Cancer Genetics and Cytogenetics 133 (2002) 160–163

0165-4608/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S0165-4608(01)00583-0

Short communication

Concomitant tetrasomy 3q and trisomy 18 in CD5

�

, CD13

�

chronic lymphocytic leukemia

Giorgina Specchia

a,b,

*, Francesco Albano

a

, Luisa Anelli

b,c

, Clelia Tiziana Storlazzi

c

,Michele Monaco

b

, Silvana Capalbo

a

, Mariano Rocchi

c

, Vincenzo Liso

a

a

Department of Hematology, University of Bari, Policlinico, 70124 Bari, Italy

b

Department of Hematology, University and Hospital of Foggia, 71100 Foggia, Italy

c

Sezione di Genetica-DAPEG, University of Bari, Policlinico, 70124 Bari, Italy

Received 30 May 2001; received in revised form 14 August 2001; accepted 15 August 2001

Abstract

Chronic lymphocytic leukemia (CLL) associated with myeloid antigens on the surface of B neoplasticcells is a recently identified immunologic variant confined to patients with CD5 negative B-CLL. We de-scribe the case of a 61-year-old female diagnosed with CD5

�

, CD13

�

B-CLL with a tetrasomy 3q re-vealed by fluorescence in situ hybridization analysis, in addition to trisomy 18. To our knowledge, this isthe first reported case of B-CLL with this kind of cytogenetic abnormality. © 2002 Elsevier Science Inc.

All rights reserved.

1. Introduction

Chronic lymphocytic leukemia (CLL) is a lymphoprolif-erative malignancy characterized by the accumulation ofmonoclonal lymphocytes. Conventional cytogenetic studieshave shown that CLL is associated with various chromo-somal aberrations in about 50% of patients [1–3]. The pre-dominant chromosomal abnormalities in CLL involve eitherthe loss or gain of genetic material. The most common cyto-genetic abnormality associated with CLL are del(13)(q14),del(11)(q23),

�

12, del(17)(p13), del(6)(q21) [4–6]. Abnor-malities of chromosome 3 have been rarely observed inlymphoproliferative disorders. Indeed, few CLL cases withinvolvement of chromosome 3 in any kind of cytogeneticabnormality have been described in literature. To the best ofour knowledge, tetrasomy 3q has never been described inCLL [7]. We report a case of CLL featuring this abnormal-ity and concomitant trisomy 18.

2. Materials and methods

Conventional cytogenetic analysis of a 24- to 48-hourculture was performed on bone marrow cells by standardtechniques and evaluated by Giemsa-Trypsin-Giemsa

(GTG) banding at about 400-band level according to theISCN criteria [8].

Whole chromosome paint probes (WCP), derived fromDOP-amplified [9] flow-sorted human chromosomes, werea gift of the Sanger Centre (Cambridge, UK). The partialchromosome painting probe (PCP) #015 derives from DNAof a somatic cell hybrid DNA amplified with dual-Alu-PCR. The hybrid contains the region 3q22

→

3qter in addi-tion to the entire chromosome X. pAE0.68 is an alphoidprobe specific for chromosome 3. For details on the probessee our web site http://www.biologia.uniba.it/rmc/.

Chromosome preparations were hybridized in situ withprobes labeled with biotin by nick translation [10]. Briefly,500 ng of labeled probe were used for fluorescence in situhybridization (FISH) experiments; hybridization was per-formed at 37

�

C in 2

�

SSC, 50% (v/v) formamide, 10% (w/v)dextran sulphate, 5

�

g COT1 DNA (BRL), and 3

�

g soni-cated salmon sperm DNA, in a volume of 10

�

l. Post-hybridization washing was at 42

�

C in 2

�

SSC-50% forma-mide (

�

3) followed by three washes in 0.1

�

SSC at 60

�

C.Biotin-labeled DNA was detected with Cy3-conjugated avi-din (Perkin Elmer Life Sciences, USA). Chromosome iden-tification was obtained by simultaneous DAPI staining, thatproduces a Q-banding pattern. In co-hybridization experi-ments the second probe was directly labeled with FluorX(Amersham, USA). Digital images were obtained using aLeica DMRXA epifluorescence microscope (Leica, Ger-

* Corresponding author. Tel.:

�

39-080-5478711; fax:

�

39-080-5428978.

E-mail address

: emadhba@cimedoc.uniba.it (G. Specchia).

