Cytogenetic Analysis in Ataxia Telangiectasia with Malignant Lymphoma Iskra PetkoviG Ivo LigutiG Mara Dominis, Dagmar Loffler-Bad .ak, Mladen ( epuliG and Melita Naki
ABSTRACT: We present the results of cytogenetic analysis in a brother and sister with ataxia telangiec-
tasia (AT), one of whom had malignant T-cell lymphoma. In both children, cytogenetic analysis of phytohemagglutinin (PHA)-stimulated lymphocytes showed chromosomal instability and inv( 7) in 10% of the cells examined. The malignant lymphoma was analyzed cytogenetically on slides obtained from short-term culture of the lymph node cells; 64 cells were analyzed. A heterogeneous cell population was noted. Fourteen cells (21.9%) had a normal male karyotype; t(7;14)(p14;q12) and inv(7)(p14q35) were observed in 6.3% and 3.1% of metaphases. Owing to low frequency, these cells are probably a characteristic of the basic disease and have no features of malignant cells. Forty cells (62.5%) had a pseudodiploid karyotype 46,XY,dup(1)(p2 2p36 ),del( 5 )(q33 ),del(12 )(p11), without cytogenetically evident aberrations of chromosomes 7 and 14. The results of these investigations suggest that the cells with rearrangements of chromosomes 1, 5, and 12 are malignant cells and did not originate by transformation of cells with inv(7) and t(7;14).
INTRODUCTION Ataxia telangiectasia (AT) is an autosomal recessive disease. The basic clinical characteristics are cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and frequent sinopulmonary infections. Cytogenetic analysis has demonstrated high frequency of chromosome anomalies with frequent clonal aberrations of chromosomes 7 and 14 [1-3]. Patients with AT have an increased risk of developing malignancies as compared with the general population. Malignancies of the lymphoreticular system such as lymphocyte leukemia and malignant lymphomas are frequent [4]. The nature of the AT association and malignancies is not clear. Malignancies can be a characteristic of the disease itself or a consequence of immunodeficiency or of chromosome instability. Immediate evidence of the importance of chromosome aberrations in the development of neoplasms can be obtained by cytogenetic analysis of normal and malignant cells in persons with AT and a malignant disease. Such analyses, however, are very rare. Although a few investigators indicate the development of malignant cell population from the cells with clonal chromosome aberrations present before manifestation of malignancy, others have failed to
From the Institute for Mother and Child Health, Clinical Hospital "Dr. O. Novosel" (I. P., I. L., D. L.-B., M. C., M. N.), and Medical Faculty. University of Zagreb (M. D.), Zagreb, Yugoslavia. Address reprint requests to Dr. sci. ing. Iskra PetkoviG znan. say. ~vearova 5, 41000 Zagreb, Yugoslavia. Received August 20, 1991; accepted January 8, 1992. 158 Cancer Genet Cytogenet58:158-163(1992} 0165-4608/92/$05.00
demonstrate such a relationship [5-8]. Thus, the role of AT chromosome aberrations in the process of malignant transformation has not been clarified and is still of great scientific interest. We performed cytogenetic analysis in two children with AT, of whom one had malignant T-cell lymphoma.
MATERIALS A N D METHODS
Our patients are the only children of healthy parents who are not related. No cases of AT have been reported in the family of either parent. AT was diagnosed on the grounds of the clinical picture and laboratory tests during the children's previous stay in our institute (Table 1). Case 1
In December 1989, the parents first noticed enlarged lymph nodes and appearance of new ones during the next few months, but no changes in the general condition of the child. In July 1990, a normal finding was obtained by cytologic puncture of an enlarged lymph node. Hematologic findings were within normal limits. In December 1990, the boy (aged 10 years) was admitted to our institute for examination and treatment of general lymphoadenopathy. Lymph nodes were 0.3-6 cm, hard, and painless on palpation. An enlarged liver was palpated 2 cm below the right rib arch. Very high a-fetoprotein values (312.2 /~g/L) and deficiency of IgA were established. The number of B lymphocytes was on low-normal, with normal values of the total number of T lymphocytes and suppressor cells and deficiency of inductor cells. Hematologic findings © 1992 Elsevier Science Publishing Co., Inc. 655 Avenueof the Americas,New York, NY 10010
AT with Malignant Lymphoma in a Child
159
Table 1 Clinical Findings in Two Children With Ataxia Telangiectasia Patient
1
2
Sex Cerebellar ataxia Oculomotor apraxia Nystagmus and stabismus Telangiectasia Dysarthria Generalized hypotonia Diminished tendon reflexes Small stature Mental retardation Thymic hypoplasia Recurrent sinopulmonary infections IgA IgMa IgG° IgE° OKT4Q
M + + + + + + + + + + + N t ~, ~
F + + ÷ + + + + + + + + 1' J,
The values are expressed as normal (N), increased ( I" ), or decreased ( ,~).
were unremarkable. Cytologic analysis of the lymph node showed hypercellularity, but no elements existed to diagnose a malignant process. To make a precise diagnosis, we analyzed the lymph node biopsy histopathologically. Pathologic analysis of the biopsy indicated a malignant lymphoma, most probably of the T-cell type. The cells corresponded morphologically to large centrocytes with a number of blasts and polymorphic histiocytes (CD2-, T6 + ). The phenotype of the proliferating cells was not easy to determine because there were many B1 (CD19) and CD2 ÷ or CD3 ÷ cells, with restricted CD4 and CD8. IgM was slightly positive in rare cells, IgG was positive in large cells, and IgE and IgA were negative. Three weeks after the boy was admitted to the hospital a bone marrow (BM) biopsy indicated transformation of malignant lymphoma in the form of acute lymphocytic leukemia (ALL) of atypical morphology. Cytostatic therapy was initiated, but the boy's response was very poor and he soon died.
cells. The cell suspension was prepared by mechanical dissociation of the node in synthetic medium minimal essential medium (MEM) with 20% human AB serum and cultivated overnight at 37°C. The cell suspension was then treated with colchicine (4 /zg/ml, 20 minutes) hypotonic solution (0.075 M KC1, 5 minutes) and finally fixed in the mixture of methanol and acetic acid (3 : 1, vol/vol). Slides were made by the air-drying method and stained for GTG banding.
