A Comprehensive FISH- and Array-Based Diagnostic Screeningstrategy for the Assessment of Clinically Relevant Genetic Parameters: A Prospective Analysis of 205 Childhood ALL Cases

Abstract Background: The detailed definition of causative genomic alterations is not only an indispensable prerequisite for the predictive and prognostic subdivision of childhood acute lymphoblastic leukemia (ALL) but increasingly also one, on which individualized treatment approaches will be based on. Apart from the already well-established genetic categories, the recent identification of several new classes of potentially relevant alterations together with the increasing availability of novel therapeutic options therefore necessitates a diagnostic workflow that is able to satisfy the ensuing clinical needs in a comprehensive manner. The two most interesting changes in this context are the therapeutically targetable recurrent but rare and heterogeneous tyrosine kinase and JAK2 pathway-activating (TKA) gene fusions and the more elusive cohort of apparently relapse risk-prone cases with hitherto only vaguely defined combinations of gene region-specific copy number alterations (CNA). Despite the availability of a multitude of applicable techniques, the fast and cost-efficient identification of the entire expectable abnormality patterns still remains a challenge, especially if one needs to perform the diagnostic work-up on a single case basis. We previously proposed that these diagnostic requirements could be covered best with a systematic hierarchical FISH screening approach for the identification of gene fusions together with array (combined SNP and non-polymorphic probes) analyses of genome-wide quantitative and qualitative large- and small-scale copy number aberrations (CNAs). Material and Methods: Since June 2015 we have therefore evaluated the feasibility of such a workflow in a prospective manner and screened so far 205 patients (i.e. 184 with B- and 21 with T-ALL, including 18 relapses with 14 diagnostic/relapse pairs) that were consecutively enrolled in the Austrian AIEOP-BFM 2009 treatment study. Cytogenetic preparations served as backup, since metaphase spreads were used for further FISH clarification of otherwise unresolvable complex rearrangements or ploidy patterns if deemed necessary. All identified gene fusions were subsequently validated with single or multiplex RT-PCR analyses. Results: FISH screening was positive in 90% (184/205) of cases and provided already the most essential diagnostic clues. CNAs were present in all T-ALL and 97% (179/184) of B-ALL cases, including 13 with an IKZF1pluspattern and three with ERG deletions, which both will be used das stratifying markers in the upcoming treatment trial. Taken together, our screening strategy allowed the unambiguous classification of the vast majority of B-ALLs: 70 hyperdiploid, 3 hypodiploid, 35 ETV6-RUNX1, 6 KMT2A-rearranged, 8 TCF3-PBX1, 2 BCR-ABL1, 4 dic(9;20), 5 iAMP21, 8 IGH-rearranged, 6 P2RY8-CRLF2, 3 PAX5-rearranged, 3 ZNF384-rearranged, 2 ETV6-rearranged, 3 TKA fusion-positive and 10 so called "B-other" cases without any apparent specific abnormality. RNA-sequencing analyses of these ten cases revealed that seven of them harbored a DUX4 gene rearrangement. Conclusions: Apart from its proven practical diagnostic value, our combined FISH/array approach has also several additional advantages, especially if one considers the amount of achievable information. Both procedures require only little amount of material and are highly standardized, reproducible and robust technologies. Moreover, arrays deliver DNA-sequence-based data, the coordinates of which can be efficiently stored, processed and analyzed. As such, they not only serve as a pure diagnostic tool but also as a valuable discovery platform. Disclosures No relevant conflicts of interest to declare.

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Chromothripsis-Mediated Structural Variations and Clonal Evolution In Recurrent Childhood High Hyperdiploid Acute Lymphoblastic Leukemia

Blood ◽

10.1182/blood.v122.21.233.233 ◽

2013 ◽

Vol 122 (21) ◽

pp. 233-233

Author(s):

Cai Chen ◽

Christoph Bartenhagen ◽

Michael Gombert ◽

Vera Okpanyi ◽

Vera Binder ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Copy Number ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Clonal Evolution ◽

