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acute lymphoblastic leukemia
Cancer Genetics and Cytogenetics 142 (2003) 60–64
Short communication
Unusual karyotype aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with non-Hodgkin lymphoma/acute lymphoblastic leukemia Roberta Okhowata, Stefan Dornerb, Wolfgang Hinterbergerb, Christa Fonatscha,* a Institut für Medizinische Biologie der Universität Wien, 1090 Wien, Austria Sozialmedizinisches Zentrum Ost der Stadt Wien, 2. Medizinische Abteilung, 1220 Wien, Austria Received 1 July 2002; accepted 9 September 2002
b
Abstract
The t(2;18)(p12;q21), known as a rare variant of the t(14;18)(q32;q21), together with t(3;14)(q27;q32), t(8;15)(q24;q22) and two other unusual translocations involving chromosomes 6, 9, 12, and 13, were demonstrated in the bone marrow cells of a 70-year-old male with suspected non-Hodgkin lymphoma/ acute lymphoblastic leukemia. The complex chromosomal aberrations were identified by chromosome banding analysis and by fluorescence in situ hybridization (FISH) with whole chromosome painting probes, centromere-specific -satellite probes, and probes specific for genomic sequences of some likely to be involved candidate genes. Several but not all of the chromosomal aberrations could be proved by multicolor FISH. Possible mechanisms leading to this unusual karyotype commonly associated with different histologic lymphoma subtypes and their prognostic implications are discussed. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Chromosomal aberrations are found in more than 90% of non-Hodgkin lymphomas (NHL) [1]. Several clonal chromosomal abnormalities were described to be commonly associated with NHL and a number of the recurring abnormalities correlate with histology and immunophenotype [2–4]. In 80%–85% of follicular lymphomas and in 30% of diffuse large-cell lymphomas the translocation t(14;18)(q32;q21) is observed [5]. Variant translocations involve chromosome bands 2p12 or 22q11, where the genes encoding (IGK) and (IGL) light chains of immunoglobulin are located. In the t(14;18), the BCL-2 gene, located on 18q21, is rearranged with the IGH (immunoglobulin heavy-chain) gene. In the variant translocations the BCL-2 gene is juxtaposed to the IGK or to the IGL gene, respectively [6]. The t(3;14)(q27;q32) has been described as specific for different large-cell lymphomas but also has been observed in follicular lymphomas [7,8]. In 3q27 the BCL-6 gene (or LAZ3) is located, which has been reported to be juxtaposed most frequently to the IGH gene in 14q32 by the t(3;14)
(q27;q32). Also, rearrangements between BCL-6 and the IGK genes in 2p12 or the IGL gene in 22q11 as the result of translocations t(2;3)(p12;q27) and t(3;22)(q27;q11) can be seen [7]. The t(8;14)(q24;q32) is found in more than 90% of Burkitt lymphomas [9]. The c-MYC gene, located in 8q24, is juxtaposed by this translocation to the IGH gene in 14q32 and in variant translocations to the IGK gene in 2p12 or to the IGL gene in 22q11 [10,11]. The variant t(2;8)(p12;q24) is found in 3%–5% of Burkitt lymphomas. The other variant involving chromosome 22 is observed in 5% of cases [12]. In our patient a translocation involving 8q24 and 15q22 was found. Therefore, the question arose as to whether this translocation might be a further variant of the t(8;14)(q24;q32). We report unusual translocations with chromosome breakpoints specifically affected in follicular and in diffuse largecell lymphomas in a patient with NHL/acute lymphoblastic leukemia (ALL). 2. Material and methods 2.1. Clinical data
* Corresponding author. Tel.: 43-1-4277-60601; fax: 43-1-4277-9606. E-mail address: [email protected] (C. Fonatsch).
A 70-year-old male was referred to hospital because of daily febrile temperatures up to 39C accompanied by night
0165-4608/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0165-4608(02)00801-4
R. Okhowat et al. / Cancer Genetics and Cytogenetics 142 (2003) 60–64
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Fig. 1. Karyotype of a G-banded bone marrow metaphase with the t(2;18)(p12;q21), t(3;14)(q27;q32), t(6;13)(p25;q14), t(8;15)(q24;q22), and t(9;12) (p22;q11)inv(12)(p13q11).
