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Chromosomal Changes Pattern and Gene Amplification in T Cell Non Leukemia (2001) 15, 1627–1632 2001 Nature Publishing Group All rights reserved 0887-6924/01 $15.00 www.nature.com/leu Chromosomal changes pattern and gene amplification in T cell non-Hodgkin’s lymphomas M Renedo3, B Martinez-Delgado1, E Arranz1,3, MJ Garcia2, M Urioste1, A Martinez-Ramirez1, C Rivas2, JC Cigudosa1 and J Benitez1 1Department of Human Genetics, Centro Nacional de Investigaciones Oncologicas, Madrid, Spain; 2Department of Pathology, Fundacio´n Jimenez Dı´az, Madrid, Spain; and 3GEMOLAB, Madrid, Spain T cell non-Hodgkin’s lymphomas are a heterogeneous group genetic techniques is that CGH does not need cell culture to of lymphomas with poor prognosis, and whose genetic alter- obtain metaphases and it allows a global genetic analysis of ations are not well understood. Comparative genomic hybridiz- ation (CGH) is a technique that allows the identification of DNA the genome by passing the examination of a minor cell clone imbalances without cytogenetic studies. We have studied 37 that was not representative of the tumoral genetic profile. samples from 29 T cell non-Hodgkin’s lymphomas (25 periph- We have performed CGH in 37 samples from 29 patients eral and four lymphoblastic lymphomas) by CGH in order to with Tcell NHL, in order to detect DNA sequence copy num- detect DNA sequence copy number changes of putative impor- ber changes along all chromosomes. Our results point to spe- tance in the biology and prognosis of these neoplasms. We cific regions previously described by cytogenetic techniques, detected abnormal CGH profiles in 16/27 (59%) of samples at diagnosis, a ratio that increased to 66% (23/37) when we as well as other new regions not previously described. Ampli- included the relapsed samples. The most common recurrent fication of some genes mapped in one of these regions, changes were gains related to the X chromosome, either the detected by Southern and FISH analysis was concordant with whole chromosome or partially the Xq26–27 bands (19%). Other the CGH results. recurrent changes included gains of bands 9q34, gains of chro- mosomes 17, 19, and 20, and complete or partial deletions of chromosome 13 (10%). Cancer-related genes located at Xq26– 28 region were analyzed by Southern blot and fluorescence in Materials and methods situ hybridization (FISH). Low level amplification of some of these genes was detected by this technique confirming the Patients results obtained by CGH in this region. The detection of abnor- mal CGH profiles in these T cell lymphomas could have clinical High molecular weight DNA was extracted from 37 samples implications. Patients with abnormal CGH profiles showed sig- belonging to 29 patients diagnosed of T-NHL; 35 fresh lymph nificant associations with advanced stage of disease, over- expression of P53, and higher proliferative index. Leukemia nodes and two bone marrow samples. According to the WHO 7 (2001) 15, 1627–1632. classification the 29 Tcell NHL included 25 peripheral Tcell Keywords: T cell lymphomas; CGH; gains and losses; gene ampli- lymphomas (PTCL) (two angioimmunoblastic, three anaplastic fication large cell, two mycosis fungoides, 17 unspecified and one unclassifiable) and four precursor Tcell lymphoblastic lym- phomas. The 17 unspecified PTCL corresponded to the pleo- Introduction morphic and lymphoepithelioid types of the Kiel classi- fication.8 In seven cases, different samples were obtained at Tcell lymphomas are a heterogeneous group of non- different stages during the evolution of the disease. A summary Hodgkin’s lymphomas (NHLs) with a highly variable mor- of clinical data is shown in Table 1. phology and controversial classification. In Spain, these tumors are less common than those of B cell origin, as occurs in the rest of Europe (10–15% of all NHLs). Moreover, com- Immunohistochemistry pared with B cell neoplasms, Tcell NHLs appear to have a worse prognosis.1 Tissue was routinely fixed in 10% formalin, embedded in par- The genetic data on T cell neoplasms are still limited. affin and stained with hematoxylin-eosin (E–H) and Giemsa Although non-random abnormalities at the cytogenetic level in thin sections (4 ␮m). have been described, such as rearrangements involving An immunohistochemical study was performed with a 14q11, 14q32, 6p23, 1p36,2 and trisomies of chromosomes streptavidin-biotin peroxidase complex technique using a X, 3 and 7q,3–6 the molecular changes that could play a role in complete panel of monoclonal antibodies against Tand B tumoural pathogenesis and progression and in drug resistance lymphocytes (CD45, CD3, CD4, CD8, CD20), histiocytes remain unknown. (CD68), dendritic cells (CD21), activation