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Acute lymphoblastic leukemia (ALL) is the most common childhood cancer. Here, using whole-genome, exome and transcriptome sequencing of 2,754 childhood patients with ALL, we find that, despite a generally low mutation burden, ALL cases harbor a median of four putative somatic driver alterations per sample, with 376 putative driver genes identified varying in prevalence across ALL subtypes. Most samples harbor at least one rare gene alteration, including 70 putative cancer driver genes associated with ubiquitination, SUMOylation, noncoding transcripts and other functions. In hyperdiploid B-ALL, chromosomal gains are acquired early and synchronously before ultraviolet-induced mutation. By contrast, ultraviolet-induced mutations precede chromosomal gains in B-ALL cases with intrachromosomal amplification of chromosome 21. We also demonstrate the prognostic significance of genetic alterations within subtypes. Intriguingly, DUX4- and KMT2A-rearranged subtypes separate into CEBPA/FLT3- or NFATC4-expressing subgroups with potential clinical implications. Together, these results deepen understanding of the ALL genomic landscape and associated outcomes.

Childhood myelodysplastic syndromes (MDS) account for less than 5% of pediatric hematologic malignancies and differ from their adult counterpart in terms of biology, genetics, and cure rates. Complete (-7) or partial loss (del7q) of chromosome 7 constitutes the most common cytogenetic abnormality and is associated with more advanced disease typically requiring timely hematopoietic stem cell transplantation (HSCT). Previously, we and others established a link between -7 and germline GATA2 mutations in pediatric MDS (37% of MDS/-7 cases are GATA2-deficient) as well as constitutional SAMD9/9L disorders where -7 is utilized as an escape mechanism from the growth-restrictive effect of SAMD9/9L mutations. To date, comprehensive sequencing studies have been performed in 96 children with primary MDS, as reported by Pastor et al, Leukemia 2017 and Schwartz et al, Nature Comm 2017. This work established mutations in SETBP1, ASXL1, PTPN11, RUNX1 and RAS pathway genes as common somatic drivers. However, little is known about the clonal development of -7 and the role of additional somatic mutations. The knowledge about clonal hierarchies is essential for the understanding of disease progression on molecular level and for mapping potential drug targets. The rationale for the current study was to i) define the most common somatic drivers in a large cohort of patients with childhood MDS, ii) identify clonal/subclonal mutations, iii) infer clonal architecture of monosomy 7 and track the changes over time. We studied a cohort of 576 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, consisting of 482 (83%) patients with refractory cytopenia of childhood (RCC) and 94 (17%) MDS with excess blasts (EB). All patients underwent deep sequencing for 30 genes relevant to pediatric MDS and additional WES was performed in 150/576 patients. Using 20 computational predictors (including CADD and REVEL), population databases and germline testing, we identified the most likely pathogenic mutations. First, we excluded germline predisposing mutations in GATA2, SAMD9/SAMD9L and RUNX1 detected in 7% (38/576), 8% (43 of 548 evaluable) and 0.7% (4/576) of patients, respectively. Then we focused on the exploration of somatic aberrations. Most common karyotype abnormalities were monosomy 7 (13%, 77/576) and trisomy 8 (3%, 17/576). A total of 104 patients carried somatic mutations, expectedly more prevalent in the MDS-EB group as compared to RCC (56%, 53/94 vs 10.6%, 51/482; pSETBP1>ASXL1; -7>SETBP1>ASXL1>PTPN11; -7>SETBP1>ASXL1>CBL, -7>EZH2>PTPN11). Finally, we tracked clonal evolution over time in 12 cases with 2-12 available serial samples using deep sequencing complemented by serial CFC-analysis. This confirmed that SETBP1 clones are rapidly expanding, while ASXL1 subclones exhibit an unstable pattern with clonal sweeping, while additional minor clones are acquired as late events. In 2 of 11 transplanted patients who experienced relapse, the original clonal architecture reappeared after HSCT. In summary, the hierarchy of clonal evolution in pediatric MDS with -7 follows a defined pattern with -7 aberrations arising as ancestral