sweats for the past 10 days. The last 2 days before admission he had experienced fever spikes of even higher temperatures. Weight loss of 7 kg had been recognized over the preceding few months. He was in reduced condition with dry mucous membranes. Liver, spleen, and lymph nodes were normal. Ten years ago he had a squamous cell carcinoma of the vocal cord. The therapy could not be evaluated, but no radiation therapy was performed. Four years ago he had a stroke with hemiplegia of the right side. Hematologic parameters of red blood cells were normal. Thrombocytopenia was significant and white blood cells were nearly normal at 10.4 109/L with 3% blasts. Lactate dehydrogenase (LDH) was eight times as high as the normal range and the C-reactive protein (crp) was significantly increased to 80 mg/L (normal: 5 mg/L), indicating an ongoing inflammation. A broad antibiotic treatment was started to which the patient responded with a decrease of crp and body temperature. Bone marrow was hypercellular, with 97% blast cells, which were CD10, CD19, CD20, CD24, CD38, and CD45, and hence immunophenotypically and morphologically the diagnosis of a B-cell malignancy, a NHL/ALL was made. Induction chemotherapy was started 2 days after admission with vincristine, daunorubicin, and methothrexate in combination with dexamethasone. According to the protocol of the German ALL Study of adults, granulocyte colony stimulating factor was administered. After 10 days the patient developed pneumonia and a cerebral stroke. The patient died despite the adapted antibiotic regimen and substitution with blood products and immunoglobulins.
2.2. Classical cytogenetics Bone marrow cells were cultured for 24, 48, and 72 hours and harvested according to standard procedures. The chromosomes subsequently were stained using a modified G-banding technique [13]. The patient’s constitutional karyotype was determined on metaphases from phytohemagglutinin-stimulated lymphocytes. The karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN 1995) [14]. 2.2. Fluorescence in situ hybridization (FISH) The following probes were used: commercially available digoxigenin- and/or biotin-labeled whole chromosome painting (WCP) probes for chromosomes 2, 6, 8, 9, 12, 13, 15, and 18 (Qbiogene, Heidelberg, Germany); -satellite probes of chromosomes 9 and 12; a digoxigenin-labeled probe specific to genomic sequences including the c-MYC locus in 8q24 (Qbiogene, Heidelberg, Germany); and an IGH dualcolor DNA probe for hybridization to the subband 14q32.3, directly green labeled on the telomeric side and red labeled on the centromeric side of the IGH locus breakpoint (Vysis, Downers Grove, IL, USA) combined with a digoxigeninlabeled probe hybridizing to centromere 3. FISH with WCP and centromere- and locus-specific probes was performed as described elsewhere [15]. Multiple FISH (M-FISH) analysis was performed with probes of MetaSystems (Altlussheim, Germany) according to the MetaSystems protocol. The denaturation procedure was adapted from Rieder et
62
R. Okhowat et al. / Cancer Genetics and Cytogenetics 142 (2003) 60–64
al. [15]. The chromosomes were counterstained with 4 -6diamidino-2-phenylindol (DAPI). For FISH analysis, a Zeiss Axiophot epifluorescence microscope (Jena, Newcastle upon Tyne, UK) was used and the signals were captured with an Applied Imaging Ultra system and Metasystems Isis (Altlusshelm).
3. Results Translocations between the short arm of chromosome 2 and the long arm of chromosome 18, t(2;18)(p12;q21); the short arm of chromosome 6 and the long arm of chromosome 13, t(6;13)(p25;q14); and the long arms of chromosomes 8 and 15 t(8;15)(q24;q22), as well as between the short arm of chromosome 9 and one arm of chromosome 12, t(9;12)(p22;?p13); were identified by chromosome banding analysis (Fig. 1); and confirmed by FISH with whole chromosome painting probes. Interestingly, the t(9;12)(p22; ?p13) (Fig. 2a) was combined with the formation of a new centromere distal in 12p13 or a pericentric inv(12)(p13q11). The latter assumption was confirmed by FISH with the centromeric probe of chromosome 12, which showed that the centromere was displaced in the telomeric direction. A 14q was identified by G-banding analysis. To find out which partner chromosome was involved, M-FISH analysis was performed (Fig. 2b) but no translocation involving chromosome 14 could be detected by this technique. FISH analysis with the IGH dual-color and chromosome 3 -satellite probes, however, showed splitting and juxtaposition of IGH to chromosome band 3q27 (Fig. 2c). Thus, the t(3;14) (q27;q32) was identified. M-FISH not only failed in detecting the t(3;14)(q27;q32) but it also produced an incorrect result concerning the t(9;12) (p22;q11)inv(12)(p13q11). Other material than chromosome 9 seemed to be attached to chromosome 12. Only the t(2;18) (p12;q21), t(6;13)(p25;q14), and t(8;15)(q24;q22) affecting large chromosomal segments were identifiable by M-FISH. To evaluate whether the c-MYC oncogene was involved in the t(8;15)(q24;q22), FISH analysis with the c-MYC probe was performed, but no rearrangement of the c-MYC gene was observed. In 30 of 30 metaphases from bone marrow cultures, these complex aberrations were identified by chromosome banding analysis (Fig. 1) and FISH investigations (Fig. 2). Even in phytohemagglutinin-stimulated lymphocyte cultures, the same complex aberrant karyotype was observed in 11 of 14 metaphases.
