Without prophylactic therapy, B-cell Acute Lymphoblastic Leukemia (B-ALL) spreads to the leptomeninges of the central nervous system (CNS) in up to 70% of patients. CNS involvement is more common in ...certain high risk B-ALL subgroups, including patients with KMT2A (MLL)-translocations, and disease relapse in the CNS carries a poor prognosis. The genetic determinants and biology of B-ALL dissemination to the CNS are poorly defined and therefore therapies targeting the drivers of CNS disease are lacking. Whereas B-ALL exhibits significant subclonal diversity that contributes to functional heterogeneity and disease relapse, recent reports suggest similar clonal composition of bone marrow (BM) and CNS disease, with the potential for CNS dissemination being a universal property of B-ALL cells (Williams et al. 2016, Bartram et al. 2018). Furthermore, functional studies of leptomeningeal disease have focused on the invasion of B-ALL cells into the CNS but limited studies have addressed the selection of genetic clones with the ability to grow within the subarachnoid space.
To better define the evolutionary history and biology of leptomeningeal B-ALL we performed targeted DNA, SNP copy number, RNA sequencing, and functional analysis on cells isolated from matched BM and CNS tissue of patient derived xenografts (PDX) generated from a cohort of paired diagnosis and relapse samples from 14 pediatric and adult B-ALL patients of varying cytogenetics. The majority of primary patient samples yielded CNS disease 20 weeks after intrafemoral injection into NSG mice. CNS disease burden was higher in PDXs derived from relapsed B-ALL samples. Human B-ALL cells isolated from the CNS of PDXs retained competence to repopulate disease in the BM, spleen, and CNS upon serial transplantation. Targeted DNA sequencing results analyzed using a Bayesian clustering method revealed different genetic clonal composition between matched BM and CNS cells in approximately half of the xenografts. PDXs from relapse samples were more likely to show genetic discordance between the BM and CNS. Copy number analysis also confirmed frequent genetic discordance between cells isolated from the BM and CNS from individual PDXs. Interestingly, in one patient all PDXs generated from the relapse sample displayed chromosome 6p and 17p hemi-deletions that were unique to the CNS. In total, PDXs from four patients showed recurrent enrichment of specific lesions in CNS-engrafting cells, suggesting that transit to and/or survival within the subarachnoid space can be the product of selection for genetic clones with increased CNS tropism.
RNA-seq of matched BM and CNS cells derived from 45 of the primary PDXs demonstrated that CNS-isolated cells were transcriptionally distinct from their matched BM. These differences were most pronounced in samples from patients with MLL-AF4 translocations, whose CNS isolated cells grouped together in multi-dimensional scaling. Using GSEA, the most highly CNS-enriched gene sets in MLL samples were related to mRNA translation initiation and polypeptide elongation. Translation-related gene sets are similarly enriched in the blasts of MLL B-ALL patients with CNS disease in the COG 9906 study. CNS-isolated cells from PDXs of MLL patients exhibited altered rates of protein synthesis compared to matched BM-isolated cells. Treatment of PDXs with the clinically-approved translational inhibitor omacetaxine mepesuccinate (OMA) effectively decreased rates of translation in CNS-engrafting cells. Moreover, OMA reduced leukemia burden nearly 4-fold in PDXs bearing established CNS infiltration generated from two MLL patients.
Our data represent an advance in the understanding of B-ALL CNS disease. We present a rich resource of genomic and transcriptomic data from xenografts spanning multiple B-ALL subgroups across diagnosis and relapse and have identified selection for genetically and biologically distinct clones in the CNS, contrary to the current model. Furthermore, we demonstrate that in MLL patients, dysregulation of protein synthesis occurs at CNS dissemination and targeting this process is a novel therapeutic paradigm that may benefit patients with CNS disease.
Mullighan:Cancer Prevention and Research Institute of Texas: Consultancy; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding; Amgen: Honoraria, Speakers Bureau; Abbvie: Research Funding.
