doi:10.

1182/blood-2007-07-092015
Prepublished online October 16, 2007;
2008 111: 387-391

Jonathan C. Strefford, Reiner Siebert, Martin J. S. Dyer and Christine J. Harrison

Harder, Alexander Claviez, Stefan Gesk, Anthony V. Moorman, Fiona Ross, Helen Mazzullo,
Lisa J. Russell, Takashi Akasaka, Aneela Majid, Kei-ji Sugimoto, E. Loraine Karran, Inga Nagel, Lana

B-cell precursor acute lymphoblastic leukemia (BCP-ALL)

in ID4 translocation involving IGH@ t(6;14)(p22;q32): a new recurrent
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Blood (print ISSN 0006-4971, online ISSN 1528-0020), is published weekly

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NEOPLASIA
Brief report
t(6;14)(p22;q32): a newrecurrent IGH@translocation involving ID4 in B-cell
precursor acute lymphoblastic leukemia (BCP-ALL)
Lisa J. Russell,
1
Takashi Akasaka,
2
Aneela Majid,
2
Kei-ji Sugimoto,
2
E. Loraine Karran,
2
Inga Nagel,
3
Lana Harder,
3
Alexander Claviez,
4
Stefan Gesk,
3
Anthony V. Moorman,
1
Fiona Ross,
5
Helen Mazzullo,
6
Jonathan C. Strefford,
1
Reiner Siebert,
3
Martin J. S. Dyer,
2
and Christine J. Harrison
1
1
Leukaemia Research Cytogenetics Group, Cancer Sciences Division, University of Southampton, Southampton General Hospital, Southampton, United
Kingdom;
2
Medical Research Council (MRC) Toxicology Unit, University of Leicester, Leicester, United Kingdom;
3
Institute of Human Genetics and
4
Department
of Pediatrics, University-Hospital Schleswig-Holstein, Campus Kiel, Kiel, Germany;
5
Wessex Regional Genetics Laboratory, Salisbury District Hospital,
Salisbury, United Kingdom; and
6
Department of Haematology and Blood Transfusion, University College Hospital, London, United Kingdom
Translocations involving the immuno-
globulin heavy chain locus (IGH@) at
chromosome band 14q32 are common in
mature B-cell neoplasms, but are rare in
B-cell precursor acute lymphoblastic leu-
kemia (BCP-ALL). Here, we report the
translocation, t(6;14)(p22;q32), involving
IGH@ as a novel recurrent translocation
in 13 BCP-ALL patients. Fluorescence in
situ hybridization and long-distance in-
verse polymerase chain reaction (PCR)
identied ID4 as the partner gene. Break-
points were scattered over a 19kb region
centromeric of ID4. Quantitative real-time
PCR showed up-regulation of ID4 mRNA.
All patients had deletions of CDKN2A
and PAX5 located on the short arm of
chromosome 9, frequently as a result of
an isochromosome, i(9)(q10) (9/13, 69%).
This study denes a new subgroup of
BCP-ALL characterized by ID4 over-
expression and CDKN2A and PAX5 dele-
tions. Preliminary survival data suggest
that this subgroup may be associated
with a good response to therapy. (Blood.
2008;111:387-391)
2008 by The American Society of Hematology
Introduction
Chromosomal translocations lead to oncogene activation in hema-
tologic malignancies, where they play an important role both at
diagnosis and as an indicator of prognosis. Rearrangements
involving the immunoglobulin heavy chain locus (IGH@) at
chromosome band 14q32 are frequently observed in mature B-cell
neoplasms,
1,2
although a number are now emerging in B-cell
precursor acute lymphoblastic leukemia (BCP-ALL).
3-10
The translocation, t(6;14)(p22;q32), has been reported in
2 independent cases of BCP-ALL.
7,8
It was shown that as a
consequence of juxtaposing to the IGH@ enhancer, the partner
gene, ID4, was overexpressed.
7
We report here a further 13 cases,
indicating the recurrent nature of this translocation in BCP-ALL.
Moreover, we note an association with other consistent genetic
features, including deletions of CDKN2A and PAX5.
Methods
Patient samples
Samples were received from patients with the translocation, t(6;14)(p22;
q32), entered in the UK childhood (UKALLXI, ALL97, or ALL2003) or
adult (UKALLXII) ALL treatment trials or the German ALL-BFM 2000
trial (Table 1), after informed consent was obtained in accordance with the
Declaration of Helsinki. Institutional Review Board approval was provided
by each participating institution (University of Southampton, University of
Leicester, and University-Hospital Schleswig-Holstein).
Cytogenetics and uorescence in situ hybridization
Cytogenetics and uorescence in situ hybridization (FISH) were
performed on the same diagnostic samples. The involvement of
IGH@ was determined by interphase FISH, using the commercially
available IGH@ break-apart probe (Abbott Diagnostics, Abbott Park,
IL). A home-grown dual-color, break-apart probe (consisting of
BACs: RP11-377N20 and RP3-498I24; all clones from Sanger Institute,
Hinxton, United Kingdom) and a dual-color, dual-fusion probe
(IGH@-ID4; Figure 1A) were designed to detect the presence of the
translocation in metaphase and interphase, respectively. A commercially
available probe to CDKN2A (Abbott Diagnostics) and a home-grown
probe to PAX5 (BACs: RP11-344B23 and RP11-297B17) were used to
evaluate deletions of 9p. FISH mapping was carried out as reported
previously.
9
Long-distance inverse polymerase chain reaction
Long-distance inverse polymerase chain reaction (LDI-PCR) from IGHJ
was carried out as previously described.
9
Quantitative real-time PCR
Total cellular RNA, extracted from the same diagnostic samples, was
available from 2 patients: 6120 and 16503. Quantitative analysis was
performed using Applied Biosytems (Foster City, CA) MGB probes, as
previously described.
9
Submitted July 2, 2007; accepted September 19, 2007. Prepublished online as
Blood First Edition paper, October 16, 2007; DOI 10.1182/blood-2007-
07-092015.
The publication costs of this article were defrayed in part by page charge
payment. Therefore, and solely to indicate this fact, this article is hereby
marked advertisement in accordance with 18 USC section 1734.
2008 by The American Society of Hematology
387 BLOOD, 1 JANUARY 2008

