Combined High-Resolution Array-Based Comparative Genomic

Combined high-resolution array-based comparative genomic hybridization and expression proﬁling of ETV6/RUNX1-positive acute lymphoblastic leukemias reveal a high incidence of cryptic Xq duplications and identify several putative target genes within the commonly gained region

H Lilljebjo¨rn1, M Heidenblad1, B Nilsson1, C Lassen1, A Horvat1, J Heldrup2, M Behrendtz3, B Johansson1, A Andersson1 and T Fioretos1

1Department of Clinical Genetics, Lund University Hospital, Lund, Sweden; 2Department of Pediatrics, Lund University Hospital, Lund, Sweden and 3Department of Pediatrics, Linko¨ping University Hospital, Linko¨ping, Sweden

Seventeen ETV6/RUNX1-positive pediatric acute lymphoblastic in mouse models.12 Thus, although t(12;21)-positive preleuke- leukemias were investigated by high-resolution array-based mic clones, at least in some cases, arise already in utero, comparative genomic hybridization (array CGH), gene expres- additional mutations are most likely required for overt leukemia. sion profiling and fluorescence in situ hybridization. Compar- Genetic changes secondary to ETV6/RUNX1 are found in ing the array CGH and gene expression patterns revealed that 13 genomic imbalances conferred a great impact on the expres- more than 80% of t(12;21)-positive ALLs, the most common sion of genes in the affected regions. The array CGH analyses being deletion of the normal ETV6 gene, seen in 70% of cases identified a high frequency of cytogenetically cryptic genetic investigated by fluorescence in situ hybridization (FISH).13 changes, for example, del(9p) and del(12p). Interestingly, a Other frequent aberrations include duplication of the normal duplication of Xq material, varying between 30 and 60 Mb in (20%)13 or derivative chromosome 21 (10%),13 deletion of 6q size, was found in 6 of 11 males (55%), but not in females. 14 14 Genes on Xq were found to have a high expression level in (18%) and deletion of 9p (7%). Apart from the additional cases with dup(Xq); a similar overexpression was confirmed in 12p and 21q abnormalities, often found by FISH with t(12;21)-positive cases in an external gene expression data probes specific for ETV6 and RUNX1, most of the secondary set. By studying the expression profile and the proposed abnormalities have been detected by chromosome banding function of genes in the minimally gained region, several analyses alone, or in some instances by multicolor FISH.15–17 candidate target genes (SPANXB, HMGB3, FAM50A, HTATSF1 Because of the limited resolution level of these methods, many and RAP2C) were identified. Among them, the testis-specific SPANXB gene was the only one showing a high and uniform small–and all cytogenetically cryptic–genomic imbalances of overexpression, irrespective of gender and presence of Xq possible prognostic and pathogenetic importance have so far duplication, suggesting that this gene plays an important gone undetected. pathogenetic role in t(12;21)-positive leukemia. In the present study, 17 pediatric ETV6/RUNX1-positive ALLs Leukemia (2007) 21, 2137–2144; doi:10.1038/sj.leu.2404879; were investigated with high-resolution array-based comparative published online 9 August 2007 genome hybridization (array CGH), making it possible to detect Keywords: array CGH; expression profiling; / ; ETV6 RUNX1 SPANXB genomic imbalances as small as 100 kb. The array CGH profiles were also compared with gene expression patterns, showing that the genomic imbalances had a strong impact on the expression level of the affected genomic regions. The most Introduction frequent genomic imbalances detected were gain of Xq material and duplication of the der(21); notably, the X duplications Acute lymphoblastic leukemia (ALL) is the most common were found only in male patients. Overexpression of genes in malignancy in children, with an incidence of 3.9 cases per the duplicated Xq region was observed in cases with dup(Xq), 100 000 per year.1 In pediatric B-cell precursor ALL, the most and this was validated in an externally produced independent common structural genetic abnormality, present in 20–25% of data set. the cases, is the t(12;21)(p13;q22), which results in the ETV6/ By further studies of the proposed function and expression RUNX1 (also known as TEL/AML1) fusion gene.2–5 ETV6/ profile of genes mapping within the minimally gained Xq region, RUNX1-positive ALLs are generally associated with a favorable a number of genes, among them the testis-specific gene outcome with an event free survival approaching 90%,6 SPANXB, that possibly are involved in the transformation although late relapses have been noted by several groups.7,8 of preleukemic t(12;21)-positive cells to overt leukemia Several lines of evidence suggest that ETV6/RUNX1 is not were identified. sufficient for leukemic transformation, such as detection of the gene fusion in Guthrie cards from patients who later developed Patients, materials and methods ETV6/RUNX1-positive ALLs,9 identical gene fusions in twins 10 with concordant leukemia, identification of the chimeric Patients transcript in normal cord blood samples11 and lack of leukemia ETV6/RUNX1-positive ALLs with available DNA were selected from a larger series of pediatric leukemias previously studied by Correspondence: H Lilljebjo¨rn, Department of Clinical Genetics, Lund cDNA microarray.18 Seventeen children (11 boys and 6 girls; University Hospital, Lund SE-221 85, Sweden. E-mail: [email protected] median age 6 years, range 1–14 years) with ALLs harboring the Received 13 March 2007; revised 8 June 2007; accepted 28 June t(12;21), as ascertained by reverse transcription polymerase 2007; published online 9 August 2007 chain reaction (RT-PCR) and/or FISH, were included in the study Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2138 (Table 1); 14 samples were obtained at diagnosis and 3 (nos. 3, 6 were t(12;21)-positive, was used.22 The high hyperdiploid and 12) at relapse. All cases were analyzed cytogenetically cases (n ¼ 17) were excluded for reasons outlined in ‘Supple- using standard protocols and were molecularly screened for the mentary Information, Data analyses’. Since no information on presence of MLL rearrangements and BCR/ABL1, TCF3/PBX1 gender was available in the external data set, hierarchical (also known as E2A/PBX1) and ETV6/RUNX1 fusion genes at the clustering analysis of seven top ranking genes determined by Department of Clinical Genetics, Lund, Sweden, as a part of significance analysis of microarrays from a k-nearest neighbor routine diagnostic procedures. The study was approved by the classifier developed by us23 was used to determine the sex Research Ethics committee of Lund University. of each case.

