PAX5 Deletions Research Articles

Evaluation of multiplex ligation‐dependent probe amplification as a method for the detection of copy number abnormalities in B‐cell precursor acute lymphoblastic leukemia

Recent genomic studies have shown that copy number abnormalities (CNA) of genes involved in lymphoid differentiation and cell cycle control are common in B-cell precursor acute lymphoblastic leukemia (BCP-ALL). We have evaluated Multiplex Ligation-dependent Probe Amplification (MLPA) on 43 BCP-ALL patients for the detection of the most common deletions among these genes and compared the results to those obtained by fluorescence in situ hybridization (FISH) and genomic quantitative PCR (qPCR). There was good correlation between methods for CDKN2A/B, IKZF1, and PAX5 deletions in the majority of cases and MLPA confirmed the presence of deletions within the PAR1 region in two of three cases identified by FISH. Small intragenic aberrations detected by MLPA, which were below the resolution of FISH for CDKN2A/B (n = 7), IKZF1 (n = 3), and PAX5 (n = 3) were confirmed by qPCR. MLPA and qPCR were unable to detect populations present at a low level (<20%) by FISH. In addition, although MLPA identified the presence of a deletion, it was unable to discern the presence of mixed cell populations which had been identified by FISH: CDKN2A/B (n = 3), IKZF1 (n = 1), PAX5 (n = 2), and PAR1 deletion (n = 1). Nevertheless, this study has demonstrated that MLPA is a robust technique for the reliable detection of CNA involving multiple targets in a single test and thus is ideal for rapid high throughput testing of large cohorts with a view to establishing incidence and prognostic significance.

The PAX5 gene is frequently rearranged in BCR-ABL1-positive acute lymphoblastic leukemia but is not associated with outcome. A report on behalf of the GIMEMA Acute Leukemia Working Party

Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric patients with acute lymphoblastic leukemia. So far the occurrence of PAX5 alterations and their clinical correlation have not been investigated in adults with BCR-ABL1-positive acute lymphoblastic leukemia. The aim of this study was to characterize the rearrangements on 9p involving PAX5 and their clinical significance in adults with BCR-ABL1-positive acute lymphoblastic leukemia. Eighty-nine adults with de novo BCR-ABL1-positive acute lymphoblastic leukemia were enrolled into institutional (n=15) or GIMEMA (Gruppo Italiano Malattie EMatologiche dell'Adulto) (n=74) clinical trials and, after obtaining informed consent, their genome was analyzed by single nucleotide polymorphism arrays (Affymetrix 250K NspI and SNP 6.0), genomic polymerase chain reaction analysis and re-sequencing. PAX5 genomic deletions were identified in 29 patients (33%) with the extent of deletions ranging from a complete loss of chromosome 9 to the loss of a subset of exons. In contrast to BCR-ABL1-negative acute lymphoblastic leukemia, no point mutations were found, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1-positive acute lymphoblastic leukemia. The deletions were predicted to result in PAX5 haploinsufficiency or expression of PAX5 isoforms with impaired DNA-binding. Deletions of PAX5 were not significantly correlated with overall survival, disease-free survival or cumulative incidence of relapse, suggesting that PAX5 deletions are not associated with outcome. PAX5 deletions are frequent in adult BCR-ABL1-positive acute lymphoblastic leukemia and are not associated with a poor outcome.

Abstract 1600: Molecular Characterization of PAX5 alterations in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL)

Abstract Background: Recently, a genome-wide analysis using single nucleotide polymorphism (SNP) microarrays, identified genetic alterations targeting PAX5 in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, the occurrence of PAX5 alterations has not been investigated in adult BCR-ABL1-positive ALL. Aim: To characterize mutations and rearrangements on 9p involving PAX5 in adult BCR-ABL1-positive ALL. Patients and Methods: Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results: SNP array analysis identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was heterozygous. Four patterns of PAX5 deletion were observed: focal deletion involving only the PAX5 gene in one case (3%); deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in PAX5 haploinsufficiency or altered gene expression. In one patient the deletion of PAX5 involved only a subset of exons (2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used: genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Normal and deleted PAX5 subgroups were also investigated for point mutations, analyzing each exon by direct sequencing, but we did not found nucleotide substitutions. Conclusion: PAX5 deletions, but not point mutations, are frequent events in adult BCR-ABL1 positive ALL and are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1600.

Wide diversity of PAX5 alterations in B-ALL: a Groupe Francophone de Cytogénétique Hématologique study

AbstractPAX5 is the main target of somatic mutations in acute B lymphoblastic leukemia (B-ALL). We analyzed 153 adult and child B-ALL harboring karyotypic abnormalities at chromosome 9p, to determine the frequency and the nature of PAX5 alterations. We found PAX5 internal rearrangements in 21% of the cases. To isolate fusion partners, we used classic and innovative techniques (rolling circle amplification-rapid amplification of cDNA ends) and single nucleotide polymorphism-comparative genomic hybridization arrays. Recurrent and novel fusion partners were identified, including NCoR1, DACH2, GOLGA6, and TAOK1 genes showing the high variability of the partners. We noted that half the fusion genes can give rise to truncated PAX5 proteins. Furthermore, malignant cells carrying PAX5 fusion genes displayed a simple karyotype. These data strongly suggest that PAX5 fusion genes are early players in leukemogenesis. In addition, PAX5 deletion was observed in 60% of B-ALL with 9p alterations. Contrary to cases with PAX5 fusions, deletions were associated with complex karyotypes and common recurrent translocations. This supports the hypothesis of the secondary nature of the deletion. Our data shed more light on the high variability of PAX5 alterations in B-ALL. Therefore, it is probable that gene fusions occur early, whereas deletions should be regarded as a late/secondary event.