Copy Number Variations ◽

Chromosome 3 ◽

Structural Variations ◽

Childhood All ◽

A Genome

Abstract High hyperdiploid acute lymphoblastic leukemia (HeH-ALL) is characterized by 51-67 chromosomes and nonrandom gains of specific chromosomes (X, 4, 6, 10, 14, 17, 18, and 21). It presents the most frequent numerical cytogenetic alteration in childhood pre B-cell ALL occurring in 25-30% of cases. Recurrent disease will affect 15-20%. Pre-leukemic HeH clones are generated in utero, but cooperating oncogenic lesions are necessary for overt leukemia and remain to be determined. Recently, a phenomenon termed chromothripsis has been described in which massive structural variations occur in a single aberrant mitosis. Whole or partial chromosomes are shattered and some fragments are lost in the process of rejoining. Thus, characteristically, chromosomal copy numbers oscillate between two copy number states. Chromothripsis has been suggested to be a tumor-driving alteration that may be present in 2-3% of all human cancers. Its role as a potential cooperating or initiating lesion in HeH-ALL has not been determined. We applied state-of-the-art whole-genome next-generation-sequencing to analyze structural variations in six pediatric patients with recurrent HeH-ALL. Matched sample sets taken at diagnosis, remission and/or relapse were compared. Paired end sequencing was carried out on a Genome Analyzer IIx or a HiSeq 2000 (Illumina), respectively. Reads were aligned against the human reference genome (GRCh37) using BWA. Translocations were detected by GASV. Copy number variations were analyzed by FREEC. Structural variations were validated by PCR/Sanger sequencing and FISH. Of the six patients analyzed, five harbored on average one interchromosomal translocation or intrachromosomal inversion, but one patient presented with massive genomic rearrangements (Figure). These affected chromosome 3, 11, 12 and 20. Ten copy number shifts on chromosome 3 oscillating between two copy number states (2 and 3) indicated that these rearrangements were caused by chromothripsis. Breakpoint sequencing revealed that one of the identified translocations (t(12;20)(p13.1;p12.3)), was indeed a three-loci-rearrangement composed of small fragments derived from chromosomes 3, 12 and 20. Characteristically for chromothripsis, the breakpoints clustered closely. Three breakpoints separated by 224 bp and 64 kb were located in the transducin (beta)-like X-linked receptor 1 (TBL1XR1) gene. Other genes repeatedly targeted included the MACRO domain-containing protein 2 (MACROD2) gene (a deacetylase involved in deacetylation of lysine residues in histones and other proteins), the KIAA1467 gene (a transmembrane protein of the integrin alpha FG-GAP repeat containing 3 (ITFG3) family), and a novel regulatory lincRNA (ENSG00000243276). MACROD2 was previously observed as a target of chromothripsis in a colorectal carcinoma. Thus, the characterized breakpoints may identify fragile genomic sites prone to chromothriptic rearrangement. DNA repair was effectuated by non-homologous-end-joining as typical addition of non-template nucleotides with microhomologies of two to four nucleotides at the breakpoints demonstrated. Copy number profiles of this patient showed that at least two distinct leukemic clones could be identified at diagnosis. One had acquired chromothriptic alterations and presented the dominant clone at relapse indicating chemotherapy resistance and tumor-driving potential. Prior whole-exome sequencing did not reveal mutations in known oncogenes or tumor suppressor genes. Therefore, loss of function or expression of genes affected by chromosomal rearrangements, such as TBL1XR1 that is recurrently mutated in childhood ALL with ETV6-RUNX1 translocation, may account for the tumor-driving effect. All leukemic cells at diagnosis showed conformity concerning number and pattern of whole chromosome gains demonstrating that chromothripsis was not an initiating oncogenic event, but occurred secondary to high hyperdiploidy. Further aberrations (t(4;7), loss of 4q) were gained by the chromothriptic clone and could be detected by FISH in minor subclones pointing at ongoing clonal evolution. Taken together, our study reveals chromothripsis as a novel assisting and tumor-driving lesion in HeH ALL. Chromothripsis in HeH-ALL. Copy number variations and translocations at diagnosis (left) and relapse (right). (magenta: chromothriptic translocations; green: other translocations) Disclosures: No relevant conflicts of interest to declare.

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Clonal Origins of 'late' Relapses in ETV6-RUNX1 Acute Lymphoblastic Leukemia..

Blood ◽

10.1182/blood.v114.22.1583.1583 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1583-1583

Author(s):

