Terminal Deoxynucleotidyl Transferase Positive Cells in Human Tonsils

Hematopathology / TDT-POSITIVE TONSIL CELLS Terminal Deoxynucleotidyl Transferase Positive Cells in Human Tonsils James A. Strauchen, MD, and Lorraine K. Miller, PhD Key Words: Terminal deoxynucleotidyl transferase; TdT; Tonsil; Lymphoblastic lymphoma Abstract To study the possible cellular origin of recently recognized indolent terminal deoxynucleotidyl transferase (TdT)-positive T-lymphoblastic proliferations of the tonsils and oropharynx, we studied normal human tonsils for the presence of TdT-positive cells. TdT-positive cells were readily demonstrated in the tonsils from 15 children and adults by immunohistochemical staining. TdT-positive cells were distributed in discrete foci at the periphery of lobules of lymphoid tissue and adjacent to fibrous septa and had the morphologic features of small to medium-sized lymphocytes. Double-antibody staining indicated the TdT-positive cells had the phenotype of uncommitted early lymphoid precursors (CD3, CD79a, CD10 ). Foci of TdT-positive cells were not identified in 6 reactive lymph nodes studied as controls. These studies indicate that tonsils, like bone marrow and thymus, are sites of lymphopoiesis. The presence of TdT-positive precursor cells in human tonsils may be a factor in the pathogenesis of recently described indolent T-lymphoblastic proliferations involving the tonsils and oropharynx. The presence of TdT-positive cells in human tonsils should not be misinterpreted as evidence of lymphoblastic lymphoma or leukemia. Terminal deoxynucleotidyl transferase (TdT) is a template independent DNA polymerase characteristic of lymphoid precursor cells. 1,2 TdT acts as a somatic mutagen in these cells resulting in the generation of additional immunologic diversity. 3,4 Detection of TdT by enzymatic, 5 immunofluorescence, 6,7 or immunohistochemical 8-10 methods is widely used as a marker for the neoplasms of precursor B- and T-cell origin, specifically acute lymphoblastic leukemia and lymphoblastic lymphoma (precursor B-lymphoblastic leukemia/lymphoma and precursor T-lymphoblastic lymphoma/leukemia in the Revised European-American classification of lymphoid neoplasms 11 ). TdT-positive cells are normally found in the thymus (cortical thymocytes) and in the bone marrow (B- and T-precursor cells) but generally are not recognized to occur in the peripheral lymphoid organs, eg, tonsils, lymph nodes, and spleen, in the absence of lymphoid neoplasia. 9,10 Strauchen 12 and others 13 have reported on the occurrence of previously unrecognized TdT-positive, indolent, T-lymphoblastic proliferations of undetermined clonality with predilection for involvement of the tonsils and oropharynx. To study the cellular origin of these TdT-positive proliferations, we studied normal human tonsils for the presence of TdT-positive cells, using immunohistochemical staining of formalin-fixed, paraffin-embedded tissue. TdT-positive cells were readily demonstrable in the tonsils from both children and adults. The possible relation of these TdT-positive cells to the pathogenesis of indolent T-lymphoblastic proliferations of the oropharynx is considered. Materials and Methods Tissues Tonsils were selected from 15 children and adults aged 1 to 40 years who had undergone tonsillectomy for benign 12 Am J Clin Pathol 2001;116:12-16 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE hypertrophy. Lymph nodes with reactive hyperplasia were selected from 6 children and adults aged 4 to 45 years as controls. Tissues were fixed in 10% neutral buffered formalin and processed for routine paraffin-embedding. Immunohistochemical Analysis Anti-TdT antibody (rabbit polyclonal) was obtained from DAKO (Carpinteria, CA) and Supertechs (Bethesda, MD). Anti-CD3 (rabbit polyclonal), and anti-cd79a were obtained from DAKO. Anti-CD10 was obtained from Novocastra Laboratories (Newcastle upon Tyne, England). Anti- Ki-67 (MIB-1) was obtained from Beckman Coulter (Fullerton, CA). Tissue sections for immunohistochemical analysis were deparaffinized in xylene and rehydrated through graded ethanol to deionized water. Immunohistochemical staining with anti-tdt (DAKO) was performed following antigen retrieval with microwaving in a 10- mmol/l concentration of citric acid at ph 6.0 for 15 minutes. Staining was performed on an autostainer (DAKO) using the EnVision+ System (DAKO) with peroxidase-diaminobenzidine (DAB) chromogen. Immunohistochemical staining with anti-tdt (Supertechs) was performed following antigen retrieval with trypsinization. Negative controls included absent first antibody, antibody diluent, and an irrelevant rabbit antibody. A positive control (thymus) was included in each staining. Double-antibody staining was performed using the EnVision Doublestain System (DAKO) with