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 Previous Article | Table of Contents | Next Article 
Blood, Vol. 91 No. 4 (February 15), 1998:
pp. 1399-1406
Fluorescence In Situ Hybridization Characterization of New
Translocations Involving TEL (ETV6) in a Wide Spectrum of
Hematologic Malignancies
By
Iwona Wlodarska,
Roberta La Starza,
Mathijs Baens,
Judith Dierlamm,
Ann Uyttebroeck,
Dominik Selleslag,
Adrien Francine,
Cristina Mecucci,
Anne Hagemeijer,
Herman Van den Berghe, and
Peter Marynen
From the Center for Human Genetics and Flanders Interuniversity
Institute of Biotechnology, and the Department of Pediatrics,
University of Leuven, Leuven, Belgium; the Hematology and Bone Marrow
Transplantation Unit, University of Perugia, Perugia, Italy; the
Department of Hematology, AZ St Jan, Brugge, Belgium; and the
Department of Hemato-Oncology, CHR Citadelle, Liege, Belgium.
 |
ABSTRACT |
The ETV6 (also known as TEL) gene on chromosome
12p13 is the target of a number of translocations associated with
various hematologic malignancies. The contribution of ETV6 to
leukemogenesis occurs through different mechanisms that involve either
its helix-loop-helix dimerization domain or its E26
transformation-specific (ETS) DNA-binding domain. Using
fluorescence in situ hybridization we characterized seven new
ETV6 rearrangements in chronic myeloid leukemia, acute myeloid
leukemia, acute lymphoblastic leukemia, and non-Hodgkin's lymphoma.
These aberrations, not always discernible at the cytogenetic level,
include a t(5;12)(q31;p13), t(6;12;17)(p21;p13;q25), t(7;12)(p15;p13), t(7;12)(p12;p13), t(7;12)(q36;p13), t(12;13)(p13;q12), and a not completely defined t(12;?)(p13;?). Loss or disruption of the second ETV6 allele by a del(12)(p12p13) or by an intragenic
ETV6 deletion was detected in two cases. In six cases the 12p13
breakpoint occurred in the 5 end of ETV6, upstream to
exons encoding the HLH domain, whereas the remaining case had a
breakpoint between the exons coding for the HLH domain and the exons
coding for the ETS domain of ETV6. These observations provide
further evidence for the multiple contributions of ETV6 in the
pathogenesis of a wide range of hematologic malignancies.
| |
INTRODUCTION |
RECENT MOLECULAR studies show that the
ETV6 gene (previously known as TEL), a member of the
E26 transformation-specific (ETS)-family of transcription
factors located at 12p13,1 is involved in different
chromosomal translocations associated with human leukemias (Fig 1A). For the t(3;12)(q26;p13), t(5;12)(q33;p13),
t(9;12)(q34;p13), t(12;21)(p13;q22), and t(12;22)(p13;q11) the
translocation partners were identified.1-6 These result in
the expression of a chimeric transcript consisting of ETV6
sequences fused to MDS1/EVI1 (3q26), PDGFRB (5q33),
ABL (9q34), AML1/CBFA2 (21q22), and MN1
(22q11), respectively. In ETV6 translocations involving
PDGFRB, ABL, and AML1/CBFA2 the
helix-loop-helix (HLH) dimerization domain of ETV6 influences or stimulates the activity of the fusion partner. In leukemias with a t(12;22), the aberrant MN1-ETV6
protein is believed to have transforming capacity and the DNA-binding
domain is thought to be the functional contribution of
ETV6.6 In myeloproliferative disorders with a
t(3;12)(q26;p13), the chimeric transcript consists of the first two
exons of ETV6, which code for no known functional domains, fused to
MDS1/EVI1 sequences suggesting that the oncogenic potential of
this translocation could result from the ETV6 promoter driving
the transcription of MDS1/EVI1.2 Molecular analysis
of the t(6;12)(q23;p13) recently described by Bohlander et
al,7 in a B-cell acute lymphoblastic leukemia (ALL) cell
line identified a novel gene on chromosome 6 named STL. The
ETV6 breakpoint was localized in intron 2, upstream to the exon
encoding the HLH domain. However, no obvious new chimeric reading
frames were found and the hypothesis that the t(6;12)(q23;p13) does not
lead to a fusion protein with oncogenic potential but to the
elimination of normal ETV6 function was presented.