•Intragenic PAX5 amplification defines a novel, relapse-prone subtype of B-cell precursor acute lymphoblastic leukemia with a poor outcome.
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Disease recurrence remains a significant cause of mortality in B-cell acute lymphoblastic leukemia (B-ALL). Genomic analysis of matched diagnosis and relapse samples have demonstrated that relapse ...arises from a minor subclone already present at diagnosis and not the dominant clone in the majority of patients. However, the reasons why only some clones survive therapy and generate relapse are obscure and elucidation of the mechanisms that underlie these differing fates may be revealed by functional analysis of isolated subclones. Previous work has shown that the subclonal diversity in B-ALL exists at the level of the leukemia-initiating cells capable of generating patient derived xenografts (Notta et al., Nature, 2011). In order to investigate the functional consequences of genetic clonal evolution during disease progression, we performed in-depth genomic and functional analysis of 14 paired diagnosis/relapse samples from adult and pediatric B-ALL patients with varying cytogenetic abnormalities. Diagnosis-specific, relapse-specific, and shared clonal and subclonal variants were identified by whole exome sequencing of the patient samples. Targeted sequencing of these variants in 372 xenografts generated by transplantation of CD19+ cells in a limiting cell dilution assay uncovered clonal variation. This analysis provided for the unequivocal identification of minor subclones ancestral to the relapse, termed diagnosis Relapse-Initiating (dRI) clones, in the diagnostic sample. Our xenografting approach enabled the physical isolation of dRI clones providing a unique opportunity to interrogate their epigenetic and transcriptional landscapes in order to unravel their relapse initiating capacity. To this end, representative diagnosis, dRI and relapse clones from 5 of the 14 patients were subjected to RNAseq and ATACseq (assay for transposase-accessible chromatin using sequencing) analysis. Despite the differences in transcriptional and chromatin openness between patients, principal component analysis of subclones from individual patients positioned the dRI clones as evolutionary intermediates between the diagnosis and relapse clones. Hierarchical clustering of the most significantly differentially expressed genes and open chromatin regions demonstrated that dRI clones shared gene expression and chromatin accessibility signatures with both the dominant diagnosis clone as well as the dominant relapse clone. To gain mechanistic insight into the data we used gene set enrichment analysis (GSEA) and identified common molecular pathways present in all patients that were enriched in dRI clones and persisted at the time of relapse in comparison to the dominant diagnosis clone. dRI and relapse clones converged in the activation of genes involved in cellular functions such as endocytosis, autophagy and innate immune response. In addition, cell surface proteins like ABC transporters and ephrins were also upregulated in dRI and relapse clones. Remarkably, functional interrogation of dRI clones in secondary xenografts, in comparison to more representative diagnosis clones, displayed increased tolerance to standard chemotherapeutic agents (dexamethasone, L-asparaginase and vincristine). Investigation of the molecular pathways and cellular receptors/transporters identified by gene expression analysis are being assessed in vitro and in vivo as potential targets for novel therapeutic approaches and disease monitoring.
Overall, we have shown evidence that minor subclones at diagnosis, ancestral to the relapse clone, possess functional advantages and unique properties over other diagnostic subclones prior to treatment exposure. In depth analysis of pathways identified in these dRI subclones will shed light on potential new therapeutic approaches for abrogating and reducing disease recurrence in B-ALL.
Mullighan:Amgen: Honoraria, Speakers Bureau; Cancer Prevention and Research Institute of Texas: Consultancy; Abbvie: Research Funding; Pfizer: Honoraria, Research Funding, Speakers Bureau; Loxo Oncology: Research Funding.