Array CGH 20,21 RT-PCR, RQ-PCR, sequencing and bisulfite sequencing The array CGH was performed as previously described. Expression of the SPANX, HMGB3, FAM50A, HTATSF1 and DNA was analyzed using 32K array slides containing 32 433 RAP2C genes was investigated by RT-PCR and real-time bacterial artificial chromosome (BAC) clones (BACPAC quantitative RT-PCR (RQ-PCR). DNA fragments amplified Resources, Oakland, CA, USA) covering 98% of the human with SPANXB-specific primers were sequenced using the genome at a 100 kb resolution (Swegene DNA Microarray BigDye Terminator v1.1 Cycle Sequencing Kit (Applied Bio- Resource Center, Lund University). Detailed information is systems, Foster City, CA, USA) with the same primers as in the available in Supplementary Information. PCR and analyzed on an ABI PRISM 3100-Avant genetic analyzer (Applied Biosystems). SPANXB promoter methylation cDNA microarray was studied using bisulfite sequencing of genomic DNA. The cDNA microarray analyses have previously been 18 Detailed information for the RT-PCR, RQ-PCR and bisulfite reported. In short, samples from 87 B-lineage ALLs, of which sequencing is available in Supplementary Information. 20 harbored the ETV6/RUNX1 fusion gene, were hybridized to 27K microarray slides containing 25 648 cDNA clones (Swe- gene DNA Microarray Resource Center) representing 13 737 FISH Unigene clusters and 11 592 Entrez gene entries, according to The array CGH findings for chromosomes X, 6 and 11 (see Unigene build 188. RNA extraction, amplification, labeling, Results) were confirmed in three cases using metaphase FISH. hybridization, scanning, post-hybridization washing and feature Lack of material precluded FISH analyses of the three remaining 18 analysis were performed as described. cases with dup(Xq). BACs (BACPAC resources) and whole chromosome paint probes for 6, 11 and X were used (Abbott, Data analyses Abbott Park, IL, USA). ETV6/RUNX1-positive metaphases were The images from the CGH and gene expression arrays were identified using the dual-color translocation probe LSI TEL/ analyzed using the GenePix Pro 4.0 software (Axon Instruments, AML1 ES (Abbott). Foster City, CA, USA), and the obtained data matrices were uploaded to the BioArray Software Environment. Detailed information is available in Supplementary Information. Results Abnormalities revealed by array CGH External data set In total, 45 imbalances (median size 24 Mb; range 1–181 Mb), To validate our findings in ALLs with ETV6/RUNX1, an external excluding copy number polymorphisms, were found in 14 data set consisting of 118 pediatric B-lineage ALLs, of which 20 (82%) of the 17 cases (Table 2). The imbalances comprised 17

