Chromosome rearrangements at telomeric level in hematologic disorders

ELSEVIER

Chromosome Rearrangements at Telomeric Level in Hematologic Disorders Paola Temperani, Francesca Giacobbi, Giovanna Gandini, Umberto Torelli, and Giovanni Emilia

ABSTRACT: Following retrospective screening of our karyotype data from 414 consecutive non-childhood, neoplastic, and preneoplastic hematologic diseases, we have isolated 11 cases with alterations involving one or two chromosome termini, including: a) nonclonal telomeric associations (tas), b) subclonal terminal rearrangements consisting of additional (add) material of unknown origin fused at the end of the chromosome, c) clonal telomere-centromere fusion (t telcen) with pseudodicentric structure. Most of these abnormalities were present in karyotypes with multiple alterations and associated to an evolutive stage of the disease (9 of 94 cases studied in progression, including three of 22 CML studied in blast crisis). The immunophenotype of the cell populations was lymphoid in eight cases, six of which were NHL, and myeloid, erythroid, and undifferentiated in the other three. More data on telomeric abnormalities may clarify whether there is ubiquitous genomic instability of neoplastic cells or an inborn cell lineage predisposition favoring rearrangements involving telomeres.

INTRODUCTION Chromosome alterations may occur at the end of a chromosome, the telomere, leading, if two telomeres are involved, to the formation of dicentric (telomeric association) and ring chromosomes, or to terminal non-reciprocal translocations, w h e n a telomere rearranges w i t h an intra-arm c h r o m o s o m e segment. End-to-end translocations [1], non-reciprocal terminal translocations, and unstable terminal translocations, k n o w n as "jumping translocations" are rarely described as constitutional karyotype abnormalities [2, 3]. A clear demonstration of the involvement of telomeric sequences in terminal chrom o s o m e abnormalities has recently been achieved by fluorescent in situ hybridization of some constitutional nonreciprocal translocations and ring c h r o m o s o m e s [4, 5]. Telomeric associations (tas) have been found as sporadic alterations in apparently normal lymphocytes and fibroblasts of subjects affected by ataxia-telangiectasia [6] and xeroderma p i g m e n t o s u m [7], two genetic disorders in w h i c h neoplasia frequently occurs. Moreover, sporadic telomeric associations are present, w i t h various degrees of recurrence, in a number of t u m o r tissues. To date, o n l y a few cases, all of w h i c h show l y m p h o i d phenotype, with nonclonal telomeric associ-

From the Department of Medical Sciences, Hematology Oncology Section, University of Modena, Modena, Italy. Address reprint requests to: Dr. Paola Temperani, Dipartimento Scienze Mediche, Oncologiche e Radiologiche, Sezione di Medicina Interna, Oncologia ed Ematologia, Via del Pozzo 71, 41100 Modena, Italia. Received May 18, 1994; accepted October 18, 1994. Cancer Genet Cytogenet 83:121-126 (1995) © Elsevier Science Inc.. 1995 655 Avenue of the Americas, New York, NY 10010

ations [8-11], frequently involving chromosome 19p telomere [12], have been described in hematologic neoplasia. In B-cell and pre-T-cell l y m p h o i d leukemia showing tas, unstable terminal rearrangements or "jumping translocations" are also present. In these cases, different telomere8 are joined w i t h various c h r o m o s o m e segments, p a r t i c u l a r l y the pericentromeric heterochromatin of c h r o m o s o m e 1 [13]. In different k i n d s of solid tumors, authors have reported a significant i n c i d e n c e of n o n c l o n a l [14-19] and clonal [20-22] tas, alt h o u g h w i t h the pattern of the telomeres different from that found in l y m p h o i d leukemia. Furthermore, there are evidences of a ta8 clustering in specific tumors, such as giant cell bone tumors and renal cell tumors, w h i c h suggest the hypothesis of a specific cell lineage susceptibility to telomeric abnormalities. At present, the real frequency of c h r o m o s o m e abnormalities at the telomeric level in t u m o r cells could be underestimated, considering the poor m o r p h o l o g y of neoplastic chrom o s o m e s a n d the c o m p l e x i t y of alterations acquired during t u m o r progression. To evaluate the frequency of abnormalities involving telomeres in hematologic diseases we have screened the karyot y p e of a group of 414 n o n - c h i l d h o o d neoplastic and preneoplastic hematologic disorders, 94 of w h i c h have been s t u d i e d at various stages of progression. MATERIALS AND METHODS Cases