RESULTS From the lymphocyte culture of the boy, 50 metaphases stained according to G-banding method were karyotyped. In 13 (26%) cells, chromosome aberrations were identified, whereas 37 cells had a normal male karyotype (Table 2). A chromosome break (7q32) was identified in only one cell, whereas other metaphases showed stable chromosome aberrations. Deletions were noted in three cells and translocations were noted in four cells. In seven cells, aberrations of chromosome 7 were observed. Inv(7) (p14q35) was identified in five (10%) cells, in one of them associated with t(3;14)(q29;q24). In one metaphase, together with t(7;7}(p14;q35), t(1;21)(q32;q22) was also present, whereas t(7;14)(p14;q12) was observed in only one metaphase (Fig. 1). Results of the cytogenetic analysis of the lymph node are shown in Table 2. Sixty-four metaphases stained by G-banding were karyotyped. In 14 (21.9%) cells, a normal karyotype was established. Most of the cells analyzed (62.5%) belong to a pseudodiploid clone with 46 chromosomes with aberrations of chromosomes 1, 5, and 12, whereas other chromosomes are of normal morphologic appearance and G-banding pattern (Fig. 2). Analysis of banding pattern of the aberrant chromosome 1 established a duplication of the short arm segment, i.e., p22-p36. Chro-
Table 2 Results of the Chromosome Analyses in Patients With Ataxia Telangiectasia and Malignant Lymphoma
Case 2 The proband's sister was admitted for a pediatric checkup at age 11 years. With regard to the basic disease, the child's condition was unchanged. Neither lymphoadenopathy nor hepatosplenomegaly was observed. Hematologic findings were within normal values.
Cytogenetic Analysis Lymphocytes of both children were analyzed cytogenetically on slides obtained from PHA-stimulated cultures. The cells were cultivated for 3 days at 37°C and then treated with colchicine (4/~g/ml, 1.5-2.5 hours), hypotonic solution (0.075 M KC1, 5 minutes) and fixed in the mixture of methanol : acetic acid (3 : 1, vol/vol). The slides were made by the flame-drying method and stained for GTG banding. Malignant cells were analyzed cytogenetically (case 1) on slides obtained from short-term culture of lymph node
Peripheral
Lymph
blood
node
Karyotype
n (%)
n (%)
46,XY c h r o m o s o m e break 46,XY,inv(7) 46,XY,inv(7),t(3;14) 46,XY,t(7;14) 46,XY,t(7;7),t(1;21) 46,XY,dup(1),del(5),del(12) 46,XY,trip(1),del(5),del(12) Other changes Total
37 (74) 1 (2) 4 (8) 1 (2) 1 (2) 1 (2)
14 (21.9)
° Includes cells with (p13;q13), t(6;21)(q21;p11).
5 a (10) 50
2
(3.1)
4
(6.3)
40 (62.5) 1 (1.6) 3 b (4.6) 64
del(18)(p11),del(10)(p13),del(1)(q31),t(10;20)
b Includes two near-tetraploid cells and dup(17).
160
I. Petkovi~ et al.
a
mosome 1 is unstable because one cell with the short arm triplication was identified by this analysis. The aberrant chromosome 5 is most probably the result of terminal deletion of the long arm with the break in region 5q33. In all aberrant clone cells, the short arm of chromosome 12 was deleted, i.e., del(12)(p11). In 9.4% of cells AT-specific aberrations of chromosome 7 were noted but with different frequency with regard to the patient's lymphocyte analysis (Table 2). Thus, t(7;14)(p14;q12), as the only chromosome aberration, was identified in four (6.3%) and inv(7)(p14q35) was identified in two (3.1%) cells examined. Cytogenetic analysis of the proband's sister (case 2) included 50 metaphases stained by G-banding method. In 14 (28%) cells, chromosome aberrations were identified; others showed a normal female karyotype. Inv(7)(p14q35) was identified as a clonal chromosome aberration in five (10%) metaphases examined.
b
Figure 1 Chromosome aberrations observed in the peripheral blood cell culture from case 1: (a) inv(7), (b) t(7;14), (c) t(7;7) associated with t(1;21).
DISCUSSION We performed cytogenetic studies of normal and malignant cells in the boy with AT and malignant T-cell lymphoma. The analyses of malignant cells showed pseudodiploid
Figure 2 G-banded karyotype of a malignant clone from the lymph node of case 1: 46,XY,dup(1)(p22p36), del(5)(q33),del(12)(p11).
