Cytogenetic abnormalities in noncutaneous peripheral T-cell lymphoma

Cytogenetic Abnormalities in Noncutaneous Peripheral T-Cell Lymphoma Warren G. Sanger, Dennis D. Weisenburger, James O. Armitage, and David T. Purtilo

ABSTRACT: Cytogenetic studies were performed on lymph nodes from eight patients with noncutaneous peripheral T-cell lymphoma. At least one chromosomally abnormal clone was identified in each patient. Chromosomes having structural abnormalities in two or more patients included chromosome #1 (four), chromosome #2 (three), chromosome #4 (two), chromosome #8 (two), chromosome #14 (two), and chromosome #17 (two). The abnormal clones from seven patients had structural abnormalities involving either chromosomes #1 or #2, and the eighth patient had an abnormal clone that was trisomic for chromosome #1. We did not observe structural rearrangements in chromosome #14 at bands q l l or q12 in any of our cases, in contrast to previous suggestions that this chromosomal region may play a critical role in the development of T-cell lymphomas. Our findings suggest that other nonrandom chromosome abnormalities are common and may be important in the development of noncutaneous peripheral T-cell lymphomas. INTRODUCTION Cytogenetic abnormalities have been reported in a small n u m b e r of western T-cell lymphomas [1-8]. However, a n o n r a n d o m pattern of chromosome abnormalities has failed to emerge from these studies. Hecht et al. [1] and Zech et al. [9] have reported that abnormalities i n v o l v i n g the proximal portion of the long arm of chromosome #14 near band q l l are c o m m o n l y present in T-cell malignancies. Hecht et al. [1] have suggested that rearrangements in this region may be related to T-cell growth and function. Recently, Croce et al. [10] have identified the gene locus for the alpha chain of the T-cell receptor at q l l - q l 2 on chromosome #14. Croce et al. [10] speculated that the alpha chain of the T-cell receptor may participate in oncogene activation and the subsequent d e v e l o p m e n t of T-cell malignancies. In this report, we describe the cytogenetic abnormalities of eight patients with n o n c u t a n e o u s peripheral T-cell lymphoma. At least one abnormal clone with a structural rearrangement of chromosomes #1 or # 2 was observed in each of seven patients, and trisomy for chromosome # a was observed in the eighth patient. In contrast to previous studies of various types of T-cell malignancies [1, 8, 9, 11], we did not find abnormalities involving the q l l - q l 2 region of chromosome # 1 4 in our patients. From the Departmentof Pediatrics (W.G.S.,D.P.T.). the Departmentof Pathology and LaboratoryMedicine (W.G.S., D.D.W., D.P.T.), and the Department of Internal Medicine (J.O.A.), University of Nebraska Medical Center, Omaha, NE. Address requests for reprints to Warren G. Sanger, Center for Human Genetics, University of Nebraska Medical Center, 42nd & Dewey Avenue, Omaha, NE 68105. Received August 30, 1985; accepted October 26, 1985.

53 © 1986 Elsevier SciencePublishingCo., Inc. 52 VanderbiltAve., New York, NY 10017

Cancer Genet Cytogenet23:53-59 (1986) 0165-4608/86/$03.50

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w . G . Sanger et al.

