Tanja M. Davidsen

Genomic profiling of pediatric acute myeloid leukemia reveals a changing mutational landscape from disease diagnosis to relapse

The genomic and clinical information used to develop and implement therapeutic approaches for acute myelogenous leukemia (AML) originated primarily from adult patients and has been generalized to patients with pediatric AML. However, age-specific molecular alterations are becoming more evident and may signify the need to age-stratify treatment regimens. The NCI/COG TARGET-AML initiative used whole exome capture sequencing (WXS) to interrogate the genomic landscape of matched trios representing specimens collected upon diagnosis, remission, and relapse from 20 cases of de novo childhood AML. One hundred forty-five somatic variants at diagnosis (median 6 mutations/patient) and 149 variants at relapse (median 6.5 mutations) were identified and verified by orthogonal methodologies. Recurrent somatic variants [in (greater than or equal to) 2 patients] were identified for 10 genes (FLT3, NRAS, PTPN11, WT1, TET2, DHX15, DHX30, KIT, ETV6, KRAS), with variable persistence at relapse. The variant allele fraction (VAF), used to measure the prevalence of somatic mutations, varied widely at diagnosis. Mutations that persisted from diagnosis to relapse had a significantly higher diagnostic VAF compared with those that resolved at relapse (median VAF 0.43 vs. 0.24, P < 0.001). Further analysis revealed that 90% of the diagnostic variants with VAF >0.4 persisted to relapse compared with 28% with VAF <0.2 (P < 0.001). This study demonstrates significant variability in the mutational profile and clonal evolution of pediatric AML from diagnosis to relapse. Furthermore, mutations with high VAF at diagnosis, representing variants shared across a leukemic clonal structure, may constrain the genomic landscape at relapse and help to define key pathways for therapeutic targeting. Cancer Res; 76(8); 2197-205. ©2016 AACR.

A Children's Oncology Group and TARGET initiative exploring the genetic landscape of Wilms tumor

We performed genome-wide sequencing and analyzed mRNA and miRNA expression, DNA copy number, and DNA methylation in 117 Wilms tumors, followed by targeted sequencing of 651 Wilms tumors. In addition to genes previously implicated in Wilms tumors (WT1, CTNNB1, AMER1, DROSHA, DGCR8, XPO5, DICER1, SIX1, SIX2, MLLT1, MYCN, and TP53), we identified mutations in genes not previously recognized as recurrently involved in Wilms tumors, the most frequent being BCOR, BCORL1, NONO, MAX, COL6A3, ASXL1, MAP3K4, and ARID1A. DNA copy number changes resulted in recurrent 1q gain, MYCN amplification, LIN28B gain, and MIRLET7A loss. Unexpected germline variants involved PALB2 and CHEK2. Integrated analyses support two major classes of genetic changes that preserve the progenitor state and/or interrupt normal development.

The molecular landscape of pediatric acute myeloid leukemia reveals recurrent structural alterations and age-specific mutational interactions

We present the molecular landscape of pediatric acute myeloid leukemia (AML) and characterize nearly 1,000 participants in Childrens Oncology Group (COG) AML trials. The COG–National Cancer Institute (NCI) TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and microRNA sequencing and CpG methylation profiling. Validated DNA variants corresponded to diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases. In contrast, somatic structural variants, including new gene fusions and focal deletions of MBNL1, ZEB2 and ELF1, were disproportionately prevalent in young individuals as compared to adults. Conversely, mutations in DNMT3A and TP53, which were common in adults, were conspicuously absent from virtually all pediatric cases. New mutations in GATA2, FLT3 and CBL and recurrent mutations in MYC-ITD, NRAS, KRAS and WT1 were frequent in pediatric AML. Deletions, mutations and promoter DNA hypermethylation convergently impacted Wnt signaling, Polycomb repression, innate immune cell interactions and a cluster of zinc finger–encoding genes associated with KMT2A rearrangements. These results highlight the need for and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML.

MLLT1 YEATS domain mutations in clinically distinctive Favourable Histology Wilms tumours.

Wilms tumour is an embryonal tumour of childhood that closely resembles the developing kidney. Genomic changes responsible for the development of the majority of Wilms tumours remain largely unknown. Here we identify recurrent mutations within Wilms tumours that involve the highly conserved YEATS domain of MLLT1 (ENL), a gene known to be involved in transcriptional elongation during early development. The mutant MLLT1 protein shows altered binding to acetylated histone tails. Moreover, MLLT1-mutant tumours show an increase in MYC gene expression and HOX dysregulation. Patients with MLLT1-mutant tumours present at a younger age and have a high prevalence of precursor intralobar nephrogenic rests. These data support a model whereby activating MLLT1 mutations early in renal development result in the development of Wilms tumour.