1
2
3
6
7
8
13
14
15
19
20
9
4
5
10
11
12
16
17
18
21
21
X¥
AT with Malignant L y m p h o m a in a Child
Table 3
Cytogenetic F i n d i n g s in Patients With AT and Malignant Disease
Sex/age
Type of malignancy
1
F/48
CLL
T-Cell
2
F/32
CLL
T-Cell
3
F/30
CLL
T-Cell
4
M
CLL
T-Cell
5
M/27
CLL
T-Cell
6
F/25
Snbacute ALL
T-Cell
Patient
161
Chromosome changes in peripheral blood
Cell type
45,XX,- 14,t(14;14) (q12;q32) 45,XX,- 14,t(14;14) (q11;q32) 46,XX,t(14;14) (q12;q32) 46,XY,inv(14) (q12q32)
45,XX,- 14,t(14;14) (q12;q32)
Chromosome changes in malignant cells
Reference
44,XX,- 14,- 20,6q- ,i(8q), 12p - ,20p + ,t(14;14)(q12;q32) t(14;14)(q11;q32)
[6]
41,XX, - 13, - 14,- 15,- 16,- 20 t(1 ;13),t(15 ;18),t(14;14) (q12;q32),10q + 46,XY,6q- ,8p + ,11p - ,inv(14) (q12q32),r(16),19p +, - 20 t(21;21), + mar 46,XY/44,X, - Y , - 20,t(4;20) (q13;q12),del(14)(q21q31), t(6;19;22)(p11;p13;q13) 43 ,XX,t(13 ;15),t(13 ;17), t(14;14)(q12;q32),18q ~-,
[12]
[7]
[5]
[13]
[14]
-13,-14,-16,-17,-21,-22,
7
F/18
ALL
8
M/5
9
F/25
Burkitt lymphoma Hodgkin's disease (HD)
10
F/20
PLL
T-Cell
11
M/10
Malignant lymphoma
T-Cell
Abbreviations: CLL, chronic
Atypical T-cell B-Cell
46,XX,t(14;14)
Mixed cellularity
46,XX/45,XX,- 14 (18 months before HD) 45,XX,- 14,del(6)(q21) (4 months before HD) 46,X,t(X;14)(q28;q11)
46,XY
46,XY/46,XY,inv(7) (p14q35)
+ 3mar 46,XX/45,XX, - 9,t(9;16) (q12;p13) 46,XY,t(8,14)(q23;q32) (cell line) Not done
t(X;14)(q28;q11),t(1;14) (p21;q11),t(8,22)(q24;q11) 46 ,XY/46,XY,t(7;14) (p14;q12)/46,XY,inv(7) (p14q35)/46,XY,dup(1) (p22p36), del(5](q33), del(12)(p11)
[8] [15] [16]
[17] Present case
lymphocytic leukemia; ALL,acute lymphocytic leukemia; PLL, prolymphocytic leukemia.
karyotype with structural rearrangement of chromosomes 1, 5, and 12, but not of c h r o m o s o m e s 7 and 14. Chromosome instability, characteristic of patients with AT, was established by cytogenetic analysis of l y m p h o c y t e s in this patient. Unlike the investigations of some researchers in patients with AT and neoplasms, we d i d not observe t(14;14) or inv(14), but inv(7) was a clonal anomaly in 10% of the cells analysed. A c c o r d i n g to literature data, large clones of 7 0 - 1 0 0 % of cells refer to t(14;14), inv(14), and t(X;14), whereas inv(14) is considered the most frequent clonal aberration of n o n m a l i g n a n t cells in patients with AT [9, 10]. Aberrations of c h r o m o s o m e 7, such as t(7;14), t(7;7), and inv(7) are a frequent characteristic of AT, but as in our patient, were not identified as large cell clones [9, 11]. Cytogenetic analysis of the l y m p h node in our patient s h o w e d a heterogeneous cell population. Apart from the cells of n o r m a l d i p l o i d karyotype, two clones with t(7;14) and inv(7) were identified as the only chromosome aberration. Considering the low frequency of aberrations, the cells
are probably a characteristic of the basic disease a n d have no features of malignant cells. The d o m i n a n t cell p o p u l a tion has the p s e u d o d i p l o i d karyotype 46,XY,dup(1), del(5),del(12), includes 62.5% of the cells e x a m i n e d , and obviously has selective proliferating advantage and is responsible for malignant d e v e l o p m e n t . C h r o m o s o m e s 7 and 14 have a normal m o r p h o l o g i c a p p e a r a n c e and G-banding pattern in all e x a m i n e d cells of the d o m i n a n t clone. These results suggest that the m a l i g n a n t cells in our patient d i d not evolve from the cells w i t h t(7;14) and inv(7). Investigations of m a l i g n a n t cells in patients w i t h AT are scarce (Table 3). Most of the cytogenetic investigations of malignant cells i n c l u d e patients w i t h chronic T-cell leukemia, only two refer to ALL, and no data exist on cytogenetic changes of n o n - H o d g k i n ' s l y m p h o m a s and solid tumours in patients with AT [5-8, 12-18]. The results of some investigations s h o w the association of t(14;14)(q11-12;q32) and inv(14)(q12q32) and m a l i g n a n t cell transformation in patients w i t h AT and T-cell malig-
162
n a n c y [5-7, 12, 14]. Aberrant regions are 14q11-12 and 14q32, i.e., the segments where genes for the a - c h a i n of T-cell receptor and the heavy chain of i m m u n o g l o b u l i n are located, whereas Croce et al. and Kennaugh et al. assume that the localization of protooncogene TCL-1 is region 14q32 [19-21]. Chromosome aberration is s u p p o s e d to cause rearrangement of T-cell receptor a-chain locus and activation of the protooncogene, followed by clonal cell expansion [22, 23]. The t(14;14) or inv(14) is not considered sufficient for malignant cell transformation, however; another genetic event is i n d i s p e n s a b l e for the malignant process to develop [241. In contrast, DOhrsen et al. reported del(14)(q21q31) together with intact region 14q11-12 in a patient with AT and ToCLL [13]. Three investigations, i n c l u d i n g ours, differ from those previously described [8, 15]. Although the results do not exclude submicroscopic rearrangements of c h r o m o s o m e 14, the malignant cells did not originate from cells with t(14;14) or inv(14). Wake et al. suggested that aberrations of chromosome 14, although frequent in patients with AT and malignant diseases, are not an essential condition for development of T-cell n e o p l a s m s [8]. The role of AT aberration of chromosome 14 m a y also vary in different types of T-cell malignancies. On the other hand, neoplasms other than T-cell leukemias and l y m p h o m a s have been described in patients with AT, indicating that different cell types are prone to involvement in malignancy. Kaiser-McCaw and Hecht reported a boy w i t h AT and Burkitt l y m p h o m a [15]. Cytogenetic analyses of pleural effusion s h o w e d t(8;14)(q23;q32) typical of Burkitt l y m p h o m a . Thus, diverse chromosome changes e v i d e n t l y occur in AT so that selection can act to favor the emergence of a malignant clone with rearrangements characteristic of that particular type of neoplasm. Because few investigations have been performed, the biologic significance of AT c h r o m o s o m e aberrations in a malignant process has not been fully clarified. Therefore, more extensive cytogenetic analyses are necessary to define more precisely the role and degree of the association of AT clonal c h r o m o s o m e aberrations and the malignant process. Cytogenetic analysis of the p r o b a n d ' s sister established karyotype instability characteristic of most patients with AT; inv(7) was identified in 10% of the cells examined. The analysis d i d not show t(14;14) or cells with inv(14), but this does not suggest a favorable prognosis. Our results indicate n e o p l a s m d e v e l o p m e n t even in the absence of a large clone with cytogenetically evident aberrations of c h r o m o s o m e 7 or 14.