event followed by the acquisition of somatic hits. SETBP1 mutations are the dominant driver while co-dominant ASXL1 mutations are unstable. The functional interdependence and potential pharmacologic targetability of such somatic lesions warrants further studies. Disclosures Niemeyer: Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Hereditary predisposition has been ever since implicated in the etiology of childhood myelodysplastic syndromes (MDS). Until recently, GATA2 deficiency prevailed as a major germline cause in pediatric primary MDS. In the past 2 years, we and others identified germline mutations in paralogue genes SAMD9 and SAMD9L residing on chromosome 7q21.2 as new systemic diseases with high propensity for MDS with monosomy 7. Although initially, mutations in SAMD9 and SAMD9L genes were associated with MIRAGE and Ataxia-Pancytopenia syndromes, respectively, with recent reports the phenotypes are becoming more intertwined. Nevertheless, the predisposition to MDS with monosomy 7 (-7) remains a common clinical denominator. Both genes are categorized as negative regulators of cellular proliferation and mutations were shown to be activating. Because of their high evolutionary divergence, classical in silico prediction is erratic, thereby establishing in vitro testing as the current gold standard for pathogenicity evaluation. The objectives of this study were to define the prevalence of SAMD9/9L germline mutations in primary pediatric MDS, and to describe the clinical phenotype and outcome. In addition, we aimed to characterize the somatic mutational architecture and develop a functional scoring system. Within the cohort of 548 children and adolescents with primary MDS diagnosed between 1998 and 2016 in Germany, 43 patients (8%) carried SAMD9/9L mutations that were mutually exclusive with GATA2 deficiency and known constitutional bone marrow (BM) failure. MDS type refractory cytopenia of childhood was diagnosed in 91% (39/43), and MDS with excess blasts in 9% (4/43) of mutated cases. Karyotype at diagnosis was normal in 58%, and -7 was detected in 37% of SAMD9/9L cohort. Within MDS subgroup with -7 (n=74), SAMD9/9L mutations accounted for 22% of patients. Notably, the demographics, familial disease, diagnostic blood and BM findings, overall survival (OS) and the outcome after HSCT were not influenced by mutational status in our study cohort (n=548). At the last follow up, 88% (38/43) of SAMD9/9L MDS patients were alive; 35/43 had been transplanted with a 5-year-OS of 85%. Next, we added 26 additional cases with SAMD9/9L mutations diagnosed in Europe within EWOG-MDS studies. In the total cohort of 69 germline mutated patients we found a total of 75 SAMD9/9L mutations, of which 67 were novel. Of those we tested 47 using a HEK293 cell in vitro system and 45/47 mutants inhibited proliferation. While 53/69 patients carried only single germline mutations (missense in 50/53 and truncating in 3/53), in the remaining 16 patients, 11 additional truncating and 7 missense mutations were found. We did not observe an association between germline mutation and phenotype. Immunological issues (e.g. recurring infections, low Ig) were described in 32%/50% of SAMD9/9L-mutated patients, while physical anomalies were very heterogeneous and reported in ~50% of patients in both mutational groups. Intriguingly, genital phenotypes occurred in 40% of SAMD9L, while neurological problems were present in 30% of SAMD9 - mutational subgroups. To elucidate the somatic mutational landscape, we performed whole exome and deep sequencing of 58 SAMD9/9L patients and identified recurrent somatic mutations in known oncogenes that were earlier associated with pediatric MDS: SETBP1 (10%), RUNX1 (7%), ASXL1 (5%), EZH2 (5%), CBL (3%). The identified somatic mutations occurred in association with monosomy 7 background (18/20). Finally, we utilized the results from functional testing of the 47 SAMD9/9L variants as our test cohort to develop combinatorial in silico scoring. The rationale was to decrease the dependency on functional validation. Based on the results of 20 in silico tools we could concatenate a matrix of 5 algorithms to resolve the pathogenicity of >80% of variants. Using this model, all variants predicted as pathogenic showed also growth-restrictive effect in vitro. In summary, pathogenic SAMD9/9L germline