Frederik W van Delft ◽

Sharon W Horsley ◽

Kristina Anderson ◽

Caroline M Bateman ◽

Susan Colman ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

B Cell ◽

Copy Number ◽

Conflicts Of Interest ◽

Fusion Gene ◽

Lymphoblastic Leukemia ◽

Leukemic Cells ◽

Childhood All ◽

Remission Duration ◽

Clonal Origin

Abstract Abstract 1583 Poster Board I-609 Approximately a quarter of B cell precursor childhood acute lymphoblastic leukemia (ALL) is characterized by an ETV6-RUNX1 (TEL-AML1) fusion gene and has an overall good prognosis. The majority of these children will be treated on the standard risk arm of the United Kingdom ALL treatment protocols. Relapse usually occurs after cessation of treatment but remarkably can present many years later. The incidence of ETV6-RUNX1 at relapse has been reported to be less than or similar to de novo ALL. Molecular studies on neonatal bloodspots and on twins with concordant ALL have demonstrated the prenatal origin of major subtypes of childhood ALL, including most ETV6-RUNX1 fusion gene positive cases. In addition these investigations have suggested the existence of a preleukaemic stem cell requiring additional mutations or ‘hits’ in order to develop frank leukemia. To understand the genetic basis and clonal origin of late relapses we have compared the profiles of genome-wide copy number alterations (CNA) at relapse versus presentation in samples matched with remission DNA from 24 patients. The selected samples had tumor cell purity >75% before DNA extraction. DNA copy number alteration data was generated using the Affymetrix 500K SNP arrays. LOH analysis was performed using CNAG 3.0 and dCHIP 2008. Overall we identified 168 CNA at presentation and 252 at relapse (excluding deletions at IgH and TCR loci), equating to 6.96 and 10.3 CNA at presentation and relapse respectively. Although the number of CNA increased at relapse, no single gene or pathway was uniquely targeted in relapse. The most frequent alterations involved loss of 12p3.2 (ETV6), 9p21.3 (CDKN2A/B), 6q16.2-3 and gain of 21q22.1-22.12. A novel observation was gain of part or whole of chromosome 16 (2 patients at presentation, 5 at relapse) and deletion of the oncogene Plasmocytoma Variant Translocation 1 (PVT1) in 3 patients. Pathway analysis demonstrated frequent involvement at presentation and relapse of genes implicated in both B cell development (44 versus 46%) and cell cycle control (46 versus 71%). In order to study the clonal origin of relapse, we devised a classification describing the change in CNA between presentation and relapse in each individual patient. The clonal relationship between the presentation and relapse clone was established by the persistence of both the ETV6-RUNX1 fusion and at least 1 Ig and/or TCR rearrangement. We used a classification focussed on ‘driver’ CNA, defined as CNA that target genes functionally involved in leukemogenesis or CNA that are recurrently targeted as described in the literature. The four categories of relapse were type 1 (the dominant clone at presentation presented unchanged at relapse), type 2 (the relapse clone was derived from the major subclone at presentation with additional CNA), type 3 (the relapse clone was derived from a minor clone at presentation with gains and losses of CNA) and type 4 (the relapse clone is derived from an ancestral or preleukemic clone at initial presentation with all CNA gained). Twenty-one of the 24 patients were classifiable in this way (Figure 1). Although comparative relapse / presentation CNA profiles cannot identify precise clonal origins of relapse, the data indicate that irrespective of time to relapse (<2 to 9.9 years), the relapse clone appeared to be derived from either a major or minor clone at diagnosis with none (0/6) of the very late relapses (>5 years) derived from pre-leukemic cells lacking CNA. This data indicate diverse clonal origins of relapse and extended periods of dormancy, possibly via quiescence, for stem cells in ETV6-RUNX1+ ALL. Relapse type Remission duration (years) < 2 2 - 5 > 5 1 • • 2 • ••••••• •• 3 •• •• ••• 4 •• Figure 1. Each patient is represented by a black dot. Each patient is classified on the basis of the relapse type and remission duration. Disclosures No relevant conflicts of interest to declare.

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Significance of Copy Number Alterations for Molecular Treatment Response in Childhood Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v110.11.1434.1434 ◽

2007 ◽

Vol 110 (11) ◽

pp. 1434-1434

Author(s):

Doris Steinemann ◽

Gunnar Cario ◽

Martin Stanulla ◽

Leonid Karawajew ◽

Marcel Tauscher ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Treatment Response ◽

Copy Number ◽

Array Cgh ◽

Residual Disease ◽

Lymphoblastic Leukemia ◽

Individual Treatment ◽

Copy Number Alterations ◽

Childhood All ◽

Significant Difference

Abstract In vivo response to initial therapy, as assessed by determination of minimal residual disease after five and 12 weeks of treatment, has evolved as one of strong prognostic factors in children with acute lymphoblastic leukemia (ALL) treated according to the BFM regime. It is currently not known if the individual treatment response might be influenced by copy number alterations (CNA) leading to altered gene expression. We compared leukemic genomic profiles of 25 treatment sensitive (MRD-SR) and 25 resistant (MRD-HR) childhood ALL patients by means of high-resolution array-CGH. CNA were found in 46 patients (92%) of both treatment response groups. Microscopic alterations affecting the whole or nearly whole chromosome arm were frequently found, e.g. gain of 21 in 11/50, loss of 9p in 5/50, loss of 8p in 3/50, loss of 20q in 3/50 and loss of 7p in 2/50 or gain of 1q in 2/50. The most significant difference was a gain of chromosome 1q23-qter due to an unbalanced t(1;19), found in 10/25 MRD-SR patients, but in none of the MRD-HR patients (p<0.002). The most frequent CNA in the MRD-HR group were deletions of genomic regions harboring the immunoglobulin genes (Ig), e.g. 2p11.2 in 15 of 25 cases (60%) compared to 7 of 25 in the MRD-SR group (28%) (p=0.045). Combining all Ig loci, significantly more MRD-HR than MRD-SR patients were affected with deletions (17 versus 8 patients, p=0.02). The frequency of other CNA, like loss of 9p21 or gains of 21q, did not differ strongly between the two patient groups. This is the first study evaluating the clinical significance of CNA as detected by array-CGH in childhood ALL and may lead to improved risk classification.