Forty-three cases of myelodisplastic s y n d r o m e (MDS), 246 cases of myeloproliferative disorders, a n d 125 cases of lym-

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phoproliferative disorders with defined karyotype have been retrospectively screened for the presence of rearrangements at the telomeric level. The 246 cases of myeloproliferative disorders (MPD) i n c l u d e d 79 patients w i t h acute myeloid leukemia (AML), 84 w i t h chronic myeloid leukemia (CML) (22 in blastic crisis), 28 with essential idiopathic thromb o c y t h e m i a (ET), 19 w i t h idiopathic myelofibrosis (IMF), nine w i t h i p e r e o s i n o p h i l i c syndrome, and eight with polycytemia vera (PV). The 125 lymphoproliferative disorders inc l u d e d 41 patients with acute l y m p h o i d leukemia (ALL), 41 w i t h n o n - H o d g k i n l y m p h o m a (NHL), 22 w i t h chronic lymphocytic leukemia (CLL), and 21 with m u l t i p l e myeloma (MM). In 11 of 414 cell p o p u l a t i o n s whose karyotypes have been screened there were different types of abnormalities involving telomeres. Seven of these occurred in patients with different types of lymphoproliferative disorders (1-ALL, patient 7; 2-CLL, patients 5 and 6; 4-NHL, patients 8-11), while the r e m a i n i n g four were present in patients with myeloproliferative disease (1 PV, patient 1; and three CML blast crisis patients 2-4) (Table 1). The immunophenotype of the CML blast crisis cell p o p u l a t i o n s was l y m p h o i d in patient 3, erythroid in patient 4, and undifferentiated in patient 2. In the last two cases, the blast crisis cell p o p u l a t i o n s were extramedullary. Cytogenetics

The cytogenetic analyses of the 11 cases were performed before chemotherapy or after a period of at least 3 months free of treatment. The karyotypes were defined at the onset of the disease in patients 1-4 and 6 (re-examined in evolution in cases 2, 3, 4), at diagnosis of NHL (stage III a n d IV in patients 11 and 10, respectively) or at various stages of the disease (indolent phase in patient 5, first relapse in patient 7, Richter syndrome in patient 8, leukemic onset of immunocytoma in patient 9) in the other patients (Table 1). Suspen-

Table 1

Cases

Clinical details Sex/age (yrs)

1

M/27

2 3 4 5

F/46 F/61 F/53 F/55

6

M/78

7 8 9 10 11

M/42 F/72 M/50 F/65 F/70

Disease°

Previous therapy

PV CML-BC CML-BC CML-BC B-CLL T-CLL c-ALL NHL NHL NHL NHL

Bloodletting IFN, T Busulfan T, UM Apheresis, Cy Chlorambucil VCR, E Cy, VCR VCR, Cy None None

" PV, polycythemia vera; CML-BC, chronic myelocytic leukemia-blastic crisis; undifferentiated (patient 2), lymphod (patient 3), erythroid (patient 4); CLL, B or T chronic lymphocytic leukemia; c-ALL, common acute lymphocytic leukemia; NHL. non-Hodgkin lymphoma; Richter syndrome evoluted from B-prolymphocytic leukemia (patient 8), leukemized lymphoplasmocytic lymphoma (patient 9), lymphoblastic lymphoma stage IV (patient 10), folicular lymphoma stage III (patient 11); IFN, ~ interferon; T, thieguanine; Cy, cyclophosphamide; tJM, uracil mustard; VCR, vin(:ristine; E, epirubicin.

