Accepted Manuscript Jumping-like translocation – a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia Iveta Sarova , Jana Brezinova , Halka Lhotska , Adela Berkova , Sarka Ransdorfova , Zuzana Zemanova , Jacqueline Soukupova , Kyra Michalova PII:
S2210-7762(14)00087-8
DOI:
10.1016/j.cancergen.2014.05.001
Reference:
CGEN 284
To appear in:
Cancer Genetics
Received Date: 21 November 2013 Revised Date:
14 April 2014
Accepted Date: 1 May 2014
Please cite this article as: Sarova I, Brezinova J, Lhotska H, Berkova A, Ransdorfova S, Zemanova Z, Soukupova J, Michalova K, Jumping-like translocation – a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Jumping-like translocation – a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia
Zuzana Zemanovab, Jacqueline Soukupovaa and Kyra Michalovaa,b
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Iveta Sarovaa,b*, Jana Brezinovaa, Halka Lhotskab, Adela Berkovab, Sarka Ransdorfovaa,
a
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
b
Center of Oncocytogenetics, Institute of Medical Biochemistry and Laboratory Diagnostics,
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General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech
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Republic
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Short running title: Jumping-like translocation
Keywords: Burkitt lymphoma/leukemia, jumping translocation, chromothripsis, 13q deletion,
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FISH
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*Corresponding author:
Iveta Sarova Cytogenetic Department, Institute of Hematology and Blood Transfusion U Nemocnice 1, 128 20 Prague 2, Czech Republic tel.: 420 221 977 236, fax: 420 224 913 728, e-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Chromosomal translocations are acquired genetic rearrangements in human cancers. Jumping translocations are rare nonreciprocal rearrangements involving the same donor chromosome segment translocated to two or more recipient chromosomes. In this report, we describe a patient with Burkitt lymphoma/leukemia (BL) and a complex karyotype, including
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t(2;8)(p12;q24), copy-neutral loss of heterozygosity at 17p13.1–p13.3 and 19q13.1–q13.2, trisomy 20 and two uncommon chromosomal aberrations. The first one was a complex rearrangement of chromosome 15 (probably the consequence of chromothripsis), masked by an apparently balanced reciprocal translocation t(11;15)(p11.2;q21). The second one was a
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special type of unbalanced “vice versa” jumping translocation, which involved the same acceptor chromosome arm (13q) and various donor chromosome segments. It is unclear, whether both untypical rearrangements are consequence of the TP53 alteration or assumed
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chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) translocation in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an
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additional change, subsequently accelerated the progression of the disease.
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Introduction Burkitt lymphoma/leukemia (BL) is a high-grade form of non-Hodgkin lymphoma with a mature B-cell phenotype (1). Alteration of the MYC gene (on 8q24) is a typical chromosomal finding in BL (2,3). The most common translocation, t(8;14)(q24;q32), occurs in
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approximately 70%–80% of BL patients and leads to the overexpression of transcription factor gene MYC when it is juxtaposed with the enhancer element of the immunoglobulin heavy chain gene (IgH) at 14q32 (1). The MYC oncogene is essential for normal cell growth and proliferation and plays a central role in the malignant transformation of BL (2).
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Translocation variants t(2;8)(p12;q24) and t(8;22)(q24;q11) are observed in 10%–15% of BL patients, where the MYC gene is joined to the immunoglobulin light chain gene region kappa (IgK at 2p11) or lambda (IgL at 22q11) (1).
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Approximately 60%–70% of sporadic BL in adults display an additional chromosomal abnormality and 30%–50% of patients exhibit complex karyotypes (4,5). The most frequent secondary aberrations include the gain of 1q, 7q, or 12q or the loss of 6q, 13q, or 17p (3,4,6). One of the described mechanisms of these gains and losses in BL is jumping translocation (JT) (7). JT is a nonreciprocal translocation involving the same donor chromosome arm or
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chromosomal segment and two or more recipient chromosomes in different cells in the same patient (8,9).
In this report, we present a patient with BL and a complex karyotype including the translocation
t(2;8)(p12;q24),
the
apparently
balanced
reciprocal
translocation
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t(11;15)(p11.2;q21), and trisomy 20. In some mitoses, the aberrations were accompanied by an unbalanced nonreciprocal translocation of chromosome 13 with multiple partner
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chromosomes. Unlike classic JT, the translocation involved the same acceptor chromosome arm, 13q, and various donor chromosomal segments. Because the systematic analyses of the chromosomal breakpoints involved in translocations and their possible genetic consequences are necessary to understand the mechanism and significance of each aberration, we studied the breakpoints on derivative chromosome 11, 13, and 15 using molecular cytogenetic methods.
Materials and methods
Case report
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ACCEPTED MANUSCRIPT In January 2013, a 28-year-old woman was examined for headache, parodontitis, and loss of weight (8 kg/month). A bone-marrow aspirate was hypercellular, with reduced granulopoiesis without maturation, erythropoiesis (normoblastic), and increased counts of myeloperoxidase (MPO)-negative blasts morphologically classified as FAB (French–American–British) subtype L3. An HIV test was negative. An ophthalmological examination showed edema of
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the optic nerve and magnetic resonance confirmed central nervous system (CNS) and head region soft-tissue infiltration. A diagnosis of leukemized Burkitt lymphoma (mature B-ALL) with CNS involvement was made. The patient was treated with protocol GMALL BALL/NHL 2002 and cranial radiation therapy. The treatment was complicated by Aspergillus
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pneumonia. After a right lower lobe lobectomy, the patient was able to continue with chemotherapy. At the time of writing, the patient was in the first complete remission.