Array comparative genomic hybridization analysis of adult acute leukemia patients

We have performed a retrospective array-based comparative hybridization (array-CGH) study on 41 acute leukemia samples [ n = 17 acute lymphoblastic leukemia (ALL) patients only at diagnosis, n = 3 ALL patients both at diagnosis and relapse; n = 20 acute myeloid leukemia (AML) patients only at diagnosis and n = 1 AML patient both at diagnosis and relapse] using an Agilent 44 K array. In addition to previously detected cytogenetic aberrations, we observed cryptic aberrations in 95% of ALL and 90.5% of AML cases. ALL-specific recurrent abnormalities were RB1 ( n = 3), PAX5 ( n = 4), and CDKN2B ( n = 3) deletions; AML-specific recurrent abnormalities were HOXA9 and HOXA10 ( n = 2) deletions and NOTCH1 duplication ( n = 2). Recurrent duplication of the ELK1 oncogene was observed in both ALL ( n = 2) and AML ( n = 3) cases. Our results demonstrate that oligo-array CGH (oaCGH) is an effective method for defining copy number alterations and identification of novel recurring unbalanced abnormalities. At least for now, however, the use of oaCGH for routine diagnosis still has some restrictions.

PAX5 Wild-Type without IKZF1 (Ikaros) Deletion Is Associated with Prolonged Disease-Free Survival and Low Rate of Cumulative Incidence of Relapse in Adult BCR-ABL1-Positive Acute Lymphoblastic Leukemia (ALL): On Behalf of GIMEMA AL Working Party.

Abstract 12 Background:Recently, in genome-wide analyses of DNA copy number abnormalities using single nucleotide polymorphism (SNP) microarrays, genetic alterations targeting PAX5 were identified in over 30% of pediatric acute lymphoblastic leukemia cases (Mullighan et al. Nature 2008). However, so far, the occurrence of PAX5 alterations and their clinical correlation has not been investigated in adults with BCR-ABL1-positive ALL. Aim:To characterize the rearrangements on 9p involving PAX5 and their clinical significance in adult BCR-ABL1-positive ALL. Patients and Methods:Eighty-nine patients with de novo adult BCR-ABL1-positive ALL were enrolled into institutional (n = 15) or Gruppo Italiano Malattie Ematologiche Maligne dell'Adulto Acute Leukemia Working Party (n = 74) clinical trials and after obtaining informed consent their genome was analyzed by SNP arrays (Affymetrix 250K NspI and SNP 6.0), FISH, genomic PCR and resequencing. Results:By SNP array analysis we identified a loss of PAX5 in 29 patients (33%). In all cases the deletion was hemizygous. Four patterns of PAX5 deletion were observed: 1) focal deletion involving only the PAX5 gene in one case (3%); 2) deletions involving only a portion of PAX5 and both telomeric and centromeric genes in 11 cases (38%) with a median size of 310 kb (range: 101 kb-16,395 kb); 3) broader deletions involving the entire PAX5 locus and a variable number of flanking genes in 10 patients (34%) with a median size of 1,999 kb (range: 567 kb-1,208 kb); 4) deletion of all chromosome 9 or 9p in 7 patients (24%). These deletions may result in either PAX5 haploinsufficiency or expression of PAX5 alleles with impaired DNA-binding or transcriptional activation activity. In one patient the deletion of PAX5 involved only a subset of exons (exons 2-6) resulting in an alternative transcript encoding a prematurely truncated protein lacking key PAX5 functional domains. To investigate the consequences of genomic PAX5 alteration, quantitative PCR (q-PCR) was used, demonstrating that genomic alterations on 9p13.2 lead to a significant down-modulation at the transcript level of Pax5. Furthermore, both normal and deleted PAX5 subgroups were investigated for point mutations by sequencing analysis but we did not found nucleotide substitutions, suggesting that deletions are the main mechanism of inactivation of PAX5 in BCR-ABL1 positive ALL. However, when we investigated the association of PAX5 deletions with clinical outcome, no association with PAX5 status was detected (overall survival: p=0.3294; disease free-survival,DFS: p=0.9249 and cumulative incidence of relapse, CIR: p=0.945), suggesting that PAX5 deletions are not associated with outcome. Twenty-three patients (26%) had deletion of both PAX5 and IKZF1, encoding for the transcription factor Ikaros. We assessed the contribution of both PAX5 and IKZF1 alterations on clinical outcome finding out that the occurrence of both normal PAX5 and IKZF1 loss is associated with a shorter DFS (p=0.04) and higher CIR cumulative incidence of relapse in comparison to patients with both normal IKZF1 and PAX5 (p = 0.009). These results were confirmed by multivariate analysis. Conclusion:PAX5 deletions are frequent events in adult BCR-ABL1 positive ALL and are often associated with IKZF1 loss. The genomic status of both PAX5 and IKZF1 can be useful to identify at diagnosis groups of patients with worse prognosis.Supported by: European LeukemiaNet, AIL, AIRC, FIRB 2006, Strategico di Ateneo, GIMEMA Onlus. Disclosures:No relevant conflicts of interest to declare.

Next Generation Transcriptomic Resequencing Identifies Novel Genetic Alterations in High-Risk (HR) Childhood Acute Lymphoblastic Leukemia (ALL): A Report From the Children's Oncology Group (COG) HR ALL TARGET Project.