sion cultures of bone marrow cells (patients 1-4), peripheral blood cells (patients 2-6, 7, 9), pleural effusion cells (patients 4 and 10), and disaggregated l y m p h node (patient 11), spleen (patient 8), and extramedullary hemopoietic mass (patient 2) were incubated at 37°C in RPMI 1640 m e d i u m , s u p p l e m e n t e d with 17% FCS, 20 mM L-glutamin, and 80 ~tg/mL gentamycin. After a period of 24-72 hours, cells were processed according to standard techniques and the slides G-banded by trypsin (GTG) or C-banded by b a r i u m hydroxide (CBG). For each patient, a sample of 24-hour cultured cells was syncronized by methotrexate 1 × 10 7 M for 17 hours and, after changing the medium, incubated with BrdU 1 × 10:3 M for a further 5 hours [23]. Chromosome preparations were then stained by the fluorochrome (Heochst 33258)-photolysis-Giemsa sequential method (FPG). The chromosome analysis has been performed examining 15-20 banded metaphases for each case, to identify abnormal clones representing more than 10-15% of the cell population. In the presence of a nonclonal chromosome rearrangement, 50 b a n d e d metaphases were microscopically analyzed. RESULTS Chromosome abnormalities involving one or two telomeric regions were found in 9.75% of 41 patients with NHL, 9.09% of 22 patients with CLL, 13.63% of 22 patients with CML in blast crisis, 2.43% of 41 patients with ALL, and 1.56% of 64 patients with chronic MPD. No telomeric rearrangements were present in patients with AML (79 cases), MDS (43 cases), MM (21 cases), and CML (84 cases) at diagnosis or in the chronic phase of the disease. In four cases, abnormalities were sporadic (nonclonal) in six evolutive (subclonal) and in one h o m o g e n e o u s (clonal). Nonclonal telomeric abnormalities essentially concerned telomeric associations present in few cells of three different l y m p h o i d neoplasia with an abnormal karyotype (Table 2, patients 6-9, Fig. la, b, and c) and in the bone marrow cells of PV (Table 2, patient 1; Fig. ld). The latter case also presented a small clone with an isochromosome, i(17q). No

1

a

i a

b

c

d

Figure 1 Nonclonal telomeric associations (tas). Detail of metaphases from a) NHL Richter syndrome (case 8) tas(6;6)(q27;q27); b) leukemized NHL (case 9) tas(2;17)(q37;25); c) T-CLL (case 6) tas(1;3)(q44;q29); and d) PV (case 1) tas(2;2)(p25;q37).

