Identification of two distinct MYC breakpoint clusters and their ...

Leukemia (2007) 21, 1739–1751 & 2007 Nature Publishing Group All rights reserved 0887-6924/07 $30.00 www.nature.com/leu

ORIGINAL ARTICLE Identification of two distinct MYC breakpoint clusters and their association with various IGH breakpoint regions in the t(8;14) translocations in sporadic Burkitt-lymphoma K Busch1, T Keller2, U Fuchs3, R-F Yeh4, J Harbott1, I Klose1, J Wiemels4, A Novosel5, A Reiter1 and A Borkhardt5 1 Department of Pediatric Hematology and Oncology, Justus-Liebig-University, Giessen, Germany; 2ACOMED Statistik, Statistical Analyses, Leipzig, Germany; 3Pediatric Hematology and Oncology, Dr V Haunersches Kinderspital, Ludwig-MaximiliansUniversity Munich, Munich, Germany; 4Department of Epidemiology and Biostatistics, Center for Bioinformatics and Molecular Biostatistics, University of California, San Fransisco, CA, USA and 5Oncology and Clinical Immunology, Clinic of Pediatric Hematology, Heinrich-Heine-University, Dusseldorf, Germany

The chromosomal translocation t(8;14) is the hallmark of Burkitt’s-lymphoma (BL) and fuses the proto-oncogene c-MYC to the IGH locus. We analyzed the genomic structure of MYC/ IGH fusions derived from a large series of 78 patients with t(8;14) and asked (i) whether distinct breakpoint clusters exist within the MYC gene and (ii) whether any pairwise association between particular IGH and MYC breakpoints exist. Identification of such associations will help elucidate the etiology of the breaks on the MYC locus. Scan statistic analyses revealed two distinct, but large clusters within c-MYC containing 60/78 (77%) of the breakpoints. Clusters 1 and 2 were 560 and 779 bp in length within a 4555 bp breakpoint cluster region. Breaks within IGH switch l and joining region did not differ with respect to their corresponding MYC breakpoints. However, there was a highly significant correlation between breakpoints 50 of MYC cluster 1 and fusions to IGH switch c region and breakpoints downstream of MYC cluster 2 and fusions to IGH switch a region (v2-test: Po0.005). Chromatin changes governing choice of IGH-Fc region recombination may parallel changes in the MYC gene 50 region chromatin leading to some degree of coordinated ontological specificity in breakpoint location. Leukemia (2007) 21, 1739–1751; doi:10.1038/sj.leu.2404753; published online 31 May 2007 Keywords: Burkitt’s lymphoma; MYC/IgH translocation; statistical analyses; V(D)J recombination; class switch recombination

Introduction B-cell development takes place in distinct stages as directed in part via signals transmitted through, and structural modifications of, the B-cell receptor when directed by an immune response. Early stages of B-cell development occur in the bone marrow including the recombination of the immunoglobulin (IG) genes via the recombination-activating gene (RAG 1/2) endonuclease complex. B-cells that express a functional B-cell receptor differentiate into mature naive B-cells and leave the bone marrow to undergo clonal expansion in the germinal centers (GC). The IG genes are further modified by both somatic hypermutation (SHM) and class-switch recombination (CSR) resulting in antigen-activated B cells.1 The molecular processes that remodel Ig genes involve distinct mechanisms of DNA double-strand breaks and DNA repair. For V(D)J recombination RAGs recognize and bind to site-specific highly defined Correspondence: Professor A Borkhardt, Pediatric Hematology, Oncology and Clinical Immunology, Heinrich-Heine University Dusseldorf, D-40225 Dusseldorf, Germany. E-mail: [email protected] Received 26 December 2005; revised 2 April 2007; accepted 17 April 2007; published online 31 May 2007

recombination sequence signals (RSSs).2,3 In contrast, the DNA cleavage during CSR and SHM is not sequence-specific and is initiated over large target regions via the activationinduced deaminase (AID) endonuclease. Mistakes during these DNA cleavage processes may lead to chromosomal translocations involving the Ig loci and a proto-oncogene. The protooncogene comes under the control of the active Ig locus creating a deregulated, constitutive expression of the oncogene and plays an important role in the pathogenesis of B-cell malignancies.4–6 Three different models have emerged to explain how RAG proteins mediate chromosomal translocations: substrate-selection errors (also called the RAG misrecognition model), the enddonation model and the transposition model (reviewed in detail in Roth D B7). With respect to the RAG-misrecognition model, the presence of RSS-like sequences (pseudo-RSS) near chromosomal breakpoints leaves little doubt that the DNA break within the antigen-receptor containing chromosome is mediated via RAG, but the cause of the DNA break within the protooncogene has been unclear.8 In most of the cloned chromosomal breakpoints the identified pseudo-RSS are not located proximal to the breakpoint and deviate so far from the consensus-RSS that the involvement of RAG seems unlikely.4,7,9,10 Thus, until recently it remained a largely unresolved question of whether the pseudo-RSS occur merely coincidental or really indicate the involvement of the V(D)J recombinase.11,12 In follicular lymphoma (FL) the BCL-2/IGH fusion is the molecular equivalent of the translocation t(14;18) and demonstrates clear involvement of RAG in the IGH-JH breakpoints. Recently, work of the Lieber laboratory shed new light on the question how the RAG complex cleaves the BCL-2 protooncogene at its well-defined major breakpoint cluster region (Mbr) located in the untranslated portion of its third exon.13–16 BCL-2 breakpoints are dependent upon the formation of stable non-B DNA secondary structure, which is cleaved by RAG activity in the absence of RSS. Therefore, the t(14;18)-associated BCL-2/IGH fusion is presumably mediated by the RAG complex only. In summary, the RAG complex is capable of acting as a nuclease based on sequence or structural specificity. Notably, the occurrence of other non-B form DNA structures (loops, cruciforms, left-handed Z-helices, triplexes or tetraplexes) in the human genome coincide with genomic rearrangements, for example, gross deletions or constitutional translocations.17 Whether these genomic instabilities are generated by RAG or perhaps other nucleases that are attracted by the unusual DNA structures is unknown. In contrast to the recently characterized RAG-mediated DNA cleavage errors, mistakes during AID-mediated CSR are less well understood, but tight regulatory control of AID is also necessary

