iAMP21 Patients Research Articles

3D Genome Organization and Single-Cell Characterization of Pediatric Intrachromosomal Amplification of Chromosome 21 (iAMP21) B-Cell Acute Lymphoblastic Leukemia

B-cell Acute Lymphoblastic Leukemia (B-ALL) is the most frequent childhood cancer, and around 2% of these cases are associated with intrachromosomal amplification of chromosome 21 (iAMP21). The iAMP21 cases represent one of the high-risk but least understood sub-group of B-ALL. The sub-group also has a significantly low survival rate and a higher risk of relapse than non-iAMP21 cases. Recent studies suggest that structural variations in iAMP21 are linked to complex rearrangements such as a chromothripsis event on chromosome 21. The consequences of chromothripsis by which it drives the tumorigenesis are still unclear. Given the limited knowledge of chromothripsis and its impact on the epigenetic landscape in pediatric iAMP21 cases, we aim to systematically provide a critical understanding of the causes and consequences of such complex genomic rearrangements. We conducted an in-depth analysis of 9 iAMP21 and 10 non-iAMP21 pediatric patient samples associated with B-ALL using different sequencing techniques. These included whole genome sequencing (WGS), long-read sequencing, RNA-sequencing, and 3D genome organization. The WGS analysis revealed that iAMP21 cases had more genomic rearrangements compared to the non-iAMP21 group. Interestingly, iAMP21 subtypes displayed a unique feature - the presence or absence of the chromothripsis signature on chromosome 21. However, all patients shared a common characteristic - amplified and frequently deleted regions on chromosome 21, suggesting these genetic changes are a key aspect of the iAMP21 subtype, regardless of chromothripsis. Genomic rearrangements, a crucial element in oncogenesis, also modify the 3D structure of the human genome, and iAMP21 is no exception. Our Hi-C data analysis showed the devastating effect of chromothripsis on the 3D organization of chromosomes in iAMP21 patients. We observed a significant reconfiguration of the genomic compartments, along with changes in cis- and trans-acting regulatory elements such as enhancers and promoters. These changes disrupt the delicate balance of gene expression profile. To better understand the impact of these genomic rearrangements on the transcriptional landscape, we examined global gene expression patterns in iAMP21 subtypes, identifying distinct upregulated and downregulated genes. Our comparison between iAMP21 and non-iAMP21 groups identified a unique set of 186 upregulated and 174 downregulated genes in iAMP21 patients. Further, tumor diversity can greatly influence cancer diagnosis and treatment responses. Therefore, analyzing gene expression at the single-cell level is essential to uncover this diversity and identify unique cellular characteristics. Our analysis provided clear evidence of iAMP21 subclones within individual patient samples, resulting from differences in genomic rearrangements in each cancer cell. The iAMP21 cases with complex chromosomal rearrangement make it a high-risk and poorly characterized subtype of pediatric ALL. We hypothesize that even though related to worse outcomes, investigating the chromosomal rearrangement and the epigenetic landscape in iAMP21 may expose specific vulnerabilities of cancer cells in these patients allowing for targeted therapies and devising new therapeutic strategies. While our findings so far are preliminary, they offer valuable insights that can guide the formulation of hypotheses for further detailed experiments and data analysis. The project has already amassed a wealth of genomic data and a consistent approach to data analysis, addressing the challenging task of identifying relevant prognostic and predictive molecular factors in patients battling aggressive forms of cancer.

Gain of chromosome 21 increases the propensity for P2RY8::CRLF2 acute lymphoblastic leukemia via increased HMGN1 expression.

Acute lymphoblastic leukemia (ALL) patients with a gain of chromosome 21, intrachromosomal amplification of chromosome 21 (iAMP21), or Down syndrome (DS), have increased expression of genes in the DS critical region (DSCR) of chromosome 21, including the high-mobility group nucleosome-binding protein 1, HMGN1. Children with DS are predisposed to develop hematologic malignancies, providing insight into the role of chromosome 21 in the development of leukemias. A 320-kb deletion in the pseudoautosomal region of the X/Y chromosome in leukemic cells, resulting in a gene fusion between the purinergic receptor and cytokine receptor-like factor-2 (P2Y Receptor Family Member 8 (P2RY8)::CRLF2), is a common feature in ~60% of DS-ALL and ~40% of iAMP21 patients, suggesting a link between chromosome 21 and P2RY8::CRLF2. In an Australian cohort of pediatric B-ALL patients with P2RY8::CRLF2 (n = 38), eight patients harbored gain of chromosome 21 (+21), and two patients had iAMP21, resulting in a significantly increased HMGN1 expression. An inducible CRISPR/Cas9 system was used to model P2RY8::CRLF2 and investigate its cooperation with HMGN1. This model was then used to validate HMGN1 as an influencing factor for P2RY8::CRLF2 development. Using Cas9 to cleave the DNA at the pseudoautosomal region without directed repair, cells expressing HMGN1 favored repair, resulting in P2RY8::CRLF2 generation, compared with cells without HMGN1. CRISPR/Cas9 P2RY8::CRLF2 cells expressing HMGN1 exhibit increased proliferation, thymic stromal lymphopoietin receptor (TSLPR) expression, and JAK/STAT signaling, consistent with cells from patients with P2RY8::CRLF2. Our patient expression data and unique CRISPR/Cas9 modeling, when taken together, suggest that HMGN1 increases the propensity for P2RY8::CRLF2 development. This has important implications for patients with DS, +21, or iAMP21.

A Comprehensive Study of Human Integrins in Pediatric Lymphoblastic Leukemia Supports a Role of CD49f (Integrin α6) in the Localization to Bone Marrow but Not Spinal Fluid