CSF3R mutations have a high degree of overlap with CEBPA mutations in pediatric AML.

Publishers Note: There is an [Inside Blood Commentary][1] on this article in this issue. To the editor: Childhood cancers represent distinct clinical entities, often with unique genomic alterations and therapeutic responses that differ from cancers arising in adults. Pediatric acute myeloid

Abstract LB-93: Demonstration of significant clonal evolution from diagnosis to relapse in childhood AML determined by exome capture sequencing: an NCI/COG TARGET AML study

Proceedings: AACR 103rd Annual Meeting 2012‐‐ Mar 31‐Apr 4, 2012; Chicago, IL Acute myeloid leukemia (AML) is a heterogeneous group of disorders with significant genomic complexity that contributes to variable clinical responses and poor outcomes despite intensive therapy. Although genomic alterations, including karyotypic alterations and somatic mutations have been identified in most patients with AML, their utility has been limited by an inability to accurately predict outcome in the majority of patients. There is also little information on the molecular evolution of AML from diagnosis to relapse. To define the genomic profile of relapse in AML and evaluate the extent of clonal evolution from diagnosis to relapse, we performed exome capture sequencing in matched trios of specimens (diagnostic, remission and relapse specimens) from 22 patients with AML (64 specimens) who were treated on COG clinical trials and lacked previously known high risk features. In the 22 matched diagnostic and 20 relapse cases, 729 somatic variants were identified with a variant allelic frequency ranging from 1% to 94% in diagnostic (N=384) or relapse (N=345) specimens (33.1 mutations/patient) of which 372 were verified with secondary deep sequencing of targeted regions. A verification rate of 86% in mutations with >20% variant allelic frequency was achieved. 335 somatic mutations (median15.5 mutations/patient, range 8-42 mutations/patient) were observed in the 20 cases with diagnostic and relapse specimens. Of these 335 somatic mutations, 221 mutations (66%) in 193 genes were identified in the diagnostic specimens, of which 107 mutations (49%) in 101 genes were present at disease recurrence. In addition to the 107 mutations that persisted from diagnosis to relapse, there was emergence of an additional 114 mutations in 106 genes at the time of relapse, possibly due to clonal selection of a rare pre-existing clone. Somatic mutations in 17 genes were detected in more than one patient at either diagnosis or relapse or both. Of the mutations that were present at both diagnosis and relapse, only 2 genes (KIT and TET2) were mutated in more than one patient, highlighting the paucity of common, novel relapse-associated mutations in the studied cohort. In addition evaluation of copy number (CN) alterations by SNP genotyping in the diagnostic and relapse specimens revealed similar clonal evolution with apparent resolution of diagnostic CN variations and appearance of novel somatic CN alterations in relapse specimens. The study results highlight the genomic complexity of childhood AML, and suggest significant clonal evolution from diagnosis to relapse, with resolution of mutations associated with chemotherapy sensitive clones. Newly evolved mutations may represent rare, chemotherapy resistant clones present at diagnosis, which are selected by exposure to chemotherapy, and may cooperate with other mutations to lead to therapy resistance and poor outcome. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr LB-93. doi:1538-7445.AM2012-LB-93

The genetic basis and cell of origin of mixed phenotype acute leukaemia

Mixed phenotype acute leukaemia (MPAL) is a high-risk subtype of leukaemia with myeloid and lymphoid features, limited genetic characterization, and a lack of consensus regarding appropriate therapy. Here we show that the two principal subtypes of MPAL, T/myeloid (T/M) and B/myeloid (B/M), are genetically distinct. Rearrangement of ZNF384 is common in B/M MPAL, and biallelic WT1 alterations are common in T/M MPAL, which shares genomic features with early T-cell precursor acute lymphoblastic leukaemia. We show that the intratumoral immunophenotypic heterogeneity characteristic of MPAL is independent of somatic genetic variation, that founding lesions arise in primitive haematopoietic progenitors, and that individual phenotypic subpopulations can reconstitute the immunophenotypic diversity in vivo. These findings indicate that the cell of origin and founding lesions, rather than an accumulation of distinct genomic alterations, prime tumour cells for lineage promiscuity. Moreover, these findings position MPAL in the spectrum of immature leukaemias and provide a genetically informed framework for future clinical trials of potential treatments for MPAL.A large-scale genomics study shows that the cell of origin and founding mutations determine disease subtype and lead to the expression of multiple haematopoietic lineage-defining antigens in mixed phenotype acute leukaemia.