Fig. 2. (A) Metaphase hybridized with WCP for chromosomes 9 (red) and 12 (green) showing the t(9;12)(p22;q11)inv(12)(p13q11) and one of each normal chromosomes 9 and 12. (B) M-FISH karyotype. (C) Metaphase hybridized with the IGH dual-color DNA probe and the chromosome 3 -satellite probe showing the juxtaposition of the IGH locus to band 3q27 as a result of the t(3;14)(q27;q32).
4. Discussison We present a patient with NHL/ALL and an uncommon combination of translocations usually associated with specific histologic subtypes of malignant lymphomas. The t(2;18)(p12;q21), a variant of the t(14;18)(q32;q21), is a specific primary chromosome aberration in follicular
R. Okhowat et al. / Cancer Genetics and Cytogenetics 142 (2003) 60–64
lymphomas [16] and in diffuse large-cell lymphomas [17]. In our patient neither a follicular lymphoma nor a diffuse large-cell lymphoma was diagnosed. The t(3;14)(q27;q32) leads to the rearrangement between the BCL-6 gene and the IGH gene. In the majority of cases, BCL-6 gene rearrangements are associated with diffuse largecell lymphomas of B-cell type [8]. In one of the first studies concerning this theme, Bastard et al. [7] reported 20 cases of NHL with translocations involving 3q27. In two of their patients, they found this rearrangement as the sole cytogenetic abnormality and suggested it to be the primary event [7]. Because 3q27 rearrangements have been observed as secondary anomalies in lymphomas with t(14;18), we assume that the t(3;14) of our patient is a secondary event. It has been reported that this telomeric reciprocal t(3;14) (q27;q32) was sometimes initially misinterpreted as a 3q27 deletion associated with a 14q chromosome of unknown origin. In our patient the 14q chromosome was identified by chromosome banding analysis. To examine the origin of the chromosomal segment added to 14q32, we performed M-FISH. Because of the obviously limited resolution power of this method, however, M-FISH identified neither the chromosomal segment translocated to 14q32 nor a reciprocal translocation involving 3q27. This translocation was clarified by FISH analysis using the IGH dual-color probe and a chromosome 3 -satellite probe. The t(8;15)(q24;q22) in our patient seemed to involve the breakpoint in 8q24, where the myelocytomatosis virus oncogene (c-MYC) is located [11]. We therefore examined whether the c-MYC gene was affected by the translocation but no rearrangement of c-MYC was detected. The involvement of 9p in the t(9;12)(p22;q11)inv(12) (p13q11) in our patient underlines the lymphoid origin of the disease. Loss of genetic material of 9p was initially described in a cytogenetic subgroup of ALL [18]. In 7%–10% of childhood ALL cases, 9p abnormalities have been found [19]. As the German ALL/acute unclassified leukemias (AUL) Study Group demonstrated, in 10% of adult ALL/AUL cases, 9p also was affected by structural rearrangements [20]. In our patient a pericentric inversion of chromosome 12 combined with a translocation with 9p22 did occur, leading to a shift of the centromere 12. It was not examined whether the ETV6 gene was involved in the inversion. In several studies of ALL, a translocation between the short arms of chromosomes 9 and 12, t/dic(9;12)(p11p12;p11p13), leading to a dicentric chromosome, has been reported [21]. In our case only the centromere 12 was found in the der(12) by FISH with centromere probes for chromosomes 9 and 12. The t(6;13)(p25;q14) we observed in our patient has not been described yet. Rearrangements involving the short arm of chromosome 6 only, as well as abnormalities concerning the long arm of chromosome 13, are well known. Mecucci et al. [22] reported 6p rearrangements in four patients with T-cell lymphomas, in three cases in the form of translocations. Other investigators also reported deletions and struc-
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tural anomalies of 6p in lymphomas showing T-lineage differentiation [23]. Therefore the correlation between structural rearrangements of 6p and T-cell malignancy seems to be strong but in recent studies 6p abnormalities have been shown also in B-cell lymphomas, though in the form of gain of 6p material [24]. Structural abnormalities of the long arm of chromosome 13, especially with involvement of band 13q14, are often described in B-cell chronic lymphocytic leukemia, but are also found in acute myelocytic leukemias, myelodysplastic syndromes, myeloproliferative disorders, NHL, and adult T-cell leukemia [12,25]. Whether the