Introduction: The overall survival of children with ALL (Acute Lymphoblastic Leukemia) has improved markedly over the last decade, yet relapse occurs in about 20% of patients. The spread of leukemic blasts to the Central Nervous System (CNS) and increased resistance to therapy due to cell adherence within the Bone Marrow (BM) or CNS constitutes major challenges to treatment. For these reasons, adhesion molecules governing the homing and adhesion of leukemic cells are perceived to be of extraordinary importance, both as potential biomarkers and therapeutic targets. Integrins constitute a large family of heterodimeric receptors composed of alpha and beta subunits, which play important roles during homing and migration of normal leucocytes by facilitating adhesion to both stromal cells and components of the extracellular matrix. Increased expression of CD49d (integrin subunit α4) is a marker for adverse prognosis in ALL and recently, CD49f (integrin subunit α6) was shown to facilitate metastasis of ALL xenografts to the central nervous system in mice (Yao H et al., Nature 2018). Methods Previous studies of integrins in BCP-ALL have focused on individual alpha integrins in xenograft models and were based on limited numbers of clinical samples. The present study was based on a large number of Danish pediatric BCP-ALL patients stratified between 2012-2018 using Minimal Residual Disease (MRD) according to the Nordic NOPHO-2008 protocol (Toft N et al., Eur J Haematol 2013). Diagnostic BM samples were subjected to flowcytometric analysis (FCM) of CD49f (n=246) and CD49d (n=135), using a backbone of lineage-specific B-cell markers (CD45, CD10, CD19, CD20). Leukemic blasts were detected in Cerebrospinal Fluid (CSF) using high-sensitivity FCM and the following markers CD45, CD10, CD19, CD20, CD34 and CD38 (n = 246, with matching BM and CSF samples). Results Our data provided us with a unique possibility to identify the role of CD49d and CD49f with respect to minimal residual disease (MRD) at the end of induction therapy (day 29), which is considered the most important prognostic factor in paediatric lymphoblastic leukemia. We found that CD49f was more highly expressed in patients with MRD ≥ 0,1% at day 29 than patients with MRD < 0,1% (p = 0,01), whereas no difference was seen with respect to CD49d. We also investigated the correlation between white blood cell (WBC) and surface expression of CD49d and CD49f in diagnostic BM blasts with respect to different cytogenetic subtypes. A Kruskall-Wallis test showed that the expression varies according to genetic subtypes (p‹0.0001). We found that the expression of CD49d was highest among the high hyperdiploid and iAMP21 (intrachromosomal amplification of chromosome 21) patients, whereas the expression of CD49f was highest among the t12,21 and iAMP21 patients. Notably, the expression of CD49f was inversely correlated to WBC (r=0,17, p=0.01), which was most pronounced among the patients in the B-other cytogenetic subgroup defined as leukaemia that could not be classified into the existing cytogenetic groups (r=0,42, P‹0.0001). In case of strong adherence to BM, lower levels of leukemic blasts might be expected in circulation resulting in high MRD but low WBC. Therefore, both MRD and WBC data are consistent with a prominent adhesive role of CD49f within BM. In contrast, we found significantly lower CD49f surface expression in diagnostic BM samples in patients with leukemic blasts within CSF (p=0.0297). Conclusions: Recently, Yao et al. (2018) showed that ALL cells in circulation are unable to breach the blood-brain barrier in mice and instead employ CD49f (integrin α6) to migrate into the CNS along vessels that connect vertebral or calvarial bone marrow and the subarachnoid space. Potentially, this mechanism could account for cases of ALL relapse within CNS. Our work shows a strong association between high MRD and the expression of CD49f which is a function that would have been anticipated for CD49d due to previous works with ALL and CLL. Furthermore, we found significantly lower CD49f surface expression in leukemic blasts within the BM in patients with CSF involvement and therefore no support for the recently proposed role of CD49f in facilitating the spread of leukemic cells to the CNS. Disclosures No relevant conflicts of interest to declare.

PS921 INACTIVATION OF SH2B3 THROUGH CN‐LOH 12Q IS UNIQUELY ASSOCIATED WITH B‐CELL PRECURSOR ALL WITH IAMP21 OR OTHER CHROMOSOME 21 GAIN

Background:In more than 30% of childhood B‐cell precursor acute lymphoblastic leukaemia (B‐ALL), chromosome 21 sequence is overrepresented through constitutional or acquired aneuploidy or structural rearrangements, exemplified by intrachromosomal amplification of chromosome 21 (iAMP21). How these abnormalities promote B‐ALL remains obscure but a mechanistic link is suggested by the existence of shared co‐occurring abnormalities.Aims:We aimed to identify and characterise acquired genetic abnormalities that cooperate with amplification or aneuploidy of chromosome 21 to promote B‐ALL.Methods:Regions of copy neutral loss of heterozygosity and deletion were defined by analysis of SNP6.0 arrays, methylation arrays were used to assess loss of imprinting. The mutational status of SH2B3 was determined by amplicon sequencing and a homology model based on the resolved structure of the Sh2 domain of Sh2b1 was used to assess the impact of an acquired in‐frame substitution. STAT activation status was determined by flow cytometric immunophenotyping.Results:Analysis of B‐ALL patients with iAMP21, other structural amplifications of chromosome 21 or constitutional or acquired additional whole copies of chromosome 21 (n = 99), revealed recurrent CN‐LOH of 6p, 9p and 12q. In patients without overrepresentation of chromosome 21, CN‐LOH 6p and 9p were also recurrent but intriguingly, CN‐LOH 12q was absent, among our own (n = 203) or published cases (n = 974). Further analysis demonstrated CN‐LOH 12q was not associated with loss of imprinting but with homozygous mutations or deletions of the adaptor protein, SH2B3. In patients without CN‐LOH 12q, bi‐allelic abnormalities of SH2B3 occurred, but only in iAMP21‐ALL, giving an overall incidence of 18% in this sub‐type. Taking into account our own cohort, analysis of archival array data and review of published cases, we conclude that in B‐ALL, SH2B3 abnormalities are invariably bi‐allelic, frequent in iAMP21‐ALL, recurrent but less common in association with acquired aneuploidy 21 but extremely rare in the absence of overrepresentation of chromosome 21. Importantly, as iAMP21 patients are now uniformly treated as high risk, preliminary analysis of 26 patients treated on UKALL97/99 (n = 5) or UKALL2003 (n = 21), linked 12q abnormalities to poor outcome in iAMP21‐ALL (p = 0.03). Homology modelling of an iAMP21‐ALL associated R392W mutation in the SH2 domain of SH2B3 demonstrated loss of critical interactions required for binding and suppression of activated JAK. Further implicating the JAK/STAT pathway as one target for SH2B3 tumour suppressor activity, stimulation with IL7 of iAMP21‐ALL patient derived xenograft cells, with but not without SH2B3 inactivation, resulted in activation of STAT5 though not other STATs.Summary/Conclusion:Although chromosome 21 copy number profiles are highly heterogeneous in iAMP21‐ALL a limited number of candidate oncogenes are consistently amplified, overexpressed and spared from disruption by structural rearrangements. From our data we infer that in iAMP21‐ALL, high expression of one or more of these candidate genes creates strong pressure for loss of SH2B3 function while lower but still elevated expression of the same gene/s, resulting from gain of one or two copies of whole chromosome 21, produces a similar though weaker selective environment. Since JAK/STAT activation is common in B‐ALL without gain of chromosome 21 and given the near‐exclusive association we observed, we are currently exploring the possibility that SH2B3 also interacts with other pathways specifically activated by increased doses of chromosome 21 genes.