I. Petkovie et al.
4. 5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18. We thank Dr. Frederick Hecht for helpful discussions. 19. REFERENCES
1. Humphreys MW, Nevin NC, Wooldridge MAW (1989): Cytogenetic investigation in a family with ataxia telangiectasia. Hum Genet 83:79-82. 2. Cohen MM, Shaham M, Dagan J, Shmueli E, Kohn G (1975): Cytogenetic investigations in families with ataxia telangiectasia. Cytogenet Cell Genet 15:338-356. 3. Kohn PH, Whang-Peng J, Levis WR (1982): Chromosomal insta-
20.
21.
bility in ataxia telangiectasia. Cancer Genet Cytogenet 6:289-302. Hecht F, Hecht BK (1990): Cancer in ataxia telangiectasia patients. Cancer Genet Cytogenet 46:9-19. Taylor AMR, Butterworth SV (1986): Clonal evolution of T-cell chronic lymphocytic leukemia in a patient with ataxia telangiectasia. Int J Cancer 37:511-516. Saxon A, Stevens RH, Golde DW (1979): Helper and suppressor T-lymphocyte leukemia in ataxia telangiectasia. N Engl J Med 300:700-704. Davey MP, Bertness V, Nakahara K, Johnson PA, McBride OW, Waldmann TA, Kitsch IR (1988): Juxtaposition of the T-cell receptor a-chain locus (14q11) and a region (14q32) of potential importance in leukemogenesis by a 14;14 translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia telangiectasia. Proc Natl Acad Sci USA 85:9287-9291. Wake N, Minowada J, Park B, Sandberg AA (1982): Chromosomes and causation of human cancer and leukemia. XLVIII. T-cell acute leukemia in ataxia telangiectasia. Cancer Genet Cytogenet 6:345-357. Aurias A, Dutrillaux B (1986): Probable involvement of immunoglobulin superfamily genes in most recurrent chromosomal rearrangements from ataxia telangiectasia. Hum Genet 72:210-214. Zhang F, Stern MH, Thomas G, Aurias A (1988): Molecular characterization of ataxia telangiectasia T cell clones. II. The clonal inv(14) in ataxia telangiectasia differs from the inv(14) in T cell lymphoma. Hum Genet 78:316-319. Hollis RJ, Kennaugh AA, Butterworth SV, Taylor AMR (1987): Growth of large chromosomally abnormal T cell clones in ataxia telangiectasia patients is associated with translocation at 14q11. A model for T cell neoplasia. Hum Genet 76:389-395. Amromin GD, Boder E, Teplitz R (1979): Ataxia telangiectasia with a 32 year survival. A clinicopathological report. J Neuropathol Exp Neurol 38:621-643. D~hrsen U, Uppenkamp M, Uppenkamp I, Becher R, Engelhard M, K6nig E, Meusers P, Meuer S, Brittinger G (1986): Chronic T cell leukemia with unusual cellular characteristics in ataxia telangiectasia. Blood 68:577-585. Levitt R, Pierre RV, White WL, Siekert RG (1978): Atypical lymphoid leukemia in ataxia telangiectasia. Blood 52:1003-1011. Kaiser-McCaw B, Hecht F (1982): Ataxia telangiectasia; Chromosomes and cancer. In: A Cellular and Molecular Link Between Cancer, Neuropathology and Immune Deficiency. BA Bridges, DG Harnden, eds. John Wiley and Sons, New York, pp 243-257. Bernstein R, Pinto M, Jenkis T (1981): Ataxia telangiectasia with evolution of monosomy 14 and emergence of Hodgkin's disease. Cancer Genet Cytogenet 4:31-37. Brito-Babapulle V, Catovsky D (1991): Inversions and tandem translocation involving chromosome 14qll and 14q32 in T-prolymphocytic leukemia and T-cell leukemias in patients with ataxia telangiectasia. Cancer Genet Cytogenet 55:1-9. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. Elsevier Science Publishing Company, New York, pp. 191-202. Croce CM, Isobe M, Palumbo A, Puck J, Ming J, Tweardy D, Erikson J, Davis M, Rovera G (1985): Gene for ~-chain of human T-cell receptor: Location on chromosome 14 region involved in T-cell neoplasms. Science 227:1044-1047. Kirsch IR, Morton CC, Nakahara K, Leder P (1982): Human immunoglobulin heavy chain genes map to a region of translocations in malignant B lymphocytes. Science 216:301-303. Kennaugh AA, Butterworth SV, Hollis R, Rabbitts TH, Taylor AMR (1986): The chromosome breakpoint at 14q32 in an ataxia telangiectasia t(14;14) T-cell clone is different from the 14q32
A T w i t h M a l i g n a n t L y m p h o m a in a C h i l d
breakpoint in Burkitt's and an inv(14) T-cell lymphoma. Hum Genet 73:254-259, 22. Stern MH, Zhang F, Griscelli C, Thomas G, Aurias A (1988): Molecular characterization of different ataxia telangiectasia T-cell clones. I. A common breakpoint at the 14q11.2 band splits the T-cell receptor ~-chain gene. Hum Genet 78:33-36. 23. Russo G, Isobe M, Pegoraro L, Finan J, Nowell PC, Croce CM
163
(1988): Molecular analysis of a t(7;14)(q35;q32) chromosome translocation in a T cell leukemia of a patient with ataxia telangiectasia. Cell 53:137-144. 24. Russo G, Isobe M, Gatti R, Finan J, Batuman O, Huebner K, Nowell PC, Croce CM (1989): Molecular analysis of a t(14;14) translocation in leukemic T-cells of an ataxia telangiectasia patient. Proc Natl Acad Sci USA 86:602-606.