Abstract 704▪▪This icon denotes an abstract that is clinically relevant.Relapsed ALL is a leading cause of childhood cancer death, and the biologic factors responsible for relapse are poorly understood, particularly in cases lacking sentinel chromosomal alterations. Recent studies from the Children's Oncology Group high risk ALL TARGET (Therapeutically Applicable Research to Generate Effective Targets) project that used genome-wide profiling of DNA copy number alterations and candidate gene resequencing have identified novel biomarkers of relapse (IKZF1 alteration) and therapeutic targets (JAK mutation). As a complementary approach to identify novel genomic alterations, we used second generation sequencing technology to sequence the tumor transcriptome of three cases from the COG P9906 high risk (HR) B-precursor ALL trial. The selected cases had previously been profiled by high resolution SNP and gene expression arrays and candidate gene resequencing, and lacked known sentinel chromosomal rearrangements. Each case bore features previously associated with poor treatment outcome: a gene expression profile (GEP) similar to that of BCR-ABL1 positive ALL (all cases), deletion or mutation of IZKF1 (two cases), and JAK mutation (JAK2 R867Q, one case). cDNA libraries were generated from poly-A enriched RNA and 36-50 base paired-end sequencing performed using the Illumina Genome Analyzer. Sequence alignment, variant detection and fusion transcript identification were performed using custom scripts and multiple published reference alignment and de-novo assembly algorithms. A total of 115-127 million total and 93-97 million mapped, unique reads were obtained per case. The average depth of coverage of Refseq exons ranged from 25- to 39-fold. A minimum of 5 putative fusion transcripts were identified per case, some of which were known from prior transcriptome sequencing to be recurring false positives. However, a novel transcript with an in-frame fusion of exon 9 of the striatin gene STRN3 to exon 18 of JAK2 (STRN3-JAK2) was identified in one case, and confirmed by RT-PCR and direct Sanger sequencing. Fusion of NUP214 to ABL1 was identified in a second case and also confirmed by direct sequencing. The NUP214-ABL1 rearrangement has previously only been identified in T-lineage ALL. In this case, the translocation was accompanied by amplification of the NUP214-ABL1 region at 9q. RT-PCR screening of an additional 60 high-risk ALL cases with GEP data suggestive of kinase alteration identified an additional two cases with NUP214-ABL1 fusion, each of which was accompanied by NUP214-ABL1 amplification. These two novel fusion transcripts are predicted to result in aberrant kinase signaling, and are candidates for novel therapeutic intervention. Both occurred in ALLs with a BCR-ABL1-like GEP that lacked known JAK mutations, suggesting that additional novel activating kinase mutations can be discovered via detailed sequence analysis of the 50% of BCR-ABL1-like ALLs that do not have JAK mutations. Aberrant splice variants and truncated isoforms arising from DNA copy number alterations, including internal deletions of PAX5 and truncating deletions of BTG1 were also identified using the transcriptome sequencing data. In addition, these data identified over 400 candidate non-synonymous single nucleotide and insertion/deletion variations in each patient. Known mutations involving PAX5, IKZF1 and JAK2 were robustly identified. Whole genome sequencing of matched normal DNA is underway to remove germline variation from the list of putative variants, and transcriptomic sequencing of additional cases of HR childhood ALL are being performed. Together, these data indicate that transcriptomic sequencing is a powerful method to identify novel genetic alterations in ALL, and may be used to identify novel targets for therapeutic intervention. Disclosures:No relevant conflicts of interest to declare.

What Is the Initiating Mechanism of iAMP21 in Childhood B Cell Precursor ALL?.

Abstract 581Patients with iAMP21 (intrachromosomal amplification of chromosome 21) represent a distinct cytogenetic subgroup of childhood B cell precursor ALL (BCP-ALL; incidence ∼2%) with a poor prognosis on standard therapy (29% EFS at 5 years). From cytogenetic studies the amplification of chromosome 21 appears to be the primary genetic change, however, whether this or associated abnormalities provide the initiating event of leukaemogenesis remains to be elucidated. Currently fluorescence in situ hybridisation (FISH) with probes directed to the RUNX1 gene provides the only reliable detection method. Knowledge of the underlying genetic mechanism will lead to improved diagnosis and determine those patients at high risk of relapse who require more intensive therapy. Our previous study using genomic arrays (1Mb aCGH, n=10; NimbleGen chromosome 21 custom oligonucleotide array, n=5) identified a common region of amplification (CRA) on 21q of 6.6 Mb and a common region of deletion (CRD) of 3.3 Mb within the telomeric region in 100% and 70% of patients, respectively. However, expression profiling (Affymetrix U133A, n=8) indicated that no important genes within the CRA or CRD were differentially expressed. We now have clinical, cytogenetic and FISH data on 94 iAMP21 patients (44 F, 50 M; median age 9.5y, range 2-30); the largest iAMP21 cohort investigated to date. Higher resolution analysis of 17/94 diagnostic samples by aCGH (Agilent 185K platform) have refined these regions to 5.1 Mb and 2.5 Mb in 100% and 88% of patients, respectively. Recent genome mapping has highlighted the presence of the microRNA miR-802 within the CRA and shown this region overlaps with the Down Syndrome Critical Region at 21q22.3 encompassing the genes DSCR1, DSCR3 and DYRK1. Detailed analysis of chromosome 21 identified recurrent breakpoints centromeric of the CRD within three genes: PDE9A (n=2), COL6A2 (n=2) and DSCAM (n=2). The role of these genes in the initiation of the chromosomal instability associated with iAMP21 is currently under investigation. Global analysis of the iAMP21 genome identified an average of 13 copy number alterations (CNA) per case with recurrent deletions of the leukaemia associated genes PAX5 (n=2), IKZF1 (n=4) and CDKN2A (n=4). To determine the incidences of PAX5, IKZF1 and CDKN2A deletions among the cohort of 94 iAMP21 patients, FISH using probes targeting these genes was carried out on availabel samples. IKZF1 (exons 3-6) was deleted at the highest incidence of 21% (13/62), while CDKN2A and PAX5 were deleted at a lower incidence of 17% (12/71) and 8% (4/48), respectively. The high incidence of IKZF1 deletions found in this patient group correlates with their association with high risk ALL. We have recently reported a deletion within the PAR1 region of the sex chromosomes, centromeric of the CRLF2 gene involving CSF2RA and IL3RA, which leads to deregulated expression of CRLF2. This deletion occurred at an incidence of 23% (17/73), as determined by FISH. Among pairs of matched diagnostic and relapse samples; one had this deletion at both diagnosis and relapse while the second had a deletion in the diagnostic sample only. Gain of an additional × chromosome was identified in 28% (18/65). Of note is that the 3 aberrations: abnormalities of chromosome 21, gain of the × chromosome and high incidence of deletions centromeric of CRLF2 in this patient group mirror the findings in Down Syndrome (DS) ALL, although we have not identified any mutations of the JAK2 kinase domain among this cohort of iAMP21 patients to date. This genome-wide study provides further support for the proposal that amplification of genes within the CRA on chromosome 21 is the primary event driving leukaemogenesis in iAMP21 patients. It has also highlighted that the 3 abnormalities in common with DS ALL and/or deletions of IKZF1, which all occur at a high incidence, contribute towards the progression of this high risk subtype of childhood BCP-ALL. Disclosures:No relevant conflicts of interest to declare.