MATERIALS AND METHODS

Lymph node biopsy specimens were obtained from patients being studied by the Lymphoma Study Group at the University of Nebraska Medical Center. With the exception of patient 6, who had received chemotherapy for 3 years before a biopsy was studied, none of the other patients had been treated previously. All lymph nodes were processed for histologic, immunologic, and cytogenetic studies according to a standard protocol. Lymph node biopsy specimens involved by a lymphoma were received and processed by the cytogenetics laboratory within 1 hour after the lymph node biopsy procedure, or the specimens were similarly received, cultured, and processed through the fixation stage by a nearby hospital laboratory before shipment. The tissue was placed directly into RPMI 1640 medium (Gibco) supplemented with 20% fetal bovine serum and gentamycin. The lymph node was mechanically minced and placed in culture at 37°C without mitogens for 24 hours. If sufficient tissue was available, a 48-hour culture was also performed. Two hours prior to the initiation of harvest, the cells were exposed to colcemid (0.05 ~gm/ml). The cells were then resuspended in 0.074 M KC1 for 10 minutes and fixed with methanol/glacial acidic acid (3:1). The fixation procedure was repeated three times and slide preparations were made. The slides were aged for a minimum of 24 hours and G-banded with Wright's stain. All metaphase plates were microscopically analyzed, recorded, and" photographed. Karyotypes were arranged according to the Paris Conference [12]. An abnormal clone was defined as either two or more cells with the same structural abnormality or the same extra chromosome, or the presence of three or more cells with the same missing chromosome. If mitoses could not be confidently analyzed, the mitotic cell was not included in the data. Portions of each lymph node biopsy specimen were fixed in B5 and formalin for routine histologic processing. Each case was classified according to the modified Rappaport classification [13] and the Working Formulation nomenclature [14]. Representative portions of fresh nodal tissue were also prepared for frozen section immunohistochemical analysis. Briefly, a 2.0-mm section of tissue was placed in OCT R embedding media (Miles Laboratories, Naperville, IL) and snap frozen at -150°C in isopentane quenched in liquid nitrogen. The tissue was then stored at -70°C until sectioned. Cryostat sections were air-dried and fixed in 4°C acetone for 10 minutes. In situ immunologic phenotyping was performed with a three stage immunoperoxidase technique [15] employing unconjugated mouse antibody in the first stage, followed by biotin conjugated goat anti-mouse F(ab')2 antibody, and then avidin conjugated horseradish peroxidase (Vector Laboratories, Inc., Burlingame, CA). Primary mouse antibodies to immunoglobulins (G,A,M,D, kappa and lambda; DAKO Corporation, Santa Barbara, CA), B-cell antigens BA1 (Hybritec, Inc., San Diego, CA), B4 (Coulter Immunology, Hialeah, FL) and Leu 14 (Becton-Dickinson, Inc., Mountain View, CA), and T-cell antigens OKT 4, 8 and 11 (Coulter) and Leu 1-4 (Becton-Dickinson) were used. The designation of T-cell lymphoma was based on an overwhelming majority of the cells staining with one or more monoclonal Tcell antisera and a low percentage of polyclonal B cells. RESULTS

All eight patients were classified as having intermediate or high grade diffuse lymphomas according to the Working Formulation (Table 1). Immunophenotypic analysis revealed that seven of the cases were of helper/inducer cell phenotype (OKT 4, Leu 3 positive). The other case (case 2) consisted of an equal mixture of two cell populations, one with a helper/inducer cell phenotype (OKT 4, Leu 3 positive) and the other with a suppressor/cytotoxic cell phenotype (OKT 8, Leu 2 positive).

59/M

86/F

37/M

60/M

20/M

76/F

77/F

2

3

4

5

6

7

8

Mixed cell/mixed cell Mixed cell/mixed cell Poorly differentiated lymphocytic/ small cleaved cell

Lymphoepithelioid/ immunoblastic with epithelioid cell component Lymphoepithelioid/ immunoblastic with epithelioid cell component "Histiocytic"/ immunoblastic, clear cell type Mixed cell/mixed cell Mixed cell/mixed cell

Helper/inducer

Helper/inducer

Helper/inducer

Helper/inducer

Helper/inducer

Helper/inducer and suppressor/ cytotoxic Helper/inducer

Helper/inducer

Immunologic phenotype

"Rappoport Classification/Working Formulation

56/M

1

Lymphoma classification a

0 0

16

0

3

0

83

19

20

Ceils with normal chromosomes

16 3

4

14

21

23

4

6

5

Cells with abnormal chromosomes

45,XX,t(2;8)(p24;q21),- 1 7 , - 17, + i(17q)/46,XX,t(2;8)(p24;q21}, - 17,- 17,+i(17q},+mar