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| Fig 1.
Genomic breakpoints of the ETV6 gene. (A) Exon
structure of the ETV6 mRNA showing the breakpoints of known
translocations involving ETV6, (B) genomic structure of the
ETV6 gene showing the positions of the cosmid probes used for
FISH, and (C) genomic regions of the ETV6 breakpoints as
determined by FISH are indicated by the shaded areas.
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It is clear that ETV6 is a versatile element at the center of a
network of genes involved in hematologic malignancies through different
molecular mechanisms that are only partially understood. Here we report
six leukemia cases and one B-cell non-Hodgkin's lymphoma (NHL) case
with new chromosomal rearrangements involving ETV6. The
breakpoints of a t(5;12)(q31;p13), t(6;12;17)(p21;p13;q25), t(7;12)(p15;p13), t(7;12)(p12;p13), t(7;12)(q36;p13),
t(12;13)(p13;q12), and t(12;?)(p13;?) were characterized by
fluorescence in situ hybridization (FISH) using a panel of DNA probes
including an ordered set of cosmids covering the entire ETV6
gene.
| |
MATERIALS AND METHODS |
Patients.
Patient material was collected at the Center for Human Genetics in
Leuven, Belgium, during the last 3 years. Some clinical and hematologic
findings of the reported cases are summarized in
Table 1.
Cytogenetics.
One-day cultures of bone marrow cells were used for cytogenetic
analysis in all cases. Ten to 31 R- and G-banded karyotypes were
analyzed and classified according to the International System for Human
Cytogenetic Nomenclature.8
FISH.
FISH was performed as previously described.9 Chromosome 12p
abnormalities were studied with the cosmids 4H9A (D12S158, assigned to 12p13.3), 123C12 (CDKN1B), and 9 cosmids for
ETV6 ordered from 5 to 3 (179A6 - 15A4 - 67C6 - 50F4 - 132B11 - 242E1 - 163E7 - 54D5 - 148B6).10 The
position of the different ETV6 exons in these cosmids is shown
in Fig 1B. Additional FISH experiments were performed using chromosome
5, 6, 9, 12, 13, 17, 19, 20, 21, and 22 painting probes labeled with
bio-16-dUTP (Cambio, Cambridge, UK) or Spectrum Orange-dUTP (Vysis,
Stuttgart, Germany). Three yeast artificial chromosomes
(YACs) assigned to 5q31q33 (773B7, 939F12, and 888A7), and 10 YACs
assigned to the 7p15p12 region (764B12, 776A4, 207E1, 881B6, 669G6,
16BC5, 959B3, 772B11, 802D6, and 908B12) were selected from the
sequence-tagged site (STS)-based map reported by Green et
al,11 Keen et al, 12 and Chumakov et
al.13 The cosB (PDGFRB),14 YAC
Y6,15 and cosmid ICRFCIO2D12118 hybridize to, respectively,
5q33, 14q32, and 21q22.3. Chromosomes 7, 12, and 13/21 were identified
by cohybridization with Texas Red-5-dUTP labeled
centromere probes (Dupont, Boston, MA) for chromosome 7 (p7t1), 12 (pBR12/D12Z3), and 13/21 (pUC 1.76) in combination with G-banding using
DAPI counterstaining. Between 5 and 12 abnormal metaphases
were studied for each experiment. The FISH data were collected on a
Leitz DMRB fluorescence microscope (E. Leitz Inc,
Wetzler, Germany) equipped with a cooled black and white CCD camera run
by SmartCapture software (Vysis, Stuttgart, Germany).