ABSTRACT: We present the results of cytogenetic analysis in a brother and sister with ataxia telangiec-
tasia (AT), one of whom had malignant T-cell lymphoma. In both children, cytogenetic analysis of phytohemagglutinin (PHA)-stimulated lymphocytes showed chromosomal instability and inv( 7) in 10% of the cells examined. The malignant lymphoma was analyzed cytogenetically on slides obtained from short-term culture of the lymph node cells; 64 cells were analyzed. A heterogeneous cell population was noted. Fourteen cells (21.9%) had a normal male karyotype; t(7;14)(p14;q12) and inv(7)(p14q35) were observed in 6.3% and 3.1% of metaphases. Owing to low frequency, these cells are probably a characteristic of the basic disease and have no features of malignant cells. Forty cells (62.5%) had a pseudodiploid karyotype 46,XY,dup(1)(p2 2p36 ),del( 5 )(q33 ),del(12 )(p11), without cytogenetically evident aberrations of chromosomes 7 and 14. The results of these investigations suggest that the cells with rearrangements of chromosomes 1, 5, and 12 are malignant cells and did not originate by transformation of cells with inv(7) and t(7;14).
INTRODUCTION Ataxia telangiectasia (AT) is an autosomal recessive disease. The basic clinical characteristics are cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, and frequent sinopulmonary infections. Cytogenetic analysis has demonstrated high frequency of chromosome anomalies with frequent clonal aberrations of chromosomes 7 and 14 [1-3]. Patients with AT have an increased risk of developing malignancies as compared with the general population. Malignancies of the lymphoreticular system such as lymphocyte leukemia and malignant lymphomas are frequent [4]. The nature of the AT association and malignancies is not clear. Malignancies can be a characteristic of the disease itself or a consequence of immunodeficiency or of chromosome instability. Immediate evidence of the importance of chromosome aberrations in the development of neoplasms can be obtained by cytogenetic analysis of normal and malignant cells in persons with AT and a malignant disease. Such analyses, however, are very rare. Although a few investigators indicate the development of malignant cell population from the cells with clonal chromosome aberrations present before manifestation of malignancy, others have failed to
From the Institute for Mother and Child Health, Clinical Hospital "Dr. O. Novosel" (I. P., I. L., D. L.-B., M. C., M. N.), and Medical Faculty. University of Zagreb (M. D.), Zagreb, Yugoslavia. Address reprint requests to Dr. sci. ing. Iskra PetkoviG znan. say. ~vearova 5, 41000 Zagreb, Yugoslavia. Received August 20, 1991; accepted January 8, 1992. 158 Cancer Genet Cytogenet58:158-163(1992} 0165-4608/92/$05.00
demonstrate such a relationship [5-8]. Thus, the role of AT chromosome aberrations in the process of malignant transformation has not been clarified and is still of great scientific interest. We performed cytogenetic analysis in two children with AT, of whom one had malignant T-cell lymphoma.
MATERIALS A N D METHODS
Our patients are the only children of healthy parents who are not related. No cases of AT have been reported in the family of either parent. AT was diagnosed on the grounds of the clinical picture and laboratory tests during the children's previous stay in our institute (Table 1). Case 1
In December 1989, the parents first noticed enlarged lymph nodes and appearance of new ones during the next few months, but no changes in the general condition of the child. In July 1990, a normal finding was obtained by cytologic puncture of an enlarged lymph node. Hematologic findings were within normal limits. In December 1990, the boy (aged 10 years) was admitted to our institute for examination and treatment of general lymphoadenopathy. Lymph nodes were 0.3-6 cm, hard, and painless on palpation. An enlarged liver was palpated 2 cm below the right rib arch. Very high a-fetoprotein values (312.2 /~g/L) and deficiency of IgA were established. The number of B lymphocytes was on low-normal, with normal values of the total number of T lymphocytes and suppressor cells and deficiency of inductor cells. Hematologic findings © 1992 Elsevier Science Publishing Co., Inc. 655 Avenueof the Americas,New York, NY 10010
AT with Malignant Lymphoma in a Child
159
Table 1 Clinical Findings in Two Children With Ataxia Telangiectasia Patient
1
2
Sex Cerebellar ataxia Oculomotor apraxia Nystagmus and stabismus Telangiectasia Dysarthria Generalized hypotonia Diminished tendon reflexes Small stature Mental retardation Thymic hypoplasia Recurrent sinopulmonary infections IgA IgMa IgG° IgE° OKT4Q
M + + + + + + + + + + + N t ~, ~
F + + ÷ + + + + + + + + 1' J,
The values are expressed as normal (N), increased ( I" ), or decreased ( ,~).
were unremarkable. Cytologic analysis of the lymph node showed hypercellularity, but no elements existed to diagnose a malignant process. To make a precise diagnosis, we analyzed the lymph node biopsy histopathologically. Pathologic analysis of the biopsy indicated a malignant lymphoma, most probably of the T-cell type. The cells corresponded morphologically to large centrocytes with a number of blasts and polymorphic histiocytes (CD2-, T6 + ). The phenotype of the proliferating cells was not easy to determine because there were many B1 (CD19) and CD2 ÷ or CD3 ÷ cells, with restricted CD4 and CD8. IgM was slightly positive in rare cells, IgG was positive in large cells, and IgE and IgA were negative. Three weeks after the boy was admitted to the hospital a bone marrow (BM) biopsy indicated transformation of malignant lymphoma in the form of acute lymphocytic leukemia (ALL) of atypical morphology. Cytostatic therapy was initiated, but the boy's response was very poor and he soon died.
cells. The cell suspension was prepared by mechanical dissociation of the node in synthetic medium minimal essential medium (MEM) with 20% human AB serum and cultivated overnight at 37°C. The cell suspension was then treated with colchicine (4 /zg/ml, 20 minutes) hypotonic solution (0.075 M KC1, 5 minutes) and finally fixed in the mixture of methanol and acetic acid (3 : 1, vol/vol). Slides were made by the air-drying method and stained for GTG banding.