Table 1 Cytogenetic ﬁndings in the 17 ETV6/RUNX1-positive ALLs

Case no. Sex Age (years) Karyotype

1 M 5 46,XY,t(12;21)(p13;q22)/47,idem,+16/47,idem,der(6)t(X;6)(?;q1?),+16 2 M 6 No mitoses 3 M 9 47,XY,t(12;21)(p13;q22),+der(21)t(12;21),inc/46,XY 4 F 1 ??,X?,del(12)(p13p13),t(12;21)(p13;q22) 5 M 6 ??,X?,t(12;21)(p13;q22),+der(21)t(12;21) 6a M 14 46–48,XY,+5,add(11)(p1?5),del(12)(p11),t(12;21)(p13;q22),inc/46,XY 7 M 8 47,XY,t(12;21)(p13;q22),+21 8 M 5 ??,X?,der(6)t(X;6),t(12;21)(p13;q22) 9 M 5 46,XY,t(12;21)(p13;q22),add(16)(q21)/46,XY 10 M 7 46,XY,der(12)t(12;21)(p13;q22),ider(21)(q10)t(12;21) 11 F 4 46,XX,del(12)(p13p13),t(12;21)(p13;q22)/46,XX 12 F 7 44,X,ÀX,?der(1)add(1)(p36)t(1;21)(q?;q?),À4,À5,del(6)(q?21),add(11)(p11),À12, del(12)(p13p13),der(21)t(12;21)(p13;q22),+der(?)t(?;12)(?;p?),+der(?)t(?;12)(?;q?),+der(?)t(?;21)(?;q?),inc/46,XX 13 F 6 46,XX,del(6)(q21),t(12;21)(p13;q22)/47,XX,t(12;21),+21/47,XX,t(12;21),+der(21)t(12;21) 14 F 3 47–48,XX,t(12;21)(p13;q22),+21,inc/46,XX 15 M 9 ??,X?,t(12;21)(p13;q22) 16 F 7 46,XX,t(12;21)(p13;q22)/46,XX 17 M 4 ??,X?,der(11)t(X;11),t(12;21)(p13;q22),+der(21)t(12;21) Abbreviations: ALLs, acute lymphoblastic leukemias; array CGH, array-based comparative genomic hybridization; FISH, ﬂuorescence in situ hybridization; M, male; F, female. Abnormalities identiﬁed by FISH following array CGH are indicated in bold type. aThe karyotype of case 6 has previously been reported.19

Leukemia Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2139 deletions and 28 gains, of which 13 deletions and 18 gains Gain of whole chromosomes was observed in four cases: þ X were not described in the original karyotypes. Of these, 12 in case 14, þ 5 in case 6, þ 16 in cases 1 and 7, and þ 21 (or imbalances were missed because of karyotypic failures (cases 2, rather 21q; gain of 21p cannot be identiﬁed by array CGH) in 8, 15 and 17) and 6 because they were too small (1–12 Mb) to be cases 7 and 14 (Figure 1; Table 2). identiﬁed by conventional chromosome banding. The remaining Deletions of 9p were observed in two (12%) of the cases (nos. 13 imbalances, varying in size between 17 and 155 Mb, most 1 and 6). Case 1 had a 17 Mb hemizygous deletion between likely escaped detection owing to either poor chromosome 9p21.2 and 9p23 with a 3 Mb homozygous deletion in 9p21.3. morphology or similar banding patterns of the involved Case 6 had a 3 Mb hemizygous deletion between 9p21.3 and chromosomes. Small deletions of 1 Mb at 2p11.2 and 9p22.1 with a 1 Mb homozygous deletion in 9p21.3. In both 14q32.33 were found in most cases; these regions contain the cases, the homozygous deletion included the tumor suppressor IGK@ and IGH@ loci, respectively, and the deletions are most CDKN2A. Hemizygous 12p deletions, including at least 50 likely the result of somatic immunoglobulin rearrangements genes as well as ETV6 and CDKN1B, were found in three (18%) clonotypic for the leukemic blasts.20 of the cases (nos. 4, 6 and 11; Figure 1; Table 2).

