ T cells. In lymph nodes, CD4+ cells were more frequent than CD8+ cells (the CD4:CD8 ratio was approximately 2:1), in contrast to peripheral blood (see below), in which CD4+ and CD8+ cells tended to be present in more equal numbers. Normally the CD4:CD8 ratio in lymph nodes is 3:1 to 4:1, in contrast to the 2:1 to 3:1 ratio seen in peripheral blood. Therefore, the observed differences between lymph nodes and blood may be a reflection of a relatively higher proportion of CD4+ T cells normally found in lymph nodes. The majority of the CD4+ T cells were identified within germinal centers, and not in the expanded paracortical regions by immunohistochemistry (Figure 2B). The paracortical lymphocytes did not show expression of the α-chain of the interleukin (IL)-2 receptor CD25. Similarly, a low percentage of T cells expressed CD25 by flow cytometry (<5% in all cases studied). The paracortical T cells stained strongly with CD3 and CD43 (Leu 22) but were negative for CD45RO and thus bore markers consistent with a naive, or virgin, phenotype.20Tedder TF Cooper MD Clement LT Human lymphocyte differentiation antigens HB-10 and HB-11. II. Differential production of B cell growth and differentiation factors by distinct helper T cell subpopulations.J Immunol. 1985; 134: 2989-2994PubMed Google Scholar Most CD45RO+lymphocytes were within germinal centers. In the cases studied by flow cytometry, CD45RA+ T cells were markedly increased (79% to 90%). A large number of the paracortical cells stained with CD57, TIA-1 (Figure 2, D and E), and perforin (Figure 2F), the latter two being granular serine proteases characteristically associated with cytotoxic T cells. Markers for natural killer (NK) cells (CD56 and CD16) were negative in both frozen sections and by flow cytometry (<3% of mononuclear cells). Notably, the lymph node histology in patients with and without CD95 mutations demonstrated similar histological features. In situ hybridization for EBV performed on lymph nodes from 5/10 patients was negative. There was no evidence of T or B cell clonal expansion as demonstrated by TCR-γ gene or IgH-PCR (data not shown). PCR assays for antigen receptor gene rearrangement will detect clonality in 50% to 75% of cases of B-cell or T-cell lymphomas with these methods.16Elenitoba-Johnson KSJ Kumar S Lim MS Kingma DW Raffeld M Jaffe E Marginal zone B-cell lymphoma with monocytoid B-cell lymphocytes in pediatric patients without immunodeficiency: a report of two cases.Am J Clin Pathol. 1997; 107: 92-98PubMed Google Scholar, 17McCarthy KP Sloane JP Kabarowski JH Matutes E Wiedermann LM A simplified method of detection of clonal rearrangement of the TCR-γ chain gene.Diagn Mol Pathol. 1992; 1: 173-179PubMed Google Scholar Three spleens were examined by routine histology, and in two cases immunophenotypic studies were performed. Patient 3 underwent splenectomy at the age of 6 years because of suspicion for lymphoma. Patient 5 underwent splenectomy at 5 years because of refractory idiopathic thrombocytopenia. The spleens weighed 620 and 856 g, respectively. (Normal pediatric splenic weights are 47 g (5 years) and 58 g (6 years).) Patient 11 underwent splenectomy at 3 years because of severe hypersplenism. The splenic weight was not available in this case. In each case, the splenic white pulp was moderately expanded with follicular hyperplasia and a prominent marginal zone (Figure 3). The red pulp was greatly expanded and contained numerous polyclonal plasma cells and increased lymphocytes and immunoblasts, which were predominantly CD3+. The lymphoid cells in the red pulp were similar in morphology to the lymphoid cells of the paracortical reaction seen in lymph nodes and, like the latter, were generally CD4− and CD8−. DN T cells were present but less conspicuous in the white pulp than they were in lymph nodes. Splenic T cells were similar to lymph node T cells in other respects, being negative for CD25. Liver biopsies were available for examination from patients 1 and 2. They demonstrated mild lymphocytic infiltrates composed of predominantly CD3+ T cells, of which greater than 50% were CD4−CD8−. Mild periportal fibrosis and extramedullary hematopoiesis was present in patient 1. Patient 2 developed chronic active hepatitis consistent with a form of autoimmune hepatitis. Aspirate smears from patients 2 and 5 demonstrated scattered interstitial aggregates of large atypical lymphocytes. The cells showed clumped chromatin and prominent nucleoli with increased mitoses. The degree of peripheral blood lymphocytosis varied considerably from patient to patient. Sequential analysis of peripheral blood counts demonstrated fluctuations in patient 2 in response to immunosuppressive treatment (data not shown). Representative values obtained at time of immunophenotypic analysis are presented in Table 3. The peripheral lymphocytosis ranged from 810 to 12,328 cells/mm3. DN T cells, expanded by definition in patients with ALPS, were increased 2- to 67-fold over normal values in all of the patients.Table 3Summary of Peripheral Blood Lymphocyte Phenotype, CD95 Mutations and Lymphocyte Apoptosis DefectsT cells (%)PatientLymphocytes (cells/mm3)CD3CD3/CD4CD3/CD8CD3/CD4−CD8−CD57CD57/CD8αβ DN T cellsγδ DN T cellsDN T/CD45RA% of DN T cells111,700662423183414141NDND25,712651116394222293297437,7406831271023126188041,8707023379391643NDND51,68055152020261553NDND612,328671526263222138NDND113,105732713335216271NDND145,90059142717158710NDND172,95074192529532324123792081076312914291693857Normal range1,173–2,64061–8432–5811–361–75–293–24<1<5<3NAActivation markers (%)B cells (%)NK cells (%)CD3/HLA-DRCD3/CD25CD20CD20/CD5% of CD20 cellsCD16/CD56CD16/CD56/CD3CD95 mutationLymphocyte apoptosis defect461127217633++441831258148++17724229253++158261869310++NDND41389247++33314750173++57517159067−+2210433890311−+56420105041++3511191474413++<15<375–161–10NA6–301–15NegativeNegativeND: Not determined; NA, not available.* Percentage of DN T cells that also express CD45RA.† Percentage of CD20 cells that also express CD5. Open table in a new tab ND: Not determined; NA, not available. Although the percentage of CD3+ T cells was within normal limits, a relative decrease in the number of CD4+ T cells, resulting in an alteration of the CD4:CD8 ratio was seen in all but one patient. In four patients, CD8+ T cells exceeded CD4+ cells, and in five patients the ratio of CD4:CD8 was approximately 1:1. T cells showed generally increased expression of HLA-DR, without a concomitant increase in CD25 expression. Of note, polyclonal B cell (CD20+) lymphocytosis (ranging from 20% to 43% of total lymphocytes) was seen in 7/10 patients. Notably, a high percentage of the B cells (50% to 92%) co-expressed CD5. There was no correlation between the extent of B cell lymphocytosis and the number of DN T cells. The B- and DN T-cell lymphocytosis did not reflect a general increase in all lymphocyte subsets, as the numbers of CD16+ or CD56+CD3− NK cells was within the normal range. In concert with the findings in lymph nodes, CD57+ cells were significantly increased, without an increase in NK cells. Markedly increased numbers of CD45RA+ T cells were identified in the peripheral blood of the four patients studied. As shown in Table 3, the two ALPS patients without CD95 gene mutations (patients 11 and 14) also showed increased numbers of DN T cells as well a B cell lymphocytosis (patient 14). Family members of ALPS patients were sur
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