Cytogenetic characterization of Epstein-Barr virus-associated T-cell malignancies

Cytogenetic Characterization of Epstein-Barr Virus-Associated T-Cell Malignancies Hwei-Fang Tien, Ih-Jen Su, Sou-Ming Chuang, Fen-Yu Lee, Ming-Chi Liu, Tsen-Fang Tsai, Kai-Hsin Lin, and Rong-Long Chen ABSTRACT: Recently, Epstein-Barr virus (EBV) infection has been found not only to be associated with Burkitt l y m p h o m a and nasopharyngeal carcinoma but also with some T-cell malignancies. Cytogenetic studies were performed on four Chinese patients with EBV-associated T-cell neoplasms: three peripheral T-cell lymphomas and one large granular lymphocyte leukemia with coexpression of T-cell antigen. Clonal chromosomal abnormalities were detected in all four patients. Rearrangements of chromosome 7 were observed in three patients: one at 7p22, one at 7q35 or 36, and the remaining one at both sites. The last patient also had a chromosomal abnormality involving 14qfl. Trisomy of part of the lq segment was detected in two patients. The results revealed that the chromosomal abnormalities in these patients were similar to those observed in other T-cell lymphomas. Further studies on more patients are necessary to find out whether there are specific chromosomal aberrations in EBV-associated T-cell neoplasms.

INTRODUCTION

MATERIALS AND METHODS

Epstein-Barr virus (EBV) infection has been found to be associated with infectious mononucleosis [1], African Burkitt lymphoma, nasopharyngeal carcinoma [2, 3], Hodgkin's disease [4], and the B-cell lymphoma which frequently occur in patients in an immunodeficient state [5, 6]. More recently, some peripheral T-cell lymphoma (PTL) and lymphoproliferative disease of granular lymphocytes have been demonstrated to contain EBV genomes [7-9]. The great majority of Burkitt lymphomas display a characteristic translocation between chromosomes 8 and 14, t(8;14)(q24;q32); a minority show a variant translocation, t(2;8)(p12;q24) or t(8;22) (q24;q11). The cytogenetic rearrangements, which cause dysregulation of the proto-oncogene c-myc, probably may be induced by EBV [10]. Though the number of reported cases is very limited, a 3q + marker chromosome is probably a nonrandom chromosomal abnormality in nasopharyngeal carcinoma [11, 12]. No cytogenetic finding of EBV-associated T-cell malignancies has been previously reported in the English literature until now. We present the results of cytogenetic study on four patients with EBV-associated T-cell neoplasms.

Patients Cytogenetic studies were done on four EBV-associated T-cell malignancies: three PTL and one large granular lymphocyte leukemia (I£;LL) with expression of T-cell antigen. The clinical and pathologic findings of these patients are summarized in Table 1. The detailed clinical features of patients 1 and 3 have already been reported [13, 14].

From the Departments of Internal Medicine (H. F. T.), Pathology (I. ]. S., E Y. L., M. C. L., S. M. C.), Dermatology (T. E T.), and Pediatrics (K. H. L., R. L. C.), National Taiwan UniversityHospital, Taipei, Taiwan, Republic of China. Address reprint requests to: Hwei-Fang Tien, M.D., Department of Internal Medicine, National Taiwan University Hospital, No. Z Chung-Shan South Road, Taipei, Taiwan, R.O.C. Received September 21, 1992; accepted April 7, 1993.