Table 2 Genomic imbalances in 17 ETV6/RUNX1-positive ALLs

Case no. Chromosome segments Position (Mba) Size (Mb) Gain/loss Identiﬁed by involved in imbalances G-banding/FISHb

1 6q14.1–q27 82–171 89 L Yes 9p21.2–p23 10–27 17 L No 9p21.3–p21.3 20–23 3 L No 16p13.3–q24.3 0–89 89 G Yes Xq21.31–q28 89–155 66 G No 2 11q22.3–q25 106–134 29 L No 12p13.2–p13.33 0–12 12 G No 21q21.2–q22.12 26–35 9 G No Xq21.32–q28 92–155 63 G No 3 12p13.2–p13.33 0–12 12 G Yes 21q11.2–q22.12 14–35 21 G Yes Xq25–q28 123–155 32 G No 4 12p12.1–13.33 2–22 20 L Yes 5 12p13.2–p13.33 0–12 12 G Yes 21q11.2–q22.12 14–35 21 G Yes 6 5p15.33–q35.3 0–181 181 G Yes 9p21.3–p22.1 19–22 3 L No 9p21.3–p21.3 21–22 1 L No 12p13.2–p13.33 0–12 12 G No 12p12.1–p13.2 12–25 13 L Yes 21q11.2–q22.12 14–35 21 G No 7 10p11.1–p15.3 0–39 39 G No 16p13.3–q24.3 0–89 89 G No 21p11.2–q22.3 14–47 33 G Yes 8 5q34–q35.3 166–181 15 G No 6q14.1–q27 82–171 89 L No 11q14.1–q25 84–134 50 L No Xq21.31–q28 87–155 68 G No 9 Xq25–q28 118–155 37 G No 10 6q14.1–q22.1 77–115 38 L No 6q24.3–q27 147–171 24 L No 8q13.3–q24.3 72–146 74 G No 13q13.1–q31.1 32–84 52 L No 12p13.2–p13.33 0–12 12 G Yes 21q11.2–q22.12 14–35 21 G Yes 11 12p12.3–p13.2 11–17 6 L Yes 19q13.32–q13.33 52–55 3 L No 12 No imbalances 13 No imbalances 14 18p11.23–18p11.32 0–7 7 L No 18p11.23–q23 8–76 68 G No 21p11.2–q22.3 14–47 33 G Yes Xp22.33–q28 0–155 155 G No 15 12p13.2–p13.33 0–12 12 G No 21q11.2–q22.12 14–35 21 G No 16 No imbalances 17 11q14.3–q25 89–134 45 L No Xq25–q28 123–155 32 G No Abbreviations: ALLs, acute lymphoblastic leukemias; BAC, bacterial artificial chromosome; FISH, fluorescence in situ hybridization. Gains involving Xq are indicated in bold type. aAccording to BAC mapping provided by BACPAC. bFISH with locus-specific probes for the ETV6 and RUNX1 genes.

Leukemia Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2140

Figure 1 DNA copy number heat map based on array CGH of 17 ETV6/RUNX1-positive childhood ALLs. Each row represents the genome-wide copy number proﬁle in respective case, indicated on the left. Data from the 32 433 BACs were ordered based on the chromosomal position of the BAC; chromosome numbers are indicated above the heat map. Red indicates gain of material, green indicates loss of material and black indicates no change from reference as illustrated in the test/reference log2-scale indicated on top left. (a) DNA copy number changes in 11 ALLs from male patients. Several imbalances previously described in t(12;21)-positive ALLs, such as an extra der(21)t(12;21) and deletions of 6q, 9p and 12p, are seen. Gain of the distal part of Xq is seen in six cases (cases 1, 2, 3, 8, 9 and 17). (b) Partial gain of Xq is not present in any of the female cases. Abbreviations: ALLs, acute lymphoblastic leukemias; array CGH, array-based comparative genomic hybridization; BACs, bacterial artiﬁcial chromosomes.

