Hematopathology / FLOW CYTOMETRIC DISTINCTION OF THYMOMA FROM PRECURSOR T-CELL ALL/ LYMPHOBLASTIC LYMPHOMA

We compared the antigen expression profile of thymocytes in lymphocyte-rich thymoma with that of precursor T-cell acute lymphoblastic leukemia/ lymphoblastic lymphoma (T-cell ALL/LBL) cells using 4-color flow cytometry. In all 15 thymoma cases, the thymocytes demonstrated 3 distinct subpopulations. The least mature cells (double-negative) expressed lowdensity CD2 and CD5, high-density CD7, CD10, CD34, and heterogeneous CD4 and CD8. They had the lowest density CD45 expression and were surface CD3–. The immature cells (double-positive) expressed CD2, CD5, CD7, CD4, CD8, heterogeneous surface CD3, and intermediate-density CD45. They were CD10– and CD34–. The mature cells (single-positive) expressed CD2, surface CD3, CD5, CD7, and CD4 or CD8. The heterogeneous expression of surface CD3, CD4, and CD8 also created a characteristic smearing pattern for these antigens. In all 15 T-cell ALL/LBL cases, the lymphoblasts formed a tight cluster without discrete subpopulations or smearing pattern. Of 5 double-negative cases, 4 demonstrated loss of CD2, CD10, or CD34 expression. Of 7 double-positive cases, 5 showed complete loss of surface CD3, CD2, and/or CD5; 4 were CD10+; and 2 were CD34+. Of 3 singlepositive cases, 2 showed loss of CD2 and/or aberrant expression of CD34. Analysis of antigen expression pattern, the presence or absence of T cell–associated antigen deletion, and the expression of CD10 and CD34 by 4-color flow cytometry can help differentiate thymoma from T-cell ALL/LBL. Thymocytes are immature T lymphocytes with varying degrees of maturation. They commonly are encountered in the study of anterior mediastinal masses and often are a significant component of hyperplastic thymus or lymphocyterich thymoma.1-3 Precursor T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma (T-cell ALL/LBL) also is a tumor frequently occurring in the anterior mediastinum.4 When a large biopsy or resection specimen is available for review, the morphologic differentiation between thymoma and T-cell ALL/LBL, in general, is straightforward. Owing to its anatomic location, however, the initial study of a mediastinal mass often commences with fine-needle aspiration (FNA) or mediastinoscopic biopsy, severely limiting the quantity of material available for histologic diagnosis. Differentiation of thymoma from T-cell ALL/LBL based on histologic examination can be confounded further by a thymoma that has a predominance of lymphocytes.5-8 Flow cytometric immunophenotyping (FCI) is one of the standard ancillary study modalities in the evaluation of lymphoid lesions, including anterior mediastinal masses. Although it has been used extensively to study the immunophenotype of thymocytes in normal and hyperplastic thymus and in thymomas, its usefulness in the differentiation of lymphocyte-rich thymoma from T-cell ALL/LBL has not been very well described.2,9-17 One reason for the lack of this information in the literature is that thymocytes and T-cell ALL/LBL cells share many of the same antigens, making flow cytometric differentiation difficult by simply analyzing the expression of these individual antigens by using 1or 2-color flow cytometry. We performed a comprehensive analysis of antigen-expression profiles of thymocytes in lymphocyte-rich thymomas and T-cell ALL/LBL lymphoblasts to determine Hematopathology / ORIGINAL ARTICLE Am J Clin Pathol 2004;121:268-274 269 269 DOI: 10.1309/K2FY1TED8GEGFLNG 269 © American Society for Clinical Pathology whether the expression pattern of