| |
RESULTS |
Seven new chromosome 12p translocations affecting the ETV6 gene
were identified in patients with different malignant hemopathies including chronic myeloid leukemia (CML), acute myeloid leukemia (AML),
ALL, and B-NHL. Cytogenetic findings of all seven cases are presented
in Table 2. In cases 1, 3, 4, 5, and 7 chromosomal aberrations were identified at diagnosis, whereas in case 2 and 6 the abnormal karyotypes appeared during the course of disease. The 12p aberrations were found to be the sole abnormality in karyotypes of three patients; in one case a t(12)(p13) was associated with a
monosomy 13, whereas the karyotypes of the three remaining cases displayed complex multichromosomal changes. The potential involvement of ETV6 was evaluated by FISH with a panel of cosmid probes
covering the complete gene.10 FISH results are summarized
in Table 3.
A t(5;12)(q31;p13) was found in a patient with atypical CML associated
with marked eosinophilia. FISH with ETV6 cosmids showed that
179A6 (exon 1 of ETV6) hybridized to the der(5),
whereas cosmid 67C6 (intron 1), 50F4 (exon 2), and 148B6 (exon 8)
hybridized to the der(12) (Fig 2A). The
chromosome 5 breakpoint was analyzed with a PDGFRB (5q33) probe
(cosB) and three YACs (773B7, 939F12, 888A7) assigned to 5q31q33. All
these probes hybridized to the der(12) indicating that the breakpoint
occurred proximal to 773B7, the most centromeric probe examined
(Fig 3A).
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| Fig 2.
FISH analysis of ETV6 rearrangements. (A) Case 1 with a t(5;12)(q31;p13); (B) case 2 with a t(6;12;17)(p21;p13;q25), (C)
case 3 with a t(7;12)(p15;p13); (D) case 4 with a t(7;12)(p12;p13); (E)
case 5 with a t(7;12)(q36;p13); (F) case 6 with a t(12;13)(p13;q12); and (G and H) case 7 with a der(12)t(5;12;?)(p11;p11p13;?). (A through
F) arrowheads indicate derivatives of particular t(12)(p13); (G and H)
arrows indicate der(5) and der(14) chromosomes, respectively. All green
signals result from probes labeled with a bio-16-dUTP, red signals are
generated by probes labeled with Texas Red-5-dUTP, yellow signals
result from a mixture of bio-16-dUTP-labeled and Texas
Red-5-dUTP-labeled probes in ratio 2;1. The probes used for FISH
include (A) cosmid 67C6 (green) and a chromosome 12 centromeric probe
pBR12 (red); (A inset) cosmid 179A6 (green); (B) cosmid 15A4 (red),
library 6 (green) and pBR12 (yellow); (C) mixture of two cosmids 50F4
and 132B11 (green) and pBR12 (red); (D) cosmid 179A6 (green) and pBR12
(red); (E) cosmid 50F4 (green) and pBR12 (red); (F) cosmid 54D5 and
library 9 (green), library 13 (red) and a chromosome 12 centromeric
probe pBR12 (yellow); (G) cosmid 148B6 (green) and pBR12 (red); (G
inset) cosmid 163E7 (green) and library 12 (red); and (H) cosmid 179A6
(green) and pBR12 (red). Note the hybridization of 179A6 and 67C6 to a
der(5) and der(12), respectively, in case 1; hybridization of 15A4 with
a der(12) and der(17) in case 2; separation of 50F4 and 132B11 cosmids
on both derivative chromosomes in case 3; split signal from 179A6 and
50F4 in cases 4 and 5, respectively; split signal from 54D5 on der(12)
and der(13), absence of a second 54D5 signal on a del(12)(p12p13), and
presence of a chromosome 13 and 9 material on a der(12) in case 6;
appearance of only one hybridization signal from cosmids 148B6 (G) and
179A6 (H) on a der(5) and a der(14), respectively, and the presence of
additional material on a der(5)t(5;12;?)(p11;p11p13;?) upstream of the
ETV6-specific cosmid 163E7 (G inset) in case 7.
