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AML Mutation Analysis Panel

Test Code

M AML

Test Synonyms

AML Mutation Panel (CEBPA*, DNMT3A, IDH1, IDH2, KIT, NPM1, RUNX1, TET2, TP53, WT1) / Acute Myeloid Leukemia Mutational Analysis Panel by Next Generation Sequencing, FLT3 and CEBPA by PCR and PCR/Sequencing, respectively.

Associations

Acute Myeloid Leukemia (AML)

Reflex testing
  • AML mutational testing will be performed simultaneously with cytogenetic testing.
  • FLT3 testing by PCR (send-out assay) will be initiated in parallel with AML mutation testing by NGS.
  • CEBPA* testing will be performed as a reflex send out test, if both AML mutation testing by NGS and FLT3 by PCR testing are negative.
Methodology

Next Generation Sequencing on the Illumina MiSeqDx

PCR and PCR/Sequencing for FLT3 and CEBPA testing, respectively

Turnaround Time

10-14 days

Specimen Requirements

5.0 mL (min 3.0 mL) peripheral blood or (min 2.0 mL) bone marrow in EDTA, Sodium Heparin or ACD

Specimen Stability
18-25°C for 1 week; 2 -8°C for up to 14 days
Storage & Handling

Ship at ambient temperature, cool in summer. (Do not allow cool/refrigerated pack to directly contact sample.)

Causes for Rejection

Improper specimen labeling; Insufficient sample volume; Clotted specimen; Specimen older than 7 days

Reference Range

SNVs and small to mid-size insertion/deletions (<100 bp mutations) in the following genes: CEBPA, DNMT3A, FLT3, IDH1, IDH2, KIT, NPM1, RUNX1, TET2, TP53, WT1.  The assay does not detect whole gene or exon insertion and deletions, copy number variants or gene rearrangements (fusions).  The potential for false negatives for genetic mutations increases in samples with <10% disease type cellularity.

Description

Genetic testing in AML has traditionally included classical cytogenetic analysis and FISH.  The role of molecular testing has been primarily limited to risk stratification in normal karyotype AMLs (FLT3, NPM1, CEBPa); KIT mutation testing has also been performed to further refine prognosis among core binding factor leukemias - AML with t(8;21), and AML with inv(16).  Given the recent updates in NCCN guidelines and WHO recommendations, multiple additional genes are now recommended / strongly recommended for testing in AML.  Given also the improved CMS reimbursement for 81450 (5-50 gene panel analysis for hematologic malignancies), targeted NGS panels provide a practical and feasible solution for optimal molecular genetic characterization of AML at initial diagnosis in the routine clinical laboratory setting.

References
  1. Arber, D. A., Orazi, A., Hasserjian, R., Thiele, J., Borowitz, M. J., Le Beau, M. M., . . . Vardiman, J. W. (2016). The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood, 127(20), 2391-2405. doi:10.1182/blood-2016-03-643544
  2. Bacher, U., Haferlach, C., Kern, W., Haferlach, T., & Schnittger, S. (2008). Prognostic relevance of FLT3-TKD mutations in AML: the combination matters—an analysis of 3082 patients. Blood, 111(5), 2527-2537. doi:10.1182/blood-2007-05-091215
  3. Daniel A. Arber, M. J. (2017). Initial Diagnostic Workup of Acute Leukemia: Guideline From the College of American Pathologists and the American Society of Hematology. Archives of Pathology & Laboratory Medicine. doi:doi.org/10.5858/arpa.2016-0504-CP
  4. Hatzimichael, E., Georgiou, G., Benetatos, L., & Briasoulis, E. (2013). Gene mutations and molecularly targeted therapies in acute myeloid leukemia. American Journal of Blood Research, 3(1), 29-51.
  5. Illumina. (2016). TruSight Myeloid Sequencing Panel. Data Sheet: Cancer Genomics.
  6. Illumina. (2016, April). TruSight Myeloid Sequencing Panel Reference Guide. Document #, 15054779 v02.
  7. Kumar, C. C. (2011). Genetic Abnormalities and Challenges in the Treatment of Acute Myeloid Leukemia. Genes&Cancer, 2(2), 95-107. doi:10.1177/1947601911408076
  8. Leukemia & Lymphoma Society. (2016, Aug 30). Facts and Statistics. Retrieved from lls.org: https://www.lls.org/http%3A/llsorg.prod.acquia-sites.com/facts-and-statistics/facts-and-statistics-overview/facts-and-statistics
  9. Muhammad Tayyab, Z. N., & Noor, N. (2014). Distinct Gene Mutations, their Prognostic Relevance and Molecularly Targeted Therapies in Acute Myeloid Leukemia (AML). Journal of Cancer Science & Therapy, 6, 337-349. doi:10.4172/1948-5956.1000292
  10. National Comprehensive Cancer Network. (2016). Acute Myeloid Leukemia. Version 2.2016. The NCCN Guidelines. Retrieved from NCCN.org
  11. Patel, J. P., Gönen, M., Figueroa, M. E., Fernandez, H., Sun, Z., Racevskis, J., . . . Ch. (2012). Prognostic Relevance of Integrated Genetic Profiling in Acute Myeloid Leukemia. New England Journal of Medicine, 366(12), 1079-1089. Retrieved from http://www.nejm.org/doi/full/10.1056/NEJMoa1112304
  12. WebMD. (2016, Aug 30). Leukemia & Lymphoma. Retrieved from WebMD: http://www.webmd.com/cancer/lymphoma/understanding-leukemia-basics