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Conventional and molecular cytogenetic studies
Standard chromosomal preparation techniques for bone-marrow cells (24 h MarrowGrow Medium, colcemid, hypotonic treatment, fixation in methanol/acetic acid, Wright stain [Gbanding]) were used. Twenty-two mitoses were analyzed with the Ikaros imaging system for karyotyping (MetaSystems, Altlussheim, Germany).
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The complex karyotype was verified with multicolor fluorescence in situ hybridization (mFISH) and the multicolor banding method (mBAND), using the 24XCyte color Kit, and XCyte11, XCyte13, and XCyte15 probes (MetaSystems). The MYC gene rearrangement was determined with FISH using the Vysis IGH/MYC/CEP 8 Tri-Color Dual Fusion FISH
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Translocation Probe Kit (Abbott, des Planes, Illinois). Metaphases were evaluated using an Axio Imager.Z1 microscope (Carl Zeiss, Jena, Germany) and the Isis computer analysis system (MetaSystems). A series of five bacterial artificial chromosome (BAC) probes (RP1113q31.1:85.8–86.0Mb,
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420F11
13q31.3:91.7–91.9Mb,
RP11-498F10
RP11-83D23
13q31.1:86.2–86.3Mb,
13q32.1:96.9–97.1Mb,
and
RP11-430K10 RP11-235O20
13q32.1:97.3–97.4Mb; BlueGnome, Cambridge, UK) and one Vysis locus-specific probe (LSI 13q34; Abbott) were used for 13q breakpoint mapping. Derivative chromosomes 11 and 15 were investigated with FISH analyses using nine BAC probes (BlueGnome) (see Table 1). DNA for the comparative genomic hybridization–single-nucleotide polymorphism (CGH– SNP) array (Cytochip Cancer SNP 180K; BlueGnome) was extracted from bone-marrow cells fixed in methanol and acetic acid using a QIAamp DNA mini Kit (Qiagen, Inc., Germantown, MD). DNA isolated from a human female lymphoblastoid cell line (NA 12878) from Coriell Repository (Camden, New Jersey, USA) was used as the normal sex-matched reference. 4
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Results Two
main
pathological
clones
were
identified
with
conventional
cytogenetics:
47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q21),+20[12]/47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q2
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1),der(13)t(13;?)(q32;?),+20[10]. mFISH confirmed the results of conventional cytogenetics and specified the donor segments (see Figure 1B). Four different pathological clones of derivative
chromosome
13
were
identified
with
mBAND:
der(13)t(13;?)(q32;?)[8]/dup(13)(q31q33)[5]/der(13)dup(13)(q31q33)t(13;?)(q32;?)[2]/hsr(13
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)(q31q33)[1] – its clonality was verified by FISH (see Figure 1C). The CGH–SNP array revealed an atypical pattern of 13q31.3–q32.1 gain with a minimal common amplicon in region 13q31.3 (ch13:91.0–92.0Mbp) and terminal deletion del(13)(q32.1) (see Figure 1C). A
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series of five BAC probes (situated from 13q31.1 to 13q32.1 region) and LSI 13q34 demonstrated the presence of more than four different pathological clones involving derivative chromosome 13 detected by mBAND. The most frequent pathological clone included the 13q31.3 gain and terminal deletion 13q32.1-13qter. Only the signal of RP11430K10 probe (13q31.3) was amplified in all clones, other probes had different pattern
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(normal, amplified or lost signal) in different mitoses demonstrated existence of many clones of derivative chromosome 13 (see Figure 1D).
We also investigated the breakpoints on derivative chromosomes 11 and 15 in apparently balanced translocation t(11;15)(p11.2;q21). mBAND analysis identified breakpoints at
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11p11.2 and 15q21 in all mitoses. The CGH–SNP array detected a cryptic deletion on chromosome 11, del(11)(p11.2p11.2), and two losses on chromosome 15, del(15)(q21.1q21.3)
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and del(15)(q21.3q22.2) (see Figure 1E). A series of nine BAC probes for chromosomes 11 and 15 confirmed the deletions of both chromosomes and revealed the translocation complexity,
which
probably
involved
mechanisms
such
as
chromothripsis
and
chromoanagenesis (see Table 1 and Figure 1F and 1G). The CGH-SNP array also proved copy-neutral loss of heterozygosity at 17p13.1–p13.3 (ch17:2.2–7.9Mbp) and 19q13.1–q13.2 (ch19:42.6–51.0Mbp).
Discussion
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ACCEPTED MANUSCRIPT In this report, we have described a special type of unbalanced “vice versa” jumping translocation in a patient with BL and translocation t(2;8)(p12;q24). Like JT, the acceptor chromosome is altered by deletion and the donor chromosome by duplication. However, unlike JT, vice versa jumping translocation involves the same acceptor chromosome arm but donor segments from several chromosomes. Acquired JTs have rarely been observed,
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particularly as a secondary aberration in hematological malignancies such as leukemias, multiple myeloma, and BL (7,8,10,11). Manola et al. (2008) suggested that JT may not contribute to the pathogenesis, but rather to the progression of the disease (10). Because the main clone did not display a jumping-like translocation, we assume that it played a similar
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pathogenetic role in our patient. Moreover, the number of various pathological cells differing in the origin of the donor segment indicates an extremely rapid cell cycle and the ongoing clonal evolution of the tumor cells. The duplicated chromosomal segment that is identical in
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all clones with JT is of the utmost interest for identifying the affected genes. In our case of jumping-like translocation, further analyses were directed toward the commonly deleted chromosome 13.