peroxidase-dab and alkaline phosphatase fast red chromogens. Briefly, deparaffinized sections were rehydrated, microwaved in a 10-mmol/L concentration of citric acid, ph 6.0, and incubated with anti-tdt antibody (DAKO) for 30 minutes at room temperature followed by the addition of peroxidase-dab chromogen. Sections were washed and incubated with anti-cd3, anti-cd10, or anti-cd79a for 30 minutes at room temperature followed by the addition of alkaline phosphatase fast red chromogen. TdT-positive cells were evaluated semiquantitatively by counting the number of positive cells per high power (40 objective) field in the areas with the most numerous positive cells. Results The tonsils from all 15 patients showed TdT-positive cells distributed in discrete foci at the periphery of lobules of lymphoid tissue and adjacent to fibrous septa Image 1. TdT-positive cells were not observed in the germinal centers or tonsil epithelium. TdT-positive cells had the morphologic features of small to medium-sized lymphocytes with round to ovoid nuclei and scant cytoplasm Image 2. TdT-positive cells were found in the tonsils of adults and children but were more numerous in children Table 1. Two tonsils were apparent outliers (Table 1); in these 2 cases, both tonsils were studied; however, only the first was included in the statistical analysis. Identical patterns of TdT staining were observed with both antibodies; no staining was observed in the absence of first antibody or with an irrelevant rabbit antibody control. The lymph nodes from 6 patients studied as Image 1 Tonsil from a 4-year-old child showing a focus of terminal deoxynucleotidyl transferase (TdT)-positive cells at the periphery of a lobule of lymphoid tissue adjacent to a fibrous septum (anti-tdt immunoperoxidase counterstained with hematoxylin, original magnification 200). Image 2 Higher magnification of the tonsil in Image 1 showing terminal deoxynucleotidyl transferase (TdT)-positive cells with the morphologic features of small to medium-sized lymphocytes (anti-tdt immunoperoxidase counterstained with hematoxylin, original magnification 600). American Society of Clinical Pathologists Am J Clin Pathol 2001;116:12-16 13

Strauchen and Miller / TDT-POSITIVE TONSIL CELLS controls showed only rare, single TdT-positive cells in the paracortex; foci of TdT-positive cells as in the tonsils were not observed. Double-antibody immunohistochemical studies were performed in 3 cases to define the phenotype of the TdT-positive cells; approximately 50 TdT-positive cells were evaluated in each. The TdT-positive cells were negative for CD3, CD79a, and CD10 Image 3. Staining for Ki-67 demonstrated numerous proliferating cells in areas containing TdT-positive cells Image 4. Discussion TdT is a marker for immature lymphocyte precursors of B- and T-cell lineage and of lymphoblastic neoplasms. 1,2 TdT-positive cells generally are considered to be restricted to the central lymphoid organs, ie, the bone marrow and thymus; however, small numbers may be detected in peripheral blood, 14,15 immature and fetal tissues, 16,17 and reactive lymph nodes. 9 In the present article, we report on the occurrence of TdT-positive cells in human tonsils demonstrated by immunohistochemical staining. The immunohistochemical staining for TdT with affinity-purified antibody seems to be specific. Although spurious staining for TdT has been reported with indirect immunofluorescence with unpurified antibody, 18,19 in the present study identical patterns of staining were obtained using 2 different anti-tdt antibodies with different antigen retrieval techniques. The results of the present study confirm the detection of TdT activity in human tonsil by biochemical assay. 20 TdT enzymatic activity was identified in extracts of tonsils from 6 of 22 children undergoing routine tonsillectomy; the enzyme was identical to reference TdT with respect to divalent cation and primer requirements and inhibition by sulfhydryl inhibitors and adenosine triphosphate, and was indistinguishable from acute lymphoblastic leukemia derived TdT by glycerol gradient centrifugation and phosphocellulose chromatography. 20 Double-antibody immunohistochemical studies were performed to define the phenotype of the TdT-positive cells. The TdT-positive cells did not stain for specific T-cell (CD3) or B-cell (CD79a) antigens or CD10, suggesting an immature or uncommitted lymphoid precursor. 21 The phenotype differs from TdT-positive cells in the thymus, which are CD3+ T-cell precursors, and from TdT-positive cells in the bone marrow, which are predominantly CD10+, CD79a+ B- cell precursors. 22 The phenotype resembles, however, the phenotype of TdT-positive cells in the peripheral blood that, except for a small proportion expressing B-cell antigens and common acute lymphoblastic leukemia antigen, lack specific T- or B-cell antigens, 14 as well as the phenotype of TdT-positive bone marrow lymphoid stem cells, which lack lineagespecific surface antigens. 