Cytogenetic studies were performed on lymphoma or leukemia cells from pleural effusion in patient 1, from BM in patient 2, from skin in patient 3, and from PB in patient 4. The cells from patient I were harvested directly as described previously [17], and those from patient 2, after 2-day culture without stimulant. Tumor tissue from the skin of patient 3 was minced by surgical blade into small fragments and the

Morphology and Immunophenotypic Studies Morphologic observation was done on B5-formalin-fixed tumor tissues, stained with hematoxylin-eosin (H-E) in the three PTL patients, and on bone marrow (BM) and peripheral blood (PB) smears, using the Romanowsky system [15], for the LGLL patient. Immunophenotypic studies were performed on sections of frozen tissues from the PTL patients using a panel of B- and T-cell specific or associated monoclonal antibodies, including CD19, CD20, CD2, CD3, CD4, CD8, CD7, CD25 (Tac), Ia(HLA-DR), and CD30(Ki-1) by an avidin-biotin-complex peroxidase method [16] and on PB cells of the LGLL patients by an indirect alkaline phosphatase method [15]. Chromosome Analysis

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Cancer Genet Cytogenet 69:25-30 (1993) 0165-4608/93/$00.00

26

H.-F. Tien et al.

Table 1

Histopathologic and clinical findings in four patients with EBV-associated malignancies

Case No.

Age/Sex

Diagnosis

Immunophenotypic studies

Clinical

CD2 CD3 CD4 CD7 CD8

manifestations

1

13/M Immunoblastic lymphoma

+

+

-

+

+

2

31/F

Immunoblastic lymphoma

+

+

-

-

+

3

69/F

+

-

+

+

-

4

14/F

Angiocentric lymphoma of medium cell size Large granular lymphocyte leukemia

+

-

-

+

-

Clinical Survivalb staginga (mo)

Fever 1 mo, jaundice, lymphadenopathy, hepatosplenomegaly, pleural effusion. Fever 9 mo, brain lesions, lymphadenopathy, hepatomegaly, bone marrow ( + ). A brown red nodule on the left lower leg for 2 mo. Hepatosplenomegaly, intermittent fever, and erythematous skin papules for 7 years.

IV

3 (D)

IV

19 (D)

I

13 (D)

-

6 (D)

Abbreviations: EBV. Epstein-Barrvirus; CD, cluster designation; mo, month; D, died.

a Clinical staging for lymphomapatients. b Survivalafter diagnosis.

separated cells were washed and set up for 1-day culture without stimulant before harvest. PB cells from patient 4 were harvested after culture for 2 days with phytohemagglutinin (PHA), interleukin-2 (IL-2), or without stimulant. Chromosomes were b a n d e d by a conventional trypsin-Giemsa banding technique [18] and karyotyped according to the ISCN [19]. Study of EBV DNA in T u m o r Tissue Tumor-tissue DNA was extracted from frozen specimens. The presence of EBV DNA was demonstrated by the method described previously [8]. Briefly, 10 ~g of t u m o r DNA was digested with BamHI restriction endonuclease (Boehringer,

Figure l a Southern blot hybridization for EBV DNA of four cases of EBV-associated T-cell malignancies using EBV BamHI-A and -W fragment probes, respectively. Control DNAswere from two EBV-containing cell lines, Raji and B95.8, and one EBV-negativeT-lymphoma cell line HUT-78 negative control (NC).

n-

re

Z

1

2

3

4

M a n n h e i m , Germany), size fractionated by electrophoresis i n a 0.8% agarose gel, and transferred to a Zeta-probe n y l o n merflbrane (AMF; Canno, Meriden, CT) with a standard Southern blot technique. After baking for 2 hours, the membrane was prehybridized and hybridized with the probes of EBV BamH1-A and -W fragments (kindly provided by Prof J.L. Strominger, Harvard University, Cambridge, MA) at a high radioactivity of 1-3 x 108 cpm/llg. For clonality analysis of EBV genomes, an EBV-termini fragment probe (BaMH1NHet fragment, 13kb; kindly provided by Dr. Nancy RaabTraub at the University of North Carolina, Chapel Hill, NC) was used for Southern blot analysis. The hybridized membranes were then washed and autoradiographed at -70°C for 1-3 days.

Figure l b Clonotypic proliferation of EBV genome was evaluated by using EBV BamHI-NHet terminal fragment probe. A single band was detected for the four cases of EBV-associated T-cell malignancies, suggesting clonal proliferation ofT cells, in contrast to the multiple hybridization bands in lymphoblastoid cell line (LBL). Negative control (NC)was from an EBV-negativeT lymphoma line, HUT-78.