Complexity of Clonal Architecture in Childhood Acute Lymphoblastic Leukaemia

Abstract LBA-5▪▪This icon denotes an abstract that is clinically relevant.Intra-clonal, mutational complexity is a hallmark feature of cancer and provides the substrate for sub-clonal selection, progression and therapeutic resistance. There is limited insight however into detailed sub-clonal, genetic architecture in cancers and their propagating 'stem' cells. Childhood acute lymphoblastic leukaemia (ALL) genotypes are characterized by chimeric fusion genes (or hyperdiploidy) coupled with recurrent, copy number alterations (CNA), primarily in genes regulating cell cycle or differentiation. For ETV6-RUNX1-positive cases of B precursor ALL, the temporal sequence of events is known with the fusion gene usually arising as a pre-natal, initiating event. Recurrent CNA, presumed to be functional or 'driver' mutations, are secondary to gene fusion and probably post-natal in origin. We have interrogated clonal architecture by multiplexed FISH analysis of single cells using probes labelled with three or four distinct fluorochromes. We pre-selected diagnostic samples from 30 ETV6-RUNX1-positive cases that we found to have deletion of ETV6 and CDKN2A or PAX5 or combinations of these. The application of multiple FISH probes allowed us to score all cells (200 per patient sample) for up to six genetic lesions: ETV6-RUNX1 fusion (and multiple copies of the fusion), deletion of the unrearranged ETV6 allele, extra copies of chromosome 21q (via RUNX1 signals), and one or two copy deletions of PAX5 and CDKN2A. The genetic classification of individual cells using this method allowed a designation of sub-clones and the assembly of putative ancestral, or evolutionary, trees. Although sub-clones were previously known to exist at diagnosis of ALL, the extent of sub-clonal diversity revealed by this study was unanticipated and very marked. Sub-clones, of varying sizes (1–90% of total cells), numbered 3 to 14 per case. This must be an under-estimate of genetic complexity as only selected mutations were included in the screen. The common CNA (ETV6, CDKN2A and PAX5 deletions, chromosome 21q gains) arose independently and recurrently in sub-clones and in no preferential order. Matched relapse and diagnosis pairs were available on 5 patients. In all of these, the clones at relapse could be matched to individual sub-clones at diagnosis, with relapse originating from a major or minor subclone at diagnosis. Importantly, the relapse clone often diversified further, reiterating the evolution pathway of the primary clones. These data indicate that sub-clonal diversification does not arise via linear, clonal succession but rather has a complex, branching architecture reminiscent of Charles Darwin's 1837 evolutionary speciation model. The ancestral trees so revealed in ALL are single time point snapshots and therefore disguise temporal or sequential dynamics. We show that sub-clonal architecture changes in the months leading up to a diagnosis of ALL and is re-ordered by treatment and subsequent relapse. An important prediction derived from this pattern of sub-clonal architecture is that leukaemia propagating or 'stem' cells in ALL (and other cancers) should themselves be genetically diverse. Preliminary transplantation experiments in NOD/SCID/IL2Rgamma(null) mice show that this is the case, since multiple, genetically distinct sub-clones regenerated in vivo (Figure1). The complexity of genetic architecture in ALL has substantial implications for the cancer stem cell concept and for the efficacy of generic or targeted treatments. Disclosures:No relevant conflicts of interest to declare. [Display omitted]

The Role of Zinc Finger Protein 521/Early Hematopoietic Zinc Finger Protein in Erythroid Cell Differentiation

ZNF521 (zinc finger protein 521) is a transcription factor with an N-terminal transcriptional repressor motif and 30 zinc finger domains. Although a high expression level of ZNF521 in human CD34+ progenitors and hematopoietic malignancies has been demonstrated, the functional role of ZNF521 in hematopoietic cell differentiation has not been clarified. In this study, we analyzed the role of ZNF521 in erythroid cell differentiation using the short hairpin RNA (shRNA)-mediated gene silencing method. Down-regulation of ZNF521 mediated by transient expression of shRNA for ZNF521 resulted in increased synthesis of hemoglobin in K562 and HEL cell lines as compared with control cells. K562-derived clones in which ZNF521 was constitutively silenced by shRNA also showed marked synthesis of hemoglobin and an increased expression level of glycophorin A. Since GATA-1 is the key regulator of erythroid differentiation, the effect of ZNF521 on transcription activity of GATA-1 was analyzed using a luciferase assay. GATA-1 activity was markedly inhibited by ZNF521 in a dose-dependent manner. Deletion analysis of ZNF521 showed that the repressive effect requires an N-terminal repression motif. Furthermore, the direct interaction of ZNF521 with GATA-1 was demonstrated. These results indicate that ZNF521 modulates erythroid cell differentiation through direct binding with GATA-1.