Abnormalities of 13q are the most frequent genetic aberrations in BL in general (4,6,12,13,14). Recurrent 13q abnormalities, primarily deletions, have been described in many
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types of B-lymphoid neoplasms and are often associated with aggressive tumor features and an unfavorable prognosis (5,6,13,14). More than four different pathological clones with derivative chromosome 13 were identified with the CGH–SNP array and FISH analyses in the bone-marrow cells of our patient. The amplified regions of 13q varied in size and location,
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ranging from 13q31.3 to 13q32, as has been reported in other studies (12). The minimal common amplicon in chromosomal band 13q31.3 (ch13:91.0–92.0Mb) contained one proteincoding gene (GPC5) and the miR-17-92 polycistron. The glypican gene GPC5 belongs to a
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family of cell-surface heparan sulfate proteoglycans, which may play roles in the control of cell division and growth regulation (15). However, expression analyses of cell lines with amplification at 13q31-q32 proved that GPC5 is not probably a candidate gene of this region in lymphomas unlike the cluster of six different miRNAs in MIR17HG (16,17). The overexpression of these miRNAs in the lymphocytes of transgenic mice resulted in increased proliferation, reduced apoptosis, and lymphoproliferative disease (17,18). In the most frequent pathological clone with derivative chromosome 13, the terminal deletion 13q32.1–13qter was involved. Chromosomal band 13q34 occurs in a region that is frequently deleted in BL (12,13,14). The 13qter part contains more than 30 genes, most of which encode transcription factors, cell-cycle regulators, tumor suppressors such as ING1 (inhibitor of 6
ACCEPTED MANUSCRIPT growth family, member 1), RAB20 (a member of the RAS oncogene family), LIG4 (ligase IV, DNA, ATP-dependent), TNFSF13B (tumor necrosis factor [ligand] superfamily, member 13b), RAP2A (a member of the RAS oncogene family), ERCC5 (xeroderma pigmentosum, complementation group G), and others. Many studies have tried to identify the critical regions of 13q loss and specific altered genes, but no causal gene has been defined as yet because
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limited data are available and the variability in the deleted regions is large (4,5,12,13). The systematic analysis of the chromosomal breakpoints in translocations is necessary if we are to understand the mechanisms and significance of these aberrations. Therefore, we mapped the breakpoints on derivative chromosomes 11 and 15. A series of BAC probes for
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chromosomes 11 and 15 revealed translocation complexity, particularly involving chromosome 15, with at least seven different breakpoints. A possible mechanism generating multiple simultaneous double-stranded DNA breaks on one chromosome is chromothripsis
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followed by chromoanagenesis (19,20). Chromothripsis is defined as a series of complex rearrangements affecting a single chromosome(s) in one catastrophic event (19). A small region within the rearranged chromosome arm is shattered and stitched together, but the position or orientation of each segment differs from that on the normal chromosomal arm, and losses or amplifications are frequent. Chromothripsis is seen in 2%–3% of all cancers, with
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higher frequencies in bone cancers (19–21). We have no evidence that a single event led to complex translocation t(11;15)(p11.2;q21) in the patient presented here, but we propose that it is a mechanism that effectively explains the FISH results. We also detected two uniparental disomies (UPDs) at 17p (5.7Mbp) and 19q (8.4Mbp) with a
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CGH–SNP array. Several important genes are associated with neoplasms, such as TP53 (17p13, tumor protein p53 [Li-Fraumeni syndrome]), BCL6B (17p13, B cell lymphoma 6, member B), BCL3 (19q13, B-cell CLL/lymphoma 3), and NAPA (19q13, N-ethylmaleimide-
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sensitive factor attachment protein, alpha). The copy-neutral loss of heterozygosity is another very important mechanism of tumorigenesis (22). In general, TP53 inactivation is one of the key events in malignant transformation which support origin of complex rearrangements including chromothripsis by accumulation of double stranded breaks (DSBs) in cells escaping apoptosis (23). Schiffman et al. (2011) observed a strong correlation of 17p13 UDP with 13q31 gain in pediatric BL (14). In conclusion, the MYC rearrangements are causal genetic events in BL. Also TP53 inactivation belongs to the frequent and crucial change in aggressive tumors and may be an initiator of origin of highly complex chromosomal aberrations like we presented here. It is unclear, whether both untypical rearrangements are consequence of the TP53 alteration and 7
ACCEPTED MANUSCRIPT are thus independent on each other, or assumed chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) translocation in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an additional change, subsequently accelerated the progression. The number of chromosomal changes and pathological clones that we detected in
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one patient are evidence of chromosomal instability and very high replication potential typical of cells involved in aggressive malignant diseases.
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Acknowledgements
This study was supported by the project (Ministry of Health, Czech Republic) for Conceptual
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Development of Research Organization 00023736, RVO-VFN64165, GACR-P302/12/G157.
