ALBERT

Description: Introduction: Despite the recent identification of the Ph-like subgroup of B-cell precursor Acute Lymphoblastic Leukemia (BCP-ALL), a large number of BCP-ALL patients lack cytogenetic and molecular defined lesions. To get a higher resolution and a broader molecular view of relapsed BCP-ALL, we designed a multi-omics study to reveal age-overriding relapse-driving alterations that may unravel novel molecular targets. Methods: We studied 150 paired samples (initial diagnosis: ID; relapse: REL; complete remission: CR) from 50 patients without known translocations. The cohort consisted of 24 adult and 26 pediatric patients with minimal residual disease 〈 0.05 % at CR. All patients were treated in population based German study trials (GMALL, BFM). We examined the mutational and copy number status via exome sequencing, obtained expression profiles and fusion-genes via RNA-sequencing and the methylation status via Illumina Methylation Array. Results: With a lenient approach detecting drivers and passengers, we identified significantly more mutations in REL compared to ID samples (adult median: 52 vs 38; pediatric median: 39 vs 27). In addition, we detected 4 hypermutators (more than 100 mutations per sample), 2 were pediatric and 2 were adult samples, 3 of which were REL samples. The most recurrently mutated genes were KRAS (n=15), NRAS (n=15), TP53 (n=13), CDC27 (n=13), KMT2D (n=11), IKZF1 (n=11), CREBBP (n=10) and FLT3 (n=6; Figure 1), with mutations present in both age cohorts. NT5C2, SYK and CHD1 were exclusively mutated in the pediatric cohort with at least 3 mutations. NT5C2 was also specific for early REL. Of all REL mutations, 225 mutations (14%, mean: 4 mutations/patient) were sub-clonal (under 〈 5% mutation frequency) at ID. Copy number alterations (CNA) varied greatly among pediatric and adult samples: 6% of pediatric and 18% of adult samples had aneuploidies and or copy neutral loss of heterozygosity of whole chromosomes. Chromosomal aberrations at ID persisted at relapse (100 %). Particular targets of CNA affected well-described genes like CDKN2A, CDKN2B, PAX5 on chr9p. Genes preferentially subjected to homozygous deletions were VPREB1 (n=6), SH2B3 (n=4), and ETV6 (n=2). All SH3B2 deletions were found in pediatric samples. On the epi-genomic level, the principal component analysis of the most variable CG-sites revealed a stable methylation profile during the course of the disease. However, we found a clear separation into a smaller pediatric-dominated cluster (n=24; 20 pediatric, 4 adult) and a larger mixed-age cluster (n=76; Fig. 1, Cluster A). Differentially methylated regions, affecting a total of 269 genes, characterized the separation of the smaller cluster, henceforth called Methylation Deregulated (MDR) cluster. The samples of the MDR cluster showed also a distinct gene expression profile by RNA-seq supporting a tight connection between the methylation status and its transcriptional program. A subset of 97 genes was differentially expressed including MAPK and PDGFR genes as most prominently deregulated. Additionally we defined a MDR expression classifier comprising 30 genes (Fig. 1). On the mutational level, the MDR samples had 20 % fewer mutations (mean: 25.3) compared to the remaining samples (mean: 31.3) and fewer CNVs for the most frequently affected genes. Characterising the non-MDR samples, a third of those were categorized as Ph-like ALL using the 15 gene classifier in an unsupervised clustering; this signature also coincided with the presence of well-known fusion-genes (Fig. 1, Cluster B). The remaining samples were defined by chromosomal instability (CI; Fig. 1, Cluster C). In the CI cluster, mutations in epigenetic regulators were twice as frequent when compared to the remaining samples. Conclusions: We describe three distinct clusters in relapsed BCP-ALL, which are characterized by a different genetic alterations: a novel MDR cluster by distinct methylation changes, the Ph-like cluster by gene fusions and the CI cluster by chromosomal instability. The cluster assignment was stable over the course of the disease. All clusters occurred in pediatric and adult patients, with the methylation-driven cluster predominantly in pediatrics. The MDR cluster showed significantly fewer mutations and CNVs compared to the other two clusters. The MDR samples showed activation of the MAPK signaling pathway pointing to actionable therapeutic targets. Figure 1 Figure 1. Disclosures Gökbuget: Pfizer: Honoraria, Research Funding; Amgen: Honoraria, Research Funding.