VOLUME 111, NUMBER 1
For personal use only. by guest on November 17, 2012. bloodjournal.hematologylibrary.org From
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388 RUSSELL et al BLOOD, 1 JANUARY 2008

VOLUME 111, NUMBER 1
For personal use only. by guest on November 17, 2012. bloodjournal.hematologylibrary.org From
Results and discussion
Patient details
Thirteen BCP-ALL patients with t(6;14)(p22;q32) were identi-
ed (Figure 1B), indicating the recurrent nature of this transloca-
tion. Clinical and demographic data are shown in Table 1. All
patients had a common/pre-B ALL immunophenotype with
positive expression of CD10, CD19, HLA-DR and TdT where
data were available. No patient showed positivity for surface
IgM, T-cell, or myeloid markers. White blood cell counts were
low (median, 3 10
9
/L; range, 1-11 10
9
/L) and patients were
older than expected for BCP-ALL (median 16 years, range
6-48). All patients for whom outcome data were available
(n 10) achieved a complete remission (CR). Two patients died
in CR, while 8 remain in CR; 5 for more than 5 years. Although
the number of patients is small, their outcome appears to be
good, especially among this age group.
Molecular characterization of 6p22 breakpoints using FISH,
LDI-PCR, and quantitative real-time PCR
The involvement of IGH@ was conrmed by a positive interphase
FISH result in 10 patients with available material (Table 1). The 5
IGH@signal relocated to 6p in metaphases.
Because a previous report had indicated involvement of ID4,
7
the dual-color, break-apart FISH probe to ID4 was applied to
metaphases (Figure 1C). This conrmed that the breakpoint was
located close to ID4. The novel dual-color, dual-fusion probe
(Figure 1A) showed a positive signal pattern in all 10 IGH@
positive cases (Table 1), indicating the reliability of this probe for
the identication of this subtle translocation in interphase.
Breakpoint cloning by LDI-PCR was successful in the 3 cases
with available high-molecular weight DNA. The breakpoints of
IGH@ were within the IGHJ segments, while those in 6p22 were
located centromeric (3) of ID4 (Figure 1A,D).
Quantitative real-time PCR conrmed over-expression of ID4
(Assay ID, Hs_00155465, Applied Biosystems) mRNA in
2 patients with t(6;14)(p22;q32) (Table 2).
Associated cytogenetic changes
An abnormality of the short arm of chromosome 9 (9p) was visible
by conventional cytogenetics in 12 patients. Interestingly, this
manifested as an isochromosome [i(9)(q10)], a rare mechanism of
9p loss, in 9 patients, also reported in one previously published
case.
8
FISH showed either a monoallelic (n 6) or biallelic
(n 4) deletion of CDKN2A in all 10 patients tested (Table 1),
including the single case (7294) without a cytogenetically visible
deletion of 9p, and an additional submicroscopic 9p deletion in
Figure 1. Involvement of ID4 in the translocation, t(6;14)(p22;q32). (A) Diagram showing the location of clones used in the dual-color, dual-fusion FISH probe and breakpoints
cloned by LDI-PCRfromIGHJ6 segments. (B) Partial G-banded karyotype showing normal and derived copies of chromosomes 6 and 14. The normal copies of each chromosome are on
theleft andtherearrangedcopies aredenotedby arrows showingthebreakpoints. (C) Metaphasechromosomes hybridizedwithclones RP11-377N20(Spectrumgreen) andRP3-498I24
(Spectrumred). Two red/green fusion signals are shown, one located on the normal chromosome 6 (red arrow) and the other on the derived chromosome 6 (yellowarrow). Ared signal is
seenonthederivedchromosome14(greenarrow) indicatingthat thebreakpoint is locatedwithinRP3-498I24closetoID4. Imagewas acquiredby stainingwithDAPI (Vector Laboratories,
Burlingame, CA), with a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) tted with a 100 oil objective, a CCD camera (Applied Imaging, Newcastle, United
Kingdom), and MacProbe image acquisition version 4.3 software (Applied Imaging). (D) Nucleotide sequences of the ID4-IGHJ junctions. Vertical lines show nucleotide identity. IGHJ
segments are shown in boldface letters. Patient 2817 had an inverted fragment froms IGHJ2-3 prior to IGHJ4 segment.
t(6;14)(p22;q32) INVOLVINGID4 IN BCP-ALL 389 BLOOD, 1 JANUARY 2008