Childhood Acute Lymphoblastic Leukemia: High Genomic Stability from Initial Diagnosis to Early Relapse.

Despite considerable improvements regarding treatment outcome about 20% of children with acute lymphoblastic leukemia (ALL) still suffer from recurrent disease. Especially patients with an early relapse occurring before 6 months of treatment cessation have a poor prognosis and, therefore, urgently need therapy optimization. As an initial step, detection of genetic aberrations that are associated with resistant disease and/or early relapse might be of great value as they could serve as prognostic markers to predict disease recurrence and, in addition, may increase our understanding of mechanisms underlying treatment resistance and early relapse. This knowledge may also help to identify new therapeutic strategies including targeted approaches. Surprisingly, almost no information is available on the genetic evolution from initial diagnosis to early relapse. As a first aim in the process of uncovering the pathomechanism of early relapse we wanted to find out if early relapse of childhood ALL reflects primarily resistant disease with little or no additional aberrations at relapse or represents evolution and selection of a new and more resistant clone possibly triggered through chemotherapy. Recently, high throughput technologies (single nucleotide polymorphism [SNP]–Arrays) have been developed to screen the whole genome with high resolution for submicroscopic, genetic alterations by simultaneously analyzing the SNP genotype and determining the copy number state. This prompted us to perform SNP-array analysis encompassing more than 100.000 SNPs in 20 childhood ALL samples collected at initial diagnosis and to compare them to their counterparts obtained at relapse. The analyzed patients belonged to the intermediate or high risk treatment group; 16 exhibited B-precursor and 4 T-precursor ALL. Exclusion criteria were translocations t(12;21), t(9;22) and (4;11) as well as pre-existing conditions (e.g. Down syndrome). At initial diagnosis, the leukemic sample of all patients already displayed a complex karyotype with multiple genetic lesions (average numbers per patient: 5.15 deletions, 1.35 amplifications, and 0.65 copy number neutral LOH (CNN-LOH)). Most alterations remained stable from initial diagnosis to relapse (99 of 103 (96%) initially observed deletions as well as 24 of 27 (89%) amplifications). Four deletions occurring in 3 different patients at initial diagnosis disappeared at relapse. Nevertheless, these patients had additional stable copy number alterations. Newly occurring aberrations at relapse were less frequent (average numbers per patient: 1.8 new deletions, 0.75 new amplifications, and 0.15 new CNN-LOH). In 3 patients genetic alterations at diagnosis and relapse were entirely identical. The most commonly affected chromosome was 9p (75% of samples). Fourteen patients had either heterozygous or homozygous deletions of CDKN2A at initial diagnosis and relapse with 2 patients progressing from heterozygous to homozygous deletions. PAX5 deletions (mainly heterozygous) were detectable in 9 patients at initial diagnosis and 10 patients at relapse. Other sides of recurrent deletions – mainly detected at relapse – included multiple areas of chromosome 7p (p21.3, p15.3, p14.3, p14.3-14.1, p14.1-p13) as well as chromosome 1q (deletion: q42.12-q44, amplification: q25.2-q31.1), chromosome 17p (deletion: p13.3-p11.2) and 17q (amplification: q21.2-q25.1). Potential pathogenetic implications of specific recurrent genetic lesions will be discussed. In conclusion, our results demonstrate a high degree of genomic stability from initial diagnosis to early relapse of childhood ALL. These results suggest that – at least in some patients – early relapse might already be predetermined by a resistant leukemic clone at the time of initial diagnosis.

Sequence of Genetic Events in ETV6-RUNX1 Positive B Precursor ALL: Insights from Identical Twins with Concordant Leukaemia

Monozygotic twin pairs with concordant ALL have provided unique insights into the molecular pathogenesis and natural history of childhood leukaemia. Data from twin pair studies and neonatal blood spot screening indicate that ETV6-RUNX1 usually arises as an early or initiating pre-natal event. Its consequence appears to be the generation of a clinically silent or covert but persistent pre-leukaemic clone. Conversion to overt, clinical ALL then requires the acquisition of one or more additional genetic lesions that functionally complement ETV6-RUNX1, often including deletions of the non-rearranged ETV6 allele. Recent genome wide single nucleotide polymorphism (SNP) array based studies have revealed considerably more genetic complexity than previously suspected, with ETV6-RUNX1 cases having an average of 6 (range 1–21) genomic losses or gains (Mullighan et al., Nature 2007, 446: 758). It is however unclear from these descriptive screens or audits when these multiple changes arise in relation to the presumed initiating gene fusion and what functional contribution they make. We have used a series of identical twin pairs with ETV6-RUNX1 positive B precursor ALL to test the proposition that, as we reported previously for ETV6 deletion (Maia et al., Blood 2001, 98: 478), all presumed functional or ‘driver’ genomic changes are post-natal in origin and therefore secondary to ETV6-RUNX1 fusion. If this were to be correct then we anticipated that genomic deletions and gains should be different or distinct within each twin pair. We used 250K Sty and 250K Nsp Affymetrix SNP mapping arrays on 5 pairs of identical twins concordant for ETV6-RUNX1 gene fusion positive ALL. We identified copy number variation using the “in-house” Genome Orientated Laboratory File v2.2.9 software package. The SNP array was performed using leukaemic DNA compared to matched remission DNA for 4 out of 5 cases. The fifth case was compared to a pool of remission DNA. The total number of genetic aberrations found was 51 (excluding T cell receptor and immunoglobulin rearrangements): 36 of these lesions were deletions (mean = 7.2) and 15 amplifications. The commonest aberration, found in 8 out of the 10 children, was a deletion on 12p13.2 involving the ETV6 gene. This was discordant in all cases, consistent with our previous reports using microsatellite markers. Other aberrations included deletions of PAX5, CDKN1B, CDKN2A and CD200/BTLA. The status of these, and other, presumed ‘drivers’ of leukaemogenesis were always different when diagnostic DNA of twins, within a pair, were compared i.e. either the genetic change was absent in one but present in the other, or the alteration was present in both but had distinct genomic boundaries. However in 2 of 5 twin pairs concordant, identical lesions were detected. These were idiosyncratic or very rare genomic changes in ALL and were either in gene sparse regions or involved loci with no known or likely contribution to B cell regulation or leukaemogenesis (e.g. CRYGD). We consider the most likely explanation for these shared genetic events in twin cases is that they arise simultaneously with (or immediately prior to) ETV6-RUNX1 fusion, and in the same incipient pre-ALL stem cell, as collateral damage or ‘passenger’ mutations. These data indicate that the common and presumed ‘driver’ genetic changes that accompany ETV6-RUNX1 in ALL are all secondary to gene fusion and most probably post-natal in origin. It remains to be established whether they contribute at all to the sustained pre-leukaemic state and whether they arise independently of each other and sequentially or as a timed suite or bolus perhaps proximate to diagnosis.