A 12 Mb duplication distal of 12p13.2 was present in six the additional chromosome X material was localized on (35%) of the cases (nos. 2, 3, 5, 6, 10 and 15). In all these, gain chromosome 6 (in cases 1 and 8) (Supplementary Figure 1) of 21q was also seen, a finding consistent with an additional and on chromosome 11 (in case 17). Array CGH showed that copy of the der(21)t(12;21), as also verified by FISH in cases 3, 5 both the cases with chromosome 6 involvement had an 89 Mb and 10. In five of the cases (all except no. 2), the gained 21q loss of material on this chromosome. Deletions involving 6q material spanned at least 21 Mb between 21q11.2 and q22.12. were also present in case 10; this case, however, lacked gain As only a few BACs map to 21p, this region was not informative. of Xq (Figure 1; Table 2). The gain of material on chromosome X In case 2, the gain involved a smaller 9 Mb region between and loss of material on chromosome 11 in case 17 were less 21q21.2 and q22.12; hence, not the entire der(21)t(12;21) was evident, with log2 values around À0.2, possibly indicating duplicated (Figure 1; Table 2). that these imbalances were not present in all cells (Figure 1). The array CGH confirmed 12 (86%) of the 14 imbalances Taken together, the FISH and array CGH results indicated the identified cytogenetically in cases 1–11 and 14–17 (see below presence of a der(6)t(X;6)(q21.31;q14.1) in cases 1 and 8 and a for nos. 12 and 13; Figure 1; Table 2). Only the two imbalances der(11)t(X;11)(q25;q14.3) in case 17. The breakpoints on 6q resulting from the þ der(21)t(12;21), inferred from interphase in nos. 1 and 8 were both mapped to the adjacent BACs RP11- FISH analysis, in case 17 could not be confirmed by array 705B7 and RP11-684M3, suggesting similar if not identical CGH. The þ der(21) may have been present only in a small breakpoints at 6q. This region, however, does not contain any subclone or the duplicated fusion gene did not arise through named genes. gain of the entire derivative chromosome 21. As regards cases In addition to case 17, with the der(11)t(X;11), two cases (nos. 12 and 13, none of the imbalances identified by G-banding and 2 and 8) had hemizygous deletions of 11q, both accompanied FISH was seen using array CGH, suggesting a selection of cells by gain of Xq material. Case 2 displayed a 29 Mb deletion distal with secondary chromosomal changes during culture before the of 11q22.3, whereas case 8 had a 50 Mb deletion starting cytogenetic analysis. at 11q14.1 (Figure 1; Table 2). Unfortunately, no material from case 2 was available for FISH analysis; thus, a possible presence of a der(11)t(X;11) in this case could not be Array CGH reveals duplication of Xq material in ascertained. In case 8, the extra Xq material was part of the males only der(6)t(X;6) described above. The previously unrecognized abnormality dup(Xq) and Only two of the six cases with gain of Xq material had similar þ der(21)t(12;21) were the two most common aberrations found proximal Xq breakpoints, but all gains included the telomeric by array CGH; they were both detected in six (35%) of the cases. end of Xq. The largest duplication was 68 Mb (Xq21q28) in case Interestingly, all patients with dup(Xq) were male. Thus, 6 (55%) 8, and the smallest was 32 Mb (Xq25q28) in cases 3 and 17. of 11 boys displayed this abnormality (nos. 1, 2, 3, 8, 9 and 17; Table 2). In contrast, none of the six girls had a similar gain, although þ X was seen in one of them (no. 14; Figure 1; Impact of genetic imbalances on gene expression Table 2). The Xq gains had not been detected in any of the Visualization of the gene expression data using the CGH-plotter cases where G-banding had been performed; in hindsight, provided a tool for observing the effect of genomic imbalances however, the extra Xq material in case 9 (Table 1) is most likely on gene expression levels. This analysis showed that on the add(16)(q21). To investigate the Xq gain further, cases both deletions and duplications had a strong impact on the with available material were studied with FISH, revealing that expression level of genes in the affected regions, as exemplified