Intrachromosomal amplification of chromosome 21 in Korean pediatric patients with B-cell precursor acute lymphoblastic leukemia in a single institution.

BackgroundIntrachromosomal amplification of chromosome 21 (iAMP21), defined as the presence of three or more RUNX1 signals on one marker chromosome, is a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that is known to have a poor prognosis when treated with standard therapy. The aim of this study was to evaluate the clinical characteristics of Korean children with iAMP21.MethodsThe cytogenetic data from BCP-ALL children were reviewed. The ETV6/RUNX1 ES Dual Color Probe was used for fluorescence in situ hybridization (FISH).ResultsIn total, 295 children were included. Of these, 10 patients (3.4%) had iAMP21. The median age of iAMP21 patients was 9 years, and the median value of white blood cell count was 5.09×109/L. Slow early treatment response was observed more in iAMP21 patients. Patients with iAMP21 had a higher incidence of relapse and worse survival rates. In patients with iAMP21, the estimated 10-year cumulative incidence of relapse was 53.3%. The estimated 10-year event-free survival and overall survival rate were 46.7% and 64.8%, respectively. Most cases of leukemic relapse developed in the late period (median, 43 mo). In multivariate analysis, high risk group was the only factor that had a significant impact on death.ConclusionThe existence of iAMP21 was related to delayed treatment response and was likely to affect increased relapse and death in the late period. Further studies are needed to reveal its effect on BCP-ALL treatment outcomes and its role as an independent prognostic factor.

New and emerging prognostic and predictive genetic biomarkers in B-cell precursor acute lymphoblastic leukemia.

Acute lymphoblastic leukemia (ALL) is a heterogeneous disease at the genetic level. Chromosomal abnormalities are used as diagnostic, prognostic and predictive biomarkers to provide subtype, outcome and drug response information. t(12;21)/ETV6-RUNX1 and high hyper-diploidy are good-risk prognostic biomarkers whereas KMT2A(MLL) translocations, t(17;19)/TCF3-HLF, haploidy or low hypodiploidy are high-risk biomarkers. t(9;22)/BCR-ABL1 patients require targeted treatment (imatinib/dasatinib), whereas iAMP21 patients achieve better outcomes when treated intensively. High-risk genetic biomarkers are four times more prevalent in adults compared to children. The application of genomic technologies to cases without an established abnormality (B-other) reveals copy number alterations which can be used either individually or in combination as prognostic biomarkers. Transcriptome sequencing studies have identified a network of fusion genes involving kinase genes -ABL1,ABL2,PDGFRB,CSF1R,CRLF2,JAK2 and EPOR in-vitro and in-vivo studies along with emerging clinical observations indicate that patients with a kinase-activating aberration may respond to treatment with small molecular inhibitors like imatinib/dasatinib and ruxolitinib. Further work is required to determine the true frequency of these abnormalities across the age spectrum and the optimal way to incorporate such inhibitors into protocols. In conclusion, genetic biomarkers are playing an increasingly important role in the management of patients with ALL.

Genetic Abnormalities and Prognosis in Pediatric B–precursor Acute Lymphoblastic Leukemia Treated on Tokyo Children′s Cancer Study Group Protocol

Introduction; Acute lymphoblastic leukemia (ALL) is the most common cancer in children. Most childhood cases of B-precursor ALL (BCP-ALL) may be subclassified by the presence of either gross or submicroscopic genetic alteration. Recent genomewide analyses have identified genetic or cytogenetic subtypes, such as Ph-like ALL, deletion of ERG, intrachromosomal amplification of chromosome 21 (iAMP21) in BCP-ALL. Deletion of ERG and iAMP21 were associated with frequent IKZF1 deletions, which were found in Ph-like ALL associated with poor prognosis. However, the frequency and prognostic implications of these genomic abnormalities have not been defined in Japan.Patients and Methods; We studied 208 children with de novo BCP-ALL enrolled on Tokyo Children Cancer Study Group (TCCSG) ALL L0416 and L0616 trials. All patients had a diagnosis of BCP-ALL without BCR-ABL1 phenotype confirmed by morphology, immunophenotyping and mRNA Real-time PCR. Alterations of ERG and iAMP21 were examined by Multiplex Ligation-dependent Probe Amplification (MLPA) kit P327-B1. To identify Ph-like ALL and BCR-ABL1 like ALL, We investigated gene expression of 235 BCP-ALL cases enrolled on TCCSG L0416 and L0616 trials by microarray GeneChip Human Genome U133 Plus 2.0 and analyzed using clustering analysis as described previously. To assess whether these genomic alterations overlap with known gene abnormalities, such as mutations of CRLF2,JAK1, JAK2 and IL7R, rearrangement of P2RY8-CRLF2, PAX5-JAK2 and PDGFRB-EBF1 genes and copy number alteration of IKZF1, CDKN2A/2B, PAX5, EBF1 and SH2B3 genes, mutational analyses of these genes were also performed using direct sequencing, RT-PCR or MLPA kit P335-A1 and P200.Results; We identified Ph-like signature in 9 (3.8%) patients and BCR-ABL1 like ALL signature in 11 (4.7%) patients. Five (55.6%) of 9 Ph-like ALL patients and 3 (27.3%) of 11 BCR-ABL1 like ALL patients have IKZF1 deletions. Five patients were classified into both Ph-like ALL and BCR-ABL1 like ALL. Among these 5 patients, one patient had CRLF2 high expression, JAK2 R683S/R683G mutation, CDKN2A/2B deletion and EBF1 deletion, and another patient have PAX5-JAK2 rearrangement. These two patients relapsed, and the latter patient died. Three of 9 Ph-like ALL patients relapsed and 2 died, while 7 of 11 BCR-ABL1 like ALL patients relapsed and 2 died. We identified iAMP21 in 5 (2.4%) patients and deletion of ERG in 8 (3.8%). One of 5 patients with iAMP21 and 3 of 8 patients with ERG deletion had IKZF1 deletion. One patient with iAMP21 had BCR-ABL1 like ALL signature, but not Ph-like ALL signature. CRLF2 high expression was identified in two of 5 patients with iAMP21, and P2RY8-CRLF2 rearrangement and SH2B3 deletion was identified in one of them. No mutations of CRLF2 and JAK2 were identified in patients with iAMP21 and ERG deletion. Among 5 iAMP21 patients, patient with BCR-ALB1 signature and IKZF1 deletion relapsed, and one patient died. No patients with ERG deletion relapsed or died.Conclusions; Using GeneChip microarray analysis, low frequency of Ph-like ALL signature and BCR-ABL1 like ALL signature was found. Using MLPA as a novel screenings technique, frequency of iAMP21 and ERG deletion in pediatric BCP-ALL was similar to that of previous studies. BCR-ABL1 like ALL and iAMP21 was associated poor prognosis, while ERG deletion was associated good prognosis in pediatric ALL. These data suggest that screening for these genetic abnormalities in pediatric patients with BCP-ALL is critical not only for outcome prediction but also for risk-adapted therapy. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Minimal residual disease-based treatment is adequate for relapse-prone childhood acute lymphoblastic leukemia with an intrachromosomal amplification of chromosome 21: the experience of the ALL-BFM 2000 trial.