Abstract 3470
Hematopoiesis is traditionally seen as the unidirectional maturation of stem cells into lineage committed cells. Recent data are suggestive for some degree of lineage flexibility in ...both normal as malignant cells. We here present a boy that presented with T-cell acute lymphoblastic leukemia (T-ALL). During T-ALL treatment, the patient developed in short time a non-Langerhans-cell histiocytose in the ileum, and subsequently a disseminated form of histiocytic sarcoma (a.o. also in the liver). Interestingly, the same clonal T cell receptor (TCR) gene rearrangements were found in all three malignancies (TCRB-VJ, TCRG-VJ and TCRB-DJ) indicating they were related. There were no immunoglobulin rearrangements. To understand the evolution from T-ALL to non-Langerhans-cell histiocytose and to histiocytic sarcoma, an extensive genetic analysis was performed. Using a SNP6.0 array platform we analyzed DNA isolated from T-ALL cells, a bone marrow sample obtained during complete remission from theT-ALL, a biopsy from the ileum tumor, the liver tumor, and DNA isolated from skin fibroblasts.
The three tumor samples were not fully concordant in the SNP analysis, with lesions acquired but also absent from subsequent samples. All three tumor samples showed identical TCR gene rearrangements as well as a loss of the CDKN2A/B region, the only gene-containing copy number aberration (CNA) present in the T-ALL. The T-ALL sample showed a homozygous loss of the CDKN2A/B region, whereas both the ileum and liver samples showed a heterozygous loss. In addition, the three tumor samples shared two 1Mb regions of loss of heterozygosity (LOH) on chromosomes 6p and 11p, containing DDB2, MADD and RUNX2, amongst others. A gain on chromosome 19q containing 12 genes was shared between the ileum and liver sample only. In addition, the ileum tumor showed 14 non-shared CNAs of which 11 losses, 2 homozygous losses, and 1 gain, affecting 11 genes including ZFAT, PTPRK and beta-catenin (CTNNB1). The liver tumor acquired gains of a large part of chromosome 5, the p-arm of chromosome 11 and the q-arm of chromosome 22 reminiscent of a chromothrypsis event. The PAX5 gene indicated in the progression of an ALL to histiocytic sarcoma was not affected in either of the samples. These results indicate that the three tumors are related but did not evolve from each other in a linear manner. Apparently, the common oncogenetic transformation occurred at an early differentiation stage with lineage plasticity, i.e. with some lineage specification but without full lineage commitment.
No relevant conflicts of interest to declare.
Abstract 2458▪▪This icon denotes a clinically relevant abstract
The bacterially derived enzyme L-Asparaginase (ASNase) is a key component in the multidrug therapy regimens used worldwide to treat ...pediatric and adult patients with acute lymphoblastic leukemia (ALL), however little is known about the molecular mechanisms that control the pharmacokinetics of this therapeutic protein. As a result, many patients who receive a standardized dose either exceed or do not reach the desired serum concentration. While elevated serum levels are associated with an increase in treatment related morbidity, underexposure seriously compromises therapeutic benefits.
In search of molecular factors that determine ASNase turnover in vivo, we investigated a patient with strongly aberrant clearance kinetics. This 3-year old female diagnosed with common ALL suffered from severe ASNase-induced adverse events upon treatment with ErwiniaSNase as a result of strongly elevated serum ASNase levels. Pharmacokinetics data showed a severely delayed ASNase clearance. As a result, serum ASNase levels accumulated to intolerable levels upon repeated administration of the drug. We isolated DNA from peripheral blood mononuclear cells and buccal cells of this patient and performed targeted sequencing on genes suggested to be involved in ASNase clearance. We identified a novel heterozygous mutation in the gene encoding Cathepsin B in the germline of this patient. The mutant allele shows a deletion of a single codon, leading to a deletion of a lysine residue in the C terminus of the protein. We generated an EBV LCL cell line from this patients which showed a 75% reduction in Cathepsin B activity, relative to controls, indicating that this heterozygous mutation has a profound effect on the total Cathepsin B activity.