Leukemia Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2141 in case 1 with a der(6)t(X;6) (Supplementary Figure 2). The the significantly higher expression of SPANXB (P ¼ 0.00078), strong impact of the Xq duplication on gene expression HMGB3 (P ¼ 0.033) and FAM50A (P ¼ 0.023) in t(12;21)- was further confirmed by plotting the distribution of t-statistics positive ALLs compared to t(12;21)-negative (one-sided for genes in the minimally gained Xq region for t(12;21)- Wilcoxon rank sum test; Supplementary Figure 5). In particular, positive cases both with and without gain of Xq (Supplementary expression of SPANXB was associated with t(12;21) status as this Figure 3). gene was clearly expressed in all t(12;21)-positive cases, but was Data from the cDNA microarrays were also used to study the not, or only barely, expressed in the other ALLs (Supplementary expression of suggested tumor suppressor genes in commonly Figure 4). To investigate if the expression of SPANXB was deleted regions. A low expression level of CDKN2A was found regulated by promoter methylation, we examined the methyl- in the two cases with homozygous 9p deletions, but also in nine ation status of the SPANXB promoter region in both SPANXB- additional cases. Two of the three cases with 11q deletions positive and -negative cases using bisulfite sequencing. had analyzable expression data for the ATM gene, suggested to Previously, the expression of SPANXB, which in normal tissues be the critical target of 11q deletions in chronic B- and T-cell is confined to testis, was shown to be regulated by demethyla- neoplasms.24 Both displayed a low expression of this gene. In tion of specific CpG dinucleotides within a 500 bp region the three cases with 12p deletions, ETV6 was underexpressed, of the SPANXB promoter.25 As expected, we observed that the whereas low expression of CDKN1B was seen in only one CpG dinucleotides in the promoter region of the SPANXB- of the cases. An additional three cases (nos. 9, 13 and 16) negative cases were all methylated. However, despite the also displayed a low ETV6 expression; array CGH data for observed expression of SPANXB in the t(12;21)-positive ALLs, these cases showed log2 ratios around À0.2 for BACs near no demethylation was seen in these cases, regardless of sex. This ETV6, suggesting deletions not present in all cells or small suggests that expression of SPANXB is regulated by other CpG intragenic deletions. dinucleotides or yet to be determined regulatory elements in the vicinity of the SPANXB promoter.

Integrative genomics identifies several putative target genes within the commonly gained Xq region Discussion Given the frequent occurrence (55%) of Xq gain in males and the strong impact of genomic imbalances on gene expression, To characterize molecularly pediatric ALLs with ETV6/RUNX1, we studied the expression of genes on the X chromosome by 17 cases were investigated with genome-wide high-resolution plotting smoothed (25-probe sliding mean) z-scores (Figure 2a). array CGH and cDNA microarray gene expression analyses. The six cases (nos. 1, 2, 3, 8, 9 and 17; Tables 1 and 2) with In accordance with previous studies, an additional copy of gain of Xq displayed a distinct overexpression of genes the normal or derivative chromosome 21 and deletions at within the duplicated segment. An overexpression of genes on 12p, 9p, and 6q13,14,26 were identified as common abnor- the entire X chromosome in case 14, with gain of the whole malities. The expression levels of suggested target genes in these chromosome by array CGH, was also seen. regions were also found to be altered. To validate the common overexpression of Xq genes in The two most common genomic imbalances in this study t(12;21)-positive ALLs, an external gene expression profiling were duplication of the der(21)t(12;21) and partial gains of Xq. data set was used.22 In this, 7 of the 20 ETV6/RUNX1-positive As regards X chromosome changes in t(12;21)-positive ALLs, cases had an enrichment of relatively overexpressed genes, previous studies have mainly reported a high frequency of consistent with a gain of Xq, in a region coinciding with losses, not gains.14 To the best of our knowledge, only eight the minimally 32 Mb gained region identified by array CGH ETV6/RUNX1-positive ALLs with partial Xq gains, as iden- (Figure 2b). Notably, six of the seven patients were predicted to tified by metaphase CGH or multicolor FISH, have been be males using an expression based model (see Materials and described.15–17,27–29 All the gains in these cases were generated methods), further demonstrating the male predominance of through unbalanced translocations involving Xq, none of which this abnormality. The external data set contained 127 of the 180 were seen by conventional cytogenetics. Notably, all cases genes with assigned gene symbols in the minimally gained with Xq gain in the present study were males, as were six region, and t-statistics on the 127 genes revealed that the most of the previously published cases;16,17,27–29 the gender of the differentially expressed genes between leukemias positive and remaining two cases was not reported.15 Hence, this abnorm- negative for ETV6/RUNX1 were several members of the highly ality seems to be quite specific for ETV6/RUNX1-positive ALLs homologous SPANX family of genes (unknown function) along occurring in boys. Of the six cases in the present study, two had with the genes HMGB3 (transcription factor), FAM50A (un- similar unbalanced translocations between chromosomes X and known function), HTATSF1 (transcription elongation factor) and 6, and one had an unbalanced der(11)t(X;11). According to the RAP2C (member of RAS oncogene family) (Supplementary Mitelman database of chromosome aberrations in cancer,26 Table 1). A similar overexpression, specific for t(12;21)-positive only four such translocations have previously been described in cases, was seen in our expression data18 for the genes HMGB3 childhood ALL.15–17,30 Interestingly, three of these cases were and FAM50A, but not HTATSF1 and RAP2C. None of the ETV6/RUNX1 positive.16,17,30 None of these abnormalities had SPANX genes were, however, present on our microarrays.18 We been identified by G-banding; hence, it is possible that, owing to therefore used RT-PCR and sequencing to identify which of the the similar banding pattern of the involved chromosome known SPANX genes were expressed in five ETV6/RUNX1- arms in combination with the poor banding quality often found positive ALLs. SPANXB was clearly expressed in all t(12;21)- in childhood ALL, der(6)t(X;6) and der(11)t(X;11) may go positive cases examined (Supplementary Figure 4), but none undetected unless specifically looked for by FISH. of the genes SPANXA1, SPANXA2, SPANXC and SPANXD While this manuscript was being prepared for submission, were expressed in any of the cases (data not shown). We then a large study of childhood ALL using high-resolution single- examined the expression of SPANXB, HMGB3, FAM50A, nucleotide polymorphism arrays was published, where altera- HTATSF1 and RAP2C in eight t(12;21)-negative cases and five tions of genes important for B lymphocyte development and t(12;21)-positive cases using RQ-PCR. This analysis confirmed differentiation were found in 40% of B-progenitor ALLs.31 Small