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Cranial Irradiation Does Not Result in Pituitary-Gonadal Axis Dysfunction in Very Long-Term Male Survivors of Childhood Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v114.22.989.989 ◽

2009 ◽

Vol 114 (22) ◽

pp. 989-989

Author(s):

Niels J. van Casteren ◽

Rob Pieters ◽

Gert Dohle ◽

Manita van Baalen ◽

Sebastian Neggers ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Cranial Irradiation ◽

Gonadal Function ◽

Free T4 ◽

Childhood All ◽

Male Survivors ◽

Igf I

Abstract Abstract 989 Poster Board I-11 Introduction: One of the risks of childhood cancer treatment is fertility impairment later in life. In the past a large proportion of children with acute lymphoblastic leukemia (ALL) has received cranial irradiation as part of their treatment. The aim of this study was to evaluate whether cranial irradiation negatively affects pituitary regulated gonadal function in male survivors of childhood ALL. Patients and Methods: We examined gonadal function, including Inhibin B, LH, FSH, testosterone, and pituitary axis function by measuring TSH, Free-T4 and IGF-I levels in 89 long-term male survivors of childhood ALL after a median follow-up time of 19 year (range 7-34 years). Results: Twenty-nine out of 89 male ALL survivors received cranial irradiation. Inhibin, FSH, LH, Testosterone, testicular volume as well as TSH and Free-T4 levels were not different in the cranial irradiated group as compared to the non-irradiated group (table 1). In contrast, IGF-I levels were significantly lower in the cranial irradiated group. Survivors treated with total body irradiation or testicular irradiation had significantly decreased gonadal function based on hormone levels. Conclusions: These data show that, in contrast to the negative influence on the growth hormone axis, cranial radiotherapy as part of ALL treatment does not have a deleterious long-term effect on the hypothalamic–pituitary-gonadal axis or pituitary-thyroid axis. Disclosures: No relevant conflicts of interest to declare.

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Prognostic Impact of Ikaros (IKZF1) Gene Alterations In Childhood ALL Treated with ALL IC-BFM 2002 Protocol: A Comparison of Gene Expression and Genomic-Based Methods.

Blood ◽

10.1182/blood.v116.21.1656.1656 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1656-1656

Author(s):

Jana Volejnikova ◽

Ester Mejstrikova ◽

Karel Svojgr ◽

Jan Stary ◽

Jan Trka ◽

...

Keyword(s):

Gene Expression ◽

Dna Binding ◽

Gene Deletion ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Treatment Protocol ◽

Induction Treatment ◽

Prognostic Impact ◽

Childhood All ◽

Gene Alterations

Abstract Abstract 1656 Introduction: Recently, Ikaros (IKZF1) gene alterations were found to predict poor prognosis in childhood acute lymphoblastic leukemia (ALL). Thus, the implementation of IKZF1 status into the risk group stratification is discussed. So far, limited data are available concerning both IKZF1 importance in different treatment protocols for Ph-negative ALL and the choice of the best diagnostic method. In this study, we compared two methods based on either genomic DNA examination or gene expression analysis, and their prognostic impact within a treatment protocol for childhood ALL. Methods: Gene expression of functional (IK1, IK2) and non-DNA binding (IK4, IK6, IK8) IKZF1 isoforms was semi-quantitatively evaluated using Lab-on-a-chip (Agilent) electrophoresis and reported either as an absolute level or relatively to the total isoform signal. The thresholds for abnormal gene expression were set based on the analysis of peripheral blood (PB) of healthy donors, remission bone marrow (BM) samples of children with ALL, and sorted B- and T-cell precursor subpopulations. MLPA (multiplex ligation-dependent probe amplification) reaction including probes for Ikaros exons 1 to 8 was performed on BM DNA samples and the products were analyzed with the Coffalyser v9.4 software. Results: Results of both gene expression and MLPA analysis in the BM were available for 120 of 193 children diagnosed with ALL between 2002 and 2005. MLPA analysis revealed a deletion of at least one exon of IKZF1 gene in 17/120 (14%) patients. Of note, two patients with this deletion underwent a lineage switch (LS) from ALL to AML during the induction treatment. The entire IKZF1 gene was mononoallelically deleted in 3 patients. The ratio between non-DNA binding (IK4, IK4del, IK4A, IK6, Ik6del, IK8) and functional (IK1 and IK2) isoform expression was significantly elevated (non-DNA binding isoforms>70%) in 21 of 120 (18%) patients. The expression of a dominant-negative IK6 isoform was significantly elevated (>20% of total) in 7 of 120 (6%) patients. Surprisingly, gene deletion on one allele was not accompanied by decrease of total IKZF1 gene expression. On the contrary, patients with IKZF1 deletion had higher total IKZF1 transcript level than those without deletion (p=0.008, Mann Whitney), but it was not possible to set any reliable expression threshold for the prediction of gene deletion. The change on a DNA level was not always reflected in relative gene expression: Of 17 patients with gene deletion, only 7 had significantly altered short/long isoform ratio and 5 patients had an increased expression of IK6. Conversely, of 7 patients with IK6 overexpression, two patients had no DNA alteration, suggesting a different mechanism of altered gene expression. We next evaluated the prognostic impact of IKZF1 alterations in 113 patients treated with ALL IC-BFM 2002 protocol (5 patients were excluded due to Ph-positive ALL with imatinib-based treatment and 2 patients due to treatment change after LS). Patients with IKZF1 gene deletion had significantly worse relapse-free survival (RFS) than other patients (5-year RFS 50.0±14.4% vs. 90.8±2.9%, p=0.0002). The presence of IKZF1 deletion did not correlate with minimal residual disease (MRD) during induction treatment (days 8, 15, 33), neither in BM nor in PB. Patients with IK6 overexpression had 5-year RFS 50.0±20.4% compared to 88.4± 3.2% in those with low IK6 expression (p=0.004). The elevated short/long isoform ratio (>70%) had no prognostic impact. The best prediction of relapse was achieved via combining two factors: the presence of IKZF1 deletion detected by MLPA or the relative IK6 overexpression (>50% of total isoform signal). The 5-year RFS was 50.0±13.4% for this group (14 pts, 7 relapses) compared to 91.6±2.8% for other patients (99 pts, 8 relapses, p<0.0001). Conclusion: This study confirmed that the presence of Ikaros gene alterations was connected with a high risk of relapse also in a BFM-based protocol for Ph-negative childhood ALL treatment. The deletion within IKZF1 locus did not necessarily correlate with an altered Ikaros gene expression. Ideally, both genomic and gene expression-based approach should be applied together for the evaluation of prognosis. However, if this is not possible, the examination of DNA changes by MLPA identifies more patients who subsequently relapse than the gene expression-based approach. Support: VZ MSM 0021620813, P301/10/1877, IGA NS/10472-3 Disclosures: No relevant conflicts of interest to declare.