Telomeric Alterations in Leukemias and Lymphomas

Table 2

Cytogenetic findings

Cases

Disease

1

PV

2

CML BC

3

CML BC CML BC

4

123

5

B-CLL

6

T-CLL

7 8

c-ALL NHL Richter

9

NHL leukemized

10

NHL IV

11

NHL III

Karyotype (no. of cells) 46,XY,i(17)(q10)[2]/46,XY,tas(1;3)(p36.3;q29)[1]/46,XY,tas(2;2)(p25;q37)[1]/ 46,XY,tas(4;6)(q35;p25)[1]/46,XY[26] 46,XX,t(9;22)(q34;q11)[15] 54-57,XX, + 1, + 4, + 8, + 8,t(9;22), + 10, + 11, + 14, + 15, + 16,add(19)(p13.3), + 21, + der(22)t(9;22)[cp17] 46,XX,t(9;22)(q34;q11)[18] 46,XX,t(9;22)[6]/46,idem, - 7, + 8,add(19)(p13.3)[9] 46,XX,t(9;22)(q34;q11)[16] 52-55,XX, + 1, + 3, + 6, + 8,t(9;22), + 11, + 12. + 16,add(17p),add(20)(p13), + 21, + der(22)t(9;22)[cp11] 42-44, - X,add(Xq),add(3p),del(5p),add(8p),add(9p), - 10,t(11;14)(q13;q32), add(14q),add(16)(p13.3), - 17, + 18, - 20, - 21[cp16] 46,XY,t(14; 14)(q32;q12)[21]/46,XY,idem,tas(1;3)(q44;q29)[1]/46,XY,idem, tas(1;20)(p36.3;q13.311]/46,XY,idem,tas(3;19)(p26;q13.4)[1]/46,XY,idem, tas(13;15)(q34;q26)[1]/46,XY,[18] 46,XY,psu dic(4;12)(q10;24.3),t(9;22)(q34;qll)[33]/47,idem,i(17)(q10), + 2112] 48-50,X,add(Xq), + 3, + 4, + del(6p),tas(6;6)(q27;q27)[1], + 7,del(7q), + 8,tas(8;11) (q24.3;p15)[1],tas(9;19)(q34;q13.3)[1],add(10q),t(14;17)(p10;q10), + 15, - 16, + 18, + 20[cp16] 46,XY,add(1p),del(14q), - 19, + r[16]/46,XY,idem,tas(11;15)(p15.5;p13)[1]/ 46,XY,idem,tas( 20;22 )(q13.3 ;q13.3 )[1]/46,XY[18]/46,XY,tas(1;18 )(p36.3; p11.32)[1]/46,XY,tas(2; 17) (q37;q25)[1] 47,X,i(X)(p10),add(2p),add(14q), - 19, + 22, + r[5]/ 47,idem,del(6q),add(16)(q24)[11] 48,XX,dup(1X)(q21q32), + 3,del(7q),add(9)(p24),add(9)(p24),t(14;18) (q32;q21), + r[17]

Abbreviations: BC, blastic crisis; IV and lII are clinical stages (Ann Arbor Conference. 1971). Nomenclature of ISCN 1991 for Cancer Cytogenetics[44].

more than one tas per cell was present, and no telomere showed a specific recurrence (Table 2). Subclonal telomeric rearrangements were found as term i n a l additional material (add) of u n k n o w n origin i n three blastic crisis cells of CML (Table 2, patients 2-4, Fig. 2d, e, a n d f), l y m p h node cells of two NHL (Table 2, patients 10 a n d 11, Fig. 2a and c) and peripheral blood cells of a B-CLL i n the i n d o l e n t stage (Table 2, patient 5, Fig. 2b). All these seven cell populations showed n u m e r i c a l and structural abnormalities, five with a composite karyotype (cp). The similarities between these subclonal rearrangements are related to the presence of one whole recipient chromosome joined to a chromosome segment w h i c h could not be identified because of the loss of the donor chromosome, probably lacking telomeric sequences. The recipient telomeres involved in these terminal rearrangements were 16p, 16q, 20p (one case), both 9p in the same cell p o p u l a t i o n (one case), and 19 ptel (two cases). In the CML karyotype the t[9;22) translocation was the only chromosome rearrangement on peripheral blood a n d bone marrow cells at diagnosis. A clonal a n d stable alteration involving the telomere was found on the peripheral blood cells of a c o m m o n ALL (Table 2, patient 7, Fig. 3a). All 35 b a n d e d metaphases studied showed a t(9;22)(q34;q11) translocation, the absence of normal chromosomes 4 and 12, the presence of a derivative chromosome 4p, and a long derivative chromosome 12 with only one primary constriction, resulting from the fusion of the long arm telomere of chromosome 12 with a pericentromeric region of chromosome 4 long arm. After CBG banding, this

chromosome showed two positive signals (Fig. 3b), defining its (pseudo)dicentric structure that may be described as derivative psu dic(4;12)(q10;q24.3). The stability of derivative chromosome 4p can be explained by a telomeric healing of a centromeric fission event. The primary or secondary origin of t(4;12), as opposed to the t(9;22) translocation, could not be established, as both rearrangements were present simultaneously in all examined cells. DISCUSSION