Analyses of MYC/IgH breakpoints K Busch et al

1740 to prevent generalized genomic mutations and genomic instability.1,18,19 Sporadic Burkitts lymphoma (sBL) carry the translocation t(8;14).20,21 Breakpoints on chromosome 14 occur either within the IGH switch regions (Sa, Sg and Sm) or in the joining region (JH). Thus, in contrast to FL, in sBL the cleavage sites of IGH suggest involvement of either RAG (JH breakpoints) or AID (switch region breakpoints), depending on the individual tumor.2,18 Whether AID or RAG also generate the DSB in MYC is unknown. If these nucleases induce the DSB in MYC, one might speculate that various breakpoint clusters within the MYC gene may also exist depending on the action of either RAG or AID. To investigate this, we analyzed a large series of sBL with MYC/IGH fusion and asked two questions: (1) Do one or more breakpoint clusters exist within the MYC gene?, (2) Do the putative MYC breakpoint cluster(s) associate with particular switch or the JH breakpoint regions? Such a putative association between the different IGH regions and the MYC breakpoint clusters may give correlative evidence for the distinct activities of either AID or RAG for generation of the breakpoints within MYC.

Materials and methods

Patients Informed consent from the guardians for each patient was obtained. Tumor biopsy material, bone marrow, lymph nodes or ascites from 76 pediatric patients suffering from sporadic Burkitt’s lymphoma/B-cell leukemia with translocation t(8;14)(q24;q32) were collected in the framework of the Berlin-Frankfurt-Muenster (BFM) study group. We analyzed two additional patients with translocation t(8;14)(q24;q32) who were not enrolled in our BFM study. From October 1996 until February 2003 1261 newly diagnosed Non-HodgkinLymphomas were enrolled. Among them, 528 children suffered from Burkitts-Lymphoma (BL) or B-ALL, respectively. The selection of cases was solely based on the availability of highmolecular weight DNA. Whenever viable tumor cells were available, standard karyotyping was performed. Patients with endemic BL were excluded from this study. The procedure for isolation and storage of the tumor cells and mononuclear cells from bone marrow we described recently.22

DNA preparation, detection of der(14) MYC/IGH and der(8) IGH/MYC breakpoints High-molecular-weight genomic DNA was prepared from frozen tumor cells and mononuclear cells by washing them twice with PBS before using the QIAamp DNA or DNA Blood Mini Kit (Qiagen, Hilden, Germany) following the instructions provided by the manufacturer. The breakpoint region involving the MYC gene on chromosome 8q24 and the IGH locus on chromosome 14q32 were determined by long-distance (LD)-PCR using the Expand Long Template PCR System (Roche, Mannheim, Germany).23 To detect the rearrangement involving the c-myc gene on chromosome 8q24 and the IgH locus on chromosome 14q32, one primer for the c-myc gene (c-myc/M6 at position 4885 in exon 2) and four primers for the IgH locus were combined: three primers for the constant region (Cm03, Cg02, Ca01) and one for the JH. Primers for both genes represent the antisense strands in reverse direction, due to the head-to-head orientation of c-myc and IgH genes. Primer sequences for isolation of the der(14) MYC/IGH fusions are indicated in Supplementary Table 1. Leukemia

The quality of the genomic DNA and adequacy of sample for the amplification of long DNA fragments were tested in each sample by using c-myc/M6 and c-myc/M9 primers together with an upstream primer c-myc up (at position 1), which yielded a PCR product of 4.9 and 8.2 kbp, respectively. Each reaction mixture (50 ml) contained 250 ng of genomic DNA, 300 nM of downstream and upstream primer, 500 mM of each dNTP, buffer III with 22.5 nM MgCl2 and 2.6 units of a polymerase mix as indicated in the Expand Long Template PCR System Kit (Roche, Mannheim, Germany). Reaction conditions were as follows: denaturation at 941C for 2 min followed by 10 cycles of denaturation at 941C for 30 s, annealing at 681C for 30 s, extension at 681C for 4 min, followed by 20 identical cycles with gradual increment of extension time (15 s/cycle) and a final extension for 10 min at 681C. PCR were performed in a Thermal Cycler 9600 (Applied Biosystems, Darmstadt, Germany). For sequence analysis the LD-PCR products were purified using the QIAquick PCR Purification Kit (Qiagen, Hilden, Germany). Sequence analysis of the PCR products was performed with MYC-specific primers (c-myc1 to c-myc16, Supplementary Table 2). The sequences thus obtained were analyzed by BLAST homology search (www.ncbi.nlm.nih.gov/ BLAST/) and the junctional nucleotide sequences from each specific MYC/IGH fusion were deposited into the EMBL Nucleotide Sequence Databank (www.ebi.ac.uk/submission/ webin.html). The assigned accession numbers are listed in Table 1 according to the MYC/IGH breakpoint. Similarly, the reciprocal der(8) IGH/MYC fusions were amplified and sequenced with the help of PCR/sequencing primers shown in Supplementary Table 3.