Variable breakpoints target PAX5 in patients with dicentric chromosomes: A model for the basis of unbalanced translocations in cancer

The search for target genes involved in unbalanced acquired chromosomal abnormalities has been largely unsuccessful, because the breakpoints of these rearrangements are too variable. Here, we use the example of dicentric chromosomes in B cell precursor acute lymphoblastic leukemia to show that, despite this heterogeneity, single genes are targeted through a variety of mechanisms. FISH showed that, although they were heterogeneous, breakpoints on 9p resulted in the partial or complete deletion of PAX5. Molecular copy number counting further delineated the breakpoints and facilitated cloning with long-distance inverse PCR. This approach identified 5 fusion gene partners with PAX5: LOC392027 (7p12.1), SLCO1B3 (12p12), ASXL1 (20q11.1), KIF3B (20q11.21), and C20orf112 (20q11.1). In each predicted fusion protein, the DNA-binding paired domain of PAX5 was present. Using quantitative PCR, we demonstrated that both the deletion and gene fusion events resulted in the same underexpression of PAX5, which extended to the differential expression of the PAX5 target genes, EBF1, ALDH1A1, ATP9A, and FLT3. Further molecular analysis showed deletion and mutation of the homologous PAX5 allele, providing further support for the key role of PAX5. Here, we show that specific gene loci may be the target of heterogeneous translocation breakpoints in human cancer, acting through a variety of mechanisms. This approach indicates an application for the identification of cancer genes in solid tumours, where unbalanced chromosomal rearrangements are particularly prevalent and few genes have been identified. It can be extrapolated that this strategy will reveal that the same mechanisms operate in cancer pathogenesis in general.

T(6;14)(p22;q32): a new recurrent IGH@ translocation involving ID4 in B-cell precursor acute lymphoblastic leukemia (BCP-ALL)

Translocations involving the immunoglobulin heavy chain locus (IGH@) at chromosome band 14q32 are common in mature B-cell neoplasms, but are rare in B-cell precursor acute lymphoblastic leukemia (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 inverse polymerase chain reaction (PCR) identified ID4 as the partner gene. Breakpoints 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 defines a new subgroup of BCP-ALL characterized by ID4 over-expression and CDKN2A and PAX5 deletions. Preliminary survival data suggest that this subgroup may be associated with a good response to therapy.

Rearrangement and Deletion of the PAX5 Gene in Pediatric Acute B-Cell Lineage Lymphoblastic Leukemia.

Pediatric acute lymphoblastic leukemia (ALL) is the most common malignant disease in children. The disease results from accumulation of mutations of tumor suppressor genes and oncogenes. Recently, higher resolution SNP-chips (50,000–500,000 probes) have been developed allowing us to identify genes involved at the start sites of deletions/duplications. This permitted us both to identify unbalanced translocations involving t(1;19)(q23;p13) (TCF3/PBX1) and t(12;21)(p13;q11) (ETV6/RUNX1), as well as, to find novel fusion genes involving PAX5 in B-cell lineage ALL. PAX5 gene was rearranged to a variety of partner genes including ETV6, FOXP1, AUTS2 and C20orf112. In each case, tthe C-terminal end of the PAX5 gene was replaced by the partner gene. The PAX5 fusion gene products suppressed transcriptional activity of PAX5 in a dominant negative fashion. We also found a point mutation of PAX5 at codon 26 (Val 26 Gly); and this mutated PAX5 had attenuated transcriptional activity. Expression of PAX5/C20orf112 fusion gene in a B-cell line suppressed endgenous expression of PAX5 target genes including BLK1 and CD19. Furthermore, deletion of PAX5 was common in B-cell lineage ALL (34/339 cases). PAX5 gene is localized on chromosome 9p and concurrent deletion of PAX5 and INK4A genes were frequently detected in B-cell linage ALL. PAX5 gene may behave as a tumor suppressor gene during early development of B-cells and its alteration by either fusion to another gene, point mutation, or deletion may be associated with leukemogenesis of B-cell lineage ALL.

Genome-Wide 500K SNP Array Analyses Reveal High Frequencies of Cryptic Genetic Abnormalities and Identify Recurrent Microdeletions Targeting Novel Genes in Adult Acute Lymphoblastic Leukemia.