Recently, the UK CCLG and COG reported that an intrachromosomal amplification of chromosome 21 (iAMP21) in acute lymphoblastic leukemia (ALL) loses its adverse prognostic impact with intensified therapy. We evaluated the prognosis of iAMP21 among patients from the ALL-BFM (Berlin-Frankfurt-Münster) 2000 trial with 46 of 2 637 (2%) patients iAMP21+. 8-year event-free-survival (EFS, 64 ± 8% vs. 81 ± 1%, p=0.0026) and cumulative incidence of relapse (CIR, 29 ± 8% vs. 14 ± 1%, p=0.008) of the iAMP21 cases were significantly worse compared with non-iAMP21 patients. Within the MRD low-risk group, iAMP21 cases (n=14) had an inferior 8-year EFS (76 ± 12% vs. 92 ± 1%, p=0.0081), but no increased CIR (10 ± 10% vs. 6 ± 1%, p=0.624). Within the MRD intermediate-risk group, iAMP21 cases (n=27) had a worse 8-year EFS (56 ± 11% vs. 78 ± 2%, p=0.0077) and CIR (44 ± 11% vs. 20 ± 2%, p=0.003) with 6/10 relapses occurring after 2 years. Conclusively, we believe that there is no necessity for enrolling all iAMP21 patients into the high-risk arm of ongoing ALL-BFM trials because MRD low-risk patients have a moderate relapse risk under current therapy. Whether the increased relapse risk in MRD intermediate-risk patients can be avoided by late treatment intensification remains to be answered by the AIEOP-BFM ALL 2009 trial randomly using protracted pegylated L-asparaginase during delayed intensification and early maintenance.

Clonal Progression Including Evolution Of An iAMP21 Chromosome In Xenograft Models Of BCP-ALL

Insight into the role of recurrent genomic changes arising in leukemia has depended largely on the functional analysis of cell lines and transgenic animals. However not all acquired abnormalities are represented in available cell lines and a few are too complex to be faithfully engineered in animal models. One such abnormality is intrachromosomal amplification of chromosome 21 (iAMP21), a heterogeneous cytogenetic rearrangement, with a distinct clinical profile, occurring in 2% of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL). The most highly amplified segment of iAMP21 varies in size and copy number but has always included a 5.1 Mb common region of amplification (CRA). Regions flanking the most highly amplified segment have profiles that may be step-like, or more complex combinations of lower level amplification, normal copy number and deletion. Abnormalities at other chromosomal locations such as deletions of IKZF1, CDKN2A/B or RB1 and rearrangements activating CRLF2 co-occur with iAMP21 but have never been shown to precede its formation.To investigate clonal evolution and to provide a resource for future functional studies, we established xenografts from three cases of iAMP21 BCP-ALL in NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ (NSG) mice. Signs of disease were seen between 6 and 8 months after intrafemoral injection and autopsy revealed marked splenomegaly in all cases. Cells isolated from the bone marrow and spleen, were positive for human B cell markers CD19 and CD10. Disease onset was more rapid (3-6 months) when primografts were transplanted and cells from these secondary mice were used to establish leukemia in a third generation. Whole body imaging of mice transplanted with xenograft cells, transduced with a lentivirus expressing luciferase, revealed the rapid spread of disease from the injection site to the contralateral femur, sternum, pelvis, ribs, spine and skull, with the spleen becoming the major site of engraftment at late stage disease.Comparison of SNP 6.0 array profiles of presentation and xenografts samples revealed several examples of post-transplant genomic progression which, with the exception of a biallelic deletion of the CDKN2A/B locus, had not previously been reported in iAMP21 patients. Additional copies of chromosomes 9 and 12 in patient samples were lost in xenografts resulting in copy number neutral loss of heterozygosity (CNN-LOH) for chromosome 12 but not 9. Analysis of further primary iAMP21 BCP-ALL cases identified two with CNN-LOH for part of the long arm of chromosome 12, suggesting involvement of an imprinted locus in this region.iAMP21 chromosomes were retained in all xenografts, supporting evidence that it is a primary abnormality. However, as these rearrangements have been considered to be stable once formed, we were surprised to observe acquisition of a non–contiguous deletion of chromosome 21 in one secondary mouse and all of its tertiary recipients. The deletion was proximal to the CRA and resulted in extension of pre-existing haploinsufficient regions by 6.6 Mb. Evolution of this iAMP21 chromosome was confirmed by fluorescence in-situ hybridisation (FISH) using probes targeting the deletion, with loss of signal observed in all cells from mice carrying the deletion but in no cells from the presentation sample or unaffected mice. Although we cannot rule out that the deletion arose in response to adaptive pressure specific to the xeno-environment, opportunities to observe similar progression in patients have been limited because FISH analysis has generally targeted only the CRA and few paired presentation/relapse samples have been analysed by genomic array. While it remains likely that overrepresentation of oncogenes within the CRA acts as a primary driver of iAMP21 BCP-ALL, the additional deletion may highlight the position of tumour suppressor genes involved in leukemia progression. As these patients are treated as a single clinical entity, an important implication of these data is that the position and extent of deletions in iAMP21 rearrangements may influence response to therapy.In conclusion, xenografts of iAMP21 BCP-ALL closely resemble human primary disease and will make valuable pre-clinical models for basic and translational research. As models for the study of clonal evolution they support previous evidence that iAMP21 is a primary abnormality and have revealed novel forms of clonal progression. Disclosures:No relevant conflicts of interest to declare.