mutations account for 8% of primary pediatric MDS and 22% of MDS/-7. The mutations identified are heterogeneous and their effect can be predicted using a combinatorial in silico - in vitro approach. Finally, the clinical outcome and somatic mutational landscape are not influenced by the mutational status. Disclosures Locatelli: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Miltenyi: Honoraria; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees. Niemeyer:Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Familial myelodysplastic syndromes (MDS), leukemias, and bone marrow failure (BMF) syndromes, typically present in children and young adults. Such cases associate with germ line (GL) mutations in DDX41, RUNX1,ETV6, GATA2 and ANKRD26 and were recently designated by WHO to constitute a separate disease. Gain-of-function (GOF) GL mutations in SAMD9 and SAMD9L associate with familial pediatric myelodysplastic syndrome (MDS) cases that often have early onsets and -7/del(7q). It was not known if this was the case in sporadic adult MDS. With this question in mind, we compiled sequencing results on 799 adults with presumably acquired MDS and BMFs, and further enhanced these results through public WES data on 349 myeloid neoplasms. We also performed cell growth assays of exemplary SAMD9 and SAMD9L GL variants identified in adult MDS. We used Broad Institute's Genome Aggregation Database (gnomAD) as healthy controls (n = 138,632). After comparing ethnicities between gnomAD and our cohort, we matched healthy controls to patients by ethnicities. Defining rare variants as those present in Conservative predictions for specific SAMD9 missense variants were made by using SAMD9 GL variants reported in previous papers or public databases of registered healthy donors, and comparing Area Under the Curve of 8 different prediction algorithms. It defined 11 missense variants as "pathogenic". 57% (4/7) of the variants identified in public WES database were also likely pathogenic by our criteria. We evaluated the growth activity of these 11 unique pathogenic missense variants and 1 frameshift variant identified in a patient with -7. Compared to WT-SAMD9, Thr205Pro-SAMD9, Ile247Thr-SAMD9, and Leu574Pro-SAMD9 showed more proliferative cell growth, whereas, I268T-SAMD9 and D550V-SAMD9L had no significant differences compared to the WT. Compared to WT-SAMD9L, 6 variants (Glu220Gly, Leu1323fs, Cys228Tyr, Trp517Arg, Gly235Ser, and Trp333Cys), fused SAMD9L separately, also had higher proliferation rates than WT-SAMD9L. In total, 9 out of 13 variants (69%) cell growth was not suppressed compared to wild-type cells. Pediatric MDS variants showing less cell growth than the wild type cells suggests that they enhance the normal function of SAMD9/SAMD9L, which is to slow cell cycle progression. In this sense they are gain of function mutations (GOF). In contrast, the majority of variants identified in adult MDS were loss-of-function (LOF) mutations. Thus, given the different distribution of variants, lack of genetic reversion, and opposing functional results, we conclude that GL SAMD9/SAMD9L variants might have different consequences in familial childhood vs. adult disease. Sporadic adult MDS patients lacked early onset and genetic reversions following acquisition of -7/del(7q) or another mutations in cis characterized in pediatric MDS patients. SAMD9/SAMD9L variants could have two conflicting consequences; i) GOF mutations in pediatric patients that result in genetic reversion or MDS associated with aberrations of chromosome 7 with an early onset, or ii) LOF variants resulting in a late development of MDS due to haplo-insufficiency of SAMD9/SAMD9L. Our findings suggest that GL SAMD9/SAMD9L variants may be linked to the pathogenesis of a subset of adult sporadic MDS patients. Disclosures Sekeres: Celgene: Membership on an entity's Board of Directors or advisory committees; Celgene: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees; Opsona: Membership on an entity's Board of Directors or advisory committees. Maciejewski:Apellis Pharmaceuticals: Consultancy; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Alexion Pharmaceuticals, Inc.: Consultancy, Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Apellis Pharmaceuticals: Consultancy; Ra Pharmaceuticals, Inc: Consultancy; Ra Pharmaceuticals, Inc: Consultancy.