RESULTS From the lymphocyte culture of the boy, 50 metaphases stained according to G-banding method were karyotyped. In 13 (26%) cells, chromosome aberrations were identified, whereas 37 cells had a normal male karyotype (Table 2). A chromosome break (7q32) was identified in only one cell, whereas other metaphases showed stable chromosome aberrations. Deletions were noted in three cells and translocations were noted in four cells. In seven cells, aberrations of chromosome 7 were observed. Inv(7) (p14q35) was identified in five (10%) cells, in one of them associated with t(3;14)(q29;q24). In one metaphase, together with t(7;7}(p14;q35), t(1;21)(q32;q22) was also present, whereas t(7;14)(p14;q12) was observed in only one metaphase (Fig. 1). Results of the cytogenetic analysis of the lymph node are shown in Table 2. Sixty-four metaphases stained by G-banding were karyotyped. In 14 (21.9%) cells, a normal karyotype was established. Most of the cells analyzed (62.5%) belong to a pseudodiploid clone with 46 chromosomes with aberrations of chromosomes 1, 5, and 12, whereas other chromosomes are of normal morphologic appearance and G-banding pattern (Fig. 2). Analysis of banding pattern of the aberrant chromosome 1 established a duplication of the short arm segment, i.e., p22-p36. Chro-
Table 2 Results of the Chromosome Analyses in Patients With Ataxia Telangiectasia and Malignant Lymphoma
Case 2 The proband's sister was admitted for a pediatric checkup at age 11 years. With regard to the basic disease, the child's condition was unchanged. Neither lymphoadenopathy nor hepatosplenomegaly was observed. Hematologic findings were within normal values.
Cytogenetic Analysis Lymphocytes of both children were analyzed cytogenetically on slides obtained from PHA-stimulated cultures. The cells were cultivated for 3 days at 37°C and then treated with colchicine (4/~g/ml, 1.5-2.5 hours), hypotonic solution (0.075 M KC1, 5 minutes) and fixed in the mixture of methanol : acetic acid (3 : 1, vol/vol). The slides were made by the flame-drying method and stained for GTG banding. Malignant cells were analyzed cytogenetically (case 1) on slides obtained from short-term culture of lymph node
Peripheral
Lymph
blood
node
Karyotype
n (%)
n (%)
46,XY c h r o m o s o m e break 46,XY,inv(7) 46,XY,inv(7),t(3;14) 46,XY,t(7;14) 46,XY,t(7;7),t(1;21) 46,XY,dup(1),del(5),del(12) 46,XY,trip(1),del(5),del(12) Other changes Total
37 (74) 1 (2) 4 (8) 1 (2) 1 (2) 1 (2)
14 (21.9)
° Includes cells with (p13;q13), t(6;21)(q21;p11).
5 a (10) 50
2
(3.1)
4
(6.3)
40 (62.5) 1 (1.6) 3 b (4.6) 64
del(18)(p11),del(10)(p13),del(1)(q31),t(10;20)
b Includes two near-tetraploid cells and dup(17).
160
I. Petkovi~ et al.
a
mosome 1 is unstable because one cell with the short arm triplication was identified by this analysis. The aberrant chromosome 5 is most probably the result of terminal deletion of the long arm with the break in region 5q33. In all aberrant clone cells, the short arm of chromosome 12 was deleted, i.e., del(12)(p11). In 9.4% of cells AT-specific aberrations of chromosome 7 were noted but with different frequency with regard to the patient's lymphocyte analysis (Table 2). Thus, t(7;14)(p14;q12), as the only chromosome aberration, was identified in four (6.3%) and inv(7)(p14q35) was identified in two (3.1%) cells examined. Cytogenetic analysis of the proband's sister (case 2) included 50 metaphases stained by G-banding method. In 14 (28%) cells, chromosome aberrations were identified; others showed a normal female karyotype. Inv(7)(p14q35) was identified as a clonal chromosome aberration in five (10%) metaphases examined.
b
Figure 1 Chromosome aberrations observed in the peripheral blood cell culture from case 1: (a) inv(7), (b) t(7;14), (c) t(7;7) associated with t(1;21).
DISCUSSION We performed cytogenetic studies of normal and malignant cells in the boy with AT and malignant T-cell lymphoma. The analyses of malignant cells showed pseudodiploid
Figure 2 G-banded karyotype of a malignant clone from the lymph node of case 1: 46,XY,dup(1)(p22p36), del(5)(q33),del(12)(p11).
1
2
3
6
7
8
13
14
15
19
20
9
4
5
10
11
12
16
17
18
21
21
X¥
AT with Malignant L y m p h o m a in a Child
Table 3
Cytogenetic F i n d i n g s in Patients With AT and Malignant Disease
Sex/age
Type of malignancy
1
F/48
CLL
T-Cell
2
F/32
CLL
T-Cell
3
F/30
CLL
T-Cell
4
M
CLL
T-Cell
5
M/27
CLL
T-Cell
6
F/25
Snbacute ALL
T-Cell
Patient
161
Chromosome changes in peripheral blood
Cell type
45,XX,- 14,t(14;14) (q12;q32) 45,XX,- 14,t(14;14) (q11;q32) 46,XX,t(14;14) (q12;q32) 46,XY,inv(14) (q12q32)
45,XX,- 14,t(14;14) (q12;q32)
Chromosome changes in malignant cells
Reference
44,XX,- 14,- 20,6q- ,i(8q), 12p - ,20p + ,t(14;14)(q12;q32) t(14;14)(q11;q32)
[6]
41,XX, - 13, - 14,- 15,- 16,- 20 t(1 ;13),t(15 ;18),t(14;14) (q12;q32),10q + 46,XY,6q- ,8p + ,11p - ,inv(14) (q12q32),r(16),19p +, - 20 t(21;21), + mar 46,XY/44,X, - Y , - 20,t(4;20) (q13;q12),del(14)(q21q31), t(6;19;22)(p11;p13;q13) 43 ,XX,t(13 ;15),t(13 ;17), t(14;14)(q12;q32),18q ~-,
[12]
[7]
[5]
[13]
[14]
-13,-14,-16,-17,-21,-22,
7
F/18
ALL
8
M/5
9
F/25
Burkitt lymphoma Hodgkin's disease (HD)
10
F/20
PLL
T-Cell
11
M/10
Malignant lymphoma
T-Cell
Abbreviations: CLL, chronic
Atypical T-cell B-Cell
46,XX,t(14;14)
Mixed cellularity
46,XX/45,XX,- 14 (18 months before HD) 45,XX,- 14,del(6)(q21) (4 months before HD) 46,X,t(X;14)(q28;q11)
46,XY
46,XY/46,XY,inv(7) (p14q35)
+ 3mar 46,XX/45,XX, - 9,t(9;16) (q12;p13) 46,XY,t(8,14)(q23;q32) (cell line) Not done
t(X;14)(q28;q11),t(1;14) (p21;q11),t(8,22)(q24;q11) 46 ,XY/46,XY,t(7;14) (p14;q12)/46,XY,inv(7) (p14q35)/46,XY,dup(1) (p22p36), del(5](q33), del(12)(p11)
[8] [15] [16]
[17] Present case
lymphocytic leukemia; ALL,acute lymphocytic leukemia; PLL, prolymphocytic leukemia.
