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AML Rapid Mutation Panel

Test Code: 12346

This panel interrogates 7 genes (FLT3, IDH1/IDH2, CEBPA, KIT, NPM1, TP53) for clinically significant mutations associated with treatment selection and/or prognosis in acute myeloid leukemia (AML). DNA extracted from peripheral blood or bone marrow is tested for pathogenic mutations in the genes listed above. FLT3 is interrogated by polymerase chain reaction (PCR) followed by capillary electrophoresis for fragment analysis. IDH1 and IDH2 are screened by Sanger sequencing. CEBPA, KIT, NPM1, and TP53 are screened using next-generation sequencing (NGS).

For FLT3, the panel is designed to detect both internal tandem duplications (ITDs) and tyrosine kinase domain (TKD) mutations involving codons 835 and 836. Exon 4 of the IDH1 gene and exon 4 of the IDH2 gene are sequenced by Sanger sequencing; these exons harbor over 90% of all reported IDH1/IDH2 cancer-associated mutations,1,2 including the most frequently encountered IDH1 R132, IDH2 R140, and IDH2 R172 mutations. Other less commonly observed IDH1/IDH2 mutations are reported as variants of unknown clinical significance. Clinically relevant regions in CEBPA (entire coding region), KIT (exons 2, 3, 8-11, 13, 14, 17-19), NPM1 (exon 11), and TP53 (all coding exons) are interrogated for potential pathogenic mutations.

The 7 component genes that comprise the AML Rapid Mutation Panel are separately orderable:

  • FLT3 Mutation Analysis (test code 39786)
  • Isocitrate Dehydrogenase 1 and 2 (IDH1/IDH2) Mutation Analysis (test code 31547)
  • CEBPA Mutation Analysis (test code 90812)
  • c-KIT Mutation Analysis, cell based (test code 19961)
  • NPM (Exon 12) Mutation Analysis, cell based (test code 16158)
  • TP53 Somatic Mutation, Prognostic (test code 16515)

The AML Rapid Mutation Panel is designed for the prompt interrogation of potential mutations in genes involved in prognosis and therapeutic decision-making in the treatment of acute myeloid leukemia (AML). The test detects FLT3 mutations, which have been described in approximately 20% to 25% of AML cases. These mutations activate FLT3 kinase activity and promote malignancy. Internal tandem duplications in FLT3 are associated with a poor prognosis, high relapse rates, and reduced overall survival.3 Patients with AML that are FLT3 mutation-positive could be eligible for FDA-approved targeted therapies such as midostaurin.

IDH1 and IDH2 (isocitrate dehydrogenase 1 and 2) encode enzymes that catalyze oxidative decarboxylation of isocitrate into α-ketoglutarate (α-KG). Gain-of-function mutations in the IDH1 and IDH2 genes are associated with aberrant conversion of α-KG to 2-hydroxyglutarate (2-HG), which is an oncogenic metabolite, and recur in myeloid malignancies and glioma.4,5,6 IDH1 exon 4 (7%-14%) and IDH2 exon 4 (8%-19%) mutations occur frequently in AML and are associated with unfavorable prognosis and inferior overall survival in some studies.2,3 IDH inhibitors have been FDA-approved for eligible patients with AML harboring IDH1 or IDH2  mutations.

CEBPA encodes a transcription factor involved in granulopoiesis. Biallelic mutations in CEBPA have been shown to have improved outcome compared to wild-type CEBPA or monoallelic mutations.7

KIT encodes a receptor tyrosine kinase for its cognate ligand stem cell factor (SCF). Mutations in KIT are associated with inferior outcome in core binding factor (CBF) AMLs.8

NPM1 (nucleophosmin 1) encodes a protein that serves a variety of functions, including nucleolar shuttling. In AML, mutations in NPM1 are associated with aberrant cytoplasmic localization, disordered function, and improved clinical response in some cases.9

TP53 is a tumor suppressor gene implicated in a wide variety of cancers. In AML, TP53 mutations are associated with complex karyotypes, monosomal karyotypes, and poor outcomes.10

The interrogation of FLT3 detects internal tandem duplications (ITDs) and tyrosine kinase domain (TKD) mutations in the FLT3 gene using polymerase chain reaction (PCR) coupled with fragment analysis by capillary electrophoresis.11,12 The analytical sensitivity is 5% mutant allele fraction within the total cell population.

The interrogation of IDH1/IDH2 detects potential mutations in the entire coding regions of IDH1 exon 4 and IDH2 exon 4. These exons are amplified, and the PCR products are purified and sequenced in both forward and reverse directions by dye-terminator Sanger sequencing on an automated platform. The analytical sensitivity is 20% mutant allele fraction within the total cell population.