The telomere is a specialized structure that maintains the stability and linear organization of eukaryotic chromosomes, ensuring complete replication of DNA during the process of discontinous strand synthesis and contributing to the organization of the nuclear architecture [24]. Recently, the telomeric tandem repeated consensus sequences (TTAGGG), the peculiar replication by a ribonucleoprotein enzyme (telomerase) and the associated specific proteins that seem to be involved in the regulation and stability of telomeric length, have been described [25]. Temporary pairing of homologous telomeres occurs as a normal event during meiosis and probably in interphase of somatic cells [26]. Shortening of telomeric repeats with respect to normal tissue has been found in a n u m b e r of solid tumors [28], i n c l u d i n g giant cell bone tumors [29], renal tumors with cytogenetic evidence of telomeric association [30], and childhood acute lymphocytic leukemia [31]. On the other hand, reduction of telomeric length has been observed

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9

9

b

16

C

16

16

t9

....¸¸¸19

19

19 ¸

d

e

20

20

Figure 2 Subclonal terminal additional material (add) a) in NHL stage III (case 11); b) in B-CLL (case 5); c) in NHL stage IV (case 10); d) in CML extramedullary erythroid BC (case 2); e) in CML lymphoid BC (case 3); and f) in CML extramedullary undifferentiated BC (case 4).

in normal h u m a n somatic cells as a function of age in vivo and of the n u m b e r of cell divisions in vitro [32]. In an immortalized neoplastic cell line, such as the HeLa cells, which contain a detectable level of telomerase, there are also exceptionally long telomeres. Furthermore, immortalization of Epstein-Barr virus-transformed B-lymphocytes has been found to be accompanied by a stabilization of telomeric repeats and activation of telomerase [33].

The frequency of abnormalities at telomeric level in our 94 hematologic disorders studied in different progression stages of the disease was 9.57% (nine of 94 cases) and in CML blast crisis was 13.63% (three of 22 cases), while at diagnosis the frequency was only 0.568% (two of 352 cases). The stability of the derivative chromosomes that follow telomeric alterations is clearly dependent on their structure. Telomeric associations, which are unstable because dicentric, are found as nonclonal abnormalities, while monocentric chromosomes with terminal rearrangements are present as stable subclonal alterations. The instability of tas derivative dicentric chromosomes can explain the low frequency of their findings and suggests that these abnormalities have a frequency greater than that indicated by their cytogenetic identification. Evolutive rearrangements of tas may lead to a more stable structure, as documented in a case with tas evolved into a ring chromosome [20]. It should be noted that in one of our cases with tas and in two with subclonal terminal addition of u n k n o w n material, a ring chromosome was also present (Table 2, patients 9-11). Stabilization of dicentric chromosomes may also occur by inactivation of the second centromere. In the case of Ph + ALL, the presence of two positive C-bands but only one primary constriction on derivative chromosome 12, is indicative of functional inactivation of the second centromere and its pseudodicentric structure (psu dic) (Table 2, patient 7, Fig. 3). Telomeric sequences may rearrange with intra-arm chromosome segments, as documented by fluorescent in situ hybridization in constitutional non-reciprocal translocations [4, 5]. This seems to be the case of three NHL and three CML blast crises, in which we found a sub-clone showing a chromosome with an addition at the telomeric level of a chromosome segment that was not identifiable because of the absence in the karyotype of a donor chromosome (Table 2, patients 2-5, 10, and 11). The telomeres involved in these cases were 16p, 16q, 20p (one case), both 9p (one case), and 19p (two cases). The involvement of telomere 19p seems to be recurrent in ALL [12]. A recombinogenic property of interspersed telomeric-like sequences has been supported by their distribution along specific regions of the vertebrate genome, such as pericentromeric heterochromatin [34], the phylogenetic fusion point in h u m a n chromosome 2, the i m m u n o g l o b u l i n heavy chain switch region, and the hot spot in the mouse major histocompatibility complex, in which meiotic of mitotic recombination events may occur [35]. Recombination has been documented in yeast for maintenance of telomeric length [36] and suggested in h u m a n s for chromosome healing [37]. Polymorphism of telomeric sequences has also been reported. In humans, polymorphic repetitive DNA elements associated to telomeres are distributed in various combinations in different individuals [38], suggesting a large possibility of telomere mispairing which can occasionally resolve into chromosome aberrations as terminal additions or cryptic translocations [37, 39]. Polymorphism of interspersed telomeric-like sequences of fragile site DNA has been identified in a leukemia-prone strain of mouse, presenting radiation-sensitive fragile sites and terminal non-reciprocal translocations in leukemic cells [40]. Moreover, one of the

