ALBERT

Description: Approximately 25% of childhood acute lymphoblastic leukemias carry the ETV6/RUNX1 fusion gene. Despite their excellent initial treatment response, up to 20% of patients relapse. To gain insight into the relapse mechanisms, we analyzed single nucleotide polymorphism arrays for DNA copy number aberrations (CNAs) in 18 matched diagnosis and relapse leukemias. CNAs were more abundant at relapse than at diagnosis (mean 12.5 vs 7.5 per case; P = .01) with 5.3 shared on average. Their patterns revealed a direct clonal relationship with exclusively new aberrations at relapse in only 21.4%, whereas 78.6% shared a common ancestor and subsequently acquired distinct CNA. Moreover, we identified recurrent, mainly nonoverlapping deletions associated with glucocorticoid-mediated apoptosis targeting the Bcl2 modifying factor (BMF) (n = 3), glucocorticoid receptor NR3C1 (n = 4), and components of the mismatch repair pathways (n = 3). Fluorescence in situ hybridization screening of additional 24 relapsed and 72 nonrelapsed ETV6/RUNX1-positive cases demonstrated that BMF deletions were significantly more common in relapse cases (16.6% vs 2.8%; P = .02). Unlike BMF deletions, which were always already present at diagnosis, NR3C1 and mismatch repair aberrations prevailed at relapse. They were all associated with leukemias, which poorly responded to treatment. These findings implicate glucocorticoid-associated drug resistance in ETV6/RUNX1-positive relapse pathogenesis and therefore might help to guide future therapies.

Description: 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.

Description: The t(12;21) chromosomal translocation generating the TEL-AML1 fusion product is the most frequent genetic aberration of childhood acute lymphoblastic leukemia. This translocation has been shown to occur in utero and thus represents an early or even initiating event in leukemia development. However, additional molecular changes are necessary for the clinical manifestation of the disease. Despite increasing knowledge on TEL and AML1, the role of the TEL-AML1 fusion gene and its contribution to the malignant transformation is largely unknown. In particular, the mechanisms by which TEL-AML1 influences the biology of the affected cell, and thus survival and expansion, are yet to be defined. The aim of this study was to investigate the functional contribution of TEL-AML1 to the leukemic cell phenotype. We particularly sought to search for TEL-AML1 dependent genes as well as for pathways that function in a fusion gene dependent manner. The t(12;21) positive B cell precursor leukemia cell line REH was used as model system and RNA interference technology, a new and highly specific approach, was employed to block the expression of the TEL-AML1 fusion gene. The successful silencing of TEL-AML1 was demonstrated by Western blot analysis. In order to gain a broad overview of the impact of TEL-AML1 silencing upon the leukemic cell, RNA was isolated and subjected to Affymetrix gene chip analysis. In addition, we studied the proteome by high-resolution 2D electrophoresis. Expression profiling resulted in a specific pattern of differentially regulated genes after TEL-AML1 suppression with a particular emphasis on genes involved in the control of apoptosis. Genes with anti-apoptotic properties were down regulated after TEL-AML1 silencing while those that contribute to apoptosis were largely unaffected. Among the affected anti-apoptotic genes were the heat shock proteins, the most prominent representatives of which were HSP90 and HSP70. Their changes were observed at the mRNA but also at the protein level. This is of special interest, since both heat shock proteins interfere with the intrinsic as well as the extrinsic pathway of apoptosis and hence provide a powerful anti-apoptotic signal. These first data support a model in which TEL-AML1, as an early event, is functionally linked to an anti-apoptotic network and especially to the members of the heat shock protein family. Engagement of these mechanisms might rescue a cell from apoptosis and in consequence give rise to the development of a pre-leukemic clone, a reservoir for the acquisition of further mutations that might eventually give rise to leukemia. Thus, decreasing the apoptosis defence by silencing TEL-AML1 or by inhibiting the heat shock proteins might sensitize TEL-AML1 positive cells to apoptosis. This would not only be an option for future therapy of leukemias but also for the eradication of the pre-leukemic clone as a proposed source of relapse.