karyotype with structural rearrangement of chromosomes 1, 5, and 12, but not of c h r o m o s o m e s 7 and 14. Chromosome instability, characteristic of patients with AT, was established by cytogenetic analysis of l y m p h o c y t e s in this patient. Unlike the investigations of some researchers in patients with AT and neoplasms, we d i d not observe t(14;14) or inv(14), but inv(7) was a clonal anomaly in 10% of the cells analysed. A c c o r d i n g to literature data, large clones of 7 0 - 1 0 0 % of cells refer to t(14;14), inv(14), and t(X;14), whereas inv(14) is considered the most frequent clonal aberration of n o n m a l i g n a n t cells in patients with AT [9, 10]. Aberrations of c h r o m o s o m e 7, such as t(7;14), t(7;7), and inv(7) are a frequent characteristic of AT, but as in our patient, were not identified as large cell clones [9, 11]. Cytogenetic analysis of the l y m p h node in our patient s h o w e d a heterogeneous cell population. Apart from the cells of n o r m a l d i p l o i d karyotype, two clones with t(7;14) and inv(7) were identified as the only chromosome aberration. Considering the low frequency of aberrations, the cells
are probably a characteristic of the basic disease a n d have no features of malignant cells. The d o m i n a n t cell p o p u l a tion has the p s e u d o d i p l o i d karyotype 46,XY,dup(1), del(5),del(12), includes 62.5% of the cells e x a m i n e d , and obviously has selective proliferating advantage and is responsible for malignant d e v e l o p m e n t . C h r o m o s o m e s 7 and 14 have a normal m o r p h o l o g i c a p p e a r a n c e and G-banding pattern in all e x a m i n e d cells of the d o m i n a n t clone. These results suggest that the m a l i g n a n t cells in our patient d i d not evolve from the cells w i t h t(7;14) and inv(7). Investigations of m a l i g n a n t cells in patients w i t h AT are scarce (Table 3). Most of the cytogenetic investigations of malignant cells i n c l u d e patients w i t h chronic T-cell leukemia, only two refer to ALL, and no data exist on cytogenetic changes of n o n - H o d g k i n ' s l y m p h o m a s and solid tumours in patients with AT [5-8, 12-18]. The results of some investigations s h o w the association of t(14;14)(q11-12;q32) and inv(14)(q12q32) and m a l i g n a n t cell transformation in patients w i t h AT and T-cell malig-
162
n a n c y [5-7, 12, 14]. Aberrant regions are 14q11-12 and 14q32, i.e., the segments where genes for the a - c h a i n of T-cell receptor and the heavy chain of i m m u n o g l o b u l i n are located, whereas Croce et al. and Kennaugh et al. assume that the localization of protooncogene TCL-1 is region 14q32 [19-21]. Chromosome aberration is s u p p o s e d to cause rearrangement of T-cell receptor a-chain locus and activation of the protooncogene, followed by clonal cell expansion [22, 23]. The t(14;14) or inv(14) is not considered sufficient for malignant cell transformation, however; another genetic event is i n d i s p e n s a b l e for the malignant process to develop [241. In contrast, DOhrsen et al. reported del(14)(q21q31) together with intact region 14q11-12 in a patient with AT and ToCLL [13]. Three investigations, i n c l u d i n g ours, differ from those previously described [8, 15]. Although the results do not exclude submicroscopic rearrangements of c h r o m o s o m e 14, the malignant cells did not originate from cells with t(14;14) or inv(14). Wake et al. suggested that aberrations of chromosome 14, although frequent in patients with AT and malignant diseases, are not an essential condition for development of T-cell n e o p l a s m s [8]. The role of AT aberration of chromosome 14 m a y also vary in different types of T-cell malignancies. On the other hand, neoplasms other than T-cell leukemias and l y m p h o m a s have been described in patients with AT, indicating that different cell types are prone to involvement in malignancy. Kaiser-McCaw and Hecht reported a boy w i t h AT and Burkitt l y m p h o m a [15]. Cytogenetic analyses of pleural effusion s h o w e d t(8;14)(q23;q32) typical of Burkitt l y m p h o m a . Thus, diverse chromosome changes e v i d e n t l y occur in AT so that selection can act to favor the emergence of a malignant clone with rearrangements characteristic of that particular type of neoplasm. Because few investigations have been performed, the biologic significance of AT c h r o m o s o m e aberrations in a malignant process has not been fully clarified. Therefore, more extensive cytogenetic analyses are necessary to define more precisely the role and degree of the association of AT clonal c h r o m o s o m e aberrations and the malignant process. Cytogenetic analysis of the p r o b a n d ' s sister established karyotype instability characteristic of most patients with AT; inv(7) was identified in 10% of the cells examined. The analysis d i d not show t(14;14) or cells with inv(14), but this does not suggest a favorable prognosis. Our results indicate n e o p l a s m d e v e l o p m e n t even in the absence of a large clone with cytogenetically evident aberrations of c h r o m o s o m e 7 or 14.