An international study of intrachromosomal amplification of chromosome 21 (iAMP21): cytogenetic characterization and outcome

Intrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct cytogenetic subgroup of childhood B-cell precursor acute lymphoblastic leukaemia (BCP-ALL). To date, fluorescence in situ hybridisation (FISH), with probes specific for the RUNX1 gene, provides the only reliable detection method (five or more RUNX1 signals per cell). Patients with iAMP21 are older (median age 9 years) with a low white cell count. Previously, we demonstrated a high relapse risk when these patients were treated as standard risk. Recent studies have shown improved outcome on intensive therapy. In view of these treatment implications, accurate identification is essential. Here we have studied the cytogenetics and outcome of 530 iAMP21 patients that highlighted the association of specific secondary chromosomal and genetic changes with iAMP21 to assist in diagnosis, including the gain of chromosome X, loss or deletion of chromosome 7, ETV6 and RB1 deletions. These iAMP21 patients when treated as high risk showed the same improved outcome as those in trial-based studies regardless of the backbone chemotherapy regimen given. This study reinforces the importance of intensified treatment to reduce the risk of relapse in iAMP21 patients. This now well-defined patient subgroup should be recognised by World Health Organisation (WHO) as a distinct entity of BCP-ALL.

Risk-Directed Treatment Intensification Significantly Reduces the Risk of Relapse Among Children and Adolescents With Acute Lymphoblastic Leukemia and Intrachromosomal Amplification of Chromosome 21: A Comparison of the MRC ALL97/99 and UKALL2003 Trials

To evaluate the effect on outcome of intensifying therapy for patients with acute lymphoblastic leukemia (ALL) and an intrachromosomal amplification of chromosome 21 (iAMP21). We report two cohorts of patients treated on Medical Research Council ALL97 or United Kingdom (UK) ALL2003. iAMP21 was identified retrospectively in ALL97 and was not used to guide therapy. However, in UKALL2003, iAMP21 was determined prospectively, and patients were allocated to the most intensive treatment arm (regimen C), which included augmented Berlin-Frankfurt-Munster consolidation, escalating Capizzi maintenance, double delayed intensification, and an option for first remission transplantation. The presence of iAMP21 was determined by fluorescence in situ hybridization using probes specific for the RUNX1 gene. iAMP21 was identified in 2% of patients with B-cell precursor ALL treated on UKALL2003 and ALL97. The event-free survival, relapse, and overall survival rates at 5 years for iAMP21 patients treated on ALL97 and UKALL2003 were 29% and 78%, 70% and 16%, and 67% and 89%, respectively (all P < .01). Patients treated on ALL97 had an increased risk of relapse compared with patients treated on UKALL2003 (hazard ratio, 7.2; 95% CI, 2.91 to 17.87; P < .001). iAMP21 patients with ALL benefitted from receiving more intensive therapy in UKALL2003. In UKALL2011, they will continue to be treated as cytogenetic high risk, receive intensive chemotherapy (regimen C), and will only be recommended for transplantation if they do not achieve a complete remission by the end of induction therapy. This study illustrates how the discovery and characterization of disease-specific genetic aberrations can be used to tailor therapy more precisely.

Intrachromosomal Amplification of Chromosome 21(iAMP21): Cytogenetic Characterisation and Outcome in Childhood B-Cell Precursor Acute Lymphoblastic Leukaemia (BCP-ALL). A Study On Behalf of the Ponte Di Legno International Childhood ALL Workshop

Abstract 293▪▪This icon denotes a clinically relevant abstractIntrachromosomal amplification of chromosome 21 (iAMP21) is a distinct cytogenetic subgroup of childhood BCP-ALL, characterised by older age, low white cell count (WCC) and poor outcome on standard therapy. It is identified by a complex structure of one copy of chromosome 21, highly variable between patients, with a common 5.1Mb region of amplification within which the RUNX1 gene is located. Thus FISH, using probes directed to RUNX1 provides a reliable detection method, defining iAMP21 as ≥5 copies of RUNX1 ≥4 on the abnormal chromosome 21. The Ponte di Legno International Childhood ALL Workshop has collected data from 513 iAMP21 patients from 15 international study groups in order to further characterise this subgroup and improve identification.iAMP21 was mutually exclusive of ETV6-RUNX1 fusion (509 tested), MLL rearrangement (391 tested), TCF3-PBX1/TCF3-HLF (151 tested). There was evidence of classical high hyperdiploidy in two of the 428 patients with a successful cytogenetic result. Four patients (393 tested) were positive for the BCR-ABL1 fusion. In addition to iAMP21 all patients had one normal chromosome 21, apart from 10 patients with two copies of normal 21 in addition to iAMP21, and 4 patients with an additional iAMP21. Other whole chromosomes commonly gained or lost among the 398 patients with abnormal karyotypes were: +X (21%, n=85), −7 (5%, n=19), +10 (4%, n=17), +14 (4%, n=17), and −15 (3%, n=11). Abnormalities of 11q (14%, n=57), 7q (9%, n=35), 9p (9%, n=36) and 12p (6%, n=24) were the most frequent visible chromosomal imbalances. Deletions of CDKN2A/B and ETV6 occurred in 16% and 34% cases, respectively, suggesting that these genes were the targets of 9p and 12p abnormalities. The incidences of deletions of ETV6 (34% vs 22%) and RB1 (40% vs 6%), as well the CRLF2 rearrangement, P2RY8-CRLF2 (19% vs 4%), were significantly higher among the iAMP21 patients than a comparator cohort of 1427 children with BCP-ALL. There were a number of patients with constitutional abnormalities involving chromosome 21: Down syndrome (n=1), ring chromosome 21, r(21)c (n=3) and Robertsonian translocation between chromosomes 15 and 21, rob(15;21)(q10;q10)c (n=4). It was intriguing that in the one case with r(21)c and those with rob(15;21)(q10;q10)c, in which an acquired abnormal karyotype was present, it was the chromosome with the constitutional abnormality which gave rise to the abnormal iAMP21 chromosome.In terms of the demographic profile of iAMP21 patients, 52% were female and 48% were male, median age was 9 years (range 2–23). Patients <10 and ≥10 years old were equally distributed (51% vs 49%). The median WCC was 5×109/L, range (<1–900×109/L), with only 3% having a high WCC >50 x109/L (n=13). The NCI Risk status was standard risk (SR) in 48% and high risk (HR) in 52%. Follow-up data were available for 283 patients diagnosed before 01/01/2009. Complete remission (CR) was achieved in 280/283 (99%) of which 90 (32%) suffered a relapse and 47 (17%) patients died. In total 43% were treated as HR, with the remainder (57%) treated as SR. There was a significant improvement in event free survival (EFS) (Figure) for patients treated as HR compared to SR. After a median follow-up time of 3.4 years, the 5 year EFS rates were 70% (95% CI 59–78) and 50% (30–59), respectively, hazard ratio 0.73 (95% CI 0.59–0.91) (p=0.005).Patients with iAMP21 display a unique spectrum of genetic abnormalities, which may be useful for improving identification of iAMP21: gain of chromosomes X, 10 or 14 in the absence of high hyperdiploidy, or monosomy 7/deletion of 7q, deletions of 11q, including MLL and ATM genes, P2RY8-CRLF2, deletions of ETV6 and RB1. These characteristic abnormalities provide useful data to ascertain the presence of iAMP21 when no metaphases are available to visualise the abnormal chromosome 21, or in the rare cases where the abnormal chromosome 21 has atypical rearrangements with other chromosomes. In rare cases, iAMP21 is associated with BCR-ABL1 and high hyperdiploidy, but is exclusive of other chromosomal abnormalities of prognostic significance. Although Down syndrome was rare, other constitutional abnormalities of chromosome 21 may be related to iAMP21 in these patients. Accurate identification of this abnormality is important due to the treatment implications. This study has shown that treatment of iAMP21 patients as HR provides a significant improvement in outcome. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Genes Commonly Deleted in Childhood B-Cell Precursor ALL (BCP-ALL) Are Associated with Major Cytogenetic Subgroups