G. Specchia et al. / Cancer Genetics and Cytogenetics 133 (2002) 160–163

161

many) equipped with a cooled CCD camera. Cy3 (red), Flu-orX (green), and DAPI (blue) fluorescence signals, detectedusing specific filters, were recorded separately as gray scaleimages. Pseudocoloring and merging of images were per-formed using the Adobe Photoshop software.

3. Case report

In November 1998, a 61-year-old female presented witha short-term history of fever, weight-loss, and night sweats.Physical examination revealed splenomegaly (18 cm) butno lymphadenopathy. The white blood cell (WBC) countwas 23.0

�

10

9

/L with 75% small lymphocytes, Hgb 13 g/dL,and platelets 210

�

10

9

/L. Serum protein electrophoresis andimmunofixation did not show a monoclonal component.Bone marrow aspirate and biopsy findings were interpretedas consistent with CLL features with an interstitial patternof lymphocyte infiltration. Immunophenotypic analysis ofthe blood by flow cytometry showed the monoclonal B-cellpopulation of B-CLL (CD5 5%, CD19 86%, CD20 88%,CD23 65%, SIg

�

dim

68%, CD13 86%, CD13

�

CD19

�

80%, CD13

�

CD20

�

82%). The diagnosis of B-CLL typi-cal morphology (Binet stage A) was established, accordingto French–American–British criteria [11] (Fig. 1). She didnot start any kind of treatment for the next year. In Decem-ber 1999, because of progressive lymphocytosis with an in-creased WBC count (55.1

�

10

9

/L, Hgb 13.5 g/dL, PLT250

�

10

9

/L), she was treated with five monthly courses ofchlorambucil (10 mg/die) and prednisone (25 mg/die) forseven days. She obtained partial remission and currently, af-ter 28 months from diagnosis, she is doing well, with stabledisease.

4. Results

Cytogenetic analysis of bone marrow preparations de-tected, in 20 of 26 metaphases, a karyotype with 48 chromo-somes. One of the two supernumerary chromosomes wasclearly a chromosome 18; the second, not fully identified,was similar in size to chromosome 1 (Fig. 2A). The othermetaphases showed a normal 46,XX karyotype.

In order to better characterize the karyotipic alterations,FISH experiments were performed using WCP specific forchromosomes 1, 3, and 18. A dual-labeled FISH experimentwith WCP 1 and 3 revealed two normal copies of chromo-some 1. The marker chromosome was entirely painted bythe chromosome 3 (Fig. 2B). Partial chromosome paint-ing probe 015, cohybridized with the centromeric probepAEO.68, clearly showed that the marker chromosome wasan i(3q)(q10) (Fig. 2C). In agreement with conventional cyto-genetic analysis, hybridization with WCP 18 confirmed tri-somy 18 (Fig. 2D). The karyotype revealed by FISH analysiswas, therefore: 48,XX,

�

i(3)(q10).ish (PCP015

�

,D3Z1

�

),

�

18.ish(WCP18

�

).

5. Discussion

A survey of the literature showed that CLL has not previ-ously been reported to be associated with tetrasomy of chro-mosome 3q. Among chronic lymphoproliferative disordersreported in literature, this cytogenetic abnormality was de-scribed only in a case of Waldenstrom macroglobulinemia[12]. It is worth noting that classical cytogenetics alone wasnot able to identify the nature of the i(3q)(q10). On the con-trary, the use of appropriate FISH probes unequivocallycharacterized the chromosome. Trisomy 3 is a rare occur-rence in CLL cases [13]; comparative genomic hybridiza-tion, however, has demonstrated overrepresentation of thetelomeric portion of 3q as a recurrent aberration in B-CLL[14]. Few cases of the involvement of chromosome 3 in anytype of cytogenetic abnormality have been reported in CLL,although structural or numeric aberrations of this chromo-some have been observed in acute myeloid leukemia(AML) and myelodysplastic syndrome (MDS) [15,16]. Arecent report established that structural and numeric abnor-malities on chromosome 3 in AML and MDS were associ-ated with mutagen exposure [17]. In our CLL case no previ-ous exposure to toxic agents nor professional risk wasascertained.

According to a previous report, it seems that CD13 ex-pression is restricted to patients with CD5 negative CLL,an immunological variant that is known for its poor prog-nosis [18]. The presence of myelomonocytic antigens onthe surface of B neoplastic cells is frequently associatedwith a diffuse pattern of BM infiltration, a feature affect-ing prognosis regardless of the clinical stage [19,20]. Ourcase showed presence of CD13 and absence of CD5 ex-pression, but neither the diffuse pattern of infiltration norclinical features characterizing aggressive disease werepresent.