retinoblastoma tumor suppressor gene, located in 13q14, was involved in the t(6;13)(p25;q14) of our patient has to be studied. References [1] Bloomfield CD, Arthur DC, Frizzera G, Levine EG, Peterson BA, Gajl-Peczalska KJ. Nonrandom chromosome abnormalities in lymphoma. Cancer Res 1983;43:2975–84. [2] Schlegelberger B, Zwingers T, Harder L, Nowotny H, Siebert R, Vesely M, Bartels H, Sonnen R, Hopfinger G, Nader A, Ott G, MüllerHermelink K, Feller A, Heinz R. Clinicopathogenetic significance of chromosomal abnormalities in patients with blastic peripheral B-cell lymphoma. Blood 1999;94:3114–20. [3] Fonatsch C. Cytogenetics of malignant lymphoma. Internist (Berl) 1993;34:114–8. [4] Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15:417–74. [5] Juneja S, Lukeis R, Tan L, Cooper I, Szelag G, Parkin JD, Ironside P, Garson OM. Cytogenetic analysis of 147 cases of non-Hodgkin’s lymphoma: nonrandom chromosomal abnormalities and histological correlation. Br J Haematol 1990;76:231–7. [6] Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci USA 1985;82:7439–44. [7] Bastard C, Tilly H, Lenormand B, Bigorgne C, Boulet D, Kunlin A, Monconduit M, Piguet H. Translocations involving band 3q27 and Ig gene regions in non-Hodgkin’s lymphoma. Blood 1992;79:2527–31. [8] Willis TG, Dyer MJS. The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood 2000;96:808–22. [9] Heim S, Mitelman F. Cancer cytogenetics. New York: Wiley-Liss, Inc., 1995. [10] Zech L, Haglund V, Nilsson K, Klein G. Characteristic chromosomal abnormalities in biopsies and lymphoid cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 1976;17:47–56. [11] Dalla Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM. Human c-myc oncogene is located in the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 1982;79:7824–7. [12] Mitelman F. Catalog of chromosome aberrations in cancer. New York: Liss, 1988. [13] Fonatsch C, Schaadt M, Kirchner HH, Diehl V. A possible correlation between the degree of karyotype aberrations and the rate of sister chromatid exchanges in lymphoma lines. Int J Cancer 1980;26:749–56. [14] ISCN. An international sytem for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [15] Rieder H, Bonwetsch C, Janssen LA, Maurer J, Janssen JW, Schwartz S, Ludwig WD, Gassmann W, Bartram CR, Thiel E, Löffler H, Gökbuget N, Hoelzer D, Fonatsch C. High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia. Leukemia 1998;9:1473–81.
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[16] Stein H. The new WHO classification of malignant lymphoma. Pathologe 2000;21:101–5. [17] Offit K, Koduru PRK, Hollis R, Filippa DA, Jhanwar SC, Clarkson BC, Chaganti RS. 18q21 rearrangement in diffuse large cell lymphoma: incidence and clinical significance. Br J Haematol 1989;72:178–83. [18] Kowalczyk J, Sandberg AA. A possible subgroup of ALL with 9p-. Cancer Genet Cytogenet 1983;9:383–5. [19] Murphy SB, Raimondi SC, Rivera GK, Crone M, Dodge RK, Behm FG, Pui CH, Williams DL. Nonrandom abnormalities of chromosome 9p in childhood acute lymphoblastic leukemia: association with highrisk clinical features. Blood 1989;74:409–15. [20] Rieder H, Ludwig WD, Gassmann W, Thiel E, Löffler H, Hoelzer D, Fonatsch C. Chromosomal abnormalities in adult acute lymphoblastic leukemia: results of the German ALL/AUL study group. Recent Results Cancer Res 1993;131:133–48. [21] Mahmoud H, Carroll AJ, Behm F, Raimondi SC, Schuster J, Borowitz M, Land V, Pullen DJ, Vietti TJ, Crist W. The non-random dic(9;12) translocation in acute lymphoblastic leukemia is associated
[22]
[23]
[24]
[25]
with B-progenitor phenotype and an excellent prognosis. Leukemia 1992;6:703–7. Mecucci C, Michaux JL, Tricot G, Louwagie A, Van Den Berghe H. Rearrangements of the short arm of chromosome no. 6 in T-cell lymphomas. Leuk Res 1985;9:1139–48. Maseki N, Kaneko Y, Sakurai M, Kurihara M, Sampi K, Shimamura K, Takayama S. Chromosome abnormalities in malignant lymphoma in patients from Saitama. Cancer Res 1987;47:6767–75. Stokke T, DeAngelis P, Smedshammer L, Galteland E, Steen HB, Smeland EB, Delabie J, Holte HH. Loss of chromosome 11q21–23.1 and 17p and gain of chromosome 6p are independent prognostic indicators in B-cell non-Hodgkin`s lymphoma. Br J Cancer 2001;85: 1900–13. Tanaka K, Arif M, Eguchi M, Guo SX, Hayashi Y, Asaoku H, Kyo T, Dohy H, Kamada N. Frequent allelic loss of the RB, D13S319 and D13S25 locus in myeloid malignancies with deletion/translocation at 13q14 of chromosome 13, but not in lymphoid malignancies. Leukemia 1999;13:1367–73.