Diamond-Blackfan anemia (DBA) is a prototypic ribosomopathy and remains the most common cause of congenital pure red cell aplasia (PRCA). In 2/3 of patients, ribosomal protein haploinsufficiency is disease-causing, while in remaining 1/3 the genetic etiology is unknown. Recently, deficiency of ADA2 (DADA2) due to biallelic CECR1 -mutations was reported in patients with systemic autoinflammatory disease presenting with early onset vasculopathy, strokes, antibody deficiency, and in some cases variable cytopenias. Based on the clinical findings in an ADA2-deficient patient with PRCA resembling DBA, we aimed to define the prevalence and clinical picture of DADA2 within DBA patient cohorts. Patients enrolled in the national observational DBA registry in Germany were evaluated for the presence of mutations in CECR1 gene; additional nonconsecutive patients from the French and Turkish registries within the European DBA (EuroDBA) consortium were part of this study. Functional studies included profiling of polysomes and pre-rRNAs in patient-derived EBV-cell lines, CECR1 RT-PCR, measurements of autophagy and apoptosis, and analysis of erythropoiesis in zebrafish embryos. Systematic mutational and copy number analysis had identified typical ribosomal haploinsufficiency in 169/242 patients (70%). Out of 73 remaining patients, full CECR1 -sequencing was accomplished in 68 cases, of which 4 (6%) carried biallelic CECR1 -mutations. Additional 3 patients with biallelic CECR1 -mutations and DBA phenotype were referred from Germany (the index PRCA case), France and Turkey. In contrast to typical autoinflammatory DADA2 (caused by missense biallelic CECR1 -mutations) all patients studied here had at least one CECR1 -allele affected by truncating/stop-gain/deletion mutation leading to mRNA degradation in patient cells. Low or missing ADA2 enzyme activity in plasma confirmed DADA2, while erythrocyte ADA (eADA) levels and MCV were normal. Transfusion-dependent hypoproliferative anemia developed at a median age of 5 weeks (birth-14 years), while hypogammaglobulinemia developed in all cases either initially or during disease course. Notably, a transient hematologic response to steroids was achieved in 5/7 patients, but no improvement was observed in 2 patients treated with TNF-inhibitor; all patients at one point became heavily transfusion-dependent. Systemic vasculitis or cerebral complications were not observed in our cohort. At the last follow-up, 6/7 patients were alive; 3 had successfully undergone hematopoietic stem cell transplantation (HSCT) with myeloablative conditioning and 1 patient had died due to septic shock. Next, we addressed the question if ribosome biogenesis is affected in ADA2-deficient patient cells. Using pre-rRNA maturation assays, polysome profiling and Western blots we established that ribosome biogenesis is normal in DADA2-related PRCA and there is no increase of TP53 stabilization over basal levels in patient LCLs. Analysis of CECR1 -morpholino zebrafish embryos revealed early anemia with lethal phenotype. Although there was no evidence for extrinsic (e.g. immune-mediated) pathomechanisms in our patients, it remains to be answered if CECR1 loss directly affects erythroid development. Finally, the association between elevated levels of eADA (=ADA1) specific to classical DBA and decreased ADA2 enzyme levels in DADA2-related PRCA remains obscure. In summary, DADA2 can phenotypically mimic DBA and thus extends the spectrum of congenital PRCA. Ribosome synthesis seems not to be affected by CECR1 mutations. DADA2 should be considered in patients with DBA-like phenotype but with normal eADA/MCV and hypogammaglobulinemia, allowing for early stratification aimed at HSCT in affected individuals. Disclosures Grosse: Addmedica: Membership on an entity's Board of Directors or advisory committees.