Description: Introduction: B cell precursor acute lymphoblastic leukemia (BCP-ALL) represents the largest immunophenotypically defined subgroup of adult ALL. Yet, targeted therapies are assigned only to the minority of patients, whereas druggable lesions for the majority of adult BCP-ALL are still unknown. Thus, it remains necessary to explore their genomic landscape to unravel molecular alterations suitable for targeted therapies. We analyzed the mutational pattern of 206 candidate genes in adult BCP-ALL patients at diagnosis and matched relapse samples, focusing on therapeutically targetable alterations and their clonal evolution. Patients and methods : We performed targeted resequencing on diagnostic samples of patients with BCR-ABL negative BCP-ALL (n=89) as well as matched relapse samples (n=53). The mean age at first diagnosis was 44 years (range 17 - 79). Relapses were categorized as early (〉 18 months from diagnosis, n=33) or late (〈 18 months, n=20). All patients were enrolled on trials of the German Acute Lymphoblastic Leukemia Multicenter Study Group (GMALL). The targeted region comprised 206 genes known to be frequently mutated in leukemia and relevant in normal hematopoiesis. Customized biotinylated RNA oligo pools (SureSelect, Agilent) were used to select the targeted regions. We performed 100-bp paired-end sequencing on an Illumina Genome HiSeq1500 sequencing system. For a variant call, we required at least a read depth of 30 and a variant allele frequency (VAF) of 10%. We obtained an average coverage of 825 reads for the target region with over 98 percent of the targeted region been covered with a minimum of 30 reads. After exclusion of polymorphism annotated in dbSNP135, 625 protein changing single nucleotide variations and small indels were identified. 139 of the 206 target genes were mutated at least once. Results: On average three (median 3.0, range 1-12) genes were mutated in the 89 diagnostic BCP-ALL samples. The most frequent mutations included alterations in NRAS (18%), PAX5 (16%), TP53 (9%), JAK1/2 (8%) and IKZF1 (6%). Categorizing mutated genes according to their functional annotation revealed that 44 (49%) of patients harbored at least one mutation affecting epigenetic regulation, followed by adhesion/matrix proteins (48%), transcription factors (46%), RAS pathway genes (29%), kinase signaling (28%), and p53/cell cycle regulation (26%). Four (KMT2D, SETD2, KDM6A, KDM6B) of the twelve most frequently mutated genes, affecting 30 patients (34%), were epigenetic regulators specifically involved in histone methylation. Mutations in these genes were nearly mutually exclusive, suggesting their functional redundancy in this context. For 53 of the 89 patients matched relapse samples were available, showing a median frequency of 4.0 (range 1-36) mutated genes per sample. The frequency of mutations across gene families was similar compared to diagnostic samples, except for those involved in epigenetic regulation that showed a higher mutation rate at relapse (55%). In 68% of matched relapse samples, mutational gains and losses indicated mechanisms of clonal evolution. Among 28 patients (53%), which gained at least one mutation, the Lysine-Specific Methyltransferase 2D (KMT2D) was the most frequently affected gene with 7 patients acquiring KMT2D alterations at relapse. Other methyl-transferases (SETD2, KMT2C) as well as demethylases (KDM5A, KDM6A, KDM6B) acquired novel mutations at relapse, contributing to a total of 11 patients (21%) that gained alterations in histone methylation regulators. Notably, mutations in methylation regulators were only gained, but not lost at relapse. Nine of these 11 patients suffered an early relapse, implicating a selection advantage for clones harboring mutations in methylation regulating genes and their involvement in a more aggressive course of the disease. Conclusion: We describe for the first time a highly heterogeneous genomic mutational spectrum in adult BCP-ALL. A high mutation rate at diagnosis and further increase at relapse identified regulators of histone methylation as a most prominent target of recurrent alterations. Functional studies are needed to determine the direct biological consequence of these alterations, facilitating targeted therapeutic interventions with epigenetically active compounds. Disclosures Baldus: Novartis: Research Funding.