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| Fig 3.
Localization of probes from chromosome 5q or 7p used for
FISH detection of the breakpoints in case 1 with a t(5;12)(q31;p13) (A), case 3 with a t(7;12)(p15;p13) (B), and case 4 with a
t(7;12)(p12;p13) (C). Abbreviations: (A) Asterisk (*) indicates
hybridization with a der(12)t(5;12); (B) plus sign (+)
indicates hybridization with a der(7)t(7;12) and s
indicates split signal on der(7) and der(12); (C) ° indicates hybridization with a der(12)t(7;12), and asterisk (*) indicates hybridization with a der(7)t(7;12);  indicates position of
breakpoint.
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A second 12p13 translocation resulting in an ETV6 rearrangement
was found in a pediatric patient with AML and a previous history of
myelodysplastic syndrome (MDS). Cytogenetically the translocation was
described as a t(6;12)(p21;p13). FISH with the cosmid 148B6 showed a
hybridization signal on a der(12), whereas the cosmid 179A6 hybridized
to 17q25. This implied a three-way translocation t(6;12;17)(p21;p13;q25), which was confirmed by FISH using chromosome 6 and 17 painting probes. Further FISH analysis showed that cosmid 15A4
(intron 1) hybridized to both the der(12) and der(17) (Fig 2B), which
locates the breakpoint on chromosome 12 in the first intron of ETV6
(Fig 1C).
Two other translocations, both cytogenetically defined as a
t(7;12)(p14;p13), were found in pediatric patients with
AML-M0 or ALL-L2. FISH analysis showed that in the AML
case the translocation breakpoint was flanked by 50F4 and 132B11 (Fig
2C), and thus occurred near exon 2 of ETV6 (Fig 1C). In the
second case, cosmid 179A6 (exon 1) spanned the breakpoint (Fig 2D), and
three cosmids 3 to 179A6 hybridized with the der(12). To
determine the breakpoint on the translocation partner, FISH analysis
with chromosome 7p YACs was performed (Fig 3B and C and
Fig 4). All three YACs (959B3, 16BC5, and
669G6) from a contig covering the Retinitis Pigmentosa 9 locus at
7p1512 hybridized to the der(7) in the AML case or the
der(12) in the ALL case, indicating a different 7p breakpoint for them.
FISH analysis was then performed with YACs centromeric (881B6, 207E1, 776A4, and 764B12) or telomeric (772B11, 802D6, and 908B12) to RP9.11,13 The breakpoint of the AML t(7;12) was
found to be spanned by two overlapping probes, 802D6 (D7S516, D7S1808,
and D7S2416/1790 kb) and 908B12 (D7S1808, D7S2416, and D7S2564/1300 kb). A third overlapping YAC, 772B11 (D7S516 and D7S1808/1500 kb),
hybridized only to the der(7) chromosome. These results suggested the
localization of the breakpoint near D7S2416 at 7p15. The breakpoint of
the ALL t(7;12) was narrowed down to the area flanked by the YACs 776A4
and 764B12 (7p12).
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| Fig 4.
Scheme of the loci on 7p (ordered from telomere to
centromere). The YACs used for this study, their STS content, and their breakpoint regions of two t(7;12) are shown.
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In a karyotype of the other pediatric patient with AML, complex
chromosomal abnormalities, including add(7)(q36) and del(12)(p12p13), were observed. FISH with 179A6 (exon 1) and 148B6 (exon 8) showed hybridization signals on add(7) and del(12) chromosomes, respectively, and implied a t(7;12)(q36;p12). The ETV6 breakpoint of this
translocation was localized in a region covered by 50F4 that was split
between both derivative chromosomes (Fig 2E). Because exon 2 is located at the 3 end of 50F4, the breakpoint could be mapped to intron 1 of ETV6 (Fig 1C).