Cathepsin B is normally synthesized as a 37 kD pre-pro enzyme and is processed in a two step process into a mature 2-chain active form. During this process, the protein is transported to the lysosome where it exerts its primary function. Using a combination of biochemical and imaging experiments we show that the mutant Cathepsin B cannot be processed into the mature form and is retained in the endoplasmatic reticulum. ASNase degradation assays demonstrate that this mutant form of Cathepsin B shows a diminished protease activity towards both E.coli and Erwinia ASNase, consistent with the reduced clearance observed in our patient.
Cathepsin B and other cellular proteases are either actively secreted or released into the serum as a result of cell lysis. Although we find a variable low but detectable activity of Cathepin B in serum samples, all tested preparations of ASNase were stable upon prolonged incubation in serum, suggesting that serum components are not contributing to ASNase clearance in vivo. Hence, we propose that cellular uptake and subsequent proteolytic degradation of ASNase is the primary mechanism of clearance.
In conclusion, we have identified a mutation in protease Cathepsin B and provide evidence that this mutation results in a loss of protease function towards ASNase, which can explain the strongly delayed clearance of ASNase in the patient. Our data suggest that differences in Cathepsin B activity may contribute to the large inter-patient variability in ASNase pharmacokinetics. Furthermore, given the role of Cathepsin proteases in antigen presentation, Cathepsin B may not only provide a target for predicting or controlling ASNase clearance kinetics but inhibition of Cathepsin may also prevent or delay the formation of inhibitory antibodies.
Boos:European Erwinase Providers (EUSAPharm): Speakers Bureau; Medac: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau. Lanvers-Kaminsky:Medac: Speakers Bureau.
Background
Isolated polycystic liver disease (ADPLD) is an autosomal dominant Mendelian disorder. Heterozygous PRKCSH (where PRKCSH is protein kinase C substrate 80K‐H (80 kDa protein, heavy chain; ...MIM*177060) mutations are the most frequent cause. Routine molecular testing using Sanger sequencing identifies pathogenic variants in the PRKCSH (15%) and SEC63 (where SEC63 is Saccharomyces cerevisiae homolog 63 (MIM*608648); 6%) genes, but about approximately 80% of patients meeting the clinical ADPLD criteria carry no PRKCSH or SEC63 mutation. Cyst tissue often shows somatic deletions with loss of heterozygosity that was recently recognized as a general mechanism in ADPLD. We hypothesized that germline deletions in the PRKCSH gene may be responsible for hepatic cystogenesis in a significant number of mutation‐negative ADPLD patients.
Methods
In this study, we designed a multiplex ligation‐dependent probe amplification (MLPA) assay to screen for deletions of PRKCSH exons. Genomic DNA from 60 patients with an ADPLD phenotype was included.
Results
MLPA analysis detected no exon deletions in mutation‐negative ADPLD patients.
Conclusion
Large copy number variations on germline level are not present in patients with a clinical diagnosis of ADPLD. MLPA analysis of the PRKCSH gene should not be considered as a diagnostic method to explain hepatic cystogenesis.
The pathogenesis of polycystic liver disease is not well understood. The putative function of the associated proteins, hepatocystin and Sec63p, do not give insight in their role in cystogenesis and ...their tissue-wide expression does not fit with the liver-specific phenotype of the disease. We designed this study with the specific aim to dissect whether pathways involved in polycystic kidney diseases are also implicated in polycystic liver disease. Therefore, we immunohistochemically stained cyst tissue specimen with antibodies directed against markers for apoptosis, proliferation, growth receptors, signaling and adhesion. We analyzed genotyped polycystic liver disease cyst tissue (n=21) compared with normal liver tissue (n=13). None of the cysts showed proliferation of epithelial cells. In addition, anti-apoptosis marker Bcl-2 revealed slight increase in expression, with variable increase of apoptosis marker active caspase 3. Growth factor receptors, EGFR and c-erbB-2, were overexpressed and mislocalized. We found EGFR staining in the nuclei of cyst epithelial cells regardless of mutational state of the patient. Further, in hepatocystin-mutant polycystic liver disease patients, apical membranous staining of c-erbB-2 and adhesion markers, MUC1 and CEA, was lost and the proteins appeared to be retained in cytoplasm of cyst epithelia. Finally, we found loss of adhesion molecules E-cadherin and Ep-CAM in cyst epithelium of all patients. Nevertheless, we observed normal beta-catenin expression. Our results show that polycystic liver disease cystogenesis is different from renal cystogenesis. Polycystic liver disease involves overexpression of growth factor receptors and loss of adhesion. In contrast, proliferation or deregulated apoptosis do not seem to be implicated. Moreover differential findings for PRKCSH- and SEC63-associated polycystic liver disease suggest a divergent mechanism for cystogenesis in these two groups.