The long-term disease-free survival rate of adult acute lymphoblastic leukemia (ALL) patients remains less than 40%, in contrast to pediatric cases where it approaches 80%. Furthermore, whereas genetic abnormalities are widely used in childhood ALL for assigning patients to prognostic risk groups, their use in adult ALL is generally restricted to the presence or absence of the t(9;22)/[BCR-ABL1]. Novel prognostic markers, allowing better treatment stratification, and new treatment targets are therefore much needed. We have investigated diagnostic samples from 45 adult ALL cases using genome-wide, high-resolution single nucleotide polymorphism (SNP) array analysis with the Affymetrix 10K, 250K Nsp, and 250K Sty chips, in total including more than 500,000 SNPs with a median inter-marker distance of <2.5 kb. This method, which has not previously been applied to adult ALL, enables the detection of genetic copy number abnormalities as well as uniparental disomies (UPDs) with a much higher resolution than cytogenetic and molecular genetic techniques. We detected 367 genetic abnormalities not corresponding to known copy number polymorphisms among the 45 cases. These comprised 94 copy number gains, 211 hemizygous deletions, 47 homozygous deletions, and 15 UPDs. All but three of the patients (93%) displayed one or more anomaly not detectable with standard genetic analyses. Most notably, we found high frequencies of deletions targeting the genes CDKN2A (P16) (21 cases; 47%), PAX5 (15 cases; 33%), IKZF1 (IKAROS) (8 cases; 18%), ETV6 (7 cases; 16%), RB1 (5 cases; 11%), and EBF1 (2 cases; 4.4%). Thirty-two cases (71%) harbored a deletion of at least one of these genes. CDKN2A deletions were homozygous in 17 cases and associated with partial UPDs in 5 cases. PAX5 deletions were always hemizygous and frequently large, including also CDKN2A in all but three patients. Notably, loss of IKZF1 was found in 5 of 10 (50%) t(9;22)/[BCR-ABL1]-positive ALL cases. Reverse transcriptase-PCR for this gene showed that some t(9;22)-positive cases without this deletion expressed a dominant-negative isoform of IKZF1, suggesting that different mechanisms for downregulation of IKZF1 occur and that this gene is frequently targeted in t(9;22)+ ALL. Furthermore, the SNP array analysis revealed novel recurrent deletions targeting the genes DLG2 (4 cases; 8.9%), LDOC1 (3 cases; 6.7%), ERBB4, and CDH13 (2 cases each; 4.4%). In conclusion, using high-resolution SNP arrays we detect a very high frequency of hidden genetic changes in adult ALL cases. Deletions, frequently cryptic, comprised 70% of the found abnormalities, suggesting that microdeletions are a characteristic feature of adult ALL. Most importantly, the analyses revealed recurrent genetic abnormalities in adult ALL targeting novel genes, not previously implicated in leukemogenesis. In addition to giving new insights into the leukemogenic process, these findings are likely to be clinically important, with the possibility of identifying new prognostic markers as well as future treatment targets in adult ALL.

Changed destiny

How the identity of mature, differentiated cell types is controlled and what degree of developmental plasticity can still be realized by fully differentiated cell types is an important and fundamental question in developmental biology and stem cell research. A central question in this field, that of whether cells are reprogrammed directly to a different cell type — by direct transdifferentiation — or take a backward step to a more immature state before moving along another pathway — called dedifferentiation — is answered by a report in this issue. Cobaleda et al. demonstrate dedifferentiation during the conversion of mature B lymphoid cells to functional T cells. Deletion of Pax5, a transcription factor important for B cell differentiation and function, caused mature B cells to dedifferentiate into progenitor cells that can give rise to T cells. Pax5 is also known to play a role in cancer, and its loss is shown to induce lymphomas that arise from progenitor cells.

Conversion of mature B cells into T cells by dedifferentiation to uncommitted progenitors

Lineage commitment and differentiation to a mature cell type are considered to be unidirectional and irreversible processes under physiological conditions. The commitment of haematopoietic progenitors to the B-cell lineage and their development to mature B lymphocytes critically depend on the transcription factor encoded by the paired box gene 5 (Pax5). Here we show that conditional Pax5 deletion in mice allowed mature B cells from peripheral lymphoid organs to dedifferentiate in vivo back to early uncommitted progenitors in the bone marrow, which rescued T lymphopoiesis in the thymus of T-cell-deficient mice. These B-cell-derived T lymphocytes carried not only immunoglobulin heavy- and light-chain gene rearrangements but also participated as functional T cells in immune reactions. Mice lacking Pax5 in mature B cells also developed aggressive lymphomas, which were identified by their gene expression profile as progenitor cell tumours. Hence, the complete loss of Pax5 in late B cells could initiate lymphoma development and uncovered an extraordinary plasticity of mature peripheral B cells despite their advanced differentiation stage.

Mutations in the B-Cell Transcription Factor PAX5 Found in B-Progenitor Acute Lymphoblastic Leukemia Impair Normal PAX5 Activity.