markers (CD30, Comparative genomic hybridization (CGH) is a technique CD15, EMA) and aggressiveness (MIB1, P53) after heat pre- allowing the identification of imbalances of the entire genome treatment for antigenic retrieval. The two monoclonal anti- in terms of DNA copy number with a putative diagnostic and bodies used to consider neoplastic aggressivity, MIB1 and p53 prognostic value that could remain undetected by conven- showed nuclear strong positivity, without stained cytoplasms. tional cytogenetics. Its main advantage with respect to cyto- Immunocytochemistry technique with previous heat antigen retrieval avoids non-specific cytoplasmic discolored patterns. The quantification of MIBI and the expression of P53 protein was determined with an image analyzer (CAS-200, Becton Correspondence: J Benı´tez, Dept Gene´tica Humana, Centro Nacional Dickinson) with proliferation index program which provides de Investigaciones Oncolo´gicas Carlos III, Ctra Majadahonda-Pozuelo Km 2,200, 28220 Majadahonda, Madrid, Spain; Fax: 34 91 5097055 an accurate and objective method for assessment. P53 over- The first two authors contributed equally to this work expression was defined when positive cells were over 10%. Received 27 February 2001; accepted 15 June 2001 P53 staining is absent in reactive lymphoid tissue, and 10% Chromosomal and gene amplifications in T-NHL M Renedo et al 1628 Table 1 Clinical and tumor status data from 29 patients with Tcell NHL Case Stage Status Sex Subtype Immunophenotype P53 PI % TCR CGH 1a IA R M PTCL (Pleomorphic) CD3+, CD4+++, CD8+ + 5MN 1b IVA 2nd R ND ND M A 2 NA D F PTCL (Pleomorphic) CD3+, CD4+++, CD8+ + 5PA 3 NA D F PTCL (Pleomorphic) CD3+, CD4+++, CD8+ − 15–20 P N 4 IA D F PTCL (Pleomorphic) CD3+, CD4+++, CD8+ + 10–30 P A 5 NA D F PTCL (Pleomorphic) CD3+, CD4+, CD8+ − 30 ND N 6 IVB D M PTCL (Pleomorphic) CD3+, CD4+++, CD8+ − 50 P A 7a IIIA D CD3+, CD4+++, CD8+ + 60 M A 7b IVA R M PTCL (Pleomorphic) ND 60 P A 7c IVB R ND 55 M A 8a NA D F PTCL (Pleomorphic) CD3+, CD4+++, CD8+, CD30+ − 20–30 M N 8b R + ND M A 9 IVB D F PTCL (Pleomorphic) CD3+, CD4++, CD8+, CD30+ − 50 M A 10 IVA D F PTCL (Pleomorphic) CD3+, CD4+++, CD8+, CD30+ ND 70 P A 11 NA D M PTCL (Pleomorphic) CD3+, CD4++, CD8+ ND 20 P N 12 NA D M PTCL (Pleomorphic) CD3+, CD4+++, CD8+, CD30+ − ND M N 13 NA D M PTCL (Lymphoepithelioid) CD3+, CD4+++, CD8+ + 5–10 M A 14 NA D F PTCL (Lymphoepithelioid) CD3+, CD4++, CD8+ − 20–30 M A 15a NA D M PTCL (Lymphoepithelioid) CD3+, CD4++, CD8+ − 15–20 ND N 15b R ND ND M N 16a IA D M PTCL (Lymphoepithelioid) CD3+, CD4++, CD8+, CD30+ − 30 M N 16b CR ND ND P N 17 NA D M PTCL (Lymphoepithelioid) CD3+, CD4+++, CD8+ ND 15–20 M N 18 NA D M PTCL (AILT) CD3+, CD4++, CD8+ − 80 M A 19 IIIB R M PTCL (AILT) CD3+, CD4++, CD8+ − 60 M A 20 IA D M PTCL (Anaplastic) CD3+, CD30+ + 30 M A 21 NA D M PTCL (Anaplastic) CD3+, CD30+, ALK1+ + ND P A 22a IIA D F PTCL (Anaplastic small CD3+, CD30+ − 80 M N 22b R cell variant) + ND M A 23 IIB D F PTCL (MF) CD3+, CD4++, CD8+ − 5–10 P A 24 NA D M PTCL (MF) CD3+, CD4++, CD8+ − 5PA 25 NA D M PTCL (Unclassifiable) ND ND M A 26a IVA D M Lymphoblastic CD3+, CD1+ − 80 M N 26b IVB R ND ND M N 27 IVA D F Lymphoblastic CD3+, CD1− + 70 P A 28 IVA D F Lymphoblastic CD3+, CD1+ − 80 ND A 29 NA D M Lymphoblastic CD3+, CD1+ ND 80 ND A PTCL, peripheral T cell lymphoma; AILT, angioimmunoblastic T cell lymphoma; MF, mycosis fungoides; D, diagnosis; R, relapse; CR, complete remission; PI, proliferative index; +, P53 protein levels Ͼ10%; −, P53 protein levels Ͻ10%; ND, no data; TCR, T cell receptor rearrangements; M, monoclonal; P, polyclonal; NA, not available; A, altered CGH profile; N, normal CGH profile. was accepted as a correct value of expression in high-grade Mannheim). The amplification cycles consisted on: 45 s at lymphomas. In relation to MIB1, we considered cases under 94°C, 45 s at 54 or 55°C (mix I or mix II) and 45 s at 72°C, and over 40% of positive cells to correlate with the presence followed by final extension of 5 min at 72°C. The PCR pro- of altered CGH profiles. There is not consensus about precise ducts were then resolved in a 9% polyacrylamide gel (19:1 quantitative values of MIB1 to differentiate low and high acrylamide:bisacrylamide ratio), and then stained with grades in Tcell lymphomas, but in our experience at over 40– ethidium bromide. 50% a major aggressiveness is expected.9 Estimates of the proportion of tumoral cells ranged from 60 to 100% in peripheral T-lymphomas and between 90 and 100% in lymphoblastic lymphomas. Comparative genomic hybridization TCR (T cell receptor) gene rearrangements Comparative genomic hybridization (CGH) was performed according to Kallionemi et al,11 with some modifications.
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