Description: The chromosomal translocation t(12;21)(p13;q22) resulting in the TEL/AML1 (also known as ETV6/ RUNX1) fusion gene is the most frequent translocation in childhood B cell precursor (BCP) ALL. This type of ALL is characterized by a unique molecular signature, which includes the overexpression of the gene for the erythropoietin receptor (EpoR). So far, it is not known what causes the overexpression of the EpoR gene or whether it has any effect on the t(12;21) positive leukemia. We therefore aimed to evaluate potential mechanisms responsible for the upregulation of the EpoR in t(12;21) leukemias and to find out whether signalling via this receptor affects survival or proliferation of leukemic cells. In addition, we planned to explore signalling pathways linked to the respective effects and to elucidate relevant mechanisms that might be essential for cell survival. We first excluded the possibility that the EpoR expression is upregulated as a consequence of high Epo levels in the plasma that are induced by the patients’ low hemoglobin (Hb) levels. While Hb levels from patients with t(12;21)+ ALL were significantly lower compared to those with other subtypes of BCP ALL (median, 6,15g/dL and 7,9g/dL, respectively; p

Description: The TEL/AML1 fusion gene results from the most frequent t(12;21)(p13;q22) translocation in childhood acute lymphoblastic leukemia (ALL). Its contribution to transformation is largely unknown, in particular with respect to survival and apoptosis. We therefore silenced TEL/AML1 expression in leukemic REH cells by RNA inhibition, which eventually led to programmed cell death. Microarray and 2D gel electrophoresis data demonstrated a differential regulation of heat-shock proteins (HSPs), among them HSP90, as well as of its client, survivin. Consistent with these findings, ectopic expression of TEL/AML1 in Ba/F3 cells increased protein levels of HSP90 and survivin and conferred resistance to apoptotic stimuli. Our data suggest that TEL/AML1 not only contributes to leukemogenesis by affecting an antiapoptotic network but also seems to be indispensable for maintaining the malignant phenotype. The functional relationship between TEL/AML1, HSP90, and survivin provides the rational for targeted therapy, be it the fusion gene or the latter 2 proteins.

Description: Background: Acute lymphoblastic leukemia (ALL) with a T cell immunophenotype accounts for about 15% of childhood ALL. While contemporary treatment protocols have improved the relapse free survival for children with T-ALL, it has for a long time been considered a malignancy with a dismal prognosis. In fact, this type of leukemia is still at a higher risk for treatment failure and early relapses than B cell precursor ALLs. Further intensification of conventional treatment or stem cell transplantation may increase the cure rate for only some subtypes of ALL but is also associated with considerable treatment-related mortality and morbidity. Thus, new treatment strategies that are less toxic and, at the same time, more effective for patients with a resistant disease, are needed. The aim of the study was to evaluate whether a specific immune reaction is triggered in children with T-ALL and to identify the respective targets. We chose SEREX (serological identification of leukemia derived antigens by recombinant expression cloning) to screen a T-ALL expression library with plasma from 18 children with T-ALL. Results: Thirteen antigens with homology to known genes that are involved in critical cellular processes were detected. Some of them have already been implicated in the induction of an immune response in a variety of cancer types. From four of these genes novel isoforms were identified and further analyzed. mRNA expression of three isoforms (HECTD1Δ, CX-ORF-15Δ and hCAP-EΔ) was restricted to more than 70% of T-ALLs (n=22) and specific antibodies against these isoforms were detected in up to 30% of patients (n=16) with the highest frequency for HECTD1Δ. The alternative splicing leads to the deletion of one glutamic acid located in a putative PEST domain thereby decreasing the PEST score suggesting a higher stability of the protein. Interestingly, the HECTD1 protein was present at high abundance in T-ALLs while is was not detectable in normal hematopoietic tissues. Since the leukemia-associated antigens detected in this study have an intracellular localization, a feature that is shared by the majority of SEREX defined antigens, the generation of immune effector responses most likely requires antigen presentation. To test this assumption, dendritic cells were loaded with HECTD1Δ protein and used for T cell stimulation. A specific T cell response was induced in vitro in all three donors, including a former T-ALL patient. Conclusion: Leukemia-associated antigens, identified by SEREX, appear to be capable of inducing both a humoral and cellular immune response in children with T-ALL. Thus, these data support further studies to establish new approaches for immunotherapy.