Using genome-wide profiling of DNA copy number abnormalities using high-resolution single nucleotide polymorphism arrays, we recently identified a high frequency of genomic aberrations involving the PAX5 gene in pediatric B-progenitor ALL. PAX5 is a critical transcriptional regulator of B lymphocyte commitment and differentiation. Mutations, including partial tandem duplication, complete and focal deletions, point mutations in the DNA-binding or transactivation domain, and three translocations that encode PAX5 fusion proteins were observed in 31.7% of B-ALL. The PAX5 deletions were mono-allelic and resulted in either loss of the entire gene, or the deletion of only a subset of the exons leading to the production of PAX5 proteins that lacked the DNA-binding paired domain (exons 2–4) and/or the transcriptional activation domain (exons 7–10). In murine systems, the complete absence of PAX5 results in the arrest of B-cell development at the pro-B-cell stage prior to immunoglobulin heavy chain rearrangement, whereas haploinsufficiency leads to a partial block in B-cell development. Importantly, in the primary leukemia samples, the mono-allelic loss of PAX5 was associated with reduced expression of PAX5 by flow cytometry and quantitative RT-PCR, suggesting that haploinsufficiency contributes to the block in differentiation characteristic of B-progenitor ALL. To determine if the other identified PAX5 mutations result in hypomorphic alleles, we analyzed the DNA-binding and transcriptional activity of the encoded proteins. DNA-binding activity was assessed by electrophoretic mobility gel-shift assays using a labeled oligonucleotide probes from the promoters of the PAX5 target genes CD19 and CD79A (mb-1), and transcriptional activity was assessed by a luciferase-based reporter assays using the PAX5-dependent reporter plasmid, luc-CD19. Analysis was performed on the paired-domain mutants P80R and P34Q, the focal deletions Δe2-5, Δe2-6, Δe2-7, Δe2-8, and Δe6-8, and the PAX5-ETV6 and PAX5-FOXP1 translocation-encoded fusion proteins. As expected, DNA-binding was abrogated in deletion mutants that lacked the paired domain (Δe2-5, Δe2-6, Δe2-7, Δe2-8). In contrast, the PAX5 Δe6-8, which retains the paired DNA binding domain but lacks a significant portion of the transcriptional regulatory domain, had normal DNA binding activity. Importantly, the paired domain point mutants impaired DNA-binding in a promoter specific manner, with P80R having a marked reduction in binding to both the CD19 and mb-1 promoters, whereas P34Q showed reduced binding only to the mb-1 promoter. Surprisingly, the PAX5-ETV6 and the PAX5-FOXP1 translocations had markedly reduced DNA-binding activity despite retention of the PAX5 paired domain. As expected each of the mutants with impaired or absent DNA-binding activity were found to have markedly reduced transcriptional activity when compared to wild type PAX5. Similarly, those mutants with altered or deleted transcriptional activation domains had reduced transcriptional activity, as did the two PAX5 translocation-encoded fusion proteins (PAX5-ETV6 and PAX5-FOXP1). Moreover, transfection of increasing amounts of PAX5-ETV6 or PAX5-FOXP1 together with a fixed amount of wild type PAX5 revealed that the fusion proteins competitively inhibit the transcriptional activation of wild type PAX5. Taken together, these data indicate that the identified PAX5 mutations impair DNA-binding and/or transcriptional activity. This loss of normal PAX5 function in turn would contribute to the observed arrest in B-cell development seen in ALL.

Genes Regulating B-Cell Development and Differentiation Are Mutated in 40% of Pediatric Acute Lymphoblastic Leukemia.

Chromosomal aberrations are a hallmark of acute lymphoblastic leukemia (ALL) but alone fail to induce leukemia. To identify cooperating oncogenic lesions, we performed genome-wide analysis of leukemic blasts from 242 pediatric ALL patients using high-resolution 100K and 250K Affymetrix single nucleotide polymorphism arrays, and genomic DNA sequencing. Remarkably, our analyses identified deletion, amplification, point mutation and structural rearrangement in genes encoding regulators of B lymphocyte development in over 40% of B-progenitor ALL. PAX5, which encodes a transcription factor critical for B cell commitment and differentiation, was the most frequent target of somatic mutation, being altered in 31.7% of cases. The most frequent PAX5 mutations were copy number alterations with mono-allelic loss in 53 cases, biallelic loss in 3 cases, and an internal amplification in 1 case. PAX5 deletions and the amplification were confirmed by FISH and/or RT-PCR, and were present in over 90% of blasts. Twenty-five of the mono-allelic deletions were confined to PAX5, with the majority deleting only a subset of PAX5 exons. Thus, of the 57 cases with PAX5 copy number changes, the majority had haploinsufficiency of PAX5 (n=30), or generated hypomorphic alleles that produce proteins that lack the DNA-binding domain (n=20) or the transcriptional activation domain (n=7). Four cases contained cryptic PAX5 translocations: PAX5-ETV6 (n=2), PAX5-FOXP1 and PAX5-ZNF521 (1 case each). In addition to the structural alterations, sequencing identified 14 B-ALLs with PAX5 point mutations. Mutations were identified in the DNA-binding paired domain, homeodomain and transactivation domains. The mutations were somatically acquired, and present in a dominant clone. Gel-shift and transcriptional reporter assays demonstrated reduced DNA binding and/or transcriptional activity for each of the identified PAX5 fusion proteins and point mutants. In addition to the PAX5 mutations, deletions were also detected in the B cell regulatory genes Ikaros (20 cases), Aiolos (3), EBF (8), E2A (1), LEF1 (3), and RAG1/2 (11). Importantly, genomic sequencing of Ikaros and EBF revealed no point mutations. Although the high frequency of mutations in genes regulating B-cell development was unexpected, even more surprising was the marked difference in the frequency and type of mutations among the various genetic subtypes of ALL. Hypodiploid ALLs had alterations in B-cell development genes in 100% of cases, with broad deletions of one PAX5 allele, mutation of the other PAX5 allele in 50% of the cases, and mono-allelic deletion of other regulators of B-cell development, frequently with multiple genes affected within a single case. In contrast ETV6-RUNX1 cases exhibited a much more restricted pattern of mutations, with 27% showing focal PAX5 deletion without alterations of the retained allele. At the other end of the spectrum were hyperdiploid ALLs, with only rare cases harboring a deletion of one of these genes. These data demonstrate that disruption of pathways controlling B cell development and differentiation contributes to the pathogenesis of ALL. Moreover, the approach used provides a rational roadmap for the application of genome-wide approaches to the identification of new molecular lesions in cancer.