Short communication
Unusual karyotype aberrations involving 2p12, 3q27, 18q21, 8q24, and 14q32 in a patient with non-Hodgkin lymphoma/acute lymphoblastic leukemia Roberta Okhowata, Stefan Dornerb, Wolfgang Hinterbergerb, Christa Fonatscha,* a Institut für Medizinische Biologie der Universität Wien, 1090 Wien, Austria Sozialmedizinisches Zentrum Ost der Stadt Wien, 2. Medizinische Abteilung, 1220 Wien, Austria Received 1 July 2002; accepted 9 September 2002
b
Abstract
The t(2;18)(p12;q21), known as a rare variant of the t(14;18)(q32;q21), together with t(3;14)(q27;q32), t(8;15)(q24;q22) and two other unusual translocations involving chromosomes 6, 9, 12, and 13, were demonstrated in the bone marrow cells of a 70-year-old male with suspected non-Hodgkin lymphoma/ acute lymphoblastic leukemia. The complex chromosomal aberrations were identified by chromosome banding analysis and by fluorescence in situ hybridization (FISH) with whole chromosome painting probes, centromere-specific -satellite probes, and probes specific for genomic sequences of some likely to be involved candidate genes. Several but not all of the chromosomal aberrations could be proved by multicolor FISH. Possible mechanisms leading to this unusual karyotype commonly associated with different histologic lymphoma subtypes and their prognostic implications are discussed. © 2003 Elsevier Science Inc. All rights reserved.
1. Introduction Chromosomal aberrations are found in more than 90% of non-Hodgkin lymphomas (NHL) [1]. Several clonal chromosomal abnormalities were described to be commonly associated with NHL and a number of the recurring abnormalities correlate with histology and immunophenotype [2–4]. In 80%–85% of follicular lymphomas and in 30% of diffuse large-cell lymphomas the translocation t(14;18)(q32;q21) is observed [5]. Variant translocations involve chromosome bands 2p12 or 22q11, where the genes encoding (IGK) and (IGL) light chains of immunoglobulin are located. In the t(14;18), the BCL-2 gene, located on 18q21, is rearranged with the IGH (immunoglobulin heavy-chain) gene. In the variant translocations the BCL-2 gene is juxtaposed to the IGK or to the IGL gene, respectively [6]. The t(3;14)(q27;q32) has been described as specific for different large-cell lymphomas but also has been observed in follicular lymphomas [7,8]. In 3q27 the BCL-6 gene (or LAZ3) is located, which has been reported to be juxtaposed most frequently to the IGH gene in 14q32 by the t(3;14)
(q27;q32). Also, rearrangements between BCL-6 and the IGK genes in 2p12 or the IGL gene in 22q11 as the result of translocations t(2;3)(p12;q27) and t(3;22)(q27;q11) can be seen [7]. The t(8;14)(q24;q32) is found in more than 90% of Burkitt lymphomas [9]. The c-MYC gene, located in 8q24, is juxtaposed by this translocation to the IGH gene in 14q32 and in variant translocations to the IGK gene in 2p12 or to the IGL gene in 22q11 [10,11]. The variant t(2;8)(p12;q24) is found in 3%–5% of Burkitt lymphomas. The other variant involving chromosome 22 is observed in 5% of cases [12]. In our patient a translocation involving 8q24 and 15q22 was found. Therefore, the question arose as to whether this translocation might be a further variant of the t(8;14)(q24;q32). We report unusual translocations with chromosome breakpoints specifically affected in follicular and in diffuse largecell lymphomas in a patient with NHL/acute lymphoblastic leukemia (ALL). 2. Material and methods 2.1. Clinical data
* Corresponding author. Tel.: 43-1-4277-60601; fax: 43-1-4277-9606. E-mail address: [email protected] (C. Fonatsch).