References
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2001;20(40):5595-5610.
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2. Boxer LM, Dang CV. Translocations involving c-myc and c-myc function. Oncogene
3. Boerma EG, Siebert R, Kluin PM, et al. Translocations involving 8q24 in Burkitt
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lymphoma and other malignant lymphomas: a historical review of cytogenetics in the light of
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todays knowledge. Leukemia 2009;23(2):225-234.
4. Onciu M, Schlette E, Zhou Y, et al. Secondary chromosomal abnormalities predict outcome in pediatric and adult high-stage Burkitt lymphoma. Cancer 2006;107(5):1084-1092.
5. Poirel HA, Cairo MS, Heerema NA, et al. Specific cytogenetic abnormalities are associated with a significantly inferior outcome in children and adolescents with mature B-cell nonHodgkin's lymphoma: results of the FAB/LMB 96 international study. Leukemia 2009;23(2):323-231.
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ACCEPTED MANUSCRIPT 6. de Souza MT, Mkrtchyan H, Hassan R, et al. Secondary abnormalities involving 1q or 13q and poor outcome in high stage Burkitt leukemia/lymphoma cases with 8q24 rearrangement at diagnosis. Int J Hematol 2011;93(2):232-236.
7. Bessenyei B, Ujfalusi A, Balogh E, et al. Jumping translocation of chromosome 1q
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associated with good clinical outcome in a case of Burkitt leukemia. Cancer Genet 2011;204(4):207-210.
8. Jamet D, Marzin Y, Douet-Guilbert N, et al. Jumping translocations in multiple myeloma.
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Cancer Genet Cytogenet 2005;161(2):159-163.
9. Berger R, Bernard OA. Jumping translocations. Genes Chromosomes Cancer
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2007;46(8):717-723.
10. Manola KN, Georgakakos VN, Stavropoulou C, et al. Jumping translocations in hematological malignancies: a cytogenetic study of five cases. Cancer Genet Cytogenet
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2008;187(2):85-94.
11. Lizcova L, Zemanova Z, Malinova E, et al. Jumping translocations in bone marrow cells of pediatric patients with hematologic malignancies: a rare cytogenetic phenomenon. Cancer
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Genet 2011;204(6):348-349.
12. Toujani S, Dessen P, Ithzar N, et al. High resolution genome-wide analysis of
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chromosomal alterations in Burkitt's lymphoma. PLoS One 2009;4(9):e7089.
13. Nelson M, Perkins SL, Dave BJ, et al. An increased frequency of 13q deletions detected by fluorescence in situ hybridization and its impact on survival in children and adolescents with Burkitt lymphoma: results from the Children's Oncology Group study CCG-5961. Br J Haematol 2010;148(4):600-610.
14. Schiffman JD, Lorimer PD, Rodic V, et al. Genome wide copy number analysis of paediatric Burkitt lymphoma using formalin-fixed tissues reveals a subset with gain of chromosome
13q
and
corresponding
miRNA
over
expression.
Br
J
Haematol
2011;155(4):477-86. 9
ACCEPTED MANUSCRIPT 15. Yu W, Inoue J, Imoto I, et al. GPC5 is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines. J Hum Genet 2003;48(6):331-335.
16. Ota A, Tagawa H, Karnan S, et al. Identification and characterization of a novel gene,
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C13orf25, as a target for 13q31-q32 amplification in malignant lymphoma. Cancer Res 2004;64(9):3087-3095.
17. Lawrie CH. MicroRNAs and lymphomagenesis: a functional review. Br J Haematol
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2013;160(5):571-581.
18. Xiao C, Srinivasan L, Calado DP, et al. Lymphoproliferative disease and autoimmunity in
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mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 2008;9(4):405-414.
19. Stephens PJ, Greenman CD, Fu B, Yang F, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell 2011;144(1):27-40.
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20. Righolt C, Mai S. Shattered and stitched chromosomes-chromothripsis and chromoanasynthesis-manifestations of a new chromosome crisis? Genes Chromosomes Cancer 2012;51(11):975-981.
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21. Maher CA, Wilson RK. Chromothripsis and human disease: piecing together the shattering process. Cell 2012;148(1-2):29-32.
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22. Makishima H, Maciejewski JP. Pathogenesis and consequences of uniparental disomy in cancer. Clin Cancer Res 2011;17(12):3913-3923.
23. Jones MJ, Jallepalli PV. Chromothripsis: chromosomes in crisis. Dev Cell. 2012;23(5):908-917.
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ACCEPTED MANUSCRIPT Figure
1.
A+B:
results
of
the
mFISH
analysis
–
a
karyotype
47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q21),+20 [A] and identification of multiple partner chromosomes of unbalanced translocation der(13)t(13;?)(q32;?) [B] C: CGH–SNP array proved an atypical pattern of 13q31.3–q32.1 gain (green line) with a minimal common amplicon in region 13q31.3 (ch13:91.0–92.0Mbp) - the highest peak and terminal deletion
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del(13)(q32.1) (red line). mBAND analyses detected four different pathological clones of derivative chromosome 13; D: FISH analyses with a LSI 13q34 and series of five BAC probes for chromosome 13 (situated from 13q31.1 to 13q32.1 region) demonstrated the presence of more than four different pathological clones of derivative chromosome 13. The
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most frequent pathological clone included the 13q31.3 gain (gain of RP11-430K10 probe) and terminal deletion 13q32.1-13qter (loss of RP11-83D23, RP11-235O20 and LSI 13q34 probes); E: CGH–SNP array detected a cryptic deletion on chromosome 11 -
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del(11)(p11.2p11.2) (red line), and two losses on chromosome 15 - del(15)(q21.1q21.3) and del(15)(q21.3q22.2) (red lines); F: mapping of breakpoints on chromosomes 11 and 15 by FISH analyses with specific BAC probes – hybridization results and localization of each probe are shown in Table 1; G: schematic illustration of complex translocation t(11;15)(p11.2;q15) – one segment of chromosome 11 and two segments of chromosome 15
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(2 and 6) were lost, segments 1 and 5 were translocated on chromosome 11 .