Comprehensive characterization of pediatric acute myeloid leukemia reveals novel molecular features and age-specific interactions

We present the molecular landscape of pediatric acute myeloid leukemia (AML), characterizing nearly 1,000 participants in Children9s Oncology Group (COG) AML trials. The COG/NCI TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA and miRNA sequencing, and CpG methylation profiling. Validated DNA variants revealed diverse, infrequent mutations, with fewer than 40 genes mutated in >2% of cases. We find that somatic structural variants, including novel gene fusions, and focal MBNL1, ZEB2, and ELF1 deletions are common in younger patients, whereas short sequence variants predominate in adults. Mutations of DNMT3A and TP53, common in adults, are conspicuously absent from virtually all pediatric patients. Pediatric AML harbors novel GATA2, FLT3, and CBL mutations, recurrent MYC-ITD, frequent NRAS, KRAS, and WT1 mutations, and recurrent promoter hypermethylation of activating NK cell ligands. Across all age groups, we find distinct molecular alterations with clinical implications, suggesting a need for age-specific targeted therapies.We present the molecular landscape of pediatric acute myeloid leukemia (AML), characterizing nearly 1,000 participants in Childrens Oncology Group (COG) AML trials. The COG/NCI TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA, miRNA sequencing and CpG methylation profiling. Validated DNA variants revealed diverse, infrequent mutations. Fewer than 40 genes are mutated in >2% of cases. In contrast, somatic structural variants, including novel gene fusions and focal MBNL1, ZEB2, and ELF1 deletions, disproportionately impact younger patients. DNMT3A and TP53 mutations, common in adults, are conspicuously absent from virtually all pediatric cases. Novel GATA2, FLT3, and CBL mutations, recurrent MYC-ITD, NRAS, KRAS, and WT1 mutations are frequent. Deletions, mutations, and promoter hypermethylation convergently impact Wnt signaling, Polycomb repression, innate immune cell interactions, and a cluster of zinc finger genes associated with KMT2A rearrangements. These results highlight the need for, and facilitate the development of, age-appropriate targeted therapies in pediatric AML.We present the molecular landscape of pediatric acute myeloid leukemia (AML), characterizing nearly 1,000 participants in Children’s Oncology Group (COG) AML trials. The COG/NCI TARGET AML initiative assessed cases by whole-genome, targeted DNA, mRNA, miRNA sequencing and CpG methylation profiling. Validated DNA variants revealed diverse, infrequent mutations with fewer than 40 genes mutated in >2% of cases. In contrast, somatic structural variants, including novel gene fusions and focal MBNL1, ZEB2, and ELF1 deletions, were disproportionately prevalent in young as compared to adult patients. Conversely, DNMT3A and TP53 mutations, common in adults, are conspicuously absent from virtually all pediatric cases. Novel GATA2, FLT3, and CBL mutations, recurrent MYC-ITD, NRAS, KRAS, and WT1 mutations are frequent in pediatric AML. Deletions, mutations, and promoter DNA hypermethylation convergently impact Wnt signaling, Polycomb repression, innate immune cell interactions, and a cluster of zinc finger genes associated with KMT2A rearrangements. These results highlight the need for, and facilitate the development of age-tailored targeted therapies for the treatment of pediatric AML.