AbstractAbstract 1374Specific chromosomal abnormalities in BCP-ALL are markers of the disease used for risk stratification. Genomic studies have shown that copy number abnormalities (CNA) of genes involved in B-cell development and cell cycle control are common in BCP-ALL. Here we report the relationship between these CNA and the major cytogenetic subgroups. The CNA of the genes indicated in Table 1 was evaluated using the P335 MLPA kit (MRC Holland, The Netherlands). The cohort comprised patients aged 1–25 years, registered on the ALL97 and ALL2003 trials. Cytogenetic analysis was successful in 1351 patients, who were classified into the major subgroups (Table 1). Patients positive for ETV6-RUNX1 showed the highest number of CNA overall. In contrast, CNA occurred at a lower than expected level in high hyperdiploid patients. The incidence of CNA was low in the MLL rearranged subgroup and higher in the other poor risk subgroups: BCR-ABL1 positive and intrachromosomal amplification of chromosome 21 (iAMP21). In the group classified as other abnormal (abnormal without established chromosomal abnormality), deletions of CDKN2A/B, PAX5 and IKZF1, as well as P2RY8-CRLF2, were seen at higher than expected levels. Overall, we showed CRLF2 rearrangements to be present in 4% of the cohort. With the exception of patients with MLL rearrangements, the presence of P2RY8-CRLF2 has now been reported in all cytogenetic subgroups; as we identified a small number in patients with ETV6-RUNX1, TCF3-PBX1 and BCR-ABL1, the latter two have not been previously reported. Several groups have reported a strong association between CRLF2 overexpression and IKZF1 alterations, here we corroborated this association.Among ETV6-RUNX1 positive patients, in addition to a high incidence of ETV6 deletions, CDKN2A/B and PAX5 were each deleted in 22% of these patients, while the incidence of IKZF1 deletions was low. Although BTG1 deletions were rare throughout the cohort, they were frequently associated with ETV6-RUNX1, found in 15% of cases (p<0.0001). There was an association between BCR-ABL1 and IKZF1 with 64% having a deletion of IKZF1. Deletions of PAX5 and CDKN2A/B were also high in this subgroup.Among iAMP21 patients, RB1 deletions and P2RY8-CRLF2 were observed at incidences (39% and 30%, respectively) significantly higher than expected (p<0.0001). iAMP21 patients also showed a high incidence of ETV6 deletions. The frequent occurrence of RB1 deletions in these patients is shown here for the first time.Deletions of IKZF1 were present in 13% of patients. The size of the deletion varied; deletions involving the whole gene (n=60) or restricted to exons 4–7 (n=61) were most frequent. Other deletions occurred at lower levels: exons 2–3 (n=15), exons 2–7 (n=20), exons 4–8 (n=13) and miscellaneous deletions (n=27). There was no significant association between the pattern of exon loss and cytogenetic subgroup, although BCR-ABL1 positive patients showed a trend towards increased loss of exon 4–7 (62% p=0.027).The extent of PAX5 deletions was variable, ranging from deletions of part or the entire gene including exon 1, predicted to result in reduced PAX5 expression (n=141), to those with partial deletions not involving exon 1, predicted to express a mutant allele (n=121). No significant association was found between the pattern of exon loss and cytogenetic subgroup. Intragenic amplifications of exons 2–5 were found exclusively in the other abnormal group (n=10), similarly predicted to express mutant alleles.In conclusion, this study presents an extensive screen for CNA in selected genes of significance in BCP-ALL. We have shown that the pattern of CNA is highly variable according to the primary genetic abnormality. While this study has confirmed the findings of others, new associations have been identified. We have also shown the heterogeneous nature of CNA in PAX5 and IKZF1. Future studies will need to incorporate cytogenetic data in order to accurately assess the prognostic effect of these aberrations.Table 1:CNA in Different Cytogenetics SubgroupsCytogenetic subgroupNumberIKZF1PAX5RB1CDKN2A/BETV6EBF1BTG1P2RY8-CRLF2%%%%%%%%Total1351131972722264ETV6-RUNX13793*2282254*415*<1*High hyperdiploidy4089*4*3*16*7*0<1*2TCF3-PBX14471816235202Other abnormal32424*36*747*162110*Normal1022094228*<161MLL rearranged28880314000BCR-ABL13364*45*948*3633iAMP2133211239*12219330**p<0.0001 Disclosures:No relevant conflicts of interest to declare.