The sixth ETV6 translocation identified by FISH as a
t(12;13)(p13;q12) was masked by complex chromosomal rearrangements not detected during cytogenetic analysis. FISH with ETV6 cosmids
179A6 (exon 1) and 148B6 (exon 8) yielded hybridization signals on a small, unidentifiable chromosome and the der(12), respectively, whereas
cosmid 54D5 (exons 5, 6, 7, and 8) spanned the breakpoint. All cosmids
5 to 54D5 hybridized with the same small marker chromosome. However, none of them, nor a cosmid for CDKN1B (123C12), showed hybridization signals on the second chromosome 12, which was
cytogenetically defined as a del(12)(p12p13). FISH analysis with the
12p13.3 cosmid 4H9A (D12S158) yielded hybridization signals on
this del(12)(p) chromosome, confirming that the deletion is
interstitial. Hybridization of the ETV6 probes to the small
marker suggested a cryptic t(12;21)(p13;q22), masked by the
cytogenetically recognized t(9;12)(q13;p13), but FISH with a chromosome
21 paint and a 21qter cosmid (ICRFC102D12118) showed no rearrangement
of chromosome 21. The use of painting probes for chromosome 9, 12, 13, and 20 allowed the identification of this small chromosome as a der(13)
from a reciprocal t(12;13)(p13;q12) masked by an additional and
probably secondary t(9;13;20)(p13;q14;p12) involving the
der(12)t(12;13)(p13;q12) (Fig 2F).
The karyotype of a seventh patient with a B-NHL showed rearrangements
of both 12p chromosomes, cytogenetically defined as a del(12)(p11) and
dic(12;17)(p11;p11). FISH analysis with cosmid 179A6 (exon 1) yielded
surprisingly only a single hybridization signal on the terminal end of
the add(14)(q32) suggesting a t(12;14)(p11;q32). This reciprocal
translocation was confirmed by FISH with a cosmid 4H9A
(D12S158) showing a signal on the add(14)(q32) and with a 14q32
YAC (Y6), which hybridized to a chromosome defined cytogenetically as a
del(12p). Only one Y6 signal was observed in all abnormal cells
analyzed, indicating a cryptic del(14)(q32). Cosmid 148B6 (exon 8) did
not hybridize with the add(14)(q32), showing an intragenic deletion of
part of ETV6, which was then shown to extend as far as
CDKN1B (123C12) (Table 3). However, cosmids 148B6 and 123C12 yielded hybridization signals with a chromosome band 5p suggesting a
5;12 translocation. As cosmid 179A6 did not hybridize with this der(5),
the second ETV6 allele also appeared to be inactivated by an
intragenic deletion. Moreover, the 12p13.3 cosmid 4H9A
(D12S158) was found to be translocated to another chromosome
identified as a der(17)t(12;17)(p13;q11) using the chromosome 17 painting probe. Chromosome 17 material was also detected on a
dic(12;17)(p11;p11), on the add(1)(p34) and a partially painted
chromosome 17 suggesting a t(1;17)(p34;q24), and on a long arm of one
of five marker chromosomes. All these data indicated the involvement of
three copies of chromosome 17 in different chromosomal aberrations.
Translocation of the telomeric 12p13.3 sequences (4H9A) to 17q11,
deletion of the 5 end of ETV6, and localization of
hybridization signals from 163E7 and 148B6 cosmids on a distal but not
terminal end of a der(5) chromosome (Fig 2G, inset) suggested a masked
translocation of unknown material to the retained 3 region of
ETV6 on a der(5). Despite using chromosome 5, 12, 19, 20, and
22 libraries, the origin of this material could not be identified by
FISH because of its small size, complex aberrations, and insufficient
material resources. Summarizing the FISH and G-banding results, the
chromosome 12 abnormalities found in this patient can be described as
follows: a der(5)t(5;12;?)(p11;p11p13;?),
der(12)del(12)(p13p13)t(12;14)(p11;q32), dic(12;17)(p11;p11)
del(12)(p13p13), der(14)t(12;14)(p11;q32), and
der(17)t(12;17)(p13;q11).