Abstract LB-180: The genetic landscape of Wilms tumor

Introduction: The National Cancer Institute9s Therapeutically Applicable Research to Generate Effective Treatments (TARGET) initiative seeks to characterize the genomes of high-risk pediatric tumors to identify therapeutic targets. The High-Risk Renal Tumor TARGET initiative includes the analysis of pre-therapy favorable histology Wilms Tumors (FHWT) that relapsed and tumors with diffuse anaplasia (unfavorable histology; DAWT). These two tumor subsets have survival rates of approximately 50% and 60%, respectively. Experimental procedures: Genomic sequencing (whole genome [WGS] or exome [WXS]), global copy number analysis, and global gene expression analysis were performed on a discovery set of 117 (78 FHWT, 39 DAWT) pre-therapy high-risk WTs treated on National Wilms Tumor Study-5 (NWTS-5). To determine the frequency of recurrent variants, targeted sequencing (Illumina HiSeq2500) was performed on a validation set of pre-therapy tumor DNA from a case-cohort of all FHWT treated on NWTS-5 (531 FHWT) and all available 118 DAWT treated on NWTS-5 (these groups include tumors from the discovery set). Results: WGS and WXS revealed an average of 21.74 ± 22.6 high-quality variants per DAWT (range, 3-131) and 13.8 ± 10.9 per FHWT (range 2-58). Genes previously reported to be recurrently mutated in WT were mutated at the following frequencies in the validation set: WTX (6%), CTNNB1 (15%), WT1 (7.5%), DROSHA (11%), DGCR8 (4.5%), XPO5 (2%), SIX1/2 (7%), and MLLT1 (3%). In addition, mutations were identified in three genes that impact the NMYC pathway, which is known to be involved in renal development. These include MYCN P44L/H (4%), MAX R60Q (2%), and novel mutations in NONO (2%); these mutations were mutually exclusive. Novel mutations in BCOR, a transcriptional corepressor that regulates both gene expression during development and chromatin modification, were found in 3% of validation set tumors. Analysis of global gene expression revealed significant up-regulation of genes associated with kidney development, extracellular matrix organization, and epithelial tube development in BCOR-mutant tumors compared with precursor lesions (5 hyperplastic perilobar nephrogenic rests). TP53 mutations were identified in 48% of DAWTs and 1% of FHWTs. The above data do not include copy number changes, which were recurrently detected in WT1, WTX, NMYC, and TP53. Conclusions: Through the TARGET initiative, we have identified several novel, potential driver mutations that occur in WT and have not been reported in other pediatric tumors. The majority of these genes are known to function in processes critical to early development and/or specifically in renal development. Many of these mutations are accompanied by Wnt activating mutations or 11p15 biallelic expression. However, approximately 50% of WTs lack clear driver mutations. Future studies will need to focus on elucidating epigenetic alterations in these tumors as well as genetic changes outside of protein-coding regions. Citation Format: Samantha L. Gadd, Amy L. Walz, Ariadne HAG Ooms, Vicki Huff, Daniela S. Gerhard, Malcolm A. Smith, Jaime M. Guidry Auvil, Leandro Hermida, Tanja Davidsen, Patee Gesuwan, Daoud Meerzaman, Yussanne Ma, Marco A. Marra, Jeffrey S. Dome, Charles G. Mullighan, David A. Wheeler, Oliver A. Hampton, Julie M. Gastier-Foster, Nicole Ross, Elizabeth J. Perlman. The genetic landscape of Wilms tumor. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-180.

Abstract 484: Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project

Proceedings: AACR 106th Annual Meeting 2015; April 18-22, 2015; Philadelphia, PA Patient-derived xenografts and cell lines have been the underpinning of functional characterization and drug discovery efforts in cancer. The use of these models is often under the assumption that these systems are renewable, faithful representations of original progenitor tumor cell populations. To test this assumption, we performed whole exome (92X median coverage) and genome sequencing (36X median coverage) analysis of cell line and patient-derived mouse xenografts (PDXs) originating from 7 neuroblastoma patients. Data are from1 primary tumor, 3 PDXs, and 15 neuroblastoma cell lines cultured from tumor, bone marrow, or blood. The cell lines consisted of 4 pre-/post-therapy pairs and 3 pairs established and maintained in either hyperoxia (room air i.e. “standard” cell culture) or physiologic (bone marrow hypoxia = 5%) oxygen. 7 lymphoblastoid or fibroblast cell lines were used as matched normals to identify somatic mutations. Subclonal population structures were inferred from somatic mutation calls calibrated for copy number state and tumor purity. In all cell lines and xenografts, we observed 1-2 additional subclonal populations, primarily supported by deep coverage from exome sequencing. In nearly every case, we observed shifts in the proportional representation of genetic subclones and many subclones showed additional mutations not evident in the progenitor tissue or cancer line derived in parallel. Comparison of three cell line pairs established in bone marrow level hypoxia versus room air found only ∼40% of coding mutations in each line were shared (average 82 mutations per line), suggesting significant genetic impact of growing tumor cells in the two different culture conditions. Matched PDXs from these cases had only ∼17% of coding mutations shared across all three models. The greatest genetic similarity was seen between paired cell lines established from tissue obtained pre-/post-therapy from the same patient (36 coding mutations shared, 14 private to diagnosis and 13 private to progression). However, a second pre/post-therapy cell line pair did not share any coding mutations, although they did have 585 non-coding mutations in common (of 4,033 and 2,480 in each line), assuring that the relapse was derived from a diagnostic tumor clone. These results highlight a need for comprehensive subclonal analysis of human cancer laboratory models to better inform design and interpretation of biological and preclinical therapeutic studies. Citation Format: Maya Schonbach, Arnavaz Danesh, Jeff Bruce, Tito Woodburn, Tanja Davidsen, Leandro Hermida, Patee Gesuwan, Jaime Guidry Auvil, Oliver Hampton, David Wheeler, Julie Gastier-Foster, Malcolm Smith, Daniela Gerhard, John M. Maris, Patrick Reynolds, Trevor J. Pugh. Fidelity of subclonal representation in human neuroblastoma-derived cell line and patient-derived xenograft models: A report from the NCI-TARGET project. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 484. doi:10.1158/1538-7445.AM2015-484