Abstract
Mutation profiling of acute leukemias is a valuable tool for identifying genetic mutations with prognostic, predictive, therapeutic, and diagnostic utility. Technological advances, such as massively parallel sequencing, have allowed laboratories to assess for variation across dozens or hundreds of genes simultaneously with relatively low cost per target.
Here, we describe a procedure for designing and using a TruSeq Custom Amplicon assay targeting genes involved in acute leukemias. This method is a fully customizable, amplicon-based assay for targeted resequencing, allowing interrogation of selected genomic regions of interest. The most readily available form of the assay allows sequencing of up to 1536 amplicons in a single reaction using a straightforward workflow. The ability to multiplex up to 1536 amplicons per reaction allows coverage of up to 650 kb of cumulative sequence and supports up to 96 samples per batch, depending on library size and desired sequencing depth.
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References
Jemal A, Siegel R, Ward E et al (2009) Cancer statistics, 2009. CA Cancer J Clin 59:225–249
Whitman SP, Archer KJ, Feng L et al (2001) Absence of the wild-type allele predicts poor prognosis in adult de novo acute myeloid leukemia with normal cytogenetics and the internal tandem duplication of FLT3: a cancer and leukemia group B study. Cancer Res 61:7233–7239
Döhner K, Schlenk RF, Habdank M et al (2005) Mutant nucleophosmin (NPM1) predicts favorable prognosis in younger adults with acute myeloid leukemia and normal cytogenetics: interaction with other gene mutations. Blood 106:3740–3746
Preudhomme C, Sagot C, Boissel N et al (2002) Favorable prognostic significance of CEBPA mutations in patients with de novo acute myeloid leukemia: a study from the acute leukemia French association (ALFA). Blood 100:2717–2723
Paschka P, Marcucci G, Rupper AS et al (2006) Adverse prognostic significance of KIT mutations in adult acute myeloid leukemia with inv(16) and t(8;21): a cancer and leukemia group B study. J Clin Oncol 24:3904–3911
Shivarov V, Gueorguieva R, Stoimenov A et al (2013) DNMT3A mutation is a poor prognosis biomarker in AML: results of a meta-analysis of 4500 AML patients. Leuk Res 37:1445–1450
Mendler JH, Maharry K, Radmacher MD et al (2012) RUNX1 mutations are associated with poor outcome in younger and older patients with cytogenetically normal acute myeloid leukemia and with distinct gene and MicroRNA expression signatures. J Clin Oncol 30:3109–3118
Gaidzik VI, Paschka P, Späth D et al (2012) TET2 mutations in acute myeloid leukemia (AML): results from a comprehensive genetic and clinical analysis of the AML study group. J Clin Oncol 30:1350–1357
Alvarado Y, Kantarjian HM, Luthra R et al (2014) Treatment with FLT3 inhibitor in patients with FLT3-mutated acute myeloid leukemia is associated with development of secondary FLT3-tyrosine kinase domain mutations. Cancer 120:2142–2149
Lee HK, Kim HW, Lee IY et al (2014) G-749, a novel FLT3 kinase inhibitor can overcome drug resistance for the treatment of acute myeloid leukemia. Blood 123:2209–2210
Yuan L, Lu L, Yang Y et al (2015) Genetic mutational profiling analysis of T cell acute lymphoblastic leukemia reveal mutant FBXW7 as a prognostic indicator for inferior survival. Ann Hematol 94:1817–1828
Zenatti PP, Ribeiro D, Li W et al (2011) Oncogenic IL7R gain-of-function mutations in childhood T-cell acute lymphoblastic leukemia. Nat Genet 43:932–939
Flex E, Petrangeli V, Stella L et al (2008) Somatically acquired JAK1 mutations in adult acute lymphoblastic leukemia. J Exp Med 205:751–758
Voelkerding KV, Dames S, Durtschi JD (2009) Next-generation sequencing: from basic research to diagnostics. Clin Chem 55:641–658
Mullighan CG, Downing JR (2009) Genome-wide profiling of genetic alterations in acute lymphoblastic leukemia: recent insights and future directions. Leukemia 23:1209–1218
Pritchard CC, Salipante SJ, Koehler K et al (2013) Validation and implementation of targeted capture and sequencing for the detection of actionable mutation, copy number variation, and Gene rearrangement in clinical cancer specimens. J Mol Diagn 16:56–67
Ye K, Schulz MH, Long Q et al (2009) Pindel: a pattern growth approach to detect break points of large deletions and medium sized insertions from paired-end short reads. Bioinformatics 25:2865–2871
Chen K, Wallis JW, McLellan MD et al (2009) BreakDancer: an algorithm for high-resolution mapping of genomic structural variation. Nat Methods 6:677–681
Wang J, Mullighan CG, Easton J et al (2011) CREST maps somatic structural variation in cancer genomes with base-pair resolution. Nat Methods 8:652–654
Li J, Lupat R, Amarasinghe KC et al (2012) CONTRA: copy number analysis for targeted resequencing. Bioinformatics 28:1307–1313
Acknowledgments
Special thanks to the members of the University of Washington Genetics and Solid Tumor Laboratory and the Molecular Hematopathology Laboratory who offered their help and insight, especially Jennifer Hempelmann, Eric Hoyle, and Lena Mulillo.
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Konnick, E., Lockwood, C.M., Wu, D. (2017). Targeted Next-Generation Sequencing of Acute Leukemia. In: Fortina, P., Londin, E., Park, J., Kricka, L. (eds) Acute Myeloid Leukemia. Methods in Molecular Biology, vol 1633. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7142-8_11
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DOI: https://doi.org/10.1007/978-1-4939-7142-8_11
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