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Implication of Genetic Variations of Ikzf1, ARIDB5, and CEBPE Genes In the Risk of Childhood Acute Lymphoblastic Leukemia In Korea

Blood ◽

10.1182/blood.v116.21.3235.3235 ◽

2010 ◽

Vol 116 (21) ◽

pp. 3235-3235

Author(s):

Dong Kyun Han ◽

Hee Nam Kim ◽

Min Ho Shin ◽

Minenori Eguchi-Ishimae ◽

Mariko Eguchi ◽

...

Keyword(s):

Acute Lymphoblastic Leukemia ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Genetic Variations ◽

Childhood Acute Lymphoblastic Leukemia ◽

Asian Countries ◽

Childhood All ◽

Genomic Variations ◽

Genotype Frequencies ◽

The Impact

Abstract Abstract 3235 Background: Recent western studies have showed the implication of the germline genomic variations in IKZF1 gene at 7p12.2, ARIDB5 gene at 10q21.2, and CEBPE gene at 14q11.2 on the risk of childhood acute lymphoblastic leukemia (ALL); the most significant association was observed in the single nucleotide polymorphism (SNP) rs4132601 which located at 3' region of the IKZF1. IKZF1 plays important role in lymphocyte differentiation, proliferation and function, ARIDB5 in embryogenesis and growth regulation, and CEBPE in regulation of myelopoiesis. Genomic variants in these genes are therefore considered to be involved in transcriptional regulation and differentiation of B cell progenitors. However, there have been no reports on the role of germline variations in leukemogenesis of childhood ALL in Asian countries. The aim of this study is to show the impact of these genetic variants on childhood ALL in Korea. Patients and Methods: To examine the association between genetic variations (IKZF1 rs4132601, ARIDB5 rs7089424, and CEBPE rs2239633) and the risk of childhood ALL, we here analyzed 228 children with ALL and 508 healthy individuals in Korea. Results: In ARIDB5 rs7089424, TG and GG genotypes were significantly associated with a risk for ALL (odds ratio [OR], 1.63; 95% confidential interval [CI], 1.07–2.48; P=0.02 for TG genotype, OR, 2.69; 95% CI, 1.42–5.07; P=0.002 for GG genotype). The allele incidence of ARIDB5 rs7089424 was also significantly associated with a risk for ALL (OR, 1.66; 95% CI, 1.24–2.22; P=0.0006). CEBPE rs2239633 TT genotype showed a significant association with a decreased risk for ALL (OR, 0.54; 95% CI, 0.33–0.90; P=0.02 for TT genotype). The allele incidence of CEBPE rs2239633 was also associated with a decreased risk for ALL (OR, 0.77; 95% CI, 0.61–0.97; P=0.02). There was no significant association between IKZF1 rs4132601 polymorphism and a risk for ALL in this study. Conclusion: These results suggest that genomic variations of ARIDB5 and CEBPE may play an important role in the risk for childhood ALL in Korea, compared with findings from western countries showing a significant relation between IKZF1 and childhood ALL. Several factors should be considered to explain a discrepancy between our results and the previous studies, which include different genotype frequencies in polymorphisms and varied susceptibility to ALL in different ethnic groups. Further studies incorporating larger number of cases and analyzing other SNPs or other Asian countries are warranted in childhood ALL. Disclosures: No relevant conflicts of interest to declare.