47,XY, - 1, + del(1)(p13), + delf1)(p13), t(6;8)(p24;q21),- 1 4 , - 15, del(17)(q23), + 2mar 47,XY,del(2)(p23),del(4)(p15), + 7, del(18)(p11} 53,XX, + 1, + 6,+ 7, + 8 , + 9, + 12, + 16

46,XY,t(2;9)(p24;p23}

60,XX, + X, + 1, + del(1)(q23}, - 2, + 3, - 4 , + 5,+ 6 , + 8 , + 9,+ 11,+ 12, + 13, +17,+ 18,+20,+21,+22

46,XY,- 1, + der(1)t(1;1)(p22;q23), t(6;14)(q23;q32)

47,XY, + i(lq}, + 2, + 3, - 4, t(1;7;11)(q22;q36;q13), - 14, t(4;14)(q26;q32}

Modal karyotype

Histologic, immunologic, and cytogenetic findings in noncutaneous peripheral T-cell lymphoma

Case Age/Sex

Table 1

2p24 8q21

2p24

8q21

2p24

lq23

lq25 6q23

lq22

nlos

N-myc

N-myc

mos

N-myc

ski

ski myb

ski

Breakpoints near assigned oncogenes Oncogenes

56

W. G. Sanger et al. No consistent pattern of chromosome abnormalities was found. All eight patients had at least one chromosomally abnormal clone with modal chromosome numbers ranging from 45 to 60 (Table 1). Clones from two or more patients included structural abnormalities of chromosome #1 (four), chromosome #2 (three), chromosome #4 (two), chromosome # 8 (two), chromosome #14 (two), or chromosome #17 (two). Seven patients had an abnormal clone with a structural abnormality involving chromosome #1 or # 2 and the eighth patient (case 7) had an abnormal clone that was trisomic for chromosome #1. Figure 1 includes a representative karyotype from case 2. Although two of the eight patients (cases 1 and 2) had a structural abnormality involving chromosome #14, neither of the abnormalities involved band q l l or 12. Instead, the abnormalities involved the q32 locus. The only possible exception to this was case 5 in which there were two small identical supernumerary marker chromosomes, which could have been derived from chromosome #14. Numerical abnormalities were found in all chromosomes except chromosomes #10, #19, and the sex chromosomes. In addition to the presence of a chromosomally abnormal clone in each patient, chromosomally normal cells were detected in cases 1, 2, 3, 5, and 7.

Figure 1

Representative karyotype from case 2. Arrows denote der(1) and t(6q;14q).

Abnormalities in T-Cell Lymphoma

57

DISCUSSION Cytogenetic studies of T-cell malignancies, in general, have failed to demonstrate a singular nonrandom pattern of chromosome abnormalities. Ueshima et al. [11] recently summarized the chromosome abnormalities in 72 T-cell malignancies of various types. Ueshima et al. [11] reported that structural abnormalities were found most often in chromosome #14 (26 cases), chromosome #2 (15 cases), chromosome #1 (14 cases), chromosome #9 (14 cases), chromosome #12 (11 cases), and chromosome #17 (11 cases), with the other chromosomes being less frequently involved. A comparison of the chromosome abnormalities of our eight cases with western noncutaneous peripheral T-cell lymphoma to those observed in 16 cases in the literature [1-8] reveals similar findings (Fig. 2). Structural abnormalities were commonly present in chromosomes #1, #2, #14, and #17 in both the reported cases and [1-8] our cases and no single chromosomal region was consistently involved. However, seven of our eight cases had a structural abnormality involving either chromosome #1 or #2. In a study of 15 cases of Japanese adult T-cell leukemia/lymphoma, Ueshima et Figure 2 Chromosome abnormalities in noncutaneous peripheral T-cell lymphoma (16 cases in the literature and our eight cases). m t