ACCEPTED MANUSCRIPT Table 1. Results of FISH analyses with BAC probes for chromosomes 11 and 15: the presence of a
RP11-165B4 RP11-390K5 RP11-17G12
ch11:46.6-46.8 ch11:47.0-47.2 ch11:47.1-47.3
RP11-39N23 RP11-430B1 RP11-106N8 RP11-81G24 RP11-252A23 RP11-231A23
ch15:51.6-51.8 ch15:52.4-52.6 ch15:55.9-56.1 ch15:56.1-56.3 ch15:59.7-59.9 ch15:60.3-60.5
FISH result der(11) der(15) + + + +
+ + -
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Localization (Mbp)
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BAC Probe
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hybridization signal (probe retained) is indicated with (+), its absence (probe deleted) with (–).
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ACCEPTED MANUSCRIPT
S2210-7762(14)00087-8
DOI:
10.1016/j.cancergen.2014.05.001
Reference:
CGEN 284
To appear in:
Cancer Genetics
Received Date: 21 November 2013 Revised Date:
14 April 2014
Accepted Date: 1 May 2014
Please cite this article as: Sarova I, Brezinova J, Lhotska H, Berkova A, Ransdorfova S, Zemanova Z, Soukupova J, Michalova K, Jumping-like translocation – a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia, Cancer Genetics (2014), doi: 10.1016/j.cancergen.2014.05.001. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT
Jumping-like translocation – a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia
Zuzana Zemanovab, Jacqueline Soukupovaa and Kyra Michalovaa,b
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Iveta Sarovaa,b*, Jana Brezinovaa, Halka Lhotskab, Adela Berkovab, Sarka Ransdorfovaa,
a
Institute of Hematology and Blood Transfusion, Prague, Czech Republic
b
Center of Oncocytogenetics, Institute of Medical Biochemistry and Laboratory Diagnostics,
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General Faculty Hospital and 1st Faculty of Medicine of Charles University, Prague, Czech
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Republic
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Short running title: Jumping-like translocation
Keywords: Burkitt lymphoma/leukemia, jumping translocation, chromothripsis, 13q deletion,
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FISH
1
*Corresponding author:
Iveta Sarova Cytogenetic Department, Institute of Hematology and Blood Transfusion U Nemocnice 1, 128 20 Prague 2, Czech Republic tel.: 420 221 977 236, fax: 420 224 913 728, e-mail: [email protected]
1
ACCEPTED MANUSCRIPT Abstract Chromosomal translocations are acquired genetic rearrangements in human cancers. Jumping translocations are rare nonreciprocal rearrangements involving the same donor chromosome segment translocated to two or more recipient chromosomes. In this report, we describe a patient with Burkitt lymphoma/leukemia (BL) and a complex karyotype, including
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t(2;8)(p12;q24), copy-neutral loss of heterozygosity at 17p13.1–p13.3 and 19q13.1–q13.2, trisomy 20 and two uncommon chromosomal aberrations. The first one was a complex rearrangement of chromosome 15 (probably the consequence of chromothripsis), masked by an apparently balanced reciprocal translocation t(11;15)(p11.2;q21). The second one was a
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special type of unbalanced “vice versa” jumping translocation, which involved the same acceptor chromosome arm (13q) and various donor chromosome segments. It is unclear, whether both untypical rearrangements are consequence of the TP53 alteration or assumed
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chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) translocation in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an
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additional change, subsequently accelerated the progression of the disease.
2
ACCEPTED MANUSCRIPT
Introduction Burkitt lymphoma/leukemia (BL) is a high-grade form of non-Hodgkin lymphoma with a mature B-cell phenotype (1). Alteration of the MYC gene (on 8q24) is a typical chromosomal finding in BL (2,3). The most common translocation, t(8;14)(q24;q32), occurs in
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approximately 70%–80% of BL patients and leads to the overexpression of transcription factor gene MYC when it is juxtaposed with the enhancer element of the immunoglobulin heavy chain gene (IgH) at 14q32 (1). The MYC oncogene is essential for normal cell growth and proliferation and plays a central role in the malignant transformation of BL (2).
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Translocation variants t(2;8)(p12;q24) and t(8;22)(q24;q11) are observed in 10%–15% of BL patients, where the MYC gene is joined to the immunoglobulin light chain gene region kappa (IgK at 2p11) or lambda (IgL at 22q11) (1).