The breakpoint of a previously reported MDS case with a
t(10;12)(q24;p13)16 was also further refined using the
ETV6 cosmids. The ETV6 breakpoint occurred in intron 2 and was flanked by cosmids 50F4 and 132B11 (Fig 1C).
| |
DISCUSSION |
Seven new chromosomal aberrations involving the ETV6 gene were
detected in patients with CML, AML, ALL, and B-NHL and were further
characterized by FISH. Although cytogenetic analysis already indicated
12p abnormalities in all these cases, FISH studies for most of them
showed new and unexpected chromosomal rearrangements.
The (5;12)(q31;p13) translocation found in atypical CML showed a
breakpoint gene in the first intron of ETV6 and a chromosome 5 breakpoint centromeric to 773B7, mapped proximally to PDGFRB. These findings clearly indicate that this translocation is
different from the t(5;12)(q33;p13) resulting in the
ETV6-PDGFRB fusion cloned by Golub et
al.1 Insufficient material did not allow to
identify the target gene at 5q31 by FISH; however, the breakpoint is
localized in a region where many growth factors and hormone receptor
genes have been mapped including members of the interleukin (IL) gene
family (IL3, IL4, IL5, IL9, IL12B, IL13), interferon regulatory
factor-1 gene (IRF1), early growth response-1 gene (EGR1), colony stimulating factor-2 gene (CSF2), CD14
antigen and cell division cycle 25C gene (CDC25C). It is interesting to note that the t(5;12)(q31;p13) presented here, as well as the translocation 5;12 resulting in the ETV6-PDGFRB fusion protein, occurred in patients with marked eosinophilia.
In the second case a three-way t(6;12;17)(p21;p13;q25) disrupting the
ETV6 gene in intron 1 was identified. The 5 end of ETV6, including exon 1, was translocated to 17q25, whereas the remaining part of the gene with the HLH and ETS DNA-binding domains was
retained on the der(12) and became juxtaposed to unknown 6p21 sequences. Among the candidate genes on 6p21 are PBX2
(pre-B-cell leukemia transcription factor 2), TNFA (tumor
necrosis factor- ), PIM1, CDKN1A/WAF1
(cyclin-dependent kinase inhibitor 1A), CBFA1/AML3 ( subunit
of a core binding factor), and CCND3 (cyclin D3).
Two apparently similar translocations, both cytogenetically defined as
t(7;12)(p14;p13), appeared to be molecularly different after FISH
analysis. Although both translocations have a breakpoint in the
5 end of ETV6, they affected different regions on
chromosome 7p (see Results). The breakpoint of the AML t(7;12) was near
the D7S2416 locus at 7p15. The breakpoint is contained in the
Centre d'études du Polymorphisme Humain YACs 802D6
and 908B2. The HOX-A gene cluster was mapped to 7p15 and can be
considered candidate genes. However, polymerase chain reaction (PCR)
analysis with expressed sequence tours developed for
HOXA1 and HOXA4 did not detect the presence of these
genes on either YAC excluding these candidates (results not shown). Of
interest in a perspective of our finding is another published case of a
t(7;12)(p15;p13) found in a 4-year-old boy with a minimally
differentiated AML (French-American-British classification
AML-M0).17 This could be a relevant association because the
AML-M0 is poorly characterized at the cytogenetic level. The largest
published series of AML-M0 comprises 21 adult patients with clonal
abnormalities,18 and although 12p chromosomal aberrations
were found in three patients, no t(7;12) was present among these cases.