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Immunoglobulin Heavy Chain Locus (IGH@) Translocations in Childhood B-Cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL): Incidence and Risk Stratification

Blood ◽

10.1182/blood.v120.21.1274.1274 ◽

2012 ◽

Vol 120 (21) ◽

pp. 1274-1274 ◽

Cited By ~ 1

Author(s):

Lisa J Russell ◽

Lisa Jones ◽

Amy Erhorn ◽

Amir Ensahei ◽

Dino Masic ◽

...

Keyword(s):

B Cell ◽

Conflicts Of Interest ◽

Risk Group ◽

Lymphoblastic Leukemia ◽

White Cell Count ◽

Scanning System ◽

Childhood All ◽

True Incidence ◽

Partner Gene ◽

Automated Scanning

Abstract Abstract 1274 Translocations involving the IGH@ locus have recently been characterised as a novel cytogenetic subgroup in BCP-ALL, involving unique partner genes including: CEBP family of transcription factors; cytokine receptors, EPOR and CRLF2; and the inhibitory transcription factor, ID4. As for the mature B-cell malignancies, their expression is always deregulated by juxtaposition of transcriptional enhancers within the locus. We have developed a high throughput FISH screening approach to ascertain the true incidence of IGH@ translocations in BCP-ALL. We screened approximately 80% (2603/3194) of patients entered to the childhood clinical trial (UKALL2003) using an IGH@ break-apart rearrangement probe with automated scanning and capture (CytoVision scanning system, Leica Microsystems). We identified IGH@ translocations in 4% (104/2603) of patients tested. This included an incidence of 4% (96/2286) in BCP-ALL and the novel observation of 2% (8/317) in T-ALL patients. The median age at diagnosis of BCP- and T-ALL IGH@ translocation patients was significantly higher at 12 and 14 years, respectively, compared to the median age of 5 years for the whole trial. The median white cell count (WCC) was low in IGH@ positive BCP-ALL (median 9.05×109/L) and higher in T-ALL (median 72.8×109/L). This study confirmed CRLF2 to be the most frequent IGH@ partner gene, observed in 20% (22/104) of BCP-ALL patients. ID4 and the CEBP family were found in 8% (8/104) and 9% (10/104), respectively. CEBPD was most common (n=6), while CEBPA (n=2), CEBPE (n=1) and CEBPG (n=1) were rare. Single patients were identified with involvement of BCL2, IGF2BP1, IGK@ and the TCRA/D locus. Novel involvement of TAL1 was identified in one T-ALL patient. In 57% of children (59/104: BCP-ALL n=52, T-ALL n=7) the IGH@ translocation partner gene remains unknown. Patients with a known partner gene did not differ from those with an unknown partner gene with respect to gender, age, WCC and National Cancer Institute (NCI) risk. However, patients with a known partner gene were strongly associated with an intermediate cytogenetic risk group while patients with an unknown partner gene were mixed (good risk, 7% v 19%, intermediate risk, 93% v 69% and poor risk, 0% v 12% p=0.01). There was no difference seen for gender, age, WCC, cytogenetic risk and NCI risk when each partner gene was investigated independently, apart from those with CRLF2 involvement who had a higher WCC (<50×109/L 59% v 81% and >50×109/l 41% v 12%, p=0.005) and were solely within the intermediate cytogenetic risk group (100% v 74%, p=0.03). We investigated copy number aberrations of genes commonly altered in BCP-ALL by Multiplex Ligation-dependent Probe Amplification (MLPA) using the Salsa P335-A1 IKZF1 kit (MRC Holland) in 60 IGH@ translocation patients. The genes investigated were IKZF1, CDKN2A/B, PAX5, EBF1, ETV6, BTG1, RB1, as well as the rearrangement, P2RY8-CRLF2. Deletions of none (31%), one (18%), two (17%), three (22%) or four (8%) genes were found. Deletions of CDKN2A, CDKN2B and IKZF1 were the most frequent, in 18%, 21% and 20%, respectively. Whilst the incidence of CDKN2A/B deletions was similar to that seen in childhood BCP-ALL, IKZF1 deletions were more prevalent (20% v 14%) and PAX5 deletions occurred at a lower incidence in IGH@ rearranged patients (13% v 19%). In the two T-ALL patients with DNA available, both showed deletions of CDKN2A with one patient also showing deletion of CDKN2B. In conclusion, IGH@ translocations are present in 4% of childhood ALL with involvement in both B- and T-cell disease. Patients belong to the intermediate cytogenetic risk group with an increased incidence of IKZF1 deletions. This differs from our recent report on adult BCP-ALL, where patients with an IGH@ translocation were associated with a worse outcome, although the increased incidence of IKZF1 deletions remained consistent across all age ranges. In over 50% of IGH@ positive children, the translocation partner remains to be elucidated, indicating the presence of as yet unidentified cryptic rearrangements. Characterisation of these partners may identify additional oncogenes involved in leukemogenesis or provide potential novel therapeutic targets, as recently demonstrated for CRLF2 in BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

