Abstract A41: Ultrasensitive detection of diverse genomic alterations in hematological malignancies using a targeted amplicon-based sequencing approach
Authors: Choudhury Yukti , Cher Chae Yin , Tan Min-Han , Lim Zi Yi 
Published: June 2020

Abstract
Background: Hematologic malignancies, including acute myeloid leukemia (AML), myelodysplastic syndromes (MDS), myeloproliferative neoplasms (MPN), acute lymphoblastic leukemia (ALL), and multiple myeloma (MM), are characterized by complex mutational patterns with great inter- and intrapatient diversity of mutations. Targeted detection of somatic cancer-associated variants by next-generation sequencing technology has demonstrated clinical utility in hematologic malignancies, with recurrent somatic mutations being identifiable in most AML patients and nearly 90% of MDS patients. DNA-level structural rearrangements, copy number variations (CNVs), and microsatellite instability (MSI), particularly in therapy-related AML, are other key targets with clinical relevance. Appropriate test sensitivity of mutation detection is necessary for the determination of relapse and emergence of resistance mutations. An ultrasensitive amplicon-based sequencing platform technology (AmpliMark) was applied for the detection of mutations in hematologic samples, including in DNA from peripheral blood, bone marrow, and in plasma cell-free DNA.
Methods: The hematologic panel covers 45 genes (representing over 90% of mutations found in patients) and 6 microsatellite loci and utilizes an optimized bioinformatics pipeline. Analytical validation was done with reference standards and admixture samples. Clinical performance was assessed in samples from adult patients (n = 65) with diagnoses including AML, ALL, MM, and MPN, both treated and untreated. Of these, 25 samples were characterized for somatic mutations using orthogonal methods and the remaining were processed as routine clinical samples.
Results: Analytical validation of the panel demonstrated lower limits of variant detection to be 0.1% for mutations, 0.5% for structural rearrangements, and 5% for MSI. A high average deduplicated depth of coverage of 9375x was achieved for DNA from bone marrow and peripheral blood. The method was able to detect all orthogonally confirmed mutations for a concordance rate of 100%. In the unselected series of patient samples, at least one somatic mutation was confirmed in 85% of cases, with TET2, DNMT3A, TP53, KIT, JAK2, RUNX1, SF3B1, ASXL1, IDH1, NPM1 mutations, FLT3 internal tandem duplication, and BCR-ABL1 fusion most frequently seen. Allele frequencies detected ranged from 0.06% to over 35%. Mutations in multiple genes were observed in 70% of cases. For 10 patients, matched bone marrow and peripheral blood samples were tested and 100% mutation match was observed in the two hematologic compartments with generally higher allele frequencies in bone marrow fraction.
Conclusion: We report the application of a technology platform (Amplimark) for comprehensive genomic profiling of hematologic malignancies. Excellent concordance to standard methods and among matched samples is seen, including at low allele frequencies, suggesting clinical utility of the method for hematologic malignancies.
Citation Format: Yukti Choudhury, Chae Yin Cher, Min-Han Tan, Zi Yi Lim. Ultrasensitive detection of diverse genomic alterations in hematological malignancies using a targeted amplicon-based sequencing approach [abstract]. In: Proceedings of the AACR Special Conference on Advances in Liquid Biopsies; Jan 13-16, 2020; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(11_Suppl):Abstract nr A41.