ALBERT

Beschreibung: Asparaginase (ASNase) is one of the cornerstones of the multi-drug treatment protocol that is used to treat acute lymphoblastic leukemia (ALL) in pediatric and adult patients. Despite the fact that ASNase has been used in ALL treatment protocols for decades, little is known about the biodistribution and the mechanism of ASNase turnover in vivo. A large inter-individual variation in ASNase pharmacokinetics is observed in patients. While elevated ASNase levels are associated with an increase in adverse events, underexposure, frequently caused by antibody mediated clearance, seriously reduces therapeutic efficacy. To date, it is not possible to predict pharmacokinetics of ASNase in individual patients and therefore current therapeutic protocols are supported by frequent monitoring of ASNase levels and adjustments of administration schemes. We used an in vivo imaging approach to study ASNase biodistribution and pharmacodynamics in a mouse model and provide in vitro and in vivo evidence that identifies the endo-lysosomal protease Cathepsin B in macrophages as a critical component of ASNase degradation. Results/Discussion Mice were injected with 111Indium-labeled ASNase and biodistribution was monitored by quantitative microSPECT/CT scans and ex vivo analysis of organs using a gamma counter. Over time, ASNase accumulated in the liver and particularly the spleen and the bone marrow. We hypothesized that macrophages in these organs, efficiently take up the ASNase, thereby rapidly clearing the active enzyme from the blood. Immunohistochemical analysis confirmed the presence of ASNase in cells positive for the murine macrophage marker F4/80. To confirm the importance of macrophage populations in ASNase clearance, we depleted mice from phagocytic cells by injection of clodronate liposomes, and studied ASNase biodistribution and kinetics. Indeed, clodronate pretreatment significantly diminished the accumulation of ASNase in the liver, spleen and the bone marrow while doubling the circulatory half-life of serum ASNase activity. We conclude from these experiments that macrophages determine the pharmacokinetics of asparaginase, which raises the question whether rapid clearance of the drug by bone marrow resident macrophages will negatively affect the depletion of asparagine in the bone marrow niche. We previously linked a germline mutation in the gene encoding endosomal protease Cathepsin B to strongly diminished asparaginase degradation in a pediatric ALL patient. To connect the macrophage mediated clearance to the proposed role of Cathepsin B in ASNase degradation, we studied the contribution of this protease in macrophage-mediated degradation of asparaginase. We used cell lines to show that Cathepsin B expression is induced during differentiation from monocytes towards macrophages. This is consistent with our finding that macrophages, but not monocytes, are capable of degrading ASNase. Furthermore, we used both chemical inhibition and RNAi mediated knockdown of Cathepsin B to show that this protease is required for ASNase degradation in these macrophages. Finally, by comparing Cathepsin B knockout mice with wildtype littermates, we demonstrated that loss of Cathepsin B activity significantly delayed clearance of serum asparaginase, consistent with a prominent role for this lysosomal protease in ASNase turnover. In conclusion, by using in vivo imaging we showed that asparaginase is efficiently cleared from the circulation by macrophages. In particular, bone marrow resident macrophages may provide a protective environment for leukemic cells by effectively removing the therapeutic protein from the bone marrow niche. However, both the prominent role of macrophages and the importance of the lysosomal protease Cathepsin B in asparaginase clearance, may allow the rational design of a next generation asparaginase. Disclosures Metselaar: Enceladus Pharmaceuticals: Employment, Equity Ownership.