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cases 2125 and 12284. A monoallelic deletion of PAX5 was
indicated in all cases, either from the presence of an i(9)(q10)
(n 9) or by FISH (n 4) (Table 1). 2 of the 4 patients tested by
FISH for PAX5 deletions showed biallelic deletions of CDKN2A.
PAX5 mRNA (Assay ID, Hs_01045950, Applied Biosystems)
expression was found to be decreased in patients 6120 and 16503
(Table 2).
ID4 is one of 4 members of the basic helix-loop-helix
(bHLH) family of transcription factors, which act as transcrip-
tion inhibitory proteins.
12
They play a role in regulation of
numerous cellular processes, including growth, differentiation,
senescence and apoptosis.
13
ID proteins have recently
been shown to play a unique role in hematopoeitic cell
development
14
and to induce apoptosis in a variety of
cell types,
15,16
including B-lymphocytes.
17
Consistent with a
tumor-suppressor role, down-regulation of ID4 has been re-
ported in both mouse and human leukemias at a high frequency,
arising from aberrant methylation within the promoter region of
the gene.
18
In contrast, ID gene family members have been
reported to show overexpression in a range of cancer types,
19-23
as well as one case of BCP-ALL with t(6;14)(p22;q32).
7
Collectively, these observations indicate that the effects of
ID4 appear to be context dependent; leading to either increased
proliferation or increased cell death according to which cell
lineage is involved. It could be that ID4 expression differentially
affects lymphoid cells compared with myeloid cells. In this
regard, ID4 appears to act in a similar manner to the CEBP
proteins, whose expression in most instances results in growth
suppression but, as we have previously postulated, aberrant
expression of CEBP proteins may lead to transformation in some
B-cell precursors.
9,10
Although CDKN2A and PAX5 deletions have been well
documented in all subgroups of ALL,
24-26
their consistent loss in
association with t(6;14)(p22;q32) indicates an interaction be-
tween these pathways and a pivotal role for one or both genes. A
recent extensive study showed a decrease in PAX5 mRNA
expression in 31.7% of childhood BCP-ALL.
26
They reported
diminished PAX5 expression or loss of PAX5 functions occur-
ring via a number of different mechanisms including: deletion
(with retention of an apparently normal second allele, suggest-
ing haploinsufciency); translocation, resulting in PAX5 fusion
genes; epigenetic silencing; or, more rarely, mutation. The
mutation status of the second PAX5 allele could not be
determined in this study due to lack of material.
This is the rst report of a recurrent, novel subgroup of IGH@
positive BCP-ALL with t(6;14)(p22;q32), resulting in deregulated
expression of ID4 in cooperation with loss of CDKN2A and PAX5.
These ndings implicate ID4 as a dominant oncogene in these
patients.
Acknowledgments
The authors would like to thank all members of the UK Cancer
Cytogenetics Group for providing cytogenetic data and the
Clinical Trial Service Unit (CTSU, University of Oxford, United
Kingdom) for providing clinical data. This study could not have
been performed without the dedication of the United Kingdom
Childrens Cancer and Leukemia Group and Adult Leukemia
Working Party and their members, who designed and coordi-
nated the clinical trials through which these patients were
identied and in which they were treated.
This work was supported by Leukaemia Research (London,
United Kingdom), Medical Research Council (London, United
Kingdom), Deutsche Krebshilfe (Bonn, Germany), and Kider-Krebs-
Initiative Buchholx (Holm-Seppensen, Germany).
Authorship
Contribution: L.J.R., C.J.H., M.J.S.D., and R.S. designed the
research and wrote the paper. L.J.R., L.H., I.N., S.G., T.A., A.M.,
K.S., and E.L.K. carried out the experiments and analyzed the data.
F.R., H.M., and A.C. provided clinical material. A.V.M provided
clinical data. J.C.S played a technical supervisory role. All authors
critically reviewed the manuscript.
Conict-of-interest disclosure: The authors declare no compet-
ing nancial interests.
Correspondence: Professor Christine J. Harrison, PhD, FRC-
Path, Leukaemia Research Cytogenetics Group, Cancer Sciences
Division, University of Southampton, MP822, Duthie Building,
Southampton General Hospital, Tremona Road, Southampton,
SO16 6YD, United Kingdom; e-mail: Harrison@soton.ac.uk.
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Fold change of
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t(6;14)(p22;q32) INVOLVINGID4 IN BCP-ALL 391 BLOOD, 1 JANUARY 2008

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