A 70-year-old male was referred to hospital because of daily febrile temperatures up to 39C accompanied by night
0165-4608/03/$ – see front matter © 2003 Elsevier Science Inc. All rights reserved. doi:10.1016/S0165-4608(02)00801-4
R. Okhowat et al. / Cancer Genetics and Cytogenetics 142 (2003) 60–64
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Fig. 1. Karyotype of a G-banded bone marrow metaphase with the t(2;18)(p12;q21), t(3;14)(q27;q32), t(6;13)(p25;q14), t(8;15)(q24;q22), and t(9;12) (p22;q11)inv(12)(p13q11).
sweats for the past 10 days. The last 2 days before admission he had experienced fever spikes of even higher temperatures. Weight loss of 7 kg had been recognized over the preceding few months. He was in reduced condition with dry mucous membranes. Liver, spleen, and lymph nodes were normal. Ten years ago he had a squamous cell carcinoma of the vocal cord. The therapy could not be evaluated, but no radiation therapy was performed. Four years ago he had a stroke with hemiplegia of the right side. Hematologic parameters of red blood cells were normal. Thrombocytopenia was significant and white blood cells were nearly normal at 10.4 109/L with 3% blasts. Lactate dehydrogenase (LDH) was eight times as high as the normal range and the C-reactive protein (crp) was significantly increased to 80 mg/L (normal: 5 mg/L), indicating an ongoing inflammation. A broad antibiotic treatment was started to which the patient responded with a decrease of crp and body temperature. Bone marrow was hypercellular, with 97% blast cells, which were CD10, CD19, CD20, CD24, CD38, and CD45, and hence immunophenotypically and morphologically the diagnosis of a B-cell malignancy, a NHL/ALL was made. Induction chemotherapy was started 2 days after admission with vincristine, daunorubicin, and methothrexate in combination with dexamethasone. According to the protocol of the German ALL Study of adults, granulocyte colony stimulating factor was administered. After 10 days the patient developed pneumonia and a cerebral stroke. The patient died despite the adapted antibiotic regimen and substitution with blood products and immunoglobulins.
2.2. Classical cytogenetics Bone marrow cells were cultured for 24, 48, and 72 hours and harvested according to standard procedures. The chromosomes subsequently were stained using a modified G-banding technique [13]. The patient’s constitutional karyotype was determined on metaphases from phytohemagglutinin-stimulated lymphocytes. The karyotype was described according to the International System for Human Cytogenetic Nomenclature (ISCN 1995) [14]. 2.2. Fluorescence in situ hybridization (FISH) The following probes were used: commercially available digoxigenin- and/or biotin-labeled whole chromosome painting (WCP) probes for chromosomes 2, 6, 8, 9, 12, 13, 15, and 18 (Qbiogene, Heidelberg, Germany); -satellite probes of chromosomes 9 and 12; a digoxigenin-labeled probe specific to genomic sequences including the c-MYC locus in 8q24 (Qbiogene, Heidelberg, Germany); and an IGH dualcolor DNA probe for hybridization to the subband 14q32.3, directly green labeled on the telomeric side and red labeled on the centromeric side of the IGH locus breakpoint (Vysis, Downers Grove, IL, USA) combined with a digoxigeninlabeled probe hybridizing to centromere 3. FISH with WCP and centromere- and locus-specific probes was performed as described elsewhere [15]. Multiple FISH (M-FISH) analysis was performed with probes of MetaSystems (Altlussheim, Germany) according to the MetaSystems protocol. The denaturation procedure was adapted from Rieder et
62
R. Okhowat et al. / Cancer Genetics and Cytogenetics 142 (2003) 60–64
al. [15]. The chromosomes were counterstained with 4 -6diamidino-2-phenylindol (DAPI). For FISH analysis, a Zeiss Axiophot epifluorescence microscope (Jena, Newcastle upon Tyne, UK) was used and the signals were captured with an Applied Imaging Ultra system and Metasystems Isis (Altlusshelm).