E

e

--

c

-! Z

Chromosome

NI

10

m

ii O c .Q .(

O C0

E ~e-

m

= Cases

J-~:

Present

from the literature cases

58

W.G. Sanger et al. al. [16] reported a high incidence of trisomy 7 or 7q (5 cases) and two cases with 14q+ marker chromosomes. In contrast, Miyamoto et al. [17, 18] did not observe chromosome #7 abnormalities in any of their 30 patients with Japanese adult Tcell leukemia/lymphoma. Instead, Miyamoto et al. [17, 18] observed an increased frequency of involvement of chromosome #1 (20 cases), chromosome #3 (20 cases), and chromosome #6 (17 cases), and aberrations of the long arm of chromosome #14 (nine cases). In Caribbean adult T-cell lymphoma/leukemia, structural abnormalities of chromosomes #1, #14, #6, and #7 have been reported, with the 7q anomaly being similar to that seen in the Japanese adult T-cell leukemia/lymphoma [19]. In contrast, the most commonly involved chromosomes in mycosis fungoides and Sezary syndrome are chromosomes #1, #2, #5, #8, #9, #10, #14, and #18 [20, 21]. Kaneko et al. [22] have reported that six of eight patients with T-lymphoblastic lymphoma had clonal abnormalities involving either chromosome #1, #6, or #19. None of their eight patients had abnormalities involving 14q. In contrast, Ueshima et al. [11] reported three patients with chronic T-cell leukemia and one patient with T-cell lymphoma having a clonal abnormality, which included a break in chromosome #14 at band q11. In addition, Clare et al. [8] have recently reported two patients with T-lymphoblastic lymphoma, one patient with peripheral T-cell lymphoma, and one patient with T-cell acute lymphoblastic leukemia, each of whom had a translocation or inversion involving chromosome #14. Zech et al. [9] have also reported inv(14)(qllq32) in four of five patients with T-cell lymphocytic leukemia and Hecht et al. [1] have recently described inv(14)(qllq32) in a childhood T-cell lymphoma cell line and in a patient with T-cell chronic lymphocytic leukemia. Hecht et al. [1] have also described a t(10;14)(q23;q11.2) in a T-cell lymphoma cell line. The frequent involvement of chromosome #14 in these latter studies has led investigators [1, 9-11] to propose that the region around 14qll may be related to T-cell growth and function, and that it may be important in the development of T-cell malignancies. Although this proposal may be true for some T-cell malignancies (i.e., T-cell chronic lymphocytic leukemia), it does not appear to be the case for T-cell malignancies in general. For example, none of our eight patients with western peripheral T-cell lymphoma and only one [8] of the 16 patients reported in the literature [1-8] had a lesion of 14q11. These findings suggest that other nonrandom chromosomal events may be important in the development of noncutaneous peripheral T-cell lymphomas. In the acute leukemias, a number of distinct subentities have been identified within disorders that previously appeared to be homogeneous on the basis of their histology and immunology. It is likely that we may be observing the same phenomonen in the T-cell lymphomas. Although oncogene activation analysis was not performed in our study, it is of interest that at least one breakpoint occurred at the locus of a previously assigned oncogene in each of the cases with a structural chromosome abnormality (Table 1). Our findings suggest that chromosomal lesions other than those involving chromosome 14qll or q12 may also be important in the genesis of noncutaneous peripheral T-cell lymphoma. However, further clinical and cytogenetic studies with oncogene activation analysis, are necessary before any firm conclusions can be drawn. Supported in part by PHS Grant no. CA-30196 from the National Cancer Institute, DHHS, Grant LB506 from the Nebraska State Department of Health, the Lymphoproliferative Research Fund, and the Cytogenetics Development Fund. The authors are grateful to Kristi Sauer and Renee Walker for typing the manuscript, Jene Pierson and Teresa Schneider for their computer assistance and table preparations, and Renee Fordyce, Susan Speaks, and Michelle Hess for their expert technical assistance.

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