Many important genes located on chromosome 3, such asthe fragile histidine triad gene (

FHIT

) (on the short arm),

EVI1

,

MDS1

,

LAZ3

, and thrombopoietin genes (on the longarm) may be involved in leukemogenic processes [21–25].

Fig. 1. Peripheral blood film stained by May-Grünwald-Giemsa showingtypical small lymphocytes (�1000).

162

G. Specchia et al. / Cancer Genetics and Cytogenetics 133 (2002) 160–163

Trisomy 18 is not typical of CLL. The trisomy of thischromosome, however, frequently accompanies other chro-mosomal abnormalities in this disease, as well as in otherlymphoproliferative disorders, suggesting a synergism inthe pathogenetic role [26–28].

Acknowledgments

This research was supported by the Minestero dell’Is-truzione, dell’Università e della Ricerca; the AssociazioneItali162ana contro le Leucemie; and the Associazione Ital-iana per la Ricerca contro il Cancro, Italy.

References

[1] Juliusson G, Friberg K, Gahrton G. Chromosomal aberrations in pro-gressive and indolent chronic B-lymphocytic leukemia. A longitudi-nal study. Acta Oncol 1988;27:473–7.

[2] Gahrton G, Juliusson G, Robert KH, Friberg K. Role of chromosomal ab-normalities in chronic lymphocytic leukemia. Blood Rev 1987;1:183–92.

[3] Han T, Henderson ES, Emrich LJ, Sandberg AA. Prognostic signifi-cance of karyotypic abnormalities in B cell chronic lymphocytic leu-kemia: an update. Semin Hematol 1987;24:257–63.

[4] Cuneo A, Bigoni R, Castoldi G. Towards a clinically relevant cytoge-netic classification of chronic lymphocytic leukemia and related dis-orders. Haematologica 1998;83:577–9.

[5] Dohner H, Stilgenbauer S, Benner A, Leupolt L, Krober A, BullingerL, Dohner K, Bentz M, Lichter P. Genomic aberrations and survivalin chronic lymphocytic leukemia. N Engl J Med 2000;343:1910–16.

Fig. 2. (A) GTG-banding karyotype showing the presence of a chromosome similar to chromosome 1 in size, in addition to trisomy 18 (arrowhead). (B) Fluo-rescence in situ hybridization experiment with whole chromosome paint (WCP) probes specific for chromosomes 3 (red) and 1 (in green). White arrows pointto chromosomes 18. (C) Fluorescence in situ hybridization experiments showing cohybridization partial chromosome painting 015 (red) with the alphoidprobe specific for chromosome 3 (green). The marker chromosome shows the presence of two 3q arms. White arrows indicate chromosomes 18. (D) Fluores-cence in situ hybridization analysis using WCP specific for chromosome 18 (red), confirming the trisomy of this chromosome.

G. Specchia et al. / Cancer Genetics and Cytogenetics 133 (2002) 160–163

163

[6] Liso V, Capalbo S, Lapietra A, Pavone V, Guarini A, Specchia G.Evaluation of trisomy 12 by fluorescence in situ hybridization in pe-ripheral blood, bone marrow and lymph nodes of patients with B-cellchronic lymphocytic leukemia. Haematologica 1999;84:212–17.

[7] Mitelman F, Johansson B, Mertens F. Mitelman Database of Chromo-some Aberrations in Cancer. Available at: http://cgap.nci.nih.gov/Chromosomes/Mitelman. Accessed May 20, 2001.

[8] ISCN. Guidelines for cancer cytogenetics. Supplement to An Interna-tional System for Human Cytogenetic Nomenclature. F Mitelman,editor. Basel: S. Karger, 1995.

[9] Telenius H, Carter NP, Bebb CE, Nordenskjold M, Ponder BAJ, Tun-nacliffe A. Degenerated oligonucleotide-primed PCR: general ampli-fication of target DNA by single degenerated primer. Genomics1992;13:718–25.

[10] Lichter P, Tang Chang CJ, Call K, Hermanson G, Evans GA, HousmanD, Ward DC. High resolution mapping of human chromosomes 11 byin situ hybridization with cosmid clones. Science 1990;247:64–9.

[11] Bennett JM, Catovsky D, Daniel MT, Flandrin G, Galton DA,Gralnick HR, Sultan C. The French-American-British (FAB) Cooper-ative Group. Proposals for the classification of chronic (mature) Band T lymphoid leukaemias. J Clin Pathol 1989;42:567–84.