Leukemia Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2142

deletions of the PAX5 gene, the majority below the resolution of Gain of Xq was not discussed as a common ﬁnding in ETV6/ the platform used in the present study, were found to be the RUNX1-positive ALLs, but on careful examination we note that most common alterations. As in our study, deletions at 12p, 11q, Xq gain was present in 10 (21%) ETV6/RUNX1-positive cases, 6q and 9p were found to be common in t(12;21)-positive ALLs. conﬁrming the high prevalence of Xq duplications in this genetic

Leukemia Analyses of ETV6/RUNX1-positive ALL reveal frequent Xq duplications H Lilljebjo¨rn et al 2143

Figure 2 Relative overexpression of genes mapping to Xq25–q28 in t(12;21)-positive ALLs. The relative expression of genes located on the X chromosome in B-lineage ALLs in the data sets of Andersson et al.18 (a) and Ross et al.22 (b–d) is presented graphically. The genetic subtype and the gender of the ALL patients in the respective data sets are indicated above images (a) and (b). NK denotes normal karyotype and n/a not available. The color scale below each heat map represents z-scores; red indicates positive values, green indicates negative values and black indicates values close to zero. (a) To emphasize spatially localized expression patterns in 58 B-lineage ALLs in the data set of Andersson et al.,18 smoothed z-scores (25-probe sliding mean) were plotted for all cases. The six cases where dup(Xq) was identified by array CGH are indicated above the heat map. In all cases with gain of Xq material, the gain results in an enrichment of relatively overexpressed genes located on distal Xq, as indicated by red segments in these cases. (b) The mean z-scores over 25 probe sets were plotted for 101 B-lineage ALLs in the data set of Ross et al.22 The seven t(12;21)-positive cases indicated by arrows exhibit an enrichment of relatively overexpressed genes distal of Xq25, consistent with a gain of Xq material. Six of these cases were predicted to be males. (c) Enlarged view of the genes located distal of Xq25, indicated by * in image (b), at the single gene level. A probe set (220922_s_at) detecting several genes in the SPANX family is marked by an arrow. This probe set displays a significant overexpression in t(12;21)-positive cases, regardless of gender. (d) The relative expression detected by the probe set is plotted for each case. The y-axis indicates z-scores, whereas each case is separated on the x axis as in images (b) and (c). Cases negative for t(12;21) are colored green and cases positive for t(12;21) are colored red. A t(12;21)-specific overexpression of one or more of the genes recognized by this probe set is clearly indicated. Abbreviations: ALLs, acute lymphoblastic leukemias; array CGH, array-based comparative genomic hybridization. subtype. Interestingly, we found that PAX5 alterations and Xq been shown to be primarily regulated by promoter methyla- duplications were mutually exclusive in this data set, possibly tion,25 providing a mechanism for the re-activation in females. indicating that the two changes represent different genetic However, examining the methylation status of the SPANXB pathways of disease evolution in t(12;21)-positive ALL. promoter in both t(12;21)-positive and negative cases, we The high prevalence of Xq gains indicates that one or several observed no correlation between promoter methylation and genes in the 32 Mb minimally gained region are important SPANXB expression. This does not, however, exclude the for leukemogenesis. The striking lack of this abnormality in possible involvement of CpG dinucleotides or yet to be t(12;21)-positive ALLs in girls may be explained by the special determined regulatory elements in the vicinity of the SPANXB characteristics of the X chromosome, where one of the X promoter as important regulators of SPANXB expression. chromosomes is inactivated in normal female cells.32 It is Sporadic expression of SPANXB has previously been demons- tempting to speculate that re-activation of one or several trated in cancer cell lines and various