The remaining genes are interrogated by NGS. Clinically relevant regions in CEBPA (entire coding region), KIT (exons 2, 3, 8-11, 13, 14, 17-19), NPM1 (exon 11), and TP53 (all coding exons), are interrogated for potential mutations by a bait-capture–based massively parallel sequencing test methodology using the Illumina NextSeq platform. The analytical sensitivity of the assay is 5% mutant allele fraction within the total cell population for both single nucleotide variants and small indels. In accordance with the Association for Molecular Pathology (AMP) and American College of Molecular Genetics (ACMG) guidelines, benign or likely benign variants will not be reported from this assay.

The preferred specimen types are whole blood (5 mL, 3 mL minimum) or bone marrow (3 mL, 1 mL minimum) in an EDTA tube. Other acceptable specimen types include whole blood or bone marrow collected in a green-top sodium heparin tube or DNA extracted from a Clinical Laboratory Improvement Amendments (CLIA) certified laboratory in a leak proof container, but these are not preferred.

The full results of the 7-gene panel will be available in 5 to 7 business days from receipt of the sample. A partial report for mutations detected in FLT3, IDH1, and IDH2 will be communicated as soon as available, typically in 3 to 4 days from sample receipt, to clients who have an EMR capable of receiving partial reports. If this capability is not allowed, our Oncology client services team can work with the client to fax a copy of the partial report or assist with partial reporting capabilities. Please contact the Oncology client services team for more information at 1.833.773.1441.

In both of the LeukoVantage® assays, next-generation sequencing is used to test for mutations in the same 7 genes as a part of a panel of additional genes with known clinical significance in myeloid neoplasms, and the same mutations of clinical significance are interrogated.

However, LeukoVantage® assays could provide better sequence-related details for the detected FLT3 mutations and higher sensitivity for the IDH1/IDH2 mutation detection; the trade-off is a longer turnaround time. In addition, due to the limitation of short-read sequencing, LeukoVantage® may not detect rare FLT3 ITD variants with insert sizes greater than 150 bp.

References

  1. Gene IDH1. COSMIC: Catalogue Of Somatic Mutations In Cancer. V97. Accessed March 15, 2023. https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=IDH1
  2. Gene IDH2. COSMIC: Catalogue Of Somatic Mutations In Cancer. V97. Accessed March 15, 2023. https://cancer.sanger.ac.uk/cosmic/gene/analysis?ln=IDH2
  3. Cuervo-Sierra J, Jaime-Pérez JC, Martínez-Hernández RA, et al. Prevalence and clinical significance of FLT3 mutation status in acute myeloid leukemia patients: a multicenter study. Arch Med Res. 2016;47(3):172‐179. doi:10.1016/j.arcmed.2016.06.003
  4. Megías-Vericat JE, Ballesta-López O, Barragán E, et al.  IDH1-mutated relapsed or refractory AML: current challenges and future prospects. Blood Lymphat Cancer. 2019;9:19-32. doi:10.2147/BLCTT.S177913
  5. Amaya, ML and Pollyea, DA. Targeting the IDH2 pathway in acute myeloid leukemia. Clin Cancer Res. 2018:24(20):4931-4936. doi:10.1158/1078-0432.CCR-18-0536
  6. Yan H, Parsons DW, Jin G, et al. IDH1 and IDH2 mutations in gliomas. N Engl J Med. 2009:360(8):765-773. doi:10.1056/NEJMoa0808710
  7. Pabst T, Eyholzer M, Fos J, et al. Heterogeneity within AML with CEBPA mutations; only CEBPA double mutations, but not single CEBPA mutations are associated with a favourable prognosis. Br J Cancer. 2009:100(8):1343-1346. doi:10.1038/sj.bjc.6604977
  8. Paschka P, Marcucci G, Ruppert AS, et al. 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. 2006:24(24):3904-3911. doi:10.1200/JCO.2006.06.9500
  9. Meani N, Alcalay M. Role of nucleophosmin in acute myeloid leukemia. Expert Rev Anticancer Ther. 2009:9(9):1283-1294. doi:10.1586/era.09.84
  10. Rücker FG, Schlenk RF, Bullinger L, et al. TP53 alterations in acute myeloid leukemia with complex karyotype correlate with specific copy number alterations, monosomal karyotype, and dismal outcome. Blood. 2012:119(9): 2114-2121. doi:10.1182/blood-2011-08-375758
  11. Murphy KM, Levis M, Hafez MJ, et al. Detection of FLT3 internal tandem duplication and D835 mutations by a multiplex polymerase chain reaction and capillary electrophoresis assay. J Mol Diagn. 2003;5(2):96-102. doi:10.1016/S1525-1578(10)60458-8
  12. Yamamoto Y, Kiyoi H, Nakano Y, et al. Activating mutation of D835 within the activation loop of FLT3 in human hematologic malignancies. Blood. 2001;97(8):2434-2439. doi:10.1182/blood.v97.8.2434

 

This FAQ is provided for informational purposes only and is not intended as medical advice. Test selection and interpretation, diagnosis, and patient management decisions should be based on the clinician’s education, clinical expertise, and assessment of the patient.

 

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