Statistical analysis Statistical significance and the number of clusters of translocation breakpoints within the MYC gene (bp 1–4555) was evaluated by scan statistics and gap statistics as described in Segal G H24 with 999 runs of permutations using the softwarepackage SaTScan V.3. 025 and custom programs implemented in the free statistical environment R (www.r-project.org). The IGHloci-specific distributions of MYC-breakpoints were compared using the Kruskal–Wallis test or Mann–Whitney U-test (pairwise comparison). Associations of MYC-clusters with IGH-regions were investigated via contingency table analysis using w2-tests.

Screening for human repetitive elements A direct search for several known signals including the repetitive elements (SINEs, LINEs, LTRs, transposon fossils), V(D)J-RSS motifs, topoisomerase II binding site, eukaryotes replication origin sequences, translin binding site, w-like sequences, scaffold/matrix attachment region, pyrimidine trait (Y12), putative triple helices and the purl-binding site was performed to analyze the sequence surrounding the identified MYC clusters. Matches to V(D)J RSS and Topoisomerase II binding site were scored using position-specific scoring matrices calculated based on the log odds ratio of known motif nucleotide frequency distribution to the uniform background. For the other motifs with limited number of known exemplar sequences, regular expression matches were done. Human repetitive elements were scanned by RepeatMasker (Smit AFA and Green P, 1996, unpublished results. RepeatMasker at http://ftp.genome.washington.edu/RM/RepeatMasker.html). In addition, an indirect search was performed using de novo statistical motif finders Gibbs Motif Sampler (GMS)26 and MEME27 to look for enriched motif in the proximity, (100, þ 100 bp), of breakpoints. Found

Table 1

Summarizing data from 78 BL patients analyzed Sex

Source

Age (years)

Breakpoint Breakpoint IGH-locus MYC gene (Acc. No. X00364)

Localisation within MYC gene

MYC cluster area

Acc. no. (breakpoint sequence of MYC/IGH)

Cytogenetic data and FISH analysis

1 2 3

M M F

Tumor Effusion Tumor

5.98 8.02 4.13

Switch a Switch a Switch a

871 1267 2037

50 exon 1 50 exon 1 50 exon 1

50 of 1 50 of 1 1

AJ630036a AJ630035a AJ586976a/b

46,XY,t(8;14)(q24;q32)

4

M

BM

4.03

Switch a

2051

50 exon 1

1

AJ630037a

5

M

BM

13.10

Switch a

2087

50 exon 1

1

AJ628948 AJ586973 + AJ630621

0

M M

LN Tumor

5.85 10.49

Switch a Switch a

2156 2504

5 exon 1 Exon 1

8 9 10

M M M

Effusion Tumor Effusion

12.07 7.72 4.07

Switch a Switch a Switch a

2740 2806 2822

Exon 1 Exon 1 Exon 1

1 Between 1 and 2 2 2 2

11

M

LN

9.74

Switch a

2838

Exon 1

2

AJ586972a/b

12 13 14

F M M

Tumor Aszies BM

15.38 2.92 8.48

Switch a Switch a Switch a

2889 2934 3065

Intron 1 Intron 1 Intron 1

2 2 2

AJ601400b AJ630620 AJ630614

15

M

Effusion

4.27

Switch a

3140

Intron 1

2

AJ601404b a

AJ628708 AJ586971a/b AJ628947a

9.41 11.79 6.14

Switch a Switch a Switch a

3198 3199 3229

Intron 1 Intron 1 Intron 1

2 2 2

AJ630041 AJ630034a AJ630622

9.32 6.58

Switch a Switch a

3361 3363

Intron 1 Intron 1

2 2

AJ628707 AJ628706a

6.77 11.94

Switch a Switch a

3454 3483

Intron 1 Intron 1

2 2

AJ628702a AJ630613

BM

5.76

Switch a

3781

Intron 1

3’ of 2

AJ630607

Effusion

6.25

Switch a

3879

Intron 1

3’ of 2

AJ586977b

16 17 18

M M M

BM BM BM

19 20

M F

Tumor Tumor

21 22

M M

Effusion BM

23

M

24

M

2nd relapse

Alive Alive Alive 5 mo

6 mo

wc

Alive Alive 44 mo

35 mo 4 mo

13 mo

46,XY,t(8;14)(q24;q32)/ 46,idem, der(13)t(7;13) (q11;q31) nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1)

Alive 19 mo Alive

wc

wc

Alive 3 mo

46,XY,t(8;14)(q24;q32)/ 46,XY 46,XY,t(2;13)(p16;q31B32), t(8;14)(q24;q32)

5 mo

8 mo Alive Alive

wc

Alive Alive Alive Alive

46,XY,?dup(1)(q21q32), t(8;14)(q24;q32)/46,idem, der(15)t(7;15)(q10;q10)

Alive 1 mo

46,XX,t(8;14)(q24;q32), del(9)(q?21q31), add(18)(q22B23)[8]/ 46B47,idem,der(17), +mar,inc[cp5] 46,XY,t(8;14)(q24;q32)/ 46,XY nuc ish 8q24(5’CMYCx2),(3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) 46,XY,der(6)t(1;6) (q12B21;q25B26), t(8;14)(q24;q32)

Outcome

Analyses of MYC/IgH breakpoints K Busch et al

6 7

nuc ish 8q24(C-MYCx23),14q32(IGHx2), (C-MYC con IGHx1-2) nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) 46,XY,dup(1)(q?); t(8;14)(q24;q32),inc

1st relapse

4 mo

1 mo Alive

wd

wd

Alive Alive

1741

Leukemia

1742

Leukemia

Table 1

Continued Source

Age (years)