Beschreibung: Background Glucocorticoids (GCs) such as prednisolone and dexamethasone are critical components of multi-agent chemotherapy regimens used in the treatment of acute lymphoblastic leukemia (ALL). Children with ALL are stratified into risk groups based on diagnostic features (i.e. age and cytogenetics) and therapy response. It has been established that the initial response to prednisolone is a major prognostic factor. Moreover, at relapse, de novo or acquired resistance to GCs is common and represents an important determinant in treatment failure. Recent studies performed by us and others have identified IKZF1 gene deletions and mutations as an independent prognostic factor that predicts prognosis and treatment outcome of children with B cell precursor ALL (BCP-ALL). These monoallelic IKZF1 gene deletions either affect the whole gene or may result in expression of dominant-negative IKZF1 isoforms due to intragenic deletions. However, it has not been established whether loss of IKZF1 function directly impacts the response to glucocorticoids. Results We examined whether haplodeficiency for Ikzf1 gene expression in mouse lymphocytes affects glucocorticoid-induced apoptosis. Splenocytes from Ikzf1+/- knockout mice were activated with lipopolysaccharide (LPS) and treated with increasing concentrations of either prednisolone or dexamethasone for 48 hours. B-lymphocytes haplodeficient for IKZF1 showed a significantly enhanced survival after treatment with GCs compared to wild type cells, as measured in an MTS assay and by AnnexinV staining. In case of prednisolone, the inhibitory concentration (IC50) was about ∼200-fold higher in the Ikzf1+/- splenocytes as compared to the wild-type cells. Gene expression analysis revealed that Ikzf1+/- splenocytes displayed lower overall expression levels as well as diminished transcriptional activation of several glucocorticoid receptor (GR)-induced target genes (i.e. Sgk1, Irs2, Zfp36L2). Furthermore, in luciferase reporter assays we established that IKZF1 overexpression enhances GR-mediated transcriptional activation in response to prednisolone. Finally, lentivirus-mediated IKZF1-shRNA expression in Nalm6 cell line, which reduces endogenous IKZF1 protein levels to around 50%, inhibits prednisolone and dexamethasone-induced apoptosis, demonstrating that also in human leukemia cells reduced IKZF1 expression levels protect against GC-induced cell death. In conclusion, our data provide evidence that loss of IKZF1 function mediates resistance to glucocorticoid-induced apoptosis, which may contribute to the poor outcome of IKZF1-deleted BCP-ALL. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Abstract 755 Second hematologic malignancies in non-syndromic children without a pronounced family history for cancer may be mistaken for relapses or therapy-related malignancies. Recently, we characterized diagnosis and presumed relapse samples of 22 patients with very late disease recurrences (〉2.5 years), and identified 8 patients with leukemic presentations that were fully discordant at the level of TCR-rearrangements and DNA copy number aberrations (J Clin Oncol 2011; 29:1643-9). One of these patients showed a germline deletion comprising the recombination activating genes RAG1 and RAG2, and regulatory sequences of LMO2, genes frequently affected somatically in T-ALL, suggesting a genetic predisposition to leukemia. In the current study, we performed exome sequencing to assess whether consecutive leukemic presentations in such patients are indeed fully discordant, also at the sequence level, and to identify candidate pathogenic germline variants that point at a genetic predisposition. We sequenced the exomes in samples obtained from 2 consecutive leukemic presentations, and intermittent complete remissions, from 2 patients with very late disease recurrences (〉2.5 years) and discordant leukemic presentations. We found on average 26,600 variants per exome. Recurrent variants recorded in the dbSNP and/or 1000 Genomes databases, or those present in our in-house database (〉300 exomes) were excluded, resulting in an average of 989 private variants per exome. We divided these variants into 3 groups (i) somatic variants shared between the consecutive leukemic samples but not detected in remission (ii) somatic variants present in only one of the leukemic samples and (iii) germline variants present in the remission samples of the patients. All candidate somatic variants shared between two consecutive leukemic samples were re-sequenced by Sanger sequencing and were shown to be either present in all three samples, and thus originally missed in the remission sample, or falsely detected in one or more leukemic samples. Therefore, we conclude that in both patients no somatic variants were shared between the first and second leukemic presentations, which confirms that these patients suffered from clonally unrelated second T-ALLs. From all somatic variants