Moreover, involvement of this region in myeloid leukemia with a typical
t(7;11)(p15;p15) or with a del(7p) has been already
reported.19,20
Another 7;12 translocation involving the long arm of chromosome 7 (q36)
and ETV6 was found in a 1-year-old boy with AML. The ETV6
breakpoint of this translocation was localized in intron 1 of ETV6 upstream to exons encoding the HLH domain. The partner gene at 7q36 remains unknown, among the candidate genes is a
cyclin-dependent kinase 5 (CDK5) involved in a regulation of
cell cycle.
FISH analysis of the sixth case diagnosed as ALL-L2 showed disruption
of ETV6 by a cryptic t(12;13)(p13;q12) masked by additional multichromosomal changes not discernible by cytogenetic analysis. The
ETV6 breakpoint of t(12;13) is covered by cosmid 54D5,
containing exons 5 to 8 of ETV6, and the translocation is
associated with loss of the second ETV6 allele as a consequence
of del(12)(p12p13). The 13q12 region involved in the t(12;13) is known
to carry the FGF9 (fibroblast growth factor 9), FLT1
and FLT3 (fms-related tyrosine kinase 1 and 3), IPF1
(insulin promoter factor 1), and BRCA2 (breast cancer 2)
genes. Some other ALL cases with an analogous t(12;13) were previously
reported.21-23 Although in some cases the breakpoints were
interpreted differently, there is a possibility that all of them
involve ETV6 and the same unknown sequences at 13q12.
The most complex karyotypic changes affecting both 12p chromosomes and
ETV6 were observed in a B-NHL case. FISH analysis showed that
one chromosome 12 was rearranged by a t(12;14)(p11;q32) with a
breakpoint typical for B-NHL lymphoma at 14q32 corresponding to the Ig
heavy chain gene locus. The second chromosome 12p was involved in three
different translocations, a t(5;12;?)(p11;p11p13;?), a
t(12;17)(p13;q11), and a dic(12;17)(p11;p11). The 12p13 breakpoint in
the t(5;12;?) that might have resulted in an ETV6 fusion
transcript was localized in the 5 end of the gene upstream to
cosmid 163E7 and upstream to the HLH coding exons. The partner
chromosome involved in this translocation could not be identified. FISH
analysis resulted in the detection of two different cryptic
microdeletions of 12p13 affecting both ETV6 alleles. One of
them, independent from a t(12;14)(p11;q32), covered the 3 end of
ETV6 (163E7 and 148B6) and CDKN1B (123C12), whereas the
second intragenic microdeletion was associated with a
t(5;12;?)(p11;p11p13;?) and involved the 5 end of ETV6
(179A6). This latter finding indicated that the putative reciprocal
chimeric transcript of a t(5;12;?)(p11;p11p13;?) containing the
5 end ETV6 domain was absent in the malignant cells and did not
play a significant role in the development and/or progression
of this lymphoma. It is noteworthy that deletion of the second
ETV6 allele is a commonly observed secondary phenomenon in
pre-B-ALL cases with a t(12;21)(p13;q22).24,25 In two
cases the deletion was shown to be intragenic in
ETV6,25,26 suggesting that the gene was probably
the actual target of the deletion. It was already hypothesized that
wild type ETV6 might reduce the oncogenic and growth-stimulating properties of the fusion proteins and that, therefore, its loss would provide an additional proliferative advantage
to the malignant cells. The characterization of two new cases with
biallelic ETV6 rearrangements [together with at least one more
leukemia case characterized by a
t(9;12;14)(q34;p13;q22)/ETV6-ABL and
del(12)(p11;p13)27; B-cell ALL cell line with a
t(6;12)(q23;p13)/ETV6-STL and a microdeletion of the other
ETV6 allele7; and a myeloid leukemia case with two
translocations involving ETV6, namely, t(3;12)(q26,p13) and
t(9;12)(p24;q15;p13)2] indicates that biallelic
aberrations of ETV6 are not exclusively associated with a
subtype of ALL with a t(12;21).