certain antigens can be used to distinguish these entities by using 4-color flow cytometry. Materials and Methods Case Selection A prospective analysis of lymphocyte-rich thymoma and T-cell ALL/LBL cases at Emory University Hospital during a 20-month period (July 2000 to March 2002) was performed. Clinicopathologic data were obtained by reviewing medical records or from referring pathologists. Morphologic Examination FNA smears were air dried for staining with the Romanowsky method and/or fixed in alcohol for staining with the Papanicolaou technique. Cytocentrifuged slides of the body fluid specimen also were stained with the Romanowsky method. Excisional biopsy specimens were fixed in buffered formalin solution, sectioned, and stained with H&E. Peripheral blood and bone marrow aspirate smears were stained with the Wright and Giemsa method. Flow Cytometric Immunophenotyping FCI was performed on peripheral blood, bone marrow aspirate, body fluid, or fresh excisional biopsy tissue samples and/or FNA specimens collected in RPMI 1640 culture medium. Specimens were processed routinely, and single-cell suspensions were stained with various 4-fluorochrome–conjugated antibody combinations (fluorescein isothiocyanate, phycoerythrin, peridinin chlorophyll protein, and allophycocyanin) according to the lymphoma panel protocol routinely used in our laboratory. The antibodies in the panel included those against leukocyte common antigen CD45, B-cell antigens (CD19, CD20, CD22, κ and λ light chains), T-cell antigens (CD2, CD3, CD4, CD5, CD7, CD8), myeloid antigens (CD11b, CD11c, CD13, CD14, CD15, CD33, CD34, CD117), and CD10, CD16, CD25, CD36, CD38, CD56, CD103, and HLA-DR (Becton Dickinson Biosciences, San Diego, CA). Testing for terminal deoxynucleotidyl transferase was performed in permeabilized cells from selected cases. Approximately 10,000 events were acquired on a flow cytometer (dual-laser FACSorter, Becton Dickinson Biosciences) and analyzed using the CellQuest computer software program (Becton Dickinson Biosciences). Immunohistochemical Analysis Immunohistochemical staining for cytokeratin AE1/3 was performed on thymoma cases with paraffin-embedded tissue samples or cell blocks by using the avidin-biotin complex method. Results In this prospective study, we analyzed the surface antigen expression profiles of thymocytes in lymphocyte-rich thymoma and lymphoblasts in T-cell ALL/LBL. Of the 15 thymoma cases, 6 were FNA specimens and 9 were excisional biopsy specimens. Of 6 FNA specimens, subsequent tissue biopsy specimens were available for 2. Of 15 T-cell ALL/LBL cases, 4 were tissue biopsy specimens, 5 were bone marrow aspirates, and 2 were peripheral blood samples; for 1, both FNA and tissue biopsy specimens were available; and 1 was a pleural fluid sample. For 2 other cases, both peripheral blood and bone marrow aspirates were submitted for FCI analysis. The clinicopathologic data are summarized in ❚Table 1❚. The age of the patients diagnosed with thymoma ranged from 12 to 80 years (Table 1). The mean age was 53 years. The male/female ratio was 2:1 (male, 10 [67%]; female, 5 [33%]). Twelve patients with thymoma had mediastinal masses. Of the 12, 8 underwent biopsy, 3 had FNA, and 1 had FNA with a subsequent tissue biopsy of the mediastinal masses. In the remaining 3 patients, 1 had a left-sided chest wall mass and underwent excisional biopsy, 1 with a lung mass had FNA only, and 1 with a paraspinal mass had both FNA and biopsy. The presence of neoplastic thymic epithelial cells was confirmed in all cases by immunohistochemical staining for cytokeratins. In