.u

Kb EBV~A

12

ii!~/i~//¸/

3

EBV (W)

/

.

O

z

1

2

3

4

27

Cytogenetics in EBV-Associated T-Cell Malignancies

Table 2 Case No.

Cytogenetic findings of four EBV-associated T-cell malignancies No. of m e t a p h a s e Sample

normal

abnormal

Representative karyotype of abnormal clones 71-75,XXY,dup(1)(q21q24),idic(6)(q21), + del(7)(q35 or q36), - 9,der(lO) t(6;10)(q15;q26), + 13, + 15, + 19, + der(19)t(5;19)(q11;p13), + 20. - 22, + mar[cp24] 69-75,XX,t(X;14)(p11.4;q11), + add(1)(p22), + add(2)(q31), - 4,t(4;11) (q13;q25), + 5, + add(7)(p22q35 or q36), + 8, + del(9)(q22q34), + der(13) t(8;13)(q11;q14), + 14, + 14, - 15, - 19, - 21, - 22, + m a r l , + mar2, + mar3[cp8] 45,X, - X,der(21)t(3;21)(p12;p12) 46,XX,der(4)t(1;4)(q23;q31),add(7)(p22) 46,XX,der(4)t(1;4)(q23;q31),add(7)(p22)

1

PE/dir

0

24

2a

BM/2d

9

8

3 46

Skin/ld PB/IL-2 2d PB/PHA 2d

0 1 13

11 31 4

a Cytogenetic study was done after chemotherapy in this patient. No metaphase chromosome could be found in samples cultured for 2 days without stimulant. Abbreviations: PE, pleural effusion; BM, bone marrow: PB, peripheral blood; dir, direct harvested; ld, 2d, harvested after 1- or

2-day culture; IL-2, interleukin-2 stimulated; PHA, phytohemagglutinin stimulated.

F i g u r e 2 Karyotype of a l y m p h o m a cell from case 1 showing hypertriploid c h r o m o s o m e s w i t h clonal structural abnormalities d u p (1)(q21q42), de 1(7)(q35 or 36), der(10)t(6 ;10) (q15; q26), der(19)t (5; 19) (q11;p13), a n d a marker. This cell also h a d a n o n c l o n a l abnormality del(6). A partial karyotype from another cell showing the same structural aberrations is o n the bottom. The idic(6)(q21) in this cell, w h i c h is not p r e s e n t in the above cell, is also a clonal abnormality. Arrows indicate rearranged chromosomes.

i:!f 1

2

iii

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M

M

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H.-F. Tien et al.

3

A

21

3

B

b

\

2!

it \

|

C

m

D

\

\ Figure 3 Partial karyotypes of four cells from case 3 showing der(21)t(3;21)(p12;p12).

RESULTS

Morphology and Immunophenotypic Studies Tumor cells from all four patients expressed pan-T markers (Table 1), two of them were of the T-cytotoxic/suppressor phenotype (CD8 positive, patients 1 and 2), one was of the T-helper/inducer phenotype (CD4 positive, patient 3), and the remaining one was neither of them (CD4 and CD8 negative, patient 4). The histology of patients I and 2 was classified as immunoblastic lymphoma and that of patient 3, as angiocentric lymphoma of medium cell size [20]. The leukemic cells of patient 4 had the characteristic features of large granular lymphocytes: a low nuclear cytoplasmic ratio and prominent azurophilic granules. The clinical manifestations of these patients are summarized in Table 1.

Study of EBV DNA in Tumor Tissue The EBV DNA could be detected in the cell or tissue DNA extracted from leukemic cells (case 4) or tumor tissues (cases 1-3) of the four patients by Southern blot hybridization, using BamH1-A and -W fragment probes (Fig. la). Clonotypic proliferation ofEBV genomes in the tumor cells was further verified by demonstrating the single hybridization band using BamHI-NHet fragment probe, representing the clonal proliferation of the EBV terminal region in the neoplastic cells (Fig. lb).