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Acute Lymphocytic Leukemia and Down Syndrome

Blood ◽

10.1182/blood.v118.21.sci-8.sci-8 ◽

2011 ◽

Vol 118 (21) ◽

pp. SCI-8-SCI-8

Author(s):

Shai Izraeli

Keyword(s):

Down Syndrome ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Mtor Inhibitors ◽

Lymphocytic Leukemia ◽

Leukemic Cells ◽

Aberrant Expression ◽

Childhood All ◽

Increased Risk ◽

The Common

Abstract SCI-8 Children with Down syndrome are at a markedly increased risk for acute lymphoblastic leukemia (DS-ALL). These leukemias are exclusively of the B lymphoid precursor phenotype and occur in a similar age to “common” sporadic ALLs with the striking absence of infant ALL. Recent studies reveal that DS-ALLs are heterogeneous and differ from sporadic ALLs. Only about a fifth of DS-ALLs carry the common cytogenetic aberrations typical to childhood ALL. Genomic rearrangements leading to the expression of a cytokine receptor, CRLF2, are detected in 60% of DS-ALL in comparison with up to 10% of sporadic ALLs. CRLF2 heterodimerizes with Interleukin 7 receptor-α (IL7R) to form the receptor to thymic stromal lymphopoietin (TSLP). This receptor is usually present in macrophages, dendritic cells, and some T lymphocytes and participates in allergic and inflammatory processes. The aberrant expression of the TSLP receptor is DS-ALL (and sporadic ALL) is often associated with additional mutations that cause constitutive activation of the downstream JAK-STAT and mTOR growth signaling pathways. These are either lymphoid specific activating mutations of JAK2 or JAK1 or mutations in CRLF2 or IL7R that cause ligand-independent receptor dimerization. The role of the trisomy in selecting these somatic abnormalities is presently unknown. Clinically, the prognosis of DS-ALL is inferior to sporadic ALL mainly because of increased treatment toxicity. However, recent data suggest that the inferior outcome may also be related to the genetic properties of the leukemic cells and that excessive chemotherapy dose reduction may not be appropriate for these patients. Therefore increased vigilance for infectious complications and optimal supportive care are required during periods of intensive chemotherapy. The common activation of the TSLP signaling pathway in DS-ALLs suggests a future for targeted therapy with JAK and/or mTOR inhibitors. Importantly, research of DS-ALL has proven relevant for the general patient population with ALL, as somatic mutations in the TSLP pathway have been discovered in children and adults with sporadic ALL. A major research challenge is the elucidation of the roles of constitutional and somatic trisomy 21 in leukemogenesis. Disclosures: No relevant conflicts of interest to declare.

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Effects of G-CSF On Acute Lymphoblastic Leukemia.

Blood ◽

10.1182/blood.v120.21.2564.2564 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2564-2564

Author(s):

Jordan Basnett ◽

Adam Cisterne ◽

Kenneth F Bradstock ◽

Linda J Bendall

Keyword(s):

Bone Marrow ◽

Disease Progression ◽

Microarray Analysis ◽

Environmental Changes ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Hematopoietic Stem ◽

Childhood All ◽

Extracellular Matrix Components ◽

The Impact

Abstract Abstract 2564 G-CSF is commonly used to treat chemotherapy-induced neutropenia and for the mobilization of hematopoietic stem cells for transplantation in patients with leukemia. Administration of G-CSF has profound effects on the bone marrow microenvironment including the cleavage of molecules required for the maintenance of lymphopoiesis, including CXCL12 and VLA-4. We have recently reported that G-CSF results in the dramatic suppression of B-lymphopoiesis. This, together with previous reports by ourselves, and others, showing that disruption of CXCL12 or VLA-4 slow the progression of B-lineage ALL lead us to consider that G-CSF may similarly antagonize the progression of ALL. To explore this possibility, we examined the impact of G-CSF administration on six human ALL xenografts using a NOD/SCID mouse model. Mice were engrafted without radiation and G-CSF commenced when 1% of the bone marrow consisted of ALL cells. G-CSF was administered twice daily for 10 days, at which time all animals were culled and leukemia assessed in the blood, bone marrow and spleens. Surprisingly G-CSF was found to increase disease progression in two of xenografts investigated (1345 and 0398, referred to as G-CSF responsive xenografts hereafter), while the remainder demonstrated a small reduction in leukemia, with one showing a statistical significant decrease. No evidence for a direct mitogenic effect of G-CSF could be demonstrated in any of the xenografts using exogenous G-CSF in vitro cultures in the presence or absence of human or murine stromal support. Consistent with these findings, and previous reports, little to no G-CSF receptor was detected by flow cytometry or microarray analysis of xenografts. Microarray analysis of the xenografts revealed significant differences in gene expression between the G-CSF responsive xenografts and the remainder of the samples. A total of 83 genes were expressed at a higher level and 127 genes at a lower level in the G-CSF responsive xenografts. The more highly expressed genes included cell cycle regulators (eg cyclin A1), adhesion molecules (eg ALCAM), extracellular matrix components and surface receptors. Perhaps the most interesting was the exclusive expression of the acetylcholine receptor (cholinergic receptor, nicotinic, beta 4, nAChRb4) in the G-CSF responsive cases. Analysis of a large public dataset of childhood ALL samples revealed significantly higher expression of this gene in ALL samples with rearranged MLL (p<0.03). However, small numbers of cases in all ALL subgroups had greater than an 2 fold higher expression compared to normal B cell progenitors. The role of nAChR in the response of ALL cells to micro-environmental changes induced by G-CSF remains to be determined, however, nAChR has known roles in cell proliferation and inhibition of apoptosis. Furthermore G-CSF is known to induce acetylcholine production in other tissues. In summary, G-CSF inhibited leukemia progression in the majority of patient xenografts, however, in a subset of samples G-CSF accelerated disease progression. Clinically, G-CSF administration to ALL patients has not been associated with any major adverse outcomes. However our data suggest that a small subset of patients may experience accelerated disease. Identification of features associated with adverse responses to G-CSF will permit the identification of patients for whom G-CSF may present a risk for increased disease progression. Disclosures: No relevant conflicts of interest to declare.