contrast, patients diagnosed with T-cell ALL/LBL were much younger. Their ages ranged from 3 to 58 years, with a mean age of about 23 years. The male/female ratio was the same as that for the patients with thymoma. Of 15 patients, 7 had mediastinal masses and 1 of them had concurrent pleural effusion. Among the 7 patients with mediastinal masses, 2 underwent biopsy of the mediastinal mass, 4 had peripheral blood drawn or bone marrow aspiration performed, and 1 underwent thoracentesis. Of the remaining 8 patients, 2 had lymphadenopathy and underwent lymph node FNA and/or biopsy, 1 had a tonsillar mass and underwent tissue biopsy, and 5 had abnormal peripheral blood findings without a mediastinal mass or lymphadenopathy. The latter 5 patients had either peripheral blood drawn and/or bone marrow aspiration performed for FCI owing to leukocytosis or pancytopenia (Table 1). Of the 7 patients with a mediastinal mass, 4 (cases 17, 23, 28, and 30) were found to have peripheral blood or bone marrow involvement. Another patient with lymphadenopathy had later recurrence involving the breast (case 21). Morphologically, T-cell ALL/LBL blasts were indistinguishable from immature thymocytes on the FNA smears ❚Image 1❚. In the forward vs 90° right-angle flow cytometric plot, the thymocytes in all cases showed low light scatter properties, indicating a relatively small size and simple cytoplasmic Li et al / FLOW CYTOMETRIC DISTINCTION OF THYMOMA FROM PRECURSOR T-CELL ALL/ LYMPHOBLASTIC LYMPHOMA 270 Am J Clin Pathol 2004;121:268-274 270 DOI: 10.1309/K2FY1TED8GEGFLNG © American Society for Clinical Pathology complexity. In the majority of thymoma cases, the thymocytes also displayed a distinct expression pattern of certain antigens. Of 15 thymoma cases, 14 exhibited a large population of thymocytes coexpressing CD4 and CD8 with tapering smaller populations of single-positive thymocytes exclusively expressing CD4 or CD8 ❚Image 2❚. This in effect created a distinctive “smear” pattern in the CD4 vs CD8 dotplot diagram (Image 2D). A smear pattern also was observed ❚Table 1❚ Clinicopathologic Data Diagnosis/Case No./Sex/Age (y) Clinical Manifestation Anatomic Site Specimen Type Thymoma 1/M/57 Mediastinal mass Mediastinum Biopsy 2/M/36 Left-sided chest wall mass Chest wall Biopsy 3/M/29 Mediastinal mass Mediastinum FNA 4/M/29 Paraspinal mass Paraspinal area FNA and biopsy 5/M/68 Mediastinal mass Mediastinum FNA 6/M/62 Mediastinal mass Mediastinum Biopsy 7/F/38 Mediastinal mass Mediastinum Biopsy 8/F/61 Mediastinal mass Mediastinum Biopsy 9/F/60 Mediastinal mass Mediastinum FNA and biopsy 10/F/42 Mediastinal mass Mediastinum Biopsy 11/M/74 Mediastinal mass Mediastinum FNA 12/M/73 Mediastinal mass Mediastinum Biopsy 13/M/69 Lung mass Lung FNA 14/M/12 Mediastinal mass Mediastinum Biopsy 15/F/80 Mediastinal mass Mediastinum Biopsy T-cell ALL/LBL 16/M/32 Leukocytosis BM Aspirate 17/M/20 Mediastinal mass BM Aspirate 18/M/58 Lymphadenopathy LN Biopsy 19/F/46 Pancytopenia BM Aspirate 20/M/19 Mediastinal mass Mediastinum Biopsy 21/F/22 Lymphadenopathy LN and breast FNA and biopsy 22/F/9 Mediastinal mass Mediastinum Biopsy 23/M/17 Mediastinal mass PB Aspirate 24/M/47 Leukocytosis BM Aspirate 25/F/12 Tonsillar mass Tonsil Biopsy 26/M/37 Leukocytosis PB and BM Aspirate 27/M/16 Leukocytosis PB and BM Aspirate 28/M/6 Mediastinal mass BM Aspirate 29/F/3 Mediastinal mass Pleural cavity Thoracentesis 30/M/7 Mediastinal mass PB Aspirate BM, bone marrow; FNA, fine-needle aspiration; LN, lymph node; PB, peripheral blood; T-cell ALL/LBL, precursor T-cell acute lymphoblastic leukemia/lymphoblastic lymphoma.