Telomeric Alterations in Leukemias and Lymphomas

125

- !iii( ! ii ;~H

i;

22

b

i 4

a

,=:

CBG

b Figure 3 Clonal telomere centromere translocation in BM and PB cells at I relapse of c-ALL (case 7) with karyotype: 46,XY,psu dic(4;12),t(9;22), a) Arrows indicate the presence of both t(4;12) and t(9;22) in the metaphase, b) cutout of chromosomes involved in t(9; 22)(q34;q11) and telomere-centromere t(4;12)(q10;q24.3). CBG bands of derivative chromosome 12 showed two positive signals defining its dicentric structure (dic) and the (peri)centromere breakpoint on chromosome 4q10.

first reports of telomeric associations concerns Epstein-Barrvirus-infected cell lines [41], and telomeres are found as one of the recurrent sites of chromosome virus integration [42]. At present, the data do not indicate that specific telomeres are involved in hematologic neoplasia. However, it should be noted that the terminal band 19p13.3 is a specific site of translocation in ALL and l y m p h o m a [43], and that tas and non-reciprocal translocations involving 19p telomere are recurrent in these types of neoplasia. Interestingly, all cases reported to date with tas in hematologic neoplasia are lymphoid malignancies. In our group of hematologic disorders, telomeric abnormalities, i n c l u d i n g terminal non-reciprocal translocations, are prevalent in lymphoproliferative diseases, but, at variance with other reports, they are also detected in two cell populations with a myeloid phenotype (7.1% v. 0.7%). A m o n g the lymphoproliferative diseases, the recurrence of chromosome abnormalities involving a telomere is even more evident in the NHLs, i n c l u d i n g CLL (9.5%). Moreover, we emphasize that n i n e of the 11 cases with telomeric alterations were in the progressive stage of disease. Further data, which might also be acquired by in situ hybridization of telomeric consensus sequences, could establish how frequently abnormalities of chromosome c o m p l e m e n t in neoplasia in-

volve telomeres, and could clarify whether there is an inborn cell lineage predisposition to these rearrangements or rather an ubiquitous instability of telomeres in neoplastic cells. This last behavior might be related to an alteration in synthesis and in length of telomeric DNA or to environmental agents such as viruses. This work was supported in part by a grant from Regione Emilia Romagna. We are grateful to Mr. Mike Hammersley for the linguistic revision of the text. REFERENCES 1. Sarto GE, Therman E (1980): Replication and inactivation of a dicentrix X formed by telomeric fusion. Clin Gen 5:904-911. 2. Sawyer JR, Rowe RA, Hassed SJ, Cunniff C (1993}: Highresolution cytogenetic characterization of telomeric associations in ring chromosome 19. Hum Genet 91: 42-44. 3. Rivera H, Zuffardi O, Cargantini L (1990): Non reciprocal and jumping translocation of 15q-qter in Prader-Willi syndrome. Am J Med Genet 37:311-317. 4. Park VM. Gustashaw KM, Wathen T (1992): The presence of interstitial telomeric sequences in constitutional chromosome abnormalities. Am J Hum Genet 50:914-923.

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