Chromosome Analysis The results of our cytogenetic studies are summarized in Table 2. Clonal chromosomal abnormalities were detected in all four patients (Figs. 2-4). Rearrangements of chromosome 7 were detected in three patients, at 7p22 in one (case 4), at

7q35 or 36 in one (case 1), and at both sites in one (case 2). Duplication of chromosome lq with or without translocation was observed in two patients (cases 1 and 4). A rearrangement of chromosome 14 at band qll was present in case 2.

DISCUSSION In this study, three of four patients with EBV-associated T-cell malignancies showed rearrangements of chromosome 7, one {case 4) at 71322, one (case 1) at 7q35 or q36, and the remaining one (case 2) at both sites. Case 2 also had chromosomal abnormality involving 14qll. Chromosomal aberrations involving 14q11, 7q35, and 7p15, where the T-cell receptor (TCR) a/8, [3, and y chain genes, respectively, are located [21-24], have been reported in various T-cell malignancies [25-27]. It is apparent that these chromosomal abnormalities are related to T-cell phenotype of the malignant cells, irrespective of whether EBV DNA is present in the tumor tissue or not. The same condition is also observed in Burkitt lymphoma. A characteristic chromosomal translocation, t(8;14), or its variant form, t(2;8) or t(8;22), is present both in the endemic African type and in nonendemic type of Burkitt lymphoma [28] and is not correlated with EBV [29, 30]. Chromosomal abnormalities involving 7p22, a site not far from the locus where TCR chain genes are mapped, were found in two of the four patients studied, but also have been reported in various hematologic malignancies including myeloid leukemia, lymphoid leukemia, myeloma, and lymphoma [31]. The role of platelet-derived growth factor, whose a-chain was mapped to 7pter-p21 [32], in tumors with 71322

Cytogenetics in EBV-Associated T-Cell Malignancies

29

i

¢ F ....

1

2

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6

13

14

19

3

4

ti

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8

9

10

11

12

Ifi

16

20

21

17

7

4

18

22

I!.'i 4

5

7

XX

c 4

7

Figure 4 Karyotype from case 4 showing 46,XX,der(4)t(1;4)[q23;q31),add(7)(p22). Partial karyotypes from three other cells are on the bottom. Arrows indicate rearranged chromosomes.

rearrangement needs further investigation. Chromosome rearrangements giving rise to trisomy of total or part of lq, as observed in two of the four cases, has been noted in many different types of tumors [33], and is possibly not a specific abnormality for EBV-associated T-cell malignancies. It has been recognized that a minority of patients who have classical EBV-induced infectious mononucleosis may develop chronic active EBV infection [34]; although it is unusual, some of them progress to T-cell l y m p h o m a [7] or LGLL [35]. About 20% of the peripheral T-cell lymphomas in Taiwan were found to contain the EBV genome in tumor cells [8]. EBV DNA was also detected in PB cells from five of seven patients with CD3-negative I ~ L L reported by Kawa-Ha et al. [9]. Sometimes it may not be easy, clinically or pathologically, to identify these patients as malignant T-cell proliferation rather than chronic infection or postviral syndrome associated with EBV or other infectious agents [8, 35, 36]. In addition to TCR ~-chain gene rearrangements, demonstration of clonal chromosomal abnormalities provides definite evidence for the clonal origin of the expanded cells. The diagnosis of T-cell l y m p h o m a was not made for case 1 until a clonal chromosomal aberration was detected in the cells of pleural effusion. Patient 4 had suffered from intermittent

fever and erythematous skin papules for 7 years before final diagnosis of I ~ L L was made. Cytogenetic study in these patients is very helpful for the differential diagnosis. Further study is necessary to correlate the chromosomal findings with prognosis for the disease.

This study was supported in part by grants from the National Science Council of the Republic of China, NSC 81-0412-B002-658.

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