In summary, seven new chromosomal abnormalities affecting ETV6
have been identified in patients with atypical CML
associated with eosinophilia in MDS, in young patients with acute
leukemias and, for the first time, in a case of B-NHL. In two leukemia
cases the ETV6 aberrations appeared during the course of the
disease suggesting that the disruption of this gene can not only
initiate the development but also be involved in the progression of the malignant disorder. The latter can be supported by the karyotypic findings in a case of B-NHL where the ETV6 affected
translocation was coexisting with a typical 14q32/IgH translocation
regarded as a primary rearrangement in lymphomagenesis. The frequency
of these new ETV6 translocations in hemopathies is difficult to
evaluate because of multichromosomal aberrations impeding cytogenetic
detection.
FISH analysis of seven ETV6 rearrangements showed that in six
of them the breakpoint occurred in the 5 end of ETV6
upstream of exons coding for the HLH domain. Together with the
previously reported t(10;12)(q24;p13),16 they might
generate chimeric proteins with features of transcriptional activators
analogous to the t(12;22)(p13;q11) fusion protein, but it is also
conceivable that the oncogenic properties of some of these
translocations result from the ETV6 promoter driving the
partner gene as suggested for a t(3;12)(q26;p13) or from the disruption
of as yet unidentified domain(s) encoded by the first two exons. In the
remaining case with a t(12;13)(p13;q12), the breakpoint was localized
in the 3 end of ETV6, probably upstream to exons coding
for its ETS DNA-binding domain as was previously reported for the
t(5;12)(q33;p13), t(9;12)(q34;p13), and t(12;21)(p13;q22), which might
lead to the expression of chimeric transcripts containing either the
HLH domain or the ETS domain of ETV6. On the other hand,
regarding that ETV6 is a candidate tumor suppressor gene, it is
possible that some of the ETV6-related translocations,
especially those associated with a complete or partial deletion of the
second ETV6 allele, might inactivate the ETV6 gene as
hypothesized for a t(6;12)(q23;p13).
The data presented here support the hypothesis of the multiple
contributions of ETV6 in the pathogenesis of hematologic
disorders and show the occurrence of ETV6 aberrations in
hemopathies as diverse as CML, AML, ALL, and B-NHL. The cryptic
deletions of the nontranslocated ETV6 allele found in two cases
further emphasize the significance of the inactivation of the wild type
ETV6 protein for the development and/or the progression
of some hematologic malignant disorders.
| |
FOOTNOTES |
Submitted July 17, 1997;
accepted October 15, 1997.
This report presents research results of the Belgian programme on
Interuniversity Poles of Attraction initiated by the Belgian State,
Prime Minister's Office, Science Policy Programming. The scientific
responsibility is assumed by its authors.
P.M. is an `Onderzoeksdirecteur' and M.B. is a `Postdoctoraal
Onderzoeker' of the F.W.O-Vlaanderen.
Supported in part by Grant No. G.0153.96 of the F.W.O.-Vlaanderen.
Address reprint requests to Peter Marynen, PhD, Center for
Human Genetics, University of Leuven, Campus Gasthuisberg O&N6, Herestraat 49, B-3000 Leuven, Belgium. The publication costs of this article were defrayed in part by page charge payment. This article
must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. section 1734 solely to indicate this fact.
| |
ACKNOWLEDGMENT |
The authors thank Dr C. Inglehearn (Institute of Ophthalmology,
University of London, London, UK) and Dr F. Matsuda (Center for
Molecular Biology and Genetics, Kyoto University, Sakyo-ku, Kyoto,
Japan) for providing us with YACs specific for RP9 and 14q32,
respectively. We are grateful to Magda Dehaen and the technicians of
the leukemia laboratory of the Center for Human Genetics for their
assistance.
| |
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Abstract
Introduction
Abstract