present in only one of the leukemic samples, we focused on variants in exons or splice junction sites. We found 4 nonsense mutations, 9 frame-shift mutations, 12 in-frame in/dels and 7 non-synonymous missense variants with a high interspecies conservation score (PhyloP〉3.0), mostly affecting genes implicated in oncogenesis like PTEN, TET3, CDKN2C, CD109, and GLRX2. Each leukemic sample harbored 2–11 of these putative deleterious variants. In the germline of the two patients, we identified 314 and 190 non-synonymous unknown variants in exons or splice junction sites, respectively. Among these were 12 nonsense mutations, 7 canonical splice-site mutations, 20 frame-shift mutations, 11 in-frame in/dels and 143 non-synonymous missense variants at highly conserved positions (PhyloP〉3.0). Filtering of these variants for known T-ALL associated genes resulted in several interesting novel candidate predisposing genes such as, among others, RANBP17 and HOXC13. Sequencing of the entire RANBP17 open reading frame in a cohort of 24 sporadic T-ALL samples revealed that this gene was somatically affected in one of them. In conclusion, we confirmed by exome sequencing that consecutive leukemic presentations in patients with late T-ALL recurrences may be fully discordant and thus represent independent leukemia occurrences, most likely caused by predisposing germline abnormalities. Disclosures: No relevant conflicts of interest to declare.

Beschreibung: Resistance to glucocorticoids (GCs) is a major clinical problem in the treatment of acute lymphoblastic leukemia (ALL), but the underlying mechanisms are not well understood. Although mutations in the glucocorticoid receptor (GR) gene can give rise to therapy resistance in vitro, acquired somatic mutations in the GR are rarely encountered in patients. Here we report that the protein encoded by the BTG1 gene, which is frequently deleted in (pediatric) ALL, is a key determinant of GC responsiveness. Using RNA interference, we show that loss of BTG1 expression causes GC resistance both by decimating GR expression and by controlling GR-mediated transcription. Conversely, reexpression of BTG1 restores GC sensitivity by potentiating GC-induced GR expression, a phenomenon known as GR autoinduction. In addition, the arginine methyltransferase PRMT1, a BTG1-binding partner and transcriptional coactivator, is recruited to the GR gene promoter in a BTG1-dependent manner. These results implicate the BTG1/PRMT1 complex in GR-mediated gene expression and reveal that deregulation of a nuclear receptor coactivator complex can give rise to GC resistance. Further characterization of this complex as part of the GR regulatory circuitry could offer novel opportunities for improving the efficacy of GC-based therapies in ALL and other hematologic malignancies.

Beschreibung: Introduction: Tay-Sachs Disease (GM2 gangliosidosis) is a lysosomal storage disorder caused by beta-hexosaminidase A (Hex-A) deficiency, which leads to severe and progressive neurological damage. BMT has been used successfully as treatment strategy in some storage disorders. GM2 gangliosidosis mouse models demonstrate that after BMT donor-derived metabolically competent cells infiltrate the CNS leading to delayed onset of symptoms and prolonged survival. Further enhancement of survival has been reported in mice receiving a combination of substrate deprivation therapy and BMT. To our knowledge data on the effects of BMT in children with Tay-Sachs disease are lacking. Patient: A three-year-old girl was diagnosed with Tay-Sachs disease with minimal clinical symptoms, after the same diagnosis was made in her symptomatic older sister. Pre-BMT conditioning consisted of busulfan, cyclophosphamide and ATG. She received T-cell depleted marrow from an HLA identical unrelated donor. Ciclosporin-A (CsA) was given as GvHD prophylaxis. Follow-up is 2.5 years. Results: Hex-A levels in leukocytes increased initially to donor levels and decreased to a steady level of 30% of controls for the last 1.5 years, indicating stable mixed chimerism. Also the enzyme levels in serum increased to 10% of controls, indicating release of enzyme into serum. Shortly after BMT, rapid progression of neurological symptoms occurred (MRI, EEG and neuro-psychological tests). Some neurological symptoms partially improved, others stabilized after CsA was stopped, suggesting that CsA had contributed to the neurological deterioration. Experimental substrate deprivation therapy with N-butyldeoxynojirimycin was started 1.5 years after BMT. No adverse effects of this drug were observed. Conclusion: BMT followed by substrate deprivation therapy in our patient with early stage of Tay-Sachs disease resulted in Hex-A enzyme levels in leukocytes and serum of 30% and 10% of normal levels respectively. Neurological deterioration occurred shortly after BMT and seemed to stabilize thereafter, but follow-up is too short to draw firm conclusions yet.