I. Petkovie et al.
4. 5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18. We thank Dr. Frederick Hecht for helpful discussions. 19. REFERENCES
1. Humphreys MW, Nevin NC, Wooldridge MAW (1989): Cytogenetic investigation in a family with ataxia telangiectasia. Hum Genet 83:79-82. 2. Cohen MM, Shaham M, Dagan J, Shmueli E, Kohn G (1975): Cytogenetic investigations in families with ataxia telangiectasia. Cytogenet Cell Genet 15:338-356. 3. Kohn PH, Whang-Peng J, Levis WR (1982): Chromosomal insta-
20.
21.
bility in ataxia telangiectasia. Cancer Genet Cytogenet 6:289-302. Hecht F, Hecht BK (1990): Cancer in ataxia telangiectasia patients. Cancer Genet Cytogenet 46:9-19. Taylor AMR, Butterworth SV (1986): Clonal evolution of T-cell chronic lymphocytic leukemia in a patient with ataxia telangiectasia. Int J Cancer 37:511-516. Saxon A, Stevens RH, Golde DW (1979): Helper and suppressor T-lymphocyte leukemia in ataxia telangiectasia. N Engl J Med 300:700-704. Davey MP, Bertness V, Nakahara K, Johnson PA, McBride OW, Waldmann TA, Kitsch IR (1988): Juxtaposition of the T-cell receptor a-chain locus (14q11) and a region (14q32) of potential importance in leukemogenesis by a 14;14 translocation in a patient with T-cell chronic lymphocytic leukemia and ataxia telangiectasia. Proc Natl Acad Sci USA 85:9287-9291. Wake N, Minowada J, Park B, Sandberg AA (1982): Chromosomes and causation of human cancer and leukemia. XLVIII. T-cell acute leukemia in ataxia telangiectasia. Cancer Genet Cytogenet 6:345-357. Aurias A, Dutrillaux B (1986): Probable involvement of immunoglobulin superfamily genes in most recurrent chromosomal rearrangements from ataxia telangiectasia. Hum Genet 72:210-214. Zhang F, Stern MH, Thomas G, Aurias A (1988): Molecular characterization of ataxia telangiectasia T cell clones. II. The clonal inv(14) in ataxia telangiectasia differs from the inv(14) in T cell lymphoma. Hum Genet 78:316-319. Hollis RJ, Kennaugh AA, Butterworth SV, Taylor AMR (1987): Growth of large chromosomally abnormal T cell clones in ataxia telangiectasia patients is associated with translocation at 14q11. A model for T cell neoplasia. Hum Genet 76:389-395. Amromin GD, Boder E, Teplitz R (1979): Ataxia telangiectasia with a 32 year survival. A clinicopathological report. J Neuropathol Exp Neurol 38:621-643. D~hrsen U, Uppenkamp M, Uppenkamp I, Becher R, Engelhard M, K6nig E, Meusers P, Meuer S, Brittinger G (1986): Chronic T cell leukemia with unusual cellular characteristics in ataxia telangiectasia. Blood 68:577-585. Levitt R, Pierre RV, White WL, Siekert RG (1978): Atypical lymphoid leukemia in ataxia telangiectasia. Blood 52:1003-1011. Kaiser-McCaw B, Hecht F (1982): Ataxia telangiectasia; Chromosomes and cancer. In: A Cellular and Molecular Link Between Cancer, Neuropathology and Immune Deficiency. BA Bridges, DG Harnden, eds. John Wiley and Sons, New York, pp 243-257. Bernstein R, Pinto M, Jenkis T (1981): Ataxia telangiectasia with evolution of monosomy 14 and emergence of Hodgkin's disease. Cancer Genet Cytogenet 4:31-37. Brito-Babapulle V, Catovsky D (1991): Inversions and tandem translocation involving chromosome 14qll and 14q32 in T-prolymphocytic leukemia and T-cell leukemias in patients with ataxia telangiectasia. Cancer Genet Cytogenet 55:1-9. Sandberg AA (1990): The Chromosomes in Human Cancer and Leukemia. Elsevier Science Publishing Company, New York, pp. 191-202. Croce CM, Isobe M, Palumbo A, Puck J, Ming J, Tweardy D, Erikson J, Davis M, Rovera G (1985): Gene for ~-chain of human T-cell receptor: Location on chromosome 14 region involved in T-cell neoplasms. Science 227:1044-1047. Kirsch IR, Morton CC, Nakahara K, Leder P (1982): Human immunoglobulin heavy chain genes map to a region of translocations in malignant B lymphocytes. Science 216:301-303. Kennaugh AA, Butterworth SV, Hollis R, Rabbitts TH, Taylor AMR (1986): The chromosome breakpoint at 14q32 in an ataxia telangiectasia t(14;14) T-cell clone is different from the 14q32
A T w i t h M a l i g n a n t L y m p h o m a in a C h i l d
breakpoint in Burkitt's and an inv(14) T-cell lymphoma. Hum Genet 73:254-259, 22. Stern MH, Zhang F, Griscelli C, Thomas G, Aurias A (1988): Molecular characterization of different ataxia telangiectasia T-cell clones. I. A common breakpoint at the 14q11.2 band splits the T-cell receptor ~-chain gene. Hum Genet 78:33-36. 23. Russo G, Isobe M, Pegoraro L, Finan J, Nowell PC, Croce CM
163
(1988): Molecular analysis of a t(7;14)(q35;q32) chromosome translocation in a T cell leukemia of a patient with ataxia telangiectasia. Cell 53:137-144. 24. Russo G, Isobe M, Gatti R, Finan J, Batuman O, Huebner K, Nowell PC, Croce CM (1989): Molecular analysis of a t(14;14) translocation in leukemic T-cells of an ataxia telangiectasia patient. Proc Natl Acad Sci USA 86:602-606.