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Approximately 60%–70% of sporadic BL in adults display an additional chromosomal abnormality and 30%–50% of patients exhibit complex karyotypes (4,5). The most frequent secondary aberrations include the gain of 1q, 7q, or 12q or the loss of 6q, 13q, or 17p (3,4,6). One of the described mechanisms of these gains and losses in BL is jumping translocation (JT) (7). JT is a nonreciprocal translocation involving the same donor chromosome arm or
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chromosomal segment and two or more recipient chromosomes in different cells in the same patient (8,9).
In this report, we present a patient with BL and a complex karyotype including the translocation
t(2;8)(p12;q24),
the
apparently
balanced
reciprocal
translocation
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t(11;15)(p11.2;q21), and trisomy 20. In some mitoses, the aberrations were accompanied by an unbalanced nonreciprocal translocation of chromosome 13 with multiple partner
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chromosomes. Unlike classic JT, the translocation involved the same acceptor chromosome arm, 13q, and various donor chromosomal segments. Because the systematic analyses of the chromosomal breakpoints involved in translocations and their possible genetic consequences are necessary to understand the mechanism and significance of each aberration, we studied the breakpoints on derivative chromosome 11, 13, and 15 using molecular cytogenetic methods.
Materials and methods
Case report
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ACCEPTED MANUSCRIPT In January 2013, a 28-year-old woman was examined for headache, parodontitis, and loss of weight (8 kg/month). A bone-marrow aspirate was hypercellular, with reduced granulopoiesis without maturation, erythropoiesis (normoblastic), and increased counts of myeloperoxidase (MPO)-negative blasts morphologically classified as FAB (French–American–British) subtype L3. An HIV test was negative. An ophthalmological examination showed edema of
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the optic nerve and magnetic resonance confirmed central nervous system (CNS) and head region soft-tissue infiltration. A diagnosis of leukemized Burkitt lymphoma (mature B-ALL) with CNS involvement was made. The patient was treated with protocol GMALL BALL/NHL 2002 and cranial radiation therapy. The treatment was complicated by Aspergillus
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pneumonia. After a right lower lobe lobectomy, the patient was able to continue with chemotherapy. At the time of writing, the patient was in the first complete remission.
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Conventional and molecular cytogenetic studies
Standard chromosomal preparation techniques for bone-marrow cells (24 h MarrowGrow Medium, colcemid, hypotonic treatment, fixation in methanol/acetic acid, Wright stain [Gbanding]) were used. Twenty-two mitoses were analyzed with the Ikaros imaging system for karyotyping (MetaSystems, Altlussheim, Germany).
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The complex karyotype was verified with multicolor fluorescence in situ hybridization (mFISH) and the multicolor banding method (mBAND), using the 24XCyte color Kit, and XCyte11, XCyte13, and XCyte15 probes (MetaSystems). The MYC gene rearrangement was determined with FISH using the Vysis IGH/MYC/CEP 8 Tri-Color Dual Fusion FISH
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Translocation Probe Kit (Abbott, des Planes, Illinois). Metaphases were evaluated using an Axio Imager.Z1 microscope (Carl Zeiss, Jena, Germany) and the Isis computer analysis system (MetaSystems). A series of five bacterial artificial chromosome (BAC) probes (RP1113q31.1:85.8–86.0Mb,
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420F11
13q31.3:91.7–91.9Mb,
RP11-498F10
RP11-83D23
13q31.1:86.2–86.3Mb,
13q32.1:96.9–97.1Mb,
and
RP11-430K10 RP11-235O20
13q32.1:97.3–97.4Mb; BlueGnome, Cambridge, UK) and one Vysis locus-specific probe (LSI 13q34; Abbott) were used for 13q breakpoint mapping. Derivative chromosomes 11 and 15 were investigated with FISH analyses using nine BAC probes (BlueGnome) (see Table 1). DNA for the comparative genomic hybridization–single-nucleotide polymorphism (CGH– SNP) array (Cytochip Cancer SNP 180K; BlueGnome) was extracted from bone-marrow cells fixed in methanol and acetic acid using a QIAamp DNA mini Kit (Qiagen, Inc., Germantown, MD). DNA isolated from a human female lymphoblastoid cell line (NA 12878) from Coriell Repository (Camden, New Jersey, USA) was used as the normal sex-matched reference. 4
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Results Two
main
pathological
clones
were
identified
with
conventional
cytogenetics:
47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q21),+20[12]/47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q2
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1),der(13)t(13;?)(q32;?),+20[10]. mFISH confirmed the results of conventional cytogenetics and specified the donor segments (see Figure 1B). Four different pathological clones of derivative
chromosome
13
were
identified
with
mBAND:
der(13)t(13;?)(q32;?)[8]/dup(13)(q31q33)[5]/der(13)dup(13)(q31q33)t(13;?)(q32;?)[2]/hsr(13
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)(q31q33)[1] – its clonality was verified by FISH (see Figure 1C). The CGH–SNP array revealed an atypical pattern of 13q31.3–q32.1 gain with a minimal common amplicon in region 13q31.3 (ch13:91.0–92.0Mbp) and terminal deletion del(13)(q32.1) (see Figure 1C). A
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series of five BAC probes (situated from 13q31.1 to 13q32.1 region) and LSI 13q34 demonstrated the presence of more than four different pathological clones involving derivative chromosome 13 detected by mBAND. The most frequent pathological clone included the 13q31.3 gain and terminal deletion 13q32.1-13qter. Only the signal of RP11430K10 probe (13q31.3) was amplified in all clones, other probes had different pattern
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(normal, amplified or lost signal) in different mitoses demonstrated existence of many clones of derivative chromosome 13 (see Figure 1D).