Breakpoint IGH-locus

Breakpoint MYC gene (Acc. No. X00364)

Localisation within MYC gene

MYC cluster area

Acc. no. (breakpoint sequence of MYC/IGH)

25 26 27 28 29 30 31 32 33

F M M M M M M M M

BM BM BM BM LN Tumor BM BM LN

13.71 9.62 4.59 11.95 2.58 5.24 13.62 13.47 3.79

Switch Switch Switch Switch Switch Switch Switch Switch Switch

a a a g g g g g g

3902 3985 4415 990 1007 1189 1628 1892 1937

Intron 1 Intron 1 Intron 1 50 exon 1 50 exon 1 50 exon 1 50 exon 1 50 exon 1 50 exon 1

3’ 3’ 3’ 5’ 5’ 5’ 5’ 1 1

AJ628704a AJ630627 AJ628949 AJ628951 AJ632276 AJ628952 AJ630040a AJ630042a AJ601401a/b

34

M

BM

Switch g

1979

50 exon 1

1

AJ628703a

35 36 37 38 39

M M M M M

Tumor Tumor Tumor Tumor BM

6.64 9.64 12.35 13.33 9.61

g g g g g

2043 2046 2066 2180 2183

50 50 50 50 50

1 1 1 1 1

1 1 1 1 1

AJ699162a AJ630608 AJ601403a/b AJ632275 AJ630615

40 41

M M

Tumor Tumor

4.56 6.92

Switch g Switch g

2288 2332

50 exon 1 Exon 1

1 1

AJ630617 AJ630612

42

M

BM

12.59

Switch g

2334

Exon 1

1

AJ630611

43

M

LN

7.61

Switch g

2690

Exon 1

44 45

M M

Tumor BM

10.49 4.01

Switch g Switch g

2849 3481

Exon 1 Intron 1

between 1 AJ628705 and 2 2 AJ630606 2 AJ628946a

46

M

Tumor

10.44

Switch m

1083

50 Exon 1

5’ of 1

6.42

Switch Switch Switch Switch Switch

exon exon exon exon exon

of of of of of of of

2 2 2 1 1 1 1

AJ630624

Cytogenetic data and FISH analysis

1st relapse

2nd relapse

46,XY 46,XY,t(8;14)(q24;q32)

6 mo

7 mo

4 mo 4 mo

7 mo

46,XY,t(8;14)(q24;q32) 46,XY,t(8;14)(q24;q32)/ 46,XY nuc ish 8q24(5’CMYCx2),(3’C-MYCx2), (5’CMYC sep 3’C-MYCx1)

46,XY,t(8;14)(q24;q32) 46,XY,t(8;14)(q24;q32)/ 47,idem,+der(1)t(1;7) (p11;q22) 46,XY 45,X,-Y,del(8)(p11),t(8;14;?) (q24;q32;?),dup(11) (q13q25),add(12)(p(11), add(13)(q14), add(18(q23)[cp15] 46,XY,dup(1)(q21q?31), der(6)t(6;?)(q15;?), t(8;14)(q24;q32), add(8)(q24),?der(12), add(13) (q34),inc[3]/ 46,XY[7] nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1)

46,XY,t(8;14) (q24;q32), del(11)(q22.3q23) nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), ’C-MYC sep 3’C-MYCx1) nuc ish 8q24(C-MYCx2), 14q32(IGHx2), (C-MYC con IGHx1)

Outcome

Alive Alive Alive Alive Alive 8 mo 5 mo Alive Alive

wc wc

Analyses of MYC/IgH breakpoints K Busch et al

Sex

Alive Alive Alive Alive Alive Alive Alive Alive

4 mo

Alive

16 mo

22 mo

35 mo

44 mo Alive

Alive

wc wc

Table 1

Continued Sex

Source

47

F

BM

48

F

49

Age (years)

Breakpoint MYC gene (Acc. No. X00364)

Localisation within MYC gene

MYC cluster area

Acc. no. (breakpoint sequence of MYC/IGH)

Cytogenetic data and FISH analysis

4.08

Switch m

1084

50 Exon 1

5’ of 1

AJ630626

BM

15.71

Switch m

1902

50 Exon 1

1

AJ628945

F

BM

5.95

Switch m

2077

50 Exon 1

1

AJ630039a

nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) 46,XX,?inv(11)(q22q23). nuc ish 8q24(C-MYCx2), 14q32(IGHx2) nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1)

50 51

M M

BM BM

17.69 2.20

Switch m Switch m

2105 2145

50 Exon 1 50 Exon 1

1 1

AJ628709 AJ628950

52

F

BM

13.00

Switch m

2184

50 Exon 1

1

AJ628954a

53 54 55

F M M

PB tumor effusion

no data 14.24 14.41

Switch m Switch m Switch m

2354 2365 2398

Exon 1 Exon 1 Exon 1

1 1 1

AJ628711 AJ601398 b AJ601399a/b

56 57

M M

aszites BM

9.10 13.82

Switch m Switch m

2452 2595

Exon 1 Exon 1

AJ601397 AJ628953

58 59 60 61

F M F F

PB aszites BM tumor

6.97 8.94 9.62 6.14

Switch Switch Switch Switch

m m m m

2717 2775 2829 2874

Exon Exon Exon Exon

1 between 1 and 2 2 2 2 2

AJ630616 AJ601396b AJ632277 AJ630605

62

M

BM

4.63

switch m

2880

Exon 1

2

AJ630033a

1 1 1 1

46,XY nuc ish 8q24(5’CMYCx2), (3’C-MYCx2), (5’C-MYC con 3’C-MYCx2) 46,XX,t(3;13) (q13;q31B32), add(8)(p12), t(8;14)(q24;q32), add(11)(q2?5), add(15)(p13)