Beschreibung: Abstract 3303 Introduction: Despite improvement in frontline therapy in childhood acute lymphoblastic leukemia (ALL), central nervous system (CNS) relapse remains a significant clinical problem. The ALLR3 trial (ISCRTN 45724312) was designed specifically to address this issue with the use of drugs known to penetrate the CNS. The trial incorporated a randomization between Mitoxantrone and Idarubicin during induction. Mitoxantrone showed an early benefit in all patients resulting in closure of the randomization in December 2007 (ASH Annual Meeting Abstracts, Nov 2009; 114:3390). Subsequently all patients now receive Mitoxantrone. Here we report on the outcome of patients with isolated CNS relapse (iCNSr) or combined CNS relapse (involvement of CNS and bone marrow, cCNSr). Methods: CNS involvement was defined as ≥5 WBC/μl with morphological evidence of blasts in the cerebrospinal fluid (CSF). Combined relapse (cCNSr) was defined as CNS disease with ≥ 5% blasts in the bone marrow. Time to relapse was classified as, Very Early: within 18 months of first diagnosis; Early: after 18 months of first diagnosis but within 6 months of stopping therapy and Late: more than 6 months after stopping therapy. All patients received 3 blocks of chemotherapy. Subsequently, allogenic stem cell transplant (allo-SCT) was offered to all very early relapses (iCNSr & cCNSr), early iCNSr (irrespective of immunophenotype), all T-cell cCNSr (irrespective of time to relapse) and early or late pre-B cCNSr that had a minimal residual disease level of ≥ 104 at the end of induction. All other patients were offered chemotherapy and cranial radiotherapy. Results: Of a total of 330 relapsed patients, 102 (31%) had CNS involvement. Of these 63 (62%) had iCNSr and 39 (38%) had cCNSr. The incidence of CNS disease was higher in males (M:F, CNS relapses 2.5:1 vs all relapses 1.5:1). CNS relapses had a higher proportion of T-cell disease (pre B:T CNS relapses 3.6:1 vs all relapses 7.8:1]. The number of patients presenting in very early, early and late phases were 19 (19%), 55 (54%) and 28 (27%) respectively. All late iCNSr patients were males. Almost all late relapses (iCNSr and cCNSr) (27/28) were of a pre B phenotype. At the end of induction phase, 91/102 (89%) achieved complete remission (CR) and 82/102 (80%) remained in CR after 3 blocks of chemotherapy. The estimated 3-year overall survival (OS) and progression free survival (PFS) for all patients with CNS disease was 45.5% (95%CI 32.9, 58.0) & 43.4% (95%CI 32.0, 54.7) respectively. There were no significant differences in survival with respect to site of the disease (combined vs isolated), gender or immunophenotype (pre B vs T). As shown in Table 1, CNS relapse patients who received Mitoxantrone had a significantly improved outcome when compared to those who received Idarubicin. This was most evident in those who had i) iCNSr, ii) pre-B phenotype and iii) allo-SCT, when analyzed on an intention to treat basis. This represents a considerable improvement in outcome compared to the results obtained in these sub-groups of patients in the previous UK ALLR2 study (Roy A et.al. Br. J. Haem. 2005;130:67-75). Conclusion: Mitoxantrone is highly effective in children with relapsed pre B ALL who have CNS involvement. As there were no other differences between patients treated on Mitoxantrone or Idarubicin, effective systemic therapy is as important as CNS directed therapy, if not more, in treating patients with CNS relapse. Disclosures: Off Label Use: Most drugs used in this protocol are off label as the majority of drugs used in childhood ALL are not liscensed for use in children.