Childhood myelodysplastic syndromes (MDS) belong to a rare group of disorders of aberrant clonal hematopoiesis manifesting throughout entire childhood and adolescence. We had previously established that GATA2 germline mutations can be considered the most common "first hit" in pediatric MDS seen in 7% of primary MDS. However the secondary somatic aberrations facilitating leukemogenesis are not elucidated in children. Previous sequencing efforts established that most somatic mutations very frequently encountered in adults, i.e. affecting TET2, DNMT3a, and the spliceosome genes, do not play a role in the pathogenesis of childhood MDS. Here we aim to define the global mutational landscape in childhood MDS using targeted next-generation sequencing (NGS) approaches. We investigated children and adolescents enrolled in the prospective studies of the European Working Group of Childhood MDS. Diverse target enrichment and NGS strategies were established including hybridization capture and Ampliseq PCR, Illumina Miseq/Hiseq and Iontorrent PGM. We first examined a pilot cohort of 68 patients for mutations in 138 myeloid leukemia genes. This allowed for the identification of recurrently mutated genes that were selected to be included in a pediatric MDS panel encompassing 28 genes. Targeted NGS using the Iontorrent PGM identified known recurrent mutations. However, the high indel error rate and coverage gaps in homopolymeric regions i.e. in ASXL1 precluded further studies. Using inhouse-adapted Ampliseq-Miseq approach we then sequenced DNA from bone marrow of 586 MDS patients (469 primary and 117 secondary MDS after radio/chemotherapy or inherited bone marrow failure syndromes) at an average depth exceeding 700 reads per amplicon. Somatic mutations were identified in 22% of primary MDS patients, with 1, 2 and 3 genes affected in 16%, 4.5%, and 1.5% of cases, respectively. In secondary MDS twice as many patients (46%) carried mutations; 1, 2, and 3 genes were concurrently mutated in 32.5%, 9.5%, and 4% of patients, respectively. Longitudinal NGS analyses and single CFU colony sequencing confirmed the presence of multiple somatic clones evolving in a hierarchical manner throughout disease course. Most frequent mutations identified in more than 1% of our study cohort of primary MDS were: SETBP1 (7%), ASXL1 (6%), NRAS/KRAS (5%), RUNX1 (3%), PTPN11 (3%) and BCOR/BCORL (1.5%); and in secondary MDS: RUNX1 (14.5%), TP53 (9%), NRAS/KRAS (8.5%), ASXL1 (8%), SETBP1 (6%), PTPN11 (6%), CBL (5%), BCOR/BCORL1 (3.4%). Other genes mutated at very low frequency of In conclusion, we established and cross-platform validated a targeted NGS-panel for pediatric MDS, allowing to identify clonal mutations at a sensitivity of at least 10%. In this, to our knowledge largest systematicaly studied cohort of children with MDS we show that mutational load and clonal complexity differs between primary and secondary MDS, and maintains specific patterns for monosomy 7. Although the biological significance of these genomic changes is currently not understood, the emerging distinctive patterns may already be helpful in establishing therapeutic subgroups. Disclosures No relevant conflicts of interest to declare.

The emergence of GATA2 deficiency as a germline predisposition to myeloid malignancies raises questions about the nature of acquired secondary genetic and epigenetic events facilitating leukemogenesis. Previously, mutations in ASXL1 were implicated as a possible somatic driver in single cases of GATA2-related MDS. However the landscape of secondary changes had not yet been systematically examined in larger MDS cohorts, and accounting for confounding factors. In this study, we used next-generation genomic platforms to investigate targeted mutational landscape and global epigenetic profiles in patients with GATA2 deficiency. In a large cohort of consecutively diagnosed children with MDS we had initially established that GATA2 deficiency accounts for 7% of primary MDS cases. Exploring the known association between GATA2 mutated (GATA2mut) cases and monosomy 7 (-7), the prevalence of GATA2 deficiency was very high in patients with -7 (37%), reaching its peak in adolescence (>70%). We next tested 60 GATA2-deficient patients with MDS for the presence of secondary mutations using targeted NGS for genes involved in myeloid malignancies. Somatic status was confirmed by matched analysis of fibroblasts, hair follicles or T-cells. Single hematopoietic CFU colonies were sequenced to identify subclonal patterns. For comparison, a GATA2 wildtype (GATA2-WT) cohort of 422 children and adolescents with MDS enrolled in the studies of the European Working Group of Childhood MDS were analyzed by targeted NGS. Somatic mutations were detected in 45% (27/60) of GATA2mut as compared to 19% (82/422) GATA2-WT MDS cases (p In summary, somatic SETBP1 and STAG2 mutations are associated with MDS arising from GATA2 deficiency. The remaining targeted clonal landscape is essentially determined by the presence of monosomy 7. Similarly, the global epigenetic changes correlate with morphological and cytogenetic subgroups, rather than with germline GATA2 status. The prospect of potential drug targetability of mutations frequently found in children, particularly in the SETBP1 oncogene, and in histone modifiers ASXL1 and EZH2, warrants further biological studies. Disclosures No relevant conflicts of interest to declare.