Abstract 399
Recurrent deletions in genes affecting key cellular pathways are a hallmark of pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). To gain more insight into the mechanism ...underlying these deletions, we have studied the occurrence and nature of abnormalities in one of these genes, the B cell translocation gene 1 (BTG1), in a large cohort of pediatric BCP- and T-lineage ALL cases.
The BTG1 gene was found to be exclusively affected by genomic microdeletions, which were detected in 65 out of 722 BCP-ALL patient samples (9%), but not in 109 T-ALL cases. Genomic sequencing of the entire BTG1 open reading frame (n=135) and the main coding second exon (n=158), as well as bisulphite sequencing of its promoter (n=25) provided no evidence for the presence of BTG1 point mutations or promoter methylation, respectively. Eight distinct deletion sizes (ranging from 101 to 557 kb) were identified, which all clustered at the telomeric site within a 30 bp stretch in the second and last exon of the BTG1 gene. The deletions resulted in almost identical truncations of the open reading frame and a concomitant loss of two conserved C-terminal protein interaction domains. Truncated BTG1 fusion transcripts specific for each type of deletion could be detected exclusively in the deletion-positive BCP-ALL cell lines and primary BCP-ALL samples, whereas wild-type BTG1 mRNA was expressed in both deletion-positive and -negative cases. The presence of V(D)J recombination signal sequences at both sites of virtually all deletions strongly suggests RAG1/RAG2-mediated recombination as the responsible mechanism. In line with these observations, BCP-ALL, but not T-ALL, cell lines showed increased levels of histone H3 trimethylation at lysine 4 (H3K4me3) at the BTG1 gene locus. This epigenetic mark is associated with actively transcribed loci and acts as a docking site for RAG2 binding, thereby facilitating V(D)J recombination.
BTG1 deletions were found to be unevenly distributed between the different cytogenetic subgroups, being present in 19% (n=27/142) of the ETV6-RUNX1 (TEL-AML) and 26% (n=6/23) of the BCR-ABL1 positive cases, and in only 3% (n=5/160) of the hyperdiploid cases (P<0.001, P=0.003 and P=0.002, respectively). In addition, targeted copy number analysis of recurrently affected genes in ALL revealed that cases with BTG1 deletions more frequently harbor deletions of ETV6, RB1 and EBF1 (P=0.007, P<0.001 and P<0.001, respectively). Using a sensitive PCR-based screening assay, we identified (multiple) additional BTG1 deletions at the subclonal level in 19 of the 65 deletion-positive BCP-ALL cases (29%), and in 21 of the 89 deletion-negative BCP-ALL cases (24%), but not in the T-ALL cases (n=77) or bone-marrow samples from healthy donors (n=26). Similar to the clonal BTG1 deletions, these subclonal events were enriched in the ETV6-RUNX1 subgroup and absent in the hyperdiploid cases.
In conclusion, our results indicate that BTG1 deletions act as ‘driver’ mutations in specific BCP-ALL subtypes, in which they can arise independently in multiple subclones in a locus that appears to be prone to aberrant RAG1/RAG2-mediated recombination events.
No relevant conflicts of interest to declare.
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.
No relevant conflicts of interest to declare.
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