Treatment Intensification Improves Outcome in Children with iAMP21 positive ALL

AbstractAbstract ▪656▪This icon denotes a clinically relevant abstractChromosomal abnormalities present in the bone marrow cells of children with acute lymphoblastic leukaemia (ALL) are used to assist diagnosis and define risk groups that guide therapy. One such primary genetic aberration is intrachromosomal amplification of chromosome 21 (iAMP21) which is defined as the presence of 3 or more extra copies of the RUNX1 gene on a single abnormal chromosome 21. FISH with probes targeting the RUNX1 gene is the commonly used detection method. Approximately 2–3% children with ALL harbour iAMP21 and it is associated with older age (median 10 years) and a low white cell count (WCC, <20×109/L).Among 28 patients treated on the MRC ALL97 trial between 1997 and 2002, 27 (96%) achieved a complete remission (CR), 22 (81%) relapsed and 13 (46%) died. The event free survival (EFS), relapse rate (RR) and overall survival (OS) rates at 5 years were 29% (14–46%), 70% (53–86%) and 67% (47–82%), respectively. There was no evidence that National Cancer Institute (NCI) high risk (HR) and standard risk (SR) patients had differential outcomes and the RR appeared constant with time (figure). On the basis of this increased risk of relapse, we chose to stratify these patients to the most intensive arm of the ALL2003 protocol (regimen C) which included augmented Berlin-Frankfurt-Munster (BFM) consolidation, two blocks of escalating Capizzi maintenance and a double delayed intensification. First remission transplants were recommended for patients who were slow early responders (SER) or minimal residual disease (MRD) positive (>0.01%) at day 29. Due to a relatively high transplant related mortality, transplants were restricted to patients not in CR by day 29 after June 2008.Prospective FISH screening of 2575 patients treated on ALL2003 between January 2003 and July 2011 identified 53 (2%) with iAMP21. The cohort showed a female predominance (30:23), had a median age of 10 years and 47 (89%) patients had a WCC <20×109/L. Follow-up information was available for 52 (98%) patients with a median follow-up time of 4.9 years. The majority of patients (48/52, 92%) were transferred to regimen C as directed by the protocol. At least 2 of the remaining 4 patients did not transfer to regimen C because of induction toxicity problems. Treatment response was measured at day 15 or day 8 according to whether patients received a 3 (NCI SR) or 4 (NCI HR) drug induction, respectively. Whilst none of the SR patients were SER, 13/28 (46%) HR patients had >25% blasts at day 8. MRD was assessed in 45 patients: 23 (51%) were positive (>0.01%) and 22 (49%) were negative (<0.01%) at day 28. All patients achieved a CR, 6 (12%) have subsequently relapsed whilst 4 (8%) died in first remission following a transplant from infection (n=3) or transplant-related complications (n=1). One patient relapsed and died after receiving a transplant; so in total, 5 (10%) patients have died. Among 20 (38%) patients who received a transplant in first CR, 18 (90%) were either MRD positive at day 28 or a SER. The EFS, RR, OS rates at 5 years were 78% (61–88%), 16% (7–34%) and 89% (76–95%), respectively (figure). There were no significant differences in outcome according to sex, age, WCC, NCI risk status or MRD levels at day 28. Interestingly, among the 5 relapses who had MRD measured at day 28 only 1 was positive (>0.01%); although 2 more had borderline levels (>0.005%).A comparison between the outcome of iAMP21 patients treated on ALL97 and ALL2003 showed a statistically significant decrease in RR (p<0.0001) with significant increases in EFS (p<0.0001) and OS (p<0.01). The key endpoint was the proportion of patients suffering a relapse which decreased nearly seven-fold (81% to 12%) between the two trials. Importantly none of the ALL2003 relapses occurred on treatment and all bar one were classified as late events (>6 months after the end of treatment). The findings presented herein clearly illustrate the benefit of this treatment intervention in improving survival in these patients who otherwise had an extremely poor outcome when given standard therapy. In light of these results, iAMP21 patients treated on our new trial, ALL2011, will continue to be classified in the high risk cytogenetic subgroup and will receive regimen C. They are recommended for transplant only if they fail to achieve a CR by day 29. This study illustrates how the discovery and characterisation of a disease-specific genetic aberration can be used to tailor therapy more precisely. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Ras Signalling Pathway and Novel Target Genes Related to Down Syndrome Contribute to the Development of B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL) in iAMP21 Patients