3. Results Translocations between the short arm of chromosome 2 and the long arm of chromosome 18, t(2;18)(p12;q21); the short arm of chromosome 6 and the long arm of chromosome 13, t(6;13)(p25;q14); and the long arms of chromosomes 8 and 15 t(8;15)(q24;q22), as well as between the short arm of chromosome 9 and one arm of chromosome 12, t(9;12)(p22;?p13); were identified by chromosome banding analysis (Fig. 1); and confirmed by FISH with whole chromosome painting probes. Interestingly, the t(9;12)(p22; ?p13) (Fig. 2a) was combined with the formation of a new centromere distal in 12p13 or a pericentric inv(12)(p13q11). The latter assumption was confirmed by FISH with the centromeric probe of chromosome 12, which showed that the centromere was displaced in the telomeric direction. A 14q was identified by G-banding analysis. To find out which partner chromosome was involved, M-FISH analysis was performed (Fig. 2b) but no translocation involving chromosome 14 could be detected by this technique. FISH analysis with the IGH dual-color and chromosome 3 -satellite probes, however, showed splitting and juxtaposition of IGH to chromosome band 3q27 (Fig. 2c). Thus, the t(3;14) (q27;q32) was identified. M-FISH not only failed in detecting the t(3;14)(q27;q32) but it also produced an incorrect result concerning the t(9;12) (p22;q11)inv(12)(p13q11). Other material than chromosome 9 seemed to be attached to chromosome 12. Only the t(2;18) (p12;q21), t(6;13)(p25;q14), and t(8;15)(q24;q22) affecting large chromosomal segments were identifiable by M-FISH. To evaluate whether the c-MYC oncogene was involved in the t(8;15)(q24;q22), FISH analysis with the c-MYC probe was performed, but no rearrangement of the c-MYC gene was observed. In 30 of 30 metaphases from bone marrow cultures, these complex aberrations were identified by chromosome banding analysis (Fig. 1) and FISH investigations (Fig. 2). Even in phytohemagglutinin-stimulated lymphocyte cultures, the same complex aberrant karyotype was observed in 11 of 14 metaphases.
Fig. 2. (A) Metaphase hybridized with WCP for chromosomes 9 (red) and 12 (green) showing the t(9;12)(p22;q11)inv(12)(p13q11) and one of each normal chromosomes 9 and 12. (B) M-FISH karyotype. (C) Metaphase hybridized with the IGH dual-color DNA probe and the chromosome 3 -satellite probe showing the juxtaposition of the IGH locus to band 3q27 as a result of the t(3;14)(q27;q32).
4. Discussison We present a patient with NHL/ALL and an uncommon combination of translocations usually associated with specific histologic subtypes of malignant lymphomas. The t(2;18)(p12;q21), a variant of the t(14;18)(q32;q21), is a specific primary chromosome aberration in follicular
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lymphomas [16] and in diffuse large-cell lymphomas [17]. In our patient neither a follicular lymphoma nor a diffuse large-cell lymphoma was diagnosed. The t(3;14)(q27;q32) leads to the rearrangement between the BCL-6 gene and the IGH gene. In the majority of cases, BCL-6 gene rearrangements are associated with diffuse largecell lymphomas of B-cell type [8]. In one of the first studies concerning this theme, Bastard et al. [7] reported 20 cases of NHL with translocations involving 3q27. In two of their patients, they found this rearrangement as the sole cytogenetic abnormality and suggested it to be the primary event [7]. Because 3q27 rearrangements have been observed as secondary anomalies in lymphomas with t(14;18), we assume that the t(3;14) of our patient is a secondary event. It has been reported that this telomeric reciprocal t(3;14) (q27;q32) was sometimes initially misinterpreted as a 3q27 deletion associated with a 14q chromosome of unknown origin. In our patient the 14q chromosome was identified by chromosome banding analysis. To examine the origin of the chromosomal segment added to 14q32, we performed M-FISH. Because of the obviously limited resolution power of this method, however, M-FISH identified neither the chromosomal segment translocated to 14q32 nor a reciprocal translocation involving 3q27. This translocation was clarified by FISH analysis using the IGH dual-color probe and a chromosome 3 -satellite probe. The t(8;15)(q24;q22) in our patient seemed to involve the breakpoint in 8q24, where the myelocytomatosis virus oncogene (c-MYC) is located [11]. We therefore examined whether the c-MYC gene was affected by the translocation but no rearrangement of c-MYC was detected. The involvement of 9p in the t(9;12)(p22;q11)inv(12) (p13q11) in our patient underlines the lymphoid origin of the disease. Loss of genetic material of 9p was initially