hematologic malignan- genes on the inactive X chromosome in girls could result in cies, such as multiple myeloma, chronic lymphocytic leukemia, similar effects as in boys with gain of Xq. Interestingly, chronic myeloid leukemia and acute myeloid leukemia, but it re-activation of a subset of genes on the inactive X chromosome has never been linked to any specific genetic subgroups.25,34 So has recently been described as an important mechanism in far, the function of SPANXB or the other SPANX genes has not basal-like breast cancer.33 been studied in detail, but all five SPANX genes are present on By investigating the expression of genes in the minimally the 750 kb Xq region linked to prostate cancer susceptibility, gained region in the data sets produced by us18 and Ross et al.,22 and deregulation of either one has been suggested as a possible we could show that several genes were specifically expressed in mechanism for malignant transformation of prostate cells.35 This ETV6/RUNX1-positive ALLs. These included a number of genes is supported by experimental models showing that ectopic in the highly homologous SPANX family (normally expressed expression of SPANXC induces transformation in mammalian only in spermatozoa) and the genes HMGB3 (transcription cells.35 The association between SPANXB expression and factor affecting hematopoietic stem cell fate and Wnt signaling), presence of ETV6/RUNX1 may also be of importance for future FAM50A (unknown function), HTATSF1 (transcription elonga- treatment modalities, as SPANXB, normally present only in the tion factor) and RAP2C (member of the RAS oncogene family). immune-privileged testis, has been suggested to be an ideal Because we deemed it unlikely that t(12;21)-positive ALL in target for tumor immunotherapy.34 males would evolve through different mechanisms than in females, we focused on genes on Xq that also showed upregulation among females. Such an expression pattern was, Acknowledgements in particular, exhibited by the genes in the SPANX family. Using RT-PCR, we could further show that SPANXB, and not the other This work has been supported by grants from the Swedish SPANX family members, was expressed in all t(12;21)-positive Children’s Cancer Foundation, the Swedish Cancer Society, cases. The genes in the SPANX family share more than 90% the Swedish Research Council and the Medical Faculty at sequence identity; therefore, the detection of SPANX genes Lund University. We thank the Swegene DNA Microarray other than SPANXB in the gene expression profiling data Resource Center in Lund, supported by the Knut and Alice set by Ross et al.22 is most likely the result of cross-hybridization Wallenberg Foundation through the Swegene consortium, for with SPANXB. providing BAC arrays. Although it is presently unclear which gene(s) within the The expression data discussed in this publication have been commonly gained Xq region that are important in the deposited in NCBI’s Gene Expression Omnibus (GEO, http:// leukemogenic process, we conclude that of the genes identified, www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO SPANXB provides an attractive target of the dup(Xq). This gene Series accession number GSE7186. is normally expressed only in spermatozoa and distinguishes itself as the most differentially expressed gene between t(12;21)- References positive and -negative ALLs. We suggest that this could be the result of a gain of Xq material, followed by a clonal selection of 1 Gustafsson G, Schmiegelow K, Forestier E, Clausen N, Glomstein cells showing an upregulation of SPANXB, in a majority of A, Jonmundsson G et al. Improving outcome through two decades in childhood ALL in the Nordic countries: the impact of high-dose males. In females, a re-activation on both alleles would be an methotrexate in the reduction of CNS irradiation. Nordic Society of alternative way by which SPANXB becomes upregulated. It is, in Pediatric Haematology and Oncology (NOPHO). Leukemia 2000; this context, noteworthy that SPANXB expression has previously 14: 2267–2275.

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