A New Recurrent IGH@ Translocation Involving ID4 in B-cell Precursor Acute Lymphoblastic Leukemia...

8/11/2019 A New Recurrent IGH@ Translocation Involving ID4 in B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL)

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doi:10.1182/blood-2007-07-092015Prepublished online October 16, 2007;2008 111: 387-391

Jonathan C. Strefford, Reiner Siebert, Martin J. S. Dyer and Christine J. HarrisonHarder, 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, LanaB-cell precursor acute lymphoblastic leukemia (BCP-ALL)

inID4translocation involvingIGH@t(6;14)(p22;q32): a new recurrent

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Results and discussion

Patient details

Thirteen BCP-ALL patients with t(6;14)(p22;q32) were identi-

fied (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, 3 109/L; range, 1-11 109/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 ofIGH@was confirmed 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 confirmed 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 identification 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 IGHJsegments, while those in 6p22 were

located centromeric (3) ofID4 (Figure 1A,D).Quantitative real-time PCR confirmed 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

clonedby LDI-PCRfrom IGHJ6segments.(B) PartialG-bandedkaryotypeshowing normaland derived copiesof chromosomes 6 and14. Thenormal copiesof each chromosome areon

the left and the rearrangedcopies are denoted by arrowsshowing the breakpoints.(C) Metaphase chromosomeshybridizedwith clonesRP11-377N20 (Spectrum green)and RP3-498I24

(Spectrum red). Two red/greenfusionsignals areshown,one locatedon thenormal chromosome 6 (red arrow)and theother on thederived chromosome 6 (yellowarrow).A redsignalis

seen onthe derived chromosome14 (greenarrow) indicatingthat thebreakpoint is locatedwithinRP3-498I24closeto ID4. Imagewas acquired by stainingwith DAPI (Vector Laboratories,

Burlingame, CA), with a Zeiss Axioskop microscope (Zeiss, Welwyn Garden City, United Kingdom) fitted 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 areshownin boldface letters. Patient2817 hadan inverted fragment froms IGHJ2-3priorto IGHJ4 segment.

t(6;14)(p22;q32) INVOLVINGID4IN BCP-ALL 389BLOOD, 1 JANUARY 2008 VOLUME 111, NUMBER 1

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cases 2125 and 12284. A monoallelic deletion of PAX5 wasindicated 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 ofCDKN2A.

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

development14

and to induce apoptos is in a var iety 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 CEBPproteins, 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 haploinsufficiency); 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 first report of a recurrent, novel subgroup ofIGH@

positive BCP-ALL with t(6;14)(p22;q32), resulting in deregulated

expression ofID4 in cooperation with loss ofCDKN2Aand PAX5.

These findings 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

identified 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.

Conflict-of-interest disclosure: The authors declare no compet-

ing financial 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: [email protected].

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t(6;14)(p22;q32) INVOLVINGID4IN BCP-ALL 391BLOOD, 1 JANUARY 2008 VOLUME 111, NUMBER 1


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