described in a cytogenetic subgroup of ALL [18]. In 7%–10% of childhood ALL cases, 9p abnormalities have been found [19]. As the German ALL/acute unclassified leukemias (AUL) Study Group demonstrated, in 10% of adult ALL/AUL cases, 9p also was affected by structural rearrangements [20]. In our patient a pericentric inversion of chromosome 12 combined with a translocation with 9p22 did occur, leading to a shift of the centromere 12. It was not examined whether the ETV6 gene was involved in the inversion. In several studies of ALL, a translocation between the short arms of chromosomes 9 and 12, t/dic(9;12)(p11p12;p11p13), leading to a dicentric chromosome, has been reported [21]. In our case only the centromere 12 was found in the der(12) by FISH with centromere probes for chromosomes 9 and 12. The t(6;13)(p25;q14) we observed in our patient has not been described yet. Rearrangements involving the short arm of chromosome 6 only, as well as abnormalities concerning the long arm of chromosome 13, are well known. Mecucci et al. [22] reported 6p rearrangements in four patients with T-cell lymphomas, in three cases in the form of translocations. Other investigators also reported deletions and struc-
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tural anomalies of 6p in lymphomas showing T-lineage differentiation [23]. Therefore the correlation between structural rearrangements of 6p and T-cell malignancy seems to be strong but in recent studies 6p abnormalities have been shown also in B-cell lymphomas, though in the form of gain of 6p material [24]. Structural abnormalities of the long arm of chromosome 13, especially with involvement of band 13q14, are often described in B-cell chronic lymphocytic leukemia, but are also found in acute myelocytic leukemias, myelodysplastic syndromes, myeloproliferative disorders, NHL, and adult T-cell leukemia [12,25]. Whether the retinoblastoma tumor suppressor gene, located in 13q14, was involved in the t(6;13)(p25;q14) of our patient has to be studied. References [1] Bloomfield CD, Arthur DC, Frizzera G, Levine EG, Peterson BA, Gajl-Peczalska KJ. Nonrandom chromosome abnormalities in lymphoma. Cancer Res 1983;43:2975–84. [2] Schlegelberger B, Zwingers T, Harder L, Nowotny H, Siebert R, Vesely M, Bartels H, Sonnen R, Hopfinger G, Nader A, Ott G, MüllerHermelink K, Feller A, Heinz R. Clinicopathogenetic significance of chromosomal abnormalities in patients with blastic peripheral B-cell lymphoma. Blood 1999;94:3114–20. [3] Fonatsch C. Cytogenetics of malignant lymphoma. Internist (Berl) 1993;34:114–8. [4] Mitelman F, Mertens F, Johansson B. A breakpoint map of recurrent chromosomal rearrangements in human neoplasia. Nat Genet 1997; 15:417–74. [5] Juneja S, Lukeis R, Tan L, Cooper I, Szelag G, Parkin JD, Ironside P, Garson OM. Cytogenetic analysis of 147 cases of non-Hodgkin’s lymphoma: nonrandom chromosomal abnormalities and histological correlation. Br J Haematol 1990;76:231–7. [6] Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci USA 1985;82:7439–44. [7] Bastard C, Tilly H, Lenormand B, Bigorgne C, Boulet D, Kunlin A, Monconduit M, Piguet H. Translocations involving band 3q27 and Ig gene regions in non-Hodgkin’s lymphoma. Blood 1992;79:2527–31. [8] Willis TG, Dyer MJS. The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood 2000;96:808–22. [9] Heim S, Mitelman F. Cancer cytogenetics. New York: Wiley-Liss, Inc., 1995. [10] Zech L, Haglund V, Nilsson K, Klein G. Characteristic chromosomal abnormalities in biopsies and lymphoid cell lines from patients with Burkitt and non-Burkitt lymphomas. Int J Cancer 1976;17:47–56. [11] Dalla Favera R, Bregni M, Erikson J, Patterson D, Gallo RC, Croce CM. Human c-myc oncogene is located in the region of chromosome 8 that is translocated in Burkitt lymphoma cells. Proc Natl Acad Sci USA 1982;79:7824–7. [12] Mitelman F. Catalog of chromosome aberrations in cancer. New York: Liss, 1988. [13] Fonatsch C, Schaadt M, Kirchner HH, Diehl V. A possible correlation between the degree of karyotype aberrations and the rate of sister chromatid exchanges in lymphoma lines. Int J Cancer 1980;26:749–56. [14] ISCN. An international sytem for human cytogenetic nomenclature. Mitelman F, editor. Basel: S. Karger, 1995. [15] Rieder H, Bonwetsch C, Janssen LA, Maurer J, Janssen JW, Schwartz S, Ludwig WD, Gassmann W, Bartram CR, Thiel E, Löffler H, Gökbuget N, Hoelzer D, Fonatsch C. High rate of chromosome abnormalities detected by fluorescence in situ hybridization using BCR and ABL probes in adult acute lymphoblastic leukemia. Leukemia 1998;9:1473–81.
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