Beschreibung: Abstract 1104 Poster Board I-126 Relapse is the most common cause of treatment failure in pediatric acute lymphoblastic leukemia (ALL), and is difficult to predict from information at diagnosis in the majority of cases. To explore the prognostic impact of recurrent copy number abnormalities on relapse in children diagnosed with precursor-B cell ALL, we performed genome-wide copy number profiling of 34 paired diagnosis-relapse samples. Lesions detected at diagnosis were often absent at relapse, including recurrent targets in precursor-B ALL like PAX5 (not preserved in 2 out of 7 cases with deletions at diagnosis), CDKN2A (not preserved in 1 out of 15 cases), and EBF (not preserved in 2 out of 5 cases), which illustrates that these lesions are often secondary events that are not present in the therapy-resistant progenitor that causes relapse. In contrast, deletions and nonsense mutations in IKZF1, which encodes the lymphoid differentiation factor IKAROS, were highly frequent (38%) and always preserved at time of relapse. Locus-specific copy number screening of IKZF1 in an additional cohort of diagnosis samples from children enrolled in the Dutch treatment protocol DCOG-ALL9 with (n=40) or without (n=51) relapse revealed that IKZF1 deletions were significantly enriched in relapse-prone cases (22.5% vs 3.9%; P=0.007). An independent and unbiased validation cohort of 150 DCOG-ALL9 cases was used to confirm these findings, which established that 28.6% of the cases with IKZF1 deletion at diagnosis developed a relapse. Together, we conclude that deletions of IKZF1 in DCOG-ALL9 treated pediatric precursor-B ALL patients provide a strong prognostic marker for relapse. Disclosures No relevant conflicts of interest to declare.

Beschreibung: Abstract 3244 Poster Board III-181 Recent genome-wide profiling studies have revealed that childhood acute lymphoblastic leukemia (ALL) is characterized by recurrent microdeletions, including the cell cycle regulator CDKN2A, the B-cell differentiation genes PAX5, EBF1 and IKZF1 (Ikaros) and the anti-proliferative gene B-cell translocation gene 1 (BTG1). In a previous study, we have shown that BTG1 is an important determinant of glucocorticoid sensitivity (Van Galen et al. Blood/ ASH Annual Meeting Abstracts, 2008). In the present study we have characterized these cases in more detail and elucidated the frequency of recurrent lesions in BTG1 deletion cases. Using locus-specific MLPA screening of an unselected cohort of 305 precursor B-ALL cases, we identified 26 microdeletions (8.5%). All deletions encompassed BTG1 only. We were able to genomically profile 22 diagnosis samples using Affimetrix SNP6.0 arrays. Of these, 12 did not develop a relapse during a minimal of 4,5 years of follow up. The mean number of CNVs was 29.6 of which 10.3 gains and 22.5 losses (median size 512 kb and 115 kb respectively). BTG1 deletions were generally focal, varying in size from 104 kb to 1,4 Mb. In all but one patient the breakpoints at the 5' end of the deletion tightly clustered and subsequent fine-mapping using qPCR revealed that this breakpoint cluster was located within intron 1 of the BTG1 gene. At the 3'end of the deletion, four breakpoint clusters could be identified. Analysis of the copy number variation (CNV) profiles showed that patients with a BTG1 deletion more often harbored a deletion in IKZF1 compared to an unselected cohort of pre-B ALL cases (27% vs 7%, chi-square p=0.042). In contrast, recurrent CNVs like PAX5, EBF1 and CDKN2A/B occur in similar frequencies (23%, 9% and 32% vs 17%, 0% and 23% respectively). In addition, the BTG1 deletion cases that developed into a relapse showed significantly more often a deletion in CDKN2A/B compared to the BTG1 deletion cases that did not develop a relapse (60% vs 8%, p=0.02). Together, these data indicate that pediatric precursor-B ALL carrying BTG1 deletions have distinct genomic profiles, showing increased frequencies of deletions in IKZF1 and CDKN2A. Disclosures No relevant conflicts of interest to declare.