Description: Introduction: In T-ALL, in contrast to BCP-ALL, molecular subgroups are less well defined. Molecular subgroups are based on aberrant expression of oncogenes often resting upon structural aberrations (chromosomal translocation, copy number variations, point mutations). So far, comprehensive studies on molecular subgroups have been predominantly performed in pediatric patients. The clinical relevance of molecular characteristics is not taken into account for risk stratification and targeted therapeutic options for T-ALL patients are limited. Risk stratification is mainly based on immunophenotype and MRD response. Thus, in the German Multicenter Study Group for Adult ALL (GMALL) protocols early and mature T-ALL and molecular failure after first consolidation are high risk features. Whole RNA transcriptome sequencing (RNAseq) enables molecular subgroup allocation profiles with potential prognostic relevance. Herein, we investigated a large cohort of 161 adult T-ALL patients with RNAseq for molecular subgroups and their clinical implication. Patients and methods : RNAseq data (Illumina HiSeq 4000, 100 or 125 bp paired-end, average read count ~30 million reads/sample) were generated from 161 adult T-ALL patients from diagnostic bone marrow samples. For 84 of these T-ALL patients, we additionally investigated DNA methylation using Infinium 450k methylation bead arrays and mutation status using deep targeted DNA sequencing with a gene panel consisting of 206 genes (HiSeq 1500, 100 bp paired-end, average ~800 reads/bp). All patients were treated likewise within pediatric inspired protocols of the GMALL. Clinical follow up and outcome data were available for 129 patients aged between 18 and 55 years. Results: Based on oncogene expression, we could assign molecular subgroups to 160 out of the 161 T-ALL patients: HOXA: n=37 (23%), TLX1: n=37 (23%), TAL/LMO: n=33 (20%), LYL1/LMO2: n=31 (19%), TLX3: n=17 (11%) , NKX2-1: n=4 (2%), TAL2: n=1 (1%). Among others, gene fusion events incorporating TAL1 (n=16), HOXA (n=6), TLX1 (n=14), TLX3 (n=2) and NKX2 (n=2) were detected as underlying mechanisms. Age distribution revealed more TAL/LMO in the younger population (16-25 years: 35% versus 〉35 years: 3%; p=0.001) and more LYL1/LMO2 and HOXA in the older patients (16-25 years: 23% vs. 〉35 years: 40%; n.s.). DNA methylation analyses for 84 of the 161 patients support the molecular classification with four distinct subgroups demonstrating a homogenous methylation profile for the TLX1 driven subgroup (methylation cluster M3, n=30). In contrast, methylation cluster M2 (n=21) showed a hypomethylation in CpG islands and comprises the majority of TAL/LMO positive cases (19/21) including all samples with STIL-TAL1 fusions. M1 (n=25) and M4 (n=7) clusters comprised TLX3 (mainly cluster M1, 8/10), HOXA and LYL1/LMO2 cases. Mutation analysis showed a high rate of NOTCH1 (n=71%) mutations in the TLX1 subgroup and an increased rate of mutations in the JAK/STAT pathway (n=29%) and epigenetic regulators (n=50%) in HOXA and LYL1/LMO2 subgroups. Regarding clinical outcome, 126 out of 129 (98%) patients achieved a morphologically complete remission (CR), two patients failed CR, one patient died during induction therapy. Regarding MRD response, 12% of the investigated T-ALL patients showed a molecular failure after consolidation. Noteworthy, we found a molecular CR for 28/30 (93%) patients in the TLX1 subgroup, but only 11/19 (58%) respectively 2/6 (33%) in the HOXA and the LYL1/LMO2 subgroup. This finding results in exceptional five year-overall survival (5y-OS) of 93% in TLX1 patients. Together with the NKX2-1 subgroup (n=4, with 100% 5y-OS), these patients build a prognostically favourable risk group in the overall cohort (94% 5y-OS versus 76% in TAL-LMO patients versus 62% in all other subgroups, p=0.007). This result could not only be found in the overall cohort, but also within the already good risk subgroup of thymic T-ALL patients (93% vs. 75%, p=0.02). Conclusion: This is to our knowledge the largest cohort of adult T-ALL patients characterized by RNAseq on molecular level. Our findings point towards an age dependent distribution of molecular subgroups contributing to age-dependent outcome differences. Patients with TLX1 reveal a molecular subgroup with extraordinary good prognosis. Within thymic T-ALL, the subgroup of patients without TLX1 (~53%) has an inferior but still good prognosis. Disclosures Fiedler: Gilead: Other: support for meeting attendance; Jazz Pharmaceuticals: Honoraria, Other: support for meeting attendance; Abbvie: Membership on an entity's Board of Directors or advisory committees; Morphosys: Consultancy, Honoraria; Celgene: Membership on an entity's Board of Directors or advisory committees; Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria; ARIAD/Incyte: Consultancy, Honoraria; Amgen: Consultancy, Honoraria, Other: support for meeting attendance, Patents & Royalties, Research Funding; Daiichi Sankyo: Other: support for meeting attendance. Alakel:Pfizer: Consultancy. Schwartz:AMGEN: Other: personal fees and non-financial support; Novartis: Other: personal fees and non-financial support; Jazz Pharmaceuticals: Other: personal fees and non-financial support; Pfizer: Other: personal fees ; BTG Intl Inc: Other: personal fees ; Gilead Sciences: Other: personal fees and non-financial support ; MSD Sharp & Dohme: Other: personal fees ; Basilea: Other: non-financial suppor. Müller-Tidow:Pfizer: Research Funding, Speakers Bureau; Daiichi Sankyo: Research Funding; Janssen-Cilag GmbH: Speakers Bureau; BiolineRx: Research Funding. Brüggemann:Regeneron: Research Funding; Affimed: Research Funding; Amgen: Consultancy, Honoraria, Research Funding; Janssen: Consultancy, Honoraria; Incyte: Consultancy; Roche: Consultancy; Celgene: Consultancy. Goekbuget:Servier: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; Gilead: Consultancy; Erytech: Consultancy; Kite: Consultancy; Jazz: Consultancy, Research Funding.

