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Sensitive and specific detection of measurable residual disease (MRD) after treatment in pediatric acute myeloid leukemia (AML) is prognostic of relapse and is important for clinical decision making. Mutation-based methods are increasingly being used, but are hampered by the limited number of common driver gene mutations to target as clone markers. Additional targets would greatly increase MRD detection power. However, even in cases with many AML-defining mutations, it is the limited accuracy of current molecular methods which establishes the lower bounds of sensitivity. Here we describe an ultrasensitive approach for disease monitoring with personalized hybrid capture panels targeting hundreds of somatic mutations identified by whole genome sequencing (WGS), and using extremely accurate Duplex Sequencing (DS) in longitudinal samples. In a pilot cohort of 13 patients we demonstrate detection sensitivities several orders of magnitude beyond currently available single locus testing or less accurate sequencing.

Higgins: TwinStrand Biosciences: Current Employment. Lo:TwinStrand Biosciences: Current Employment. Hipp:TwinStrand Biosciences: Current Employment. Valentine:TwinStrand Biosciences: Current Employment. Williams:TwinStrand Biosciences: Current Employment. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Current Employment.

Higgins: TwinStrand Biosciences: Employment. Pratt:TwinStrand Biosciences: Employment. Valentine:TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Radich:Novartis: Other: RNA Sequencing; TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Employment.

DNA standards are a critical resource in diagnostic and research labs. Positive and negative controls are essential for validating the sensitivity and specificity of next generation sequencing (NGS) and other genetic assays. Several commercial DNA standards have been widely used to validate clinical oncology assays. However, as genomic technologies have become more sensitive, the metrics defining what a “gold standard” entails have not been carefully re-evaluated. Cell culture and synthetic chemical means of generating DNA standards have the potential to artificially introduce mutations. Cell lines are often exposed to supraphysiologic levels of reactive oxygen. Oligonucleotide synthesis has an error rate orders of magnitude above biological levels. This background may not interfere with detection of clonal or sub clonal variants with moderately sensitive assays, but it will obscure the presence of very rare mutations when assessing with extremely sensitive ones and it will decrease apparent assay performance.

Hematopoietic cell transplantation (HCT) is used in the treatment of hematologic malignancies (HM). Clonal hematopoiesis of indeterminate potential (CHIP) is the age-associated process whereby healthy individuals accumulate low-frequency clones driven by mutations in genes recurrently mutated in myeloid cancers; CHIP is associated with an increased risk of HM and cardiovascular disease. It has been hypothesized that CHIP clones present in an HCT donor might disproportionately proliferate in the recipient due to a relative growth advantage during immune reconstitution. Here we use Duplex Sequencing (DS) to investigate, years after HCT, whether the clonal makeup of CHIP in prior donors differs from that of the donor-derived hematopoietic system long after allogeneic transfer to a recipient.