Beschreibung: We and others have shown that the B cell Translocation Gene 1 (BTG1) locus is affected by genomic deletions in 9% of pediatric acute lymphoblastic leukemia (ALL) patients. The fact that multiple subclones carrying distinct deletions can be present in individual patients suggests that interfering with normal BTG1 function provides a selective growth advantage to leukemic cells. However, it remains unclear how loss of BTG1 promotes clonal outgrowth. We detected an up to 15-fold increases of BTG1 expression when lymphoid cells were exposed to various challenge conditions, including nutrient limitation and ER stress induction. To test for a functional role for BTG1 in the cellular response to stress, we cultured BTG1 knockout cells in medium without glucose or amino acid (Figure 1) and found that BTG1 knockout cells show a 20-30% improved survival rate as compared to wildtype cells.Figure 1BTG1 knockout cells are resistant to Asparaginase treatment.Figure 1. BTG1 knockout cells are resistant to Asparaginase treatment. As Activating Transcription Factor 4 (ATF4) is a master regulator of cellular stress signaling, we hypothesized that the improved survival after BTG1 loss is regulated via ATF4. By immunoprecipitation experiments, we showed that BTG1 complexes with ATF4. In addition, co-expression of BTG1 attenuates ATF4 transcriptional activity on target gene promoters and suppresses both recombinant and endogenous ATF4 function in these promoter reporter assays (Figure 2).Figure 2BTG1 attenuates ATF4 transcriptional activity.Figure 2. BTG1 attenuates ATF4 transcriptional activity. Although BTG1 possesses no catalytic activity, it functions as a transcriptional co-regulator that acts by recruiting Protein Arginine Methyl Transferase 1 (PRMT1) to transcription factor complexes. By in vitro methylation assays with purified proteins we showed that ATF4 is directly methylated by PRMT1 on a single arginine residue. In addition we found that the PRMT1 interacting domain in BTG1, while dispensable for the BTG1-ATF4 interaction, is essential for the BTG1 mediated suppression of ATF4 function. In search for additional evidence for the functional interaction between BTG1 and ATF4 we performed global expression analysis on murine cells expressing the B cell marker B220. This revealed a significant deregulation of ATF4 target genes in BTG1 knockout cells when compared to wildtype cells. Together, our data indicate that BTG1 suppresses activation of ATF4 in response to cellular stress. Loss of BTG1 function, as it occurs during leukemia development, enhances ATF4 activity, thereby promoting cell survival under cellular stress conditions such as nutrient deprivation or ER stress. Leukemic cells carrying BTG1 deletions may thus benefit from this increased resistance to cellular stress, not only during leukemia development but also during treatment. Hence, targeting the ATF4 stress response pathway may prevent relapse of therapy-resistant leukemic clones. Cells were treated with 2 IU/L Asparaginase for 24 hours. After treatment, cell viability was measured using an MTT assay. The average of 4 independent experiments is plotted with error bars representing the standard error of the mean. A luciferase reporter gene controlled by the ATF4 responsive ASNS promoter region was transfected into HEK293 cells. Asparaginase treatment induces endogenous ATF4 expression, which results in an increase in luciferase signal (Mock transfected cells). Co-expression of BTG1 represses both endogenous ATF4 activity as well as ectopically expressed ATF4 activity as detected by a decrease in luciferase signal. The average of 2 independent experiments is plotted with error bars representing the standard deviation. Disclosures: No relevant conflicts of interest to declare.