Description: Targeted interventions and new risk stratification approaches for the improvement of outcomes in B cell precursor acute lymphoblastic leukemia (BCP-ALL) require an understanding of high-risk driver subtypes, their clinical phenotypes and actionable targets. Currently, over 20 BCP-ALL subtypes have been identified based on genomic driver alterations and corresponding gene expression signatures, in particular in pediatric ALL. This complex molecular landscape is so far only partially defined in adult BCP-ALL patients. To characterize driver subtypes and their clinical phenotypes we performed transcriptome sequencing (Illumina) on Ph-negative adult BCP-ALL patients (n=376) treated on subsequent pediatric inspired protocols of the German Acute Lymphoblastic Leukemia Study Group (GMALL). Using random forest analyses of a well-defined patient subset with available WES/gene panel and DNA-methylation data, we established a 300-gene classifier which together with fusion calling and hotspot mutation calling reliably allocated samples to driver subtypes. Subgroup allocation was feasible for n=267 / 308 Ph-negative patients (86.7%) with Ph-like ALL (27.7%) being the most frequently observed subtype followed by DUX4 (14.6%), KMT2A (10.1%), PAX5-plus (9.7%), ZNF384 (9.0%), Near haploid - High hyperdiploid (NH-HeH, 6.0%), Low haploid - Near triploid (LH-NT, 6.0%) and TCF3-PBX1 (5.6%). With frequencies below 5% were observed: PAX5-alt, MEF2D, BCL2/MYC, ETV6-RUNX1, CEBP family member fusions and TCF3-HLF. The age distribution of these driver subtypes varied across our cohort (median age: 37 years, range: 16 - 81; left panel). Adult patients harboring ETV6-RUNX1 fusions (n=4) were 25 years or younger. NH-HeH, PAX5-plus and DUX4 ALL were also observed predominantly in younger patients (median ages: 24, 24, 30 years respectively; p

Description: High-throughput sequencing describes multiple alterations in individual tumors, but their functional relevance is often unclear. Clinic-close, individualized molecular model systems are required for functional validation and to identify therapeutic targets of high significance for each patient. Here, we establish a Cre-ERT2-loxP (causes recombination, estrogen receptor mutant T2, locus of X-over P1) based inducible RNAi- (ribonucleic acid interference) mediated gene silencing system in patient-derived xenograft (PDX) models of acute leukemias in vivo. Mimicking anti-cancer therapy in patients, gene inhibition is initiated in mice harboring orthotopic tumors. In fluorochrome guided, competitive in vivo trials, silencing of the apoptosis regulator MCL1 (myeloid cell leukemia sequence 1) correlates to pharmacological MCL1 inhibition in patients´ tumors, demonstrating the ability of the method to detect therapeutic vulnerabilities. The technique identifies a major tumor-maintaining potency of the MLL-AF4 (mixed lineage leukemia, ALL1-fused gene from chromosome 4) fusion, restricted to samples carrying the translocation. DUX4 (double homeobox 4) plays an essential role in patients’ leukemias carrying the recently described DUX4-IGH (immunoglobulin heavy chain) translocation, while the downstream mediator DDIT4L (DNA-damage-inducible transcript 4 like) is identified as therapeutic vulnerability. By individualizing functional genomics in established tumors in vivo, our technique decisively complements the value chain of precision oncology. Being broadly applicable to tumors of all kinds, it will considerably reinforce personalizing anti-cancer treatment in the future.