21 Table 1 Immunohistochemical Staining for Terminal Deoxynucleotidyl Transferase (TdT) Tissue/Sex/Age (y) TdT-Positive Cells per High-Power Field * Tonsils M/1 80.5 M/2 20.5 M/4 186.0 M/6 84.0 M/6 9.5/55.5 F/8 161.5 M/9 118.5 F/11 223.0 F/15 107.5 F/19 38.0 F/23 190.0/128.0 F/33 8.0 M/36 61.0 F/37 57.5 M/40 11.0 Mean 90.4 (±71.8) Lymph nodes M/4 1.0 M/9 3.0 M/19 2.5 F/33 13.0 F/44 8.0 M/45 4.5 Mean 5.3 (±4.4) * Areas with most numerous TdT-positive cells. Both tonsils. P <.01 by the Student t test. SD is given in parentheses. Image 3 Double-antibody immunohistochemical stain with anti terminal deoxynucleotidyl transferase (TdT; brown) and anti-cd79a (red) showing TdT-positive cells negative for the B-cell antigen CD79a. TdT-positive cells also were negative for the T-cell antigen CD3 and CD10 (not shown) (anti-tdt immunoperoxidase with diaminobenzidine chromogen and anti-cd79a immunoalkaline phosphatase with fast red chromogen, original magnification 600). 14 Am J Clin Pathol 2001;116:12-16 American Society of Clinical Pathologists

Hematopathology / ORIGINAL ARTICLE A B Image 4 A, Tonsil showing histologic features of a focus containing terminal deoxynucleotidyl transferase positive cells adjacent to a fibrous septum (H&E, original magnification 200). B, Same focus stained for proliferating cell nuclear antigen with anti Ki-67 showing proliferating cells (anti Ki-67 immunoperoxidase counterstained with hematoxylin, original magnification 200). The origin of the TdT-positive cells in the tonsils is uncertain. The presence of numerous Ki-67 positive cells indicates a high proliferative index suggesting active lymphopoiesis. TdT messenger RNA and V(D)J recombination-associated genes are expressed in a fraction of mature tonsil B cells in the germinal center centrocyte fraction. 23 Under some conditions, TdT-positive T-cell precursors 24 and T- and B-cell precursors 25 may be mobilized from the central lymphoid organs and take up residence in peripheral lymphoid organs. The derivation of the thymus from the third and fourth pharyngeal pouches raises the speculation that pharyngeal epithelium may share with thymic epithelium tropism for bone marrow derived T-cell precursors. The TdT-positive cells in our study did not express T-cell markers; however, uncommitted lymphoid precursors capable of T-cell differentiation or early T-cell precursors that have not yet acquired CD3 cannot be ruled out. 14,21 The occurrence of TdT-positive T-cell precursors in the tonsil may be related to the recently reported indolent T- lymphoblastic proliferations involving the tonsils and oropharynx. 12,13 These proliferations of undetermined clonality exhibit a peculiarly indolent course, remaining confined to the tonsils, oropharynx, and regional lymph nodes for prolonged periods, even in the absence of therapy. 13 These findings would not be inconsistent with a proliferation of TdT-positive lymphoid precursor cells intrinsic to the tonsil. TdT-positive cells are part of the lymphoid population of the tonsil, which, like the bone marrow and thymus, seems to be a site of postnatal lymphopoiesis. TdT-positive cells in the tonsil should not be misinterpreted as lymphoblastic lymphoma or leukemia. From the Department of Pathology, Mount Sinai School of Medicine, New York, NY. Address reprint requests to Dr Strauchen: Dept of Pathology, Mount Sinai School of Medicine, One Gustave Levy Place, New York, NY 10029. References 1. Barr RD, Sarin PS, Perry SM. Terminal transferase in human bone-marrow lymphocytes. Lancet. 1976;1:508-509. 2. Bollum FJ. Terminal deoxynucleotidyl transferase as a hematopoietic cell marker. Blood. 1979;54:1203-1215. 3. Baltimore D. Is terminal deoxynucleotidyl transferase a somatic mutagen in lymphocytes? Nature. 1974;248:409-411. 4. Komori T, Okada A, Stewart V, et al. Lack of N regions of antigen receptor variable region genes of TdT-deficient lymphocytes. Science. 1993;261:1171-1175. 5. Kung PC, Long JC, McCaffrey RP, et al. Terminal deoxynucleotidyl transferase in the diagnosis of leukemia and malignant lymphoma. Am J Med. 1978;64:788-794. 6. Cibull ML, Coleman MS, Nelson O, et al. Evaluation of methods of detecting terminal deoxynucleotidyl transferase in human hematologic malignancies: comparison of immunofluorescence and enzymatic assays. Am J Clin Pathol. 1982;77:420-423. 7. Stass SA, Schumacher HR, Keneklis TP, et al. Terminal deoxynucleotidyl transferase immunofluorescence of bone marrow smear: experience in 156 cases. Am J Clin Pathol. 1979;72:898-903. American Society of Clinical Pathologists Am J Clin Pathol 2001;116:12-16 15

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