We also investigated the breakpoints on derivative chromosomes 11 and 15 in apparently balanced translocation t(11;15)(p11.2;q21). mBAND analysis identified breakpoints at
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11p11.2 and 15q21 in all mitoses. The CGH–SNP array detected a cryptic deletion on chromosome 11, del(11)(p11.2p11.2), and two losses on chromosome 15, del(15)(q21.1q21.3)
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and del(15)(q21.3q22.2) (see Figure 1E). A series of nine BAC probes for chromosomes 11 and 15 confirmed the deletions of both chromosomes and revealed the translocation complexity,
which
probably
involved
mechanisms
such
as
chromothripsis
and
chromoanagenesis (see Table 1 and Figure 1F and 1G). The CGH-SNP array also proved copy-neutral loss of heterozygosity at 17p13.1–p13.3 (ch17:2.2–7.9Mbp) and 19q13.1–q13.2 (ch19:42.6–51.0Mbp).
Discussion
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ACCEPTED MANUSCRIPT In this report, we have described a special type of unbalanced “vice versa” jumping translocation in a patient with BL and translocation t(2;8)(p12;q24). Like JT, the acceptor chromosome is altered by deletion and the donor chromosome by duplication. However, unlike JT, vice versa jumping translocation involves the same acceptor chromosome arm but donor segments from several chromosomes. Acquired JTs have rarely been observed,
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particularly as a secondary aberration in hematological malignancies such as leukemias, multiple myeloma, and BL (7,8,10,11). Manola et al. (2008) suggested that JT may not contribute to the pathogenesis, but rather to the progression of the disease (10). Because the main clone did not display a jumping-like translocation, we assume that it played a similar
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pathogenetic role in our patient. Moreover, the number of various pathological cells differing in the origin of the donor segment indicates an extremely rapid cell cycle and the ongoing clonal evolution of the tumor cells. The duplicated chromosomal segment that is identical in
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all clones with JT is of the utmost interest for identifying the affected genes. In our case of jumping-like translocation, further analyses were directed toward the commonly deleted chromosome 13.
Abnormalities of 13q are the most frequent genetic aberrations in BL in general (4,6,12,13,14). Recurrent 13q abnormalities, primarily deletions, have been described in many
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types of B-lymphoid neoplasms and are often associated with aggressive tumor features and an unfavorable prognosis (5,6,13,14). More than four different pathological clones with derivative chromosome 13 were identified with the CGH–SNP array and FISH analyses in the bone-marrow cells of our patient. The amplified regions of 13q varied in size and location,
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ranging from 13q31.3 to 13q32, as has been reported in other studies (12). The minimal common amplicon in chromosomal band 13q31.3 (ch13:91.0–92.0Mb) contained one proteincoding gene (GPC5) and the miR-17-92 polycistron. The glypican gene GPC5 belongs to a
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family of cell-surface heparan sulfate proteoglycans, which may play roles in the control of cell division and growth regulation (15). However, expression analyses of cell lines with amplification at 13q31-q32 proved that GPC5 is not probably a candidate gene of this region in lymphomas unlike the cluster of six different miRNAs in MIR17HG (16,17). The overexpression of these miRNAs in the lymphocytes of transgenic mice resulted in increased proliferation, reduced apoptosis, and lymphoproliferative disease (17,18). In the most frequent pathological clone with derivative chromosome 13, the terminal deletion 13q32.1–13qter was involved. Chromosomal band 13q34 occurs in a region that is frequently deleted in BL (12,13,14). The 13qter part contains more than 30 genes, most of which encode transcription factors, cell-cycle regulators, tumor suppressors such as ING1 (inhibitor of 6
ACCEPTED MANUSCRIPT growth family, member 1), RAB20 (a member of the RAS oncogene family), LIG4 (ligase IV, DNA, ATP-dependent), TNFSF13B (tumor necrosis factor [ligand] superfamily, member 13b), RAP2A (a member of the RAS oncogene family), ERCC5 (xeroderma pigmentosum, complementation group G), and others. Many studies have tried to identify the critical regions of 13q loss and specific altered genes, but no causal gene has been defined as yet because
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limited data are available and the variability in the deleted regions is large (4,5,12,13). The systematic analysis of the chromosomal breakpoints in translocations is necessary if we are to understand the mechanisms and significance of these aberrations. Therefore, we mapped the breakpoints on derivative chromosomes 11 and 15. A series of BAC probes for
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chromosomes 11 and 15 revealed translocation complexity, particularly involving chromosome 15, with at least seven different breakpoints. A possible mechanism generating multiple simultaneous double-stranded DNA breaks on one chromosome is chromothripsis
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followed by chromoanagenesis (19,20). Chromothripsis is defined as a series of complex rearrangements affecting a single chromosome(s) in one catastrophic event (19). A small region within the rearranged chromosome arm is shattered and stitched together, but the position or orientation of each segment differs from that on the normal chromosomal arm, and losses or amplifications are frequent. Chromothripsis is seen in 2%–3% of all cancers, with