1st relapse

2nd relapse

Alive

Alive Alive

Alive Alive

5 mo

Alive

1 mo Alive Alive

46,XY,?ins(1;11) (q21;q23q25), t(8;14)(q24;q32) [4]/ 46,XY[5]. nuc ish 11q23(5’MLLx2), (3’MLLx2), (5’MLL con 3’MLLx2) 5 mo

7 mo Alive Alive Alive 3 mo Alive

2 mo nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) 43-44,XY, inv(1)(p13q41), t(8;14)(q24;q32), add(12)(q24);?hsr(15)(q22), +2mar[cp4]

Outcome

4 mo

7 mo

7 mo

Analyses of MYC/IgH breakpoints K Busch et al

Breakpoint IGH-locus

wd

wc

wc

wc

1743

Leukemia

1744

Leukemia

Table 1

Continued Source

Age (years)

Breakpoint IGH-locus

Breakpoint MYC gene (Acc. No. X00364)

Localisation within MYC gene

MYC cluster area

Acc. no. (breakpoint sequence of MYC/IGH)

63 64 65

M M M

PB tumor tumor

5.86 7.34 14.25

Switch m Switch m Switch m

2888 2977 3018

Intron 1 Intron 1 Intron 1

2 2 2

AJ632278 AJ601402a/b AJ630604

66 67 68 69

F M M M

BM tumor aszites BM

9.29 12.23 4.80 4.31

Switch m Switch m Switch m JH

3401 3467 3496 381

Intron 1 Intron 1 Intron 1 50 Exon 1

2 2 2 5’ of 1

AJ630625 AJ586978b AJ630038a AJ630623

70 71 72

M M M

BM BM tumor

10.81 6.30 5.34

JH JH JH

1444 2020 2365

50 Exon 1 50 Exon 1 Exon 1

5’ of 1 1 1

AJ630619 AJ630609 AJ586975b

73

F

tumor

8.35

JH

2427

Exon 1

1

AJ632274

74

M

BM

9.92

JH

2723

Exon 1

2

AJ630610

75

M

tumor

11.13

JH

2726

Exon 1

2

AJ630618

76

M

effusion

8.65

JH

2872

Exon 1

2

AJ601405b

77 78

M M

BM tumor

15.55 10.10

JH JH

2980 3281

Intron 1 Intron 1

2 2

AJ628710 AJ586974b

Abbreviations: BM, bone marrow; JH, joining region; LN, lymph node; pB, peripheral blood; effusion, pleural effusion. a Already reported in reference Wilda M et al.23 b Already reported in reference Busch K.22 wc Death from progression of disease. wd Death from complications of toxicity of chemotherapy.

Cytogenetic data and FISH analysis

nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1)

1st relapse

46,XY.nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC con 3’C-MYCx2)

nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1)

Outcome

Alive Alive Alive

41 mo

7 mo

nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) ish (IGHx2), (IGHspx1) nuc ish 8q24(5’C-MYCx2), (3’C-MYCx2), (5’C-MYC sep 3’C-MYCx1) ish (C-MYCx2), (IgHx2),(C-MYC con IGHx1) 46,XY,t(8;14) (q24;q32)/46,XY

2nd relapse

12 mo

Alive 14 mo Alive Alive

wc

Alive Alive Alive

Alive Alive

4 mo

6 mo Alive Alive Alive

wc

Analyses of MYC/IgH breakpoints K Busch et al

Sex

Analyses of MYC/IgH breakpoints K Busch et al

1745 motifs from 100 runs GMS were then aggregated into a consensus motif.

Results

Pattern of the 78 MYC/IGH breakpoints Using the LD-PCR we determined the MYC/IGH rearrangement from 78 pediatric sBL patients enrolled in the trial NHL-BFM 95 (Table 1). The pattern of the breakpoint locations within the MYC gene and the IGH regions is shown in Figure 1. The breakpoint locations within the MYC gene were evenly distributed, 50 of exon 1 (37%), in exon 1 (31%) and in intron 1 (32%). The breakpoint locations within the IGH regions revealed 88% of breakpoints within the switch a (35%), switch g (23%) and switch m (29%) region, whereas 13% of breakpoints were identified in the joining region (Figure 1).

MYC breakpoint cluster analysis Based on the structure of MYC, the allowable range of DNA nucleotides was 4555 bp, to just before the second exon, which

contains the first translated codon. The scan statistic identified two distinct clusters, one from 1892 to 2452 and the other from nucleotides 2717 to 3496, containing 60 of 78 (77%) assessed breakpoints (P ¼ 0.004). The remaining 18 (located between the nucleotides 381 and 4415 of MYC gene) did not partition into defined clusters (Figure 2 and Table 2).

MYC breakpoint clusters associated with various breakpoints of IGH The IGH-specific distributions of the MYC breakpoints are graphed in Figure 3 and are shown to be significantly different (Kruskal–Wallis test, P ¼ 0.0007). Importantly, the pairwise comparison revealed no differences between the IGH switch m and the JH region with respect to their corresponding MYC breakpoints. Therefore, these two IGH regions were combined to determine an association between breakpoints within the MYC clusters and the IGH areas using cross tabulation. A clear relationship could be demonstrated for IGH regions Sa and Sg and the clusters 1 and 2 (P ¼ 0.002, contingency table). Whereas breakpoints 50 of cluster 1 were found to be over-represented with a link to IGH region switch g (P ¼ 0.0013), MYC breakpoints 30 of cluster 2 are strongly related to the IGH loci switch a (P ¼ 0.0025). Moreover, MYC breakpoints within the two clusters and in between were significantly associated to the combined IGH loci switch m and JH (P ¼ 0.001). Thus, the breakpoints within cluster 1 and with smaller MYC nucleotide numbers are related to IGH region Sg, whereas breakpoints within cluster 2 and with higher MYC nucleotide numbers are related to IGH region Sa. (Table 2 and Figure 4).