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Distinctive Genotypes In Infants With T-Cell Acute Lymphoblastic Leukemia (T-ALL)

Blood ◽

10.1182/blood.v122.21.1377.1377 ◽

2013 ◽

Vol 122 (21) ◽

pp. 1377-1377

Author(s):

Marcela B Mansur ◽

Frederik W Van Delft ◽

Caroline L Furness ◽

Mariana Emerenciano ◽

Susan M Colman ◽

...

Keyword(s):

T Cell ◽

Copy Number ◽

Gene Rearrangement ◽

Conflicts Of Interest ◽

Lymphoblastic Leukemia ◽

Snp Array ◽

Genetic Alterations ◽

Driver Gene ◽

A Genome ◽

Overt Disease

Abstract Acute Lymphoblastic Leukaemia (ALL) in children is a disease strongly categorised by age at diagnosis of overt disease, leukaemic subtype and recurrent genetic alterations. ALL in infants is a rare subset often associated with MLL rearrangements, B cell precursor lineage lacking CD10 (pro-B ALL) and a prenatal origin in utero. Infant T-cell ALL (T-ALL) is a very rare and poorly defined entity with a worse outcome in general. The availability to us of a unique series of 13 infant T-ALL cases (≤12 months) along with 12 remission cases allowed us to perform a genome-wide study of the clonotypic alterations present in these leukaemias and to elucidate more definitively the critical genomic hits required to drive the emergence of overt disease. The main clinical and demographic characteristics observed in our series were a median age of 9 months, no predominance of gender and a high WBC count (≥ 50,000) in 12 out of 13 cases at diagnosis. The infants were treated according to Brazilian GBTLI protocol (n=3), BFM protocol (n=2), EORTC (n=3) and INTERFANT (n=5). All samples were analysed at diagnosis for the main T-ALL associated abnormalities and our results showed 4 cases mutated for NOTCH1. One case also presented with an FBXW7 mutation concomitant with wild type NOTCH1. Two cases showed PTEN mutations and all cases were germ line for IL7R. Furthermore, we did not observe any case with SIL-TAL1 or TLX3 rearrangements. TCRG & D were both rearranged in 7 cases while 1 case presented with only TCRG rearrangement and 1 case with only TCRD rearranged. Seven cases also showed rearrangement of TCRB and 1 case showed no receptor gene rearrangement at all. This latter case also had a classical ETP profile. MLL rearrangements were detected by FISH in 4 out of 13 cases. All diagnostic samples were analysed by SNP-array to identify genomic losses and gains. As a summary of the SNP data, a recurrent somatic chromosome 3 deletion was observed in 3 cases. The losses, similar in size, result in the complete deletion of MLF1 and have not previously been described in T-ALL. MLF1 plays a key role in AML and myelodysplastic syndrome leukaemogenesis. Q-PCR and FISH were used to confirm the copy number of MLF1 in those cases analysed. Akin to paediatric T-ALL we observed a PTEN deletion in 1 patient and 2 further cases with an 11p13 deletion that could lead to activation of LMO2. One case harboured a large 11q14.1-11q23.2 deletion that includes the important ‘driver’ gene ATM. The deletion of 11q including ATM has been reported in 30% of patients with CLL and at a low rate in ALL. One patient also harboured a TCRB-MYB translocation known to be associated with a young age of onset of T-ALL and an expression signature of proliferation/mitosis. Five out of 13 cases that were negative for MLL gene rearrangement, did show deletions of CDKN2A. These results were verified by Q-PCR copy number analysis and FISH. Overall, the current data suggest that infant T-ALL presents both different and infrequent genetic abnormalities in general when compared to T-ALL in older children and adults. Disclosures: No relevant conflicts of interest to declare.

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