Short: Takeda Oncology: Consultancy, Research Funding; AstraZeneca: Consultancy; Amgen: Honoraria. Kantarjian:Ariad: Research Funding; AbbVie: Honoraria, Research Funding; Takeda: Honoraria; Agios: Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Cyclacel: Research Funding; Daiichi-Sankyo: Research Funding; Astex: Research Funding; Jazz Pharma: Research Funding; Pfizer: Honoraria, Research Funding; Immunogen: Research Funding; BMS: Research Funding; Actinium: Honoraria, Membership on an entity's Board of Directors or advisory committees; Novartis: Research Funding. Sasaki:Otsuka: Honoraria; Pfizer: Consultancy. Ravandi:Macrogenix: Consultancy, Research Funding; Cyclacel LTD: Research Funding; Menarini Ricerche: Research Funding; Selvita: Research Funding; Xencor: Consultancy, Research Funding; Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Cortes:Astellas Pharma: Consultancy, Honoraria, Research Funding; Immunogen: Consultancy, Honoraria, Research Funding; Sun Pharma: Research Funding; Merus: Consultancy, Honoraria, Research Funding; BiolineRx: Consultancy; Bristol-Myers Squibb: Consultancy, Research Funding; Biopath Holdings: Consultancy, Honoraria; Takeda: Consultancy, Research Funding; Pfizer: Consultancy, Honoraria, Research Funding; Jazz Pharmaceuticals: Consultancy, Research Funding; Forma Therapeutics: Consultancy, Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Research Funding; Daiichi Sankyo: Consultancy, Honoraria, Research Funding. Konopleva:Reata Pharmaceuticals: Equity Ownership, Patents & Royalties; Astra Zeneca: Research Funding; Agios: Research Funding; Genentech: Honoraria, Research Funding; Ascentage: Research Funding; Kisoji: Consultancy, Honoraria; Calithera: Research Funding; Stemline Therapeutics: Consultancy, Honoraria, Research Funding; Forty-Seven: Consultancy, Honoraria; Eli Lilly: Research Funding; AbbVie: Consultancy, Honoraria, Research Funding; Cellectis: Research Funding; Amgen: Consultancy, Honoraria; Ablynx: Research Funding; F. Hoffman La-Roche: Consultancy, Honoraria, Research Funding. Garcia-Manero:Novartis: Research Funding; Amphivena: Consultancy, Research Funding; Helsinn: Research Funding; AbbVie: Research Funding; Celgene: Consultancy, Research Funding; Astex: Consultancy, Research Funding; Onconova: Research Funding; H3 Biomedicine: Research Funding; Merck: Research Funding. Pratt:TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Valentine:TwinStrand Biosciences: Employment. Salk:TwinStrand Biosciences: Employment. Radich:TwinStrand Biosciences: Research Funding; Novartis: Other: RNA Sequencing. Jabbour:Pfizer: Consultancy, Research Funding; AbbVie: Consultancy, Research Funding; Takeda: Consultancy, Research Funding; Amgen: Consultancy, Research Funding; Adaptive: Consultancy, Research Funding; BMS: Consultancy, Research Funding; Cyclacel LTD: Research Funding.

Higgins: TwinStrand Biosciences: Employment. Pratt:TwinStrand Biosciences: Employment. Valentine:TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Salk:TwinStrand Biosciences: Employment.

Background:Some previous studies in Ph+ ALL suggested that T315I mutations of the ABL1KD are present in many pts prior to tyrosine kinase inhibitor (TKI) treatment. However, these reports used RT-PCR to generate complementary DNA (cDNA) prior to analysis, which may introduce random errors and lead to false positive results. We hypothesized that ultrasensitive DS of genomic DNA (gDNA) would more accurately detect low-level pretreatment ABL1mutations in Ph+ ALL because it does not rely upon error prone RT-PCR.

Acute myeloid leukemia (AML) is a challenging disease to treat: most patients achieve remission after induction chemotherapy, but the majority eventually relapse. Minimal residual disease (MRD) after initial treatment is the best predictor of relapse and is thus a critical metric around which to develop new treatments. However, conventional MRD diagnostics, including cytology and flow cytometry, are of variable sensitivity and often only perform well in specialty centers, and there is no gold standard. Molecular tests developed to measure trace MRD in other hematological malignancies (i.e. CML) are high-resolution but assay a single, universally-present mutation, while many different genetic drivers exist in AML and these are spread among dozens of genes. Virtually every AML patient harbors a unique combination of mutations, making it difficult to design an effective universal assay. As such, most reported molecular AML MRD assays are either sensitive for mutations that are only found in a narrow subset of patients, or can screen many potential sites of mutation, but with low sensitivity.

Higgins: TwinStrand Biosciences: Employment. Williams:TwinStrand Biosciences: Employment. Buckley:CTI Biopharma: Employment; TwinStrand Biosciences: Consultancy. Radich:TwinStrand Biosciences: Research Funding. Salk:TwinStrand Biosciences: Employment, Equity Ownership.