Searching for breakpoint-associated sequence motifs Figure 1 Distribution of the 78 genomic MYC/IGH breakpoints. The locations of the MYC/IGH breakpoints within the MYC gene and the IGH loci are indicated with arrows in the model of a MYC/IGH rearrangement.

In the search for known recombination-facilitating motifs (Supplementary Table 4), a polypyramidine tract was repeatedly found near translocation breakpoints on MYC. Such pyrimidine tracts are known to facilitate integration of simian virus (SV

Figure 2 Schematic presentation of the MYC clusters. The positions of the breakpoints within the MYC gene are indicated with lines and spans from position 381 located 50 of exon 1 until 4415 of intron 1. In addition, the distribution is visualized by a density plot using a Gaussian kernel with a bandwidth of 191.5 points. The two clusters are presented with a 95% confidence limit and marked in black lines indicating their regions. The distribution of the breakpoints within the IGH regions are indicated schematically in light and dark gray to point out the relevant association with the MYC clusters. Leukemia

Analyses of MYC/IgH breakpoints K Busch et al

1746 Table 2

Contigency table analyses: association between MYC clusters (highlighted in gray) and breaks within the IGH loci

Scan statistics IGHgrp * satscan2 cross-classified table Amount MYC area

IGHgrp

Satscan2

Sa Sg Sm+JH

Total

Total

1

2 cluster 1 1892–2452

3

4 cluster 2 2717–3496

5

2 4 4 10

4 11 12 27

1 1 1 3

15 2 16 33

5 0 0 5

27 18 33 78

Abbreviation: JH, joining region. P ¼ 0.002.

70%

IGH

60%

Sγ

Sα

Sµ+ JH

percentage

50% 40% 30% 20% 10% PC2

C2

>C2

Myc area

Figure 4 Bar diagram of the contingency table analyses. Association between the MYC clusters 1 (indicated as C1) and 2 (indicated as C2) and the breakpoints located within the IGH switch a, g and m region as well as in the joining region (JH) analyzed via contingency table are shown in vertical bar graphs. Figure 3 IGH-loci-specific density plots for the nucleotide numbers 700–4200 of c-MYC. The association between the MYC breakpoints and the location of the breaks within the different regions of the IGH locus is shown as a IGH-loci-specific density plot. The lowest panel shows the summarized IGH-specific density blot from all IGH regions.

40).28 Furthermore, an undirected motif search using de novo motif finding algorithms (Gibbs motif sampler and MEME) provided additional evidence for a pyrimidine tract motif, or its reverse complement, a GAGA motif. Figure 5 depicts the sequence logo of the composite motif (and its reverse complement) from 100 runs of Gibbs Motif Sampler. MEME runs yield qualitative similar sequence motifs (data not shown). A polypyrimidine motif of this sort is known to form paranemic (base unpaired) structure, which is known to form Z-DNA (left handed helix) and other structure in supercoiled DNA as occurs during transcription (Figure 5).

Determination of der(8) reciprocal IGH/MYC breakpoints in a subset of patients To study the exact configuration the reciprocal breakpoints on der(8) we isolated the IGH/MYC fusion site in a subset of Leukemia

patients. The cases were selected on the availability of DNA and quality of the reciprocal PCR products obtained. We wanted to know whether the breakpoints are perfectly reciprocal or whether deletions or insertions of nucleotides can be found. The sequences derived from 17 patients revealed four types of translocations: (i) perfectly reciprocal at least for MYC (two cases), (ii) with loss of material in both fusion partners (four cases), (iii) with gain of material derived either from IGH or MYC (four cases) or (iv) with loss of material of IGH and/or MYC and gain of material of unknown origin (seven cases) (Figure 6). These sequences without any homology to known human sequences seem to be incorporated during the translocation process by chance, since they are not derived from inverted duplications of MYC or IGH regions. Possibly the nucleotides incorporated at these sites originate from the nucleolytic processing of broken DNA ends during the NHEJ process or can be attributed to the action of pol m or pol l, both involved in the filling of small gaps before DNA end joining (reviewed in Hefferin M L29). The different types of translocation do not seem to be associated with one of the MYC breakpoint clusters identified in this study. In addition to the accession numbers of either MYC/IGH or IGH/MYC breakpoints the flanking

Analyses of MYC/IgH breakpoints K Busch et al

1747 Pos:

1

2

3

4

5

6

7

8

9

10

0.3

0.2

0.3

0.3

0.5

0.2

0.3

0.5

0.7

0.2

Total Bits: Pos:

3.4 1

2

3

4

5

6

7

8

9

10

0.6

0.7

0.1

0.4

0.5

0.2

0.3

0.2

0.2

0.8

Total Bits:

4.1

Figure 5 De novo polypyrimidine and its reverse complement motif identified in the proximity of the breakpoints.

sequences of all breakpoints are provided in Supplementary Table 5.