Abstract 70Intrachromosomal amplification of chromosome 21 (iAMP21) represents a distinct cytogenetic subgroup of BCP-ALL, in which patients experience a high-risk of relapse on standard treatment protocols. The abnormal chromosome 21 defining iAMP21 has a heterogeneous, complex profile at the genomic level. This complexity has made it difficult to elucidate target genes or the initiating mechanism giving rise to iAMP21 using standard genomic approaches. In this study, detailed genomic and mutational analysis has highlighted potential novel targets in the development of iAMP21 BCP-ALL.DNA was available from 45 iAMP21 patient samples. Patient 1 was a 10 year old female; her diagnostic karyotype was 47,XX,+10,der(21)dup(21)(q?)r(21)(q?). Fluorescence in situ hybridization detected multiple copies of RUNX1, thus defining iAMP21. SNP 6.0 arrays indicated the characteristic genomic profile of chromosome 21, comprising a ∼30Mb (from 17–47Mb) region of copy number gain/amplification. Many of the breakpoints occurred within the Down Syndrome Critical Region (DSCR), specifically within the gene, DSCAM at 41.4Mb, and a telomeric deletion was identified with a breakpoint within the gene, TSPEAR at 45.9Mb. Chromosome 7 abnormalities were frequent, with one deletion including IKZF1 at 50.3Mb. The IKZF1, ETV6 and RB1 deletions seen by SNP 6.0 arrays were confirmed by Multiplex Ligation Probe-dependent Amplification (MLPA) and quantitative PCR.Whole-exome sequencing of the diagnostic and remission DNA of patient 1 identified 44 somatic mutations; 21 were computationally predicted to be potentially damaging to the function of the protein. The variant frequency of the individual somatic mutations ranged from 1.1% − 65.2%, indicating heterogeneity within the iAMP21 genome. The majority of the variants were detected at a frequency of <10%, emphasising the importance of in depth coverage for the detection of mutations. A number (n=8) were confirmed by direct Sanger sequencing.Novel somatic mutations were identified in the B-cell development (ENPEP) and histone modification (SMYD3, RPL24, SIN3B) pathways. Genomic abnormalities in components of these pathways have been previously associated with BCP-ALL development.Six of the potentially-damaging mutations occurred within 3 genes: SIM2, SYNJ1, UMODL1, located within the DSCR, which have been implicated in the development of Down syndrome. We have previously demonstrated that Down syndrome-ALL and iAMP21 BCP-ALL share common genetic abnormalities: gain of an X chromosome and a high incidence of P2RY8-CRLF2 fusion.Furthermore, we identified a mutation in NF1, a component of the Ras signalling pathway. The mutation (P1496S) occurred in the kinase-binding domain at a variant frequency of 27%. Activation of the Ras signalling pathway was confirmed by pERK expression shown by Western blot analysis. This is the first report of deregulation of the Ras signalling pathway in iAMP21 BCP-ALL. Additional studies using a combination of target-enrichment strategies and massively parallel deep-sequencing identified NRAS and KRAS mutations in 18 of the remaining 44 iAMP21 patients. These mutations were frequently identified in hotspot regions: NRAS G12D, KRAS G13D. Dual mutations were identified in both NRAS and KRAS in two patients, suggesting a cooperative effect of Ras signalling pathway defects.In conclusion, genomic analysis and whole exome sequencing of a single patient has identified novel mutations: ENPEP and SMYD3, RPL24, SIN3B; in the B-cell development and histone modification pathways, respectively, indicating the involvement of pathways in iAMP21 patients that are common to other BCP-ALL subtypes. Although previously reported at a high level in high-risk childhood BCP-ALL, the involvement of the Ras signalling pathway was observed for the first time in iAMP21 BCP-ALL, through the identification of a NF1 mutation in patient 1. The involvement of the Ras signalling pathway in 41% of iAMP21 patients was confirmed by the finding of NRAS and KRAS mutations in a larger iAMP21 cohort. The novel identification of 3 mutated genes (SIM2, SYNJ1, UMODL1) and breakpoints within 2 others (DSCAM, TSPEAR) located within the DSCR reinforces common features between Down syndrome-ALL and iAMP21 BCP-ALL, which may further our understanding of the increased leukemia risk of Down syndrome individuals. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Grossmann:MLL Munich Leukemia Laboratory: Employment. Kohlmann:MLL Munich Leukemia Laboratory: Employment.

Genomic characterization implicates iAMP21 as a likely primary genetic event in childhood B-cell precursor acute lymphoblastic leukemia

AbstractIntrachromosomal amplification of chromosome 21 (iAMP21) defines a distinct subgroup of childhood B-cell precursor acute lymphoblastic leukemia (BCP-ALL) that has a dismal outcome when treated with standard therapy. For improved diagnosis and risk stratification, the initiating genetic events need to be elucidated. To investigate the genetic basis of BCP-ALL, genomes of 94 iAMP21 patients were interrogated by arrays, FISH, and multiplex ligation-dependent probe amplification. Most copy number alterations targeted chromosome 21, reinforcing the complexity of this chromosome. The common region of amplification on chromosome 21 was refined to a 5.1-mb region that included RUNX1, miR-802, and genes mapping to the Down syndrome critical region. Recurrent abnormalities affecting genes in key pathways were identified: IKZF1 (22%), CDKN2A/B (17%), PAX5 (8%), ETV6 (19%), and RB1 (37%). Investigation of clonal architecture provided evidence that these abnormalities, and P2RY8-CRLF2, were secondary to chromosome 21 rearrangements. Patient outcome was uniformly poor with standard therapy irrespective of the presence or absence of these changes. This study has provided evidence that chromosome 21 instability is the only anomaly among those so far investigated that is common to all iAMP21 patients, and therefore the initiating event is likely to be found among the complex structural rearrangements of this abnormal chromosome.

Prevalence and prognostic significance of chromosome 21 amplifications in children with relapsed acute lymphoblastic leukemia: the ALL-REZ BFM study group

Survival after relapse of childhood ALL is currently predicted from clinical factors; however, the underlying molecular determinants are poorly understood. To identify chromosomal alterations associated with prognosis, we screened 223 patients with first relapse of B-cell precursor ALL by multiplex ligation-dependent probe amplification (MLPA). Gains of chromosome 21 (chr21) were the most frequent alterations (41%) that were further classified into 3 groups: gain of chr21 (+21, 15%), gain of chr21 and a pattern of additional gains consistent with high hyperdiploidy (+21/HHD, 21%) as well as intrachromosomal amplification (iAMP21, 5%). Concerning time of relapse, all 3 groups had less early relapses than patients without an amplification of chr21. iAMP21 patients showed entirely late relapses. All 3 groups had a significant higher rate of treatment related death. The cumulative incidence of relapses was higher in patients with +21 or iAMP21 than in +21/HHD patients indicating that HHD positively influences outcome. Collectively, our results show that chr21 amplifications are the most frequent chromosomal alterations in relapsed ALL and are associated with prognosis and outcome.

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.

Complex genomic alterations and gene expression in acute lymphoblastic leukemia with intrachromosomal amplification of chromosome 21

We have previously identified a unique subtype of acute lymphoblastic leukemia (ALL) associated with a poor outcome and characterized by intrachromosomal amplification of chromosome 21 including the RUNX1 gene (iAMP21). In this study, array-based comparative genomic hybridization (aCGH) (n = 10) detected a common region of amplification (CRA) between 33.192 and 39.796 Mb and a common region of deletion (CRD) between 43.7 and 47 Mb in 100% and 70% of iAMP21 patients, respectively. High-resolution genotypic analysis (n = 3) identified allelic imbalances in the CRA. Supervised gene expression analysis showed a distinct signature for eight patients with iAMP21, with 10% of overexpressed genes located within the CRA. The mean expression of these genes was significantly higher in iAMP21 when compared to other ALL samples (n = 45). Although genomic copy number correlated with overall gene expression levels within areas of loss or gain, there was considerable individual variation. A unique subset of differentially expressed genes, outside the CRA and CRD, were identified when gene expression signatures of iAMP21 were compared to ALL samples with ETV6-RUNX1 fusion (n = 21) or high hyperdiploidy with additional chromosomes 21 (n = 23). From this analysis, LGMN was shown to be overexpressed in patients with iAMP21 (P = 0.0012). Genomic and expression data has further characterized this ALL subtype, demonstrating high levels of 21q instability in these patients leading to proposals for mechanisms underlying this clinical phenotype and plausible alternative treatments.