[12] Wong KF, Kwong YL, Wong TK. Concomitant partial tetrasomy 3qand trisomy 18 in Waldenstrom Macroglobulinemia. Cancer GenetCytogenet 1995;81:92–3.

[13] Ross FM, Stockdill G. Clonal chromosome abnormalities in chroniclymphocytic leukemia patients revealed by TPA stimulation of wholeblood cultures. Cancer Genet Cytogenet 1987;25:109–21.

[14] Bentz M, Huck K, du Manoir S, Joos S, Werner CA, Fischer K,Dohner H, Lichter P. Comparative genomic hybridization in chronicB-cell leukemias reveals a high incidence of chromosomal gains andlosses. Blood 1995;85:3610–18.

[15] Devald G. Fourth International Workshop on Chromosomes in Leu-kemia 1982: abnormalities of chromosome 3 among 24 patients withde novo ANLL. Cancer Genet Cytogenet 1984;11:306.

[16] Pedersen-Bjergaard J, Pedersen M, Roulstow D, Philip P. Differentgenetic pathways in leukemogenesis for patients presenting with ther-apy-related myelodysplasia and therapy-related acute myeloid leuke-mia. Blood 1995;86:3542–52.

[17] Lindquist R, Forsblom AM, Ost A, Gahrton G. Mutagen exposuresand chromosome 3 aberrations in acute myelocytic leukemia. Leuke-mia 2000;14:112–18.

[18] Ikematsu W, Ikematsu H, Okamura S, Otsuka T, Harada M, Niho Y.Surface phenotype and Ig heavy-chain usage in chronic lymphocyticleukemias: expression of myelomonocytic surface markers in CD5

�

negative chronic B-cell leukemia. Blood 1994;83:2602–10.[19] Pinto A, Del Vecchio L, Carbone A, Roncadin M, Volpe R, Serraino

D, Monfardini S, Colombatti A, Zagonel V. Expression of my-elomonocytic antigens is associated with unfavourable clinicoprog-nostic factors in B-cell chronic lymphocytic leukemia. Ann Oncol1991;2:107–13.

[20] Molica S, Dattilo A, Alberti A. Myelomonocytic associated antigensin B-chronic lymphocytic leukemia: analysis of clinical significance.Leuk Lymphoma 1991;5:139–44.

[21] Suzukawa K, Satoh H, Taniwaki M, Yokota J, Morishita K. The hu-man thrombopoietin gene is located on chromosome 3q26.33–q27,but is not transcriptionally activated in leukemia cells with 3q21 and3q26 abnormalities (3q21q26 syndrome). Leukemia 1995;9:1328–31.

[22] Fichelson S, Dreyfus F, Berger R, Melle J, Bastard C, Miclea JM,Gisselbrecht S. EVI-1 expression in leukemic patients with rear-rangements of the 3q25–q28 chromosomal region. Leukemia 1992;6:93–9.

[23] Oval J, Taetle R, Valentine MB, Ihle JN. Activation of EVI1 gene ex-pression in human acute myelogenous leukemias by translocationsspanning 300–400 kilobases on chromosome band 3q26. Proc NatlAcad Sci USA 1992;89:3937–41.

[24] Inoue H, Ishii H, Alder H, Snyder E, Druck T, Huebner K, CroceCM. Sequence of the FRA3B common fragile region: implicationsfor the mechanism of FHIT deletion. Proc Natl Acad Sci USA 1997;94:14584–9.

[25] Sugimoto K, Yamada K, Miyagawa K, Hirai H, Oshimi K. Decreasedor altered expression of the FHIT gene in human leukemias. StemCell 1997;15:223–8.

[26] Criel A, Wlodarska I, Meeus P, Stul M, Louwagie A, Van Hoof A,Hidajat M, Mecucci C, Van den Berghe H. Trisomy 12 is uncommonin typical chronic lymphocytic leukaemias. Br J Haematol 1994;87:523–8.

[27] Huret JL, Mossafa H, Brizard A, Dreyfus B, Guilhot F, Xue, XQ,Babin P, Tanzer J. Karyotypes of 33 patients with clonal aberrationsin chronic lymphocytic leukaemia. Review of 216 abnormal karyo-types in chronic lymphocytic leukaemia. Ann Genet 1989;32:155–9.

[28] Contrafatto G. Marker chromosome of macroglobulinemia identifiedby G-banding. Cytogenet Cell Genet 1977;18:370–3.