Clinical features Primary involvement of the central nervous system or bone marrow was diagnosed in 10 of our 37 patients, respectively. An abdominal mass was seen in 58 of our patients. Of the 78 BL patients 18 patients suffered a recurrence of disease and three patients perished from complications of chemotherapy (Table 1). Among the 18 patients with a relapse (17 out of 76 from the BFM study group), the IGH breakpoint was evenly located in the switch a (five patients), switch g (six patients) and switch m (six patients), whereas from the 10 patients with a break in the JH region, only one patient relapsed (Table 1). We next asked whether the der14 MYC/JGH breakpoint configuration is associated with specific clinical parameters. For this purpose, we categorized the patients into three different MYC/JGH breakpoint groups so that at least 15 patients belong to each category (Table 3). No statistically significant findings emerged from these analysis, neither staging according to Murphy30 nor lactate dehydrogenase (LDH) levels before therapy were associated with one of the defined der14 MYC/IGH categories. Notably, out of the four patients with the rare der14 breakpoint configuration (50 to MYC cluster 1 and IGH switch g) three relapsed. In contrast, all five patients survived who had the der14 breakpoint configuration 30 to MYC cluster 2 and IGH switch a. For the two patients who were not enrolled in the BFM study only sparse clinical information was available.

Discussion As part of the maturation process, B cells enter the GC and become susceptible to enzymatic activities that alter the genome by hypermutation, Ig class switching and secondary V(D)J rearrangements. The GC is a crucible of somatic genetic change and hence a plausible site for oncogenetic errors.4,9,31,32

In accordance with other studies, we found that most breakpoints within the IGH region in sporadic BLs with t(8;14)/MYCIGH translocations map to the switch regions and only a minority to the JH locus (Figure 1 and Supplementary Table 6). We demonstrate evidence of two clusters within the MYC breakpoint region (Figure 2). Given that different nucleases, RAG or AID, are responsible for the various IGH cleavage sites, the fact that the JH breakpoints and switch m breakpoints did not differ with respect to their corresponding MYC breakpoint regions is unexpected and may suggest that a third mechanism is responsible for DNA cleavage in MYC; or that RAG and AID may cause cleavage in MYC breakpoint regions equivalently, possibly due to structural attributes of the MYC promoter region (see below). Duquette,19 recently demonstrated that AID targets to G-loops, co-transcriptional RNA: DNA hybrids on the C-rich strand and single-stranded regions and G4 DNA on the G-rich strand.19 Similar distributions of RAG induced JH and AID induced switch m breakpoints does not necessarily argue against a prominent role of these G-loops. The clusters on MYC were quite wide and diffuse (560 and 779 bp in a 4555 bp region) compared to three sharply circumscribed peaks (10–15 bp in a region of 150 bp) described previously within Mbr of BCL-2 and attributed to RAG activity.15,16 This diffuse nature of MYC clusters additionally suggests that the mechanism for DNA cleavage is not restricted to a site-specific mechanism such as RAG-mediated DSB. Interestingly, the breakpoints within the MYC clusters are significantly associated with breaks in the various IGH regions. Whereas breakpoints 50 of MYC cluster 1 were preferentially paired with breaks in the IGH region switch g (Po0.01), breakpoints 30 of MYC cluster 2 were associated with the most 30 located IGH loci, switch a (Po0.02). MYC breakpoints within the two clusters were significantly associated to the combined IGH loci switch m and JH (Po0.001) (Figures 3 and 4). The significant association between particular IGH switch regions and different MYC breakpoint regions also may suggest that another cleavage activity exists for the MYC locus. Could such Leukemia

Analyses of MYC/IgH breakpoints K Busch et al

1748

a c-myc

Exon 1

Exon 2

Exon 3

c-myc inserted

c-myc deleted

Sµ IGH

VH

Sγ

Sα

Eµ

DH

Cµ Cδ

JH

Cγ

Cα

IGH inserted

IGH deleted

unknown

Cγ

Cδ Cµ

JH

P1

Eµ

DH

VH

Sµ

Sγ

4 4 12

b

Cγ P9

Cδ Cµ Eµ

32 55

DH

VH

DH

VH

Sµ

Sγ 46

Cγ P12

JH

Ex1

Cδ Cµ

JH

Ex1

Eµ Sµ

Sγ 29 2 20

P22

Exno1

263

?? Cγ Cµ

JH Eµ

P32

DH

VH

Sγ 10 42 Figure 6 (a–e) Schematic representation of MYC-IGH fusions on chromosome der(8) in a subset of patients. Patient id corresponds to Table 1. In all fusions IGH is invertedly fused to MYC in 30 –30 orientation. Insets show partial magnification of the breakpoint with numbers indicating the number of basepairs deleted or inserted, respectively.

an association between the MYC and IGH switch breakpoints be explained by the action of AID-recombinase exclusively? Isotype-specific AID co-factors that target AID to their specific Leukemia

switch regions may clearly contribute to the MYC breakpoint pattern. For the physiological CSR process, there must be factors that explain isotype-specific switching activities and thus

Analyses of MYC/IgH breakpoints K Busch et al

1749

c

Cδ Cµ

JH Eµ

P36

d DH

JH Eµ

P46

VH

DH

VH

DH

VH

DH

VH

D DH

VH

Sµ ?

?

Cδ Cµ Eµ

P37

JH

JH DH

Eµ

P48

VH

Sµ 7 10

37 ?