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higher frequencies in bone cancers (19–21). We have no evidence that a single event led to complex translocation t(11;15)(p11.2;q21) in the patient presented here, but we propose that it is a mechanism that effectively explains the FISH results. We also detected two uniparental disomies (UPDs) at 17p (5.7Mbp) and 19q (8.4Mbp) with a
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CGH–SNP array. Several important genes are associated with neoplasms, such as TP53 (17p13, tumor protein p53 [Li-Fraumeni syndrome]), BCL6B (17p13, B cell lymphoma 6, member B), BCL3 (19q13, B-cell CLL/lymphoma 3), and NAPA (19q13, N-ethylmaleimide-
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sensitive factor attachment protein, alpha). The copy-neutral loss of heterozygosity is another very important mechanism of tumorigenesis (22). In general, TP53 inactivation is one of the key events in malignant transformation which support origin of complex rearrangements including chromothripsis by accumulation of double stranded breaks (DSBs) in cells escaping apoptosis (23). Schiffman et al. (2011) observed a strong correlation of 17p13 UDP with 13q31 gain in pediatric BL (14). In conclusion, the MYC rearrangements are causal genetic events in BL. Also TP53 inactivation belongs to the frequent and crucial change in aggressive tumors and may be an initiator of origin of highly complex chromosomal aberrations like we presented here. It is unclear, whether both untypical rearrangements are consequence of the TP53 alteration and 7
ACCEPTED MANUSCRIPT are thus independent on each other, or assumed chromothripsis influenced the development of the jumping-like translocation. However, the presence of the t(11;15)(p11.2;q21) translocation in all pathological cells suggests that it occurred in the early stage of the disease, whereas the jumping-like translocation, as an additional change, subsequently accelerated the progression. The number of chromosomal changes and pathological clones that we detected in
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one patient are evidence of chromosomal instability and very high replication potential typical of cells involved in aggressive malignant diseases.
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Acknowledgements
This study was supported by the project (Ministry of Health, Czech Republic) for Conceptual
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Development of Research Organization 00023736, RVO-VFN64165, GACR-P302/12/G157.
References
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ACCEPTED MANUSCRIPT 6. de Souza MT, Mkrtchyan H, Hassan R, et al. Secondary abnormalities involving 1q or 13q and poor outcome in high stage Burkitt leukemia/lymphoma cases with 8q24 rearrangement at diagnosis. Int J Hematol 2011;93(2):232-236.
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chromosomal alterations in Burkitt's lymphoma. PLoS One 2009;4(9):e7089.
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14. Schiffman JD, Lorimer PD, Rodic V, et al. Genome wide copy number analysis of paediatric Burkitt lymphoma using formalin-fixed tissues reveals a subset with gain of chromosome
13q
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2011;155(4):477-86. 9
ACCEPTED MANUSCRIPT 15. Yu W, Inoue J, Imoto I, et al. GPC5 is a possible target for the 13q31-q32 amplification detected in lymphoma cell lines. J Hum Genet 2003;48(6):331-335.
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ACCEPTED MANUSCRIPT Figure
1.
A+B:
results
of
the
mFISH
analysis
–
a
karyotype
47,XX,t(2;8)(p12;q24),t(11;15)(p11.2;q21),+20 [A] and identification of multiple partner chromosomes of unbalanced translocation der(13)t(13;?)(q32;?) [B] C: CGH–SNP array proved an atypical pattern of 13q31.3–q32.1 gain (green line) with a minimal common amplicon in region 13q31.3 (ch13:91.0–92.0Mbp) - the highest peak and terminal deletion
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del(13)(q32.1) (red line). mBAND analyses detected four different pathological clones of derivative chromosome 13; D: FISH analyses with a LSI 13q34 and series of five BAC probes for chromosome 13 (situated from 13q31.1 to 13q32.1 region) demonstrated the presence of more than four different pathological clones of derivative chromosome 13. The
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most frequent pathological clone included the 13q31.3 gain (gain of RP11-430K10 probe) and terminal deletion 13q32.1-13qter (loss of RP11-83D23, RP11-235O20 and LSI 13q34 probes); E: CGH–SNP array detected a cryptic deletion on chromosome 11 -
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del(11)(p11.2p11.2) (red line), and two losses on chromosome 15 - del(15)(q21.1q21.3) and del(15)(q21.3q22.2) (red lines); F: mapping of breakpoints on chromosomes 11 and 15 by FISH analyses with specific BAC probes – hybridization results and localization of each probe are shown in Table 1; G: schematic illustration of complex translocation t(11;15)(p11.2;q15) – one segment of chromosome 11 and two segments of chromosome 15
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(2 and 6) were lost, segments 1 and 5 were translocated on chromosome 11 .
ACCEPTED MANUSCRIPT Table 1. Results of FISH analyses with BAC probes for chromosomes 11 and 15: the presence of a
RP11-165B4 RP11-390K5 RP11-17G12
ch11:46.6-46.8 ch11:47.0-47.2 ch11:47.1-47.3
RP11-39N23 RP11-430B1 RP11-106N8 RP11-81G24 RP11-252A23 RP11-231A23
ch15:51.6-51.8 ch15:52.4-52.6 ch15:55.9-56.1 ch15:56.1-56.3 ch15:59.7-59.9 ch15:60.3-60.5
FISH result der(11) der(15) + + + +
+ + -
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Localization (Mbp)
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BAC Probe
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hybridization signal (probe retained) is indicated with (+), its absence (probe deleted) with (–).
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ACCEPTED MANUSCRIPT