Cδ Cµ P41

Ex1

JH

JH Eµ

DH

P56

VH

Ex1

Eµ

Sµ 25 17 39

? Cδ Cµ P42

Ex1

JH

JH Eµ

DH

P59

VH

Ex1

Eµ

Sµ 72

8

52 ?

e P61

281

JH Ex1

Eµ

D DH

VH

D DH

VH

2010 91 JH P66

Exon1

Eµ

33 19 107

P72

Ex1

347 P74

Ex1

DH

VH

? DH

VH

12 39

Figure 6 Continued.

provide an accessory layer of specificity for the CSR reaction, potentially at the level of chromatin organization. Transcription is absolutely required for AID-mediated CSR and AID deaminates only single-stranded DNA. Thus, the MYC gene may be transcribed at a different rate leading to distinct DNA or chromatin structure in an isotype-specific pattern, leading to isotype-specific open regions of MYC chromatin. Notably the clusters in MYC represent the coverage of 2–4 nucleosomes. Therefore, an obvious explanation for our data is that the distinctive collections of transcription factors in B cells switching to g or a modulates transcription of different regions of MYC. There is also some other specificity to CSR switching activities.33,34 B-cells that lack the trans-activation domain of the c-Rel are able to carry out only m to a but not m to e CSR.35 Plasmid switching assays also revealed the existence of different

switching activities that mediate m to g3, m to a, m to g1 and m to e.34,36,37 However, all these isotype related switching activities seem to act very specifically whereas the MYC/IGH breakpoint pattern found in our study is only an association, albeit highly statistically significant. There are also significant differences between CSR and V(D)J recombination in terms of repairing DNA DSB. For instance, 53BP1 is recruited to sites of DNA damage and is fully dispensable for V(D)J recombination, but required for CSR.38 Similarly, in the absence of the damage-response protein H2AX, CSR is very much impaired but the V(D)J recombination is not.39–41 However, again, these functional differences between the CSR and the V(D)J recombination cannot be recapitulated in our correlative study of the MYC/IGH breakpoints. The presence of polypyrimidine tracts in correspondence with the MYC Leukemia

Analyses of MYC/IgH breakpoints K Busch et al

1750 Table 3

Clinical and biological characteristics, treatment outcome from the 76 patients enrolled in the NHL-BFM95 study IgH Breakpoints

No. of patients Female Age o10 years Stage I+II Stage III IV+ B-ALL BM disease CNS disease Abdominal involvement LDHX1000 U/L# l light chainb k light chainb Relapse of disease

Total

JH

Sm

Sa

Sg

76 13 50 12 25 39 37 10 58

10 1 6 2 3 5 4 1 6

22 8 13 2 7 13 13 3 17

26 4 20 3 9 14 14 3 20

18 0 11 5 6 7 6 3 15

36 17 25 17

3 2 3 1

13 4 5 5

13 5 12 5

7 6 5 6

Myc breakpoints clusters P

0.002 0.5 0.7 0.3 0.8 0.5 0.4 0.6 0.5

50 of cluster1

Cluster 1

Cluster 2

30 of cluster 2

10 1 6 2 2 6 6 1 7

25 4 16 7 8 10 10 1 20

33 6 23 3 14 16 15 6 24

5 2 4 0 1 4 4 1 4

5 3 4 3

8 6 5 4

18 6 12 8

3 1 3 0

Associations myc – IgH breakpoints P

0.5 0.8 0.3 0.3 0.6 0.9 0.4 0.6

Myc pclustera 1 - IgH sg

Myc Xclustera 2 – IgH sa

Myc cluster 1+2 – IgH JH+sm

15 0 9 5 6 4 4 2 12

20 4 15 2 8 10 10 3 15

27 8 17 3 10 14 13 4 20

5 5 3 4

11 10 4 3

13 6 6 6

P

0.06 0.5 0.3 0.3 0.7 0.9 0.4 0.2

Abbreviations: ALL, acute lymphoid leukaemia; BM, bone marrow; CNS, central nervous system; JH, joining region; LDH, lactate dehydrogenase. a p Clustera 1 indicates Myc breakpoints within cluster 1 and 50 of cluster 1; Xcluster 2 indicates Myc breakpoints within cluster 2 and 30 of cluster 2 # LDH not available in one patient. b Light chain expression not available from 34 patients.

clusters further indicates the fragility of the locus. This sequence motif can form slippage structures with extruded single-strand loops, and triple helices (H-DNA).42,43 Secondary structural features of DNA are known to attract activity of the nuclease activity of RAG and AID and may be involved in other nucleases such as topoisomerase II.44,45 We did not find any significant matches to the V(D)J RSS motif; however, this does not completely rule out its potential role. An analysis of DNA motifs (Supplementary Table 4) did not reveal any significant associations between known motifs and breakpoints within the MYC gene. Using an ‘undirected’ motif search, we scanned for motifs that appeared more common in the near vicinity ( þ 100 bp) and discovered the presence of the polypyrimidine track considered above, roughly a repeating ‘CT’ dinucleotide motif. Such features have been associated with non-B DNA structures particularly slippage with loop exclusion, and triplex;42 however, it should be noted that this feature was in proximity and not directly at the breakpoints. Stem-loop structures, similar in some respects to DNA loops that are formed in dinucleotide repeat slippage structures, are known to attract CSR (AID) which are relevant to the formation of MYC breaks described herein.46

Acknowledgements We are indebted to the technical staff of the Department of Pediatric Hematology and Oncology, Giessen, especially to Jutta Schieferstein and Franziska Mu¨ller for excellent technical assistance. We thank Martin Zimmermann (Department of General pediatrics, Medical School, Kiel, Germany) for statistical analysis of the clinical features. The study was funded in part by grants from the network of competence ‘Pediatric Oncology’ of the Bundesministerium fu¨r Bildung und Forschung (BMBF) No. 01GI9963 and the Forschungshilfe Station Peiper. AB and UF were supported by a Grant from the German Cancer Association, the Else-Kro¨ner-Fresenius-Foundation and the Research Foundation of the University of Munich, Germany. JW is a Scholar of the Leukemia and Lymphoma Society of America. Leukemia

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Supplementary Information accompanies the paper on the Leukemia website (http://www.nature.com/leu)

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