Hematologic Diseases

Detection of mutations in key genes recurrently mutated in myeloid malignancies. Genomic DNA is isolated from bone marrow aspirates or peripheral blood and the DNA sequence of targeted regions of the ASXL1, BCOR, BRAF, CALR, CBL, CEBPA, CSF3R, DDX41, DNMT3A, ETNK1, ETV6, EZH2, GATA2, GNAS, GNB1, IDH1, IDH2, JAK2, KIT, KRAS, MPL, NF1, NPM1, NRAS, PDGFRA, PHF6, PPM1D, PTPN11, RAD21, RUNX1, SETBP1, SF3B1, SH2B3, SMC1A, SMC3, SRSF2, STAG2, STAT3, STAT5B, TET2, TP53, U2AF1, WT1, ZRSR2 genes is determined using next-generation sequencing (NGS) technology.

disease state indication(s)
Myelodysplastic syndrome (MDS), myeloproliferative neoplasms (MPN), myelodysplastic/myeloproliferative neoplasms (MDS/MPN) including chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML)

clinical use
Myelodysplastic syndromes (MDS) are a group of clonal hematopoietic stem cell disorders characterized by cytopenias, ineffective hematopoiesis, morphologic dysplasia, and a variable risk of transformation to acute myeloid leukemia (AML). Establishing a diagnosis of MDS in a cytopenic patient is often challenging as quantification of dysplasia and blasts can be subjective and prone to wide interobserver variation even among expert hematopathologists [1]. Targeted sequencing can identify 1 or more somatic mutation in 80-90% of MDS patients, and the National Comprehensive Cancer Network (NCCN) Guidelines recommends molecular testing of bone marrow or peripheral blood for MDS-associated gene mutations in appropriate clinical contexts as an aid in diagnosis and risk stratification [2-4].
• Mutations in TP53, EZH1, ETV6, RUNX1, or ASXL1 are predictors of poor overall survival in patients with MDS, independent of established risk factors [5].
• Mutations in BCOR, DNMT3A, IDH1, NRAS, splicing factor genes (SRSF2, U2AF1, ZRSR2), or cohesion complex genes (RAD21, SMC1A, SMC3, STAG2); as well as a high total number of mutations; have also been shown to be associated with an unfavorable prognosis in MDS. SF3B1 mutations are highly predictive for the presence of ring sideroblasts, and are associated with a favorable prognosis [2-9].
• TET2 mutations are associated with an increased response to hypomethylating agents in patients with MDS when the allele frequency is >10% and ASXL1 is not mutated [10].
• TP53 mutations are associated with an unfavorable prognosis and decreased response to lenalidomide in patients with MDS with isolated del(5q), and TP53 mutation evaluation is recommended in the World Health Organization (WHO) classification of hematopoietic neoplasms to help identify an adverse prognostic subgroup in this generally favorable prognosis MDS entity [11-12].
• Mutations in TP53, TET2, or DNMT3A are predictive of poor outcomes in patients with MDS after hematopoietic stem cell transplantation [13].
• MDS-associated gene mutations are also commonly found in patients with potential pre-phase of MDS, including clonal cytopenias of undetermined significance (CCUS) and preclinical MDS [14,15]. Molecular profiling can also help in the diagnostic evaluation and risk stratification of other myeloid neoplasms.
• Mutations in key driver genes are included as diagnostic criteria for myeloproliferative neoplasms (MPN) in the WHO classification: JAK2 V617F or JAK2 exon 12 for polycythemia vera; JAK2, CALR, or MPL for primary myelofibrosis (PMF) and essential thrombocythemia; and CSF3R for chronic neutrophilia leukemia [12].
• Mutations in ASXL1, EZH2, SRSF3, or IDH1/2 are associated with an unfavorable prognosis in PMF [16].
• Targeted sequencing can identify 1 or more somatic mutation in 90% of chronic myelomonocytic leukemia (CMML) patients, and the presence of mutations is included as one of the diagnostic criteria for CMML in the WHO classification. Mutations in RUNX1, NRAS, SETBP1, or ASXL1 (nonsense and frameshift) are associated with an unfavorable prognosis [12,17].
• Mutations in SETBP1 or ETNK1 are found in up to a third of patients with atypical chronic myeloid leukemia (aCML), a rare and difficult to diagnosis MDS/MPN subtype [12].
• Myeloid neoplasms that occur on the background of a predisposing germline CEBPA, DDX41, RUNX1, ETV6, or GATA2 mutation are included as entities in the WHO classification [12].
• Mutations in key driver genes define distinct genomic subgroups of AML. These include the currently defined AML subgroups in the WHO classification as well as 3 other genomic subgroups: AML with mutations in genes encoding chromatin, RNA splicing regulators, or both; AML with TP53 mutations, chromosomal aneuoploidies, or both; and AML with IDH2R172 mutations. Chromatin-spliceosome and TP53-aneuploidy AML are associated with poor outcomes [12,18].
• Mutations in SFSF2, SF3B1, U2AF1, ZRSR2, ASXL1, EZH2, BCOR, or STAG2 are reported to be highly specific for secondary AML, and may also be helpful in identifying a subset of elderly patients with de novo AML with worse clinical outcomes [19].
References:
1. Font P, et al. Inter-observer variance with the diagnosis of myelodysplastic syndromes (MDS) following the 2008 WHO classification. Ann Hematol 2013;92:19-24.
2. National Comprehensive Cancer Network (NCCN) Practice Guidelines in Oncology, Myelodysplastic Syndromes
3. Papaemmanuil E, et al. Clinical and biological implications of driver mutations in myelodysplastic syndromes. Blood 2013;122:3616-27.
4. Haferlach T, et al. Landscape of genetic lesions in 944 patients with myelodysplastic syndromes. Leukemia 2014;28:241-247.
5. Bejar R, et al. Clinical effects of point mutations in myelodysplastic syndromes. N Engl J Med 2011;364:2496-506.
6. Damm F, et al. BCOR and BCORL1 mutations in myelodysplastic syndromes and related disorders. Blood 2013;122:3169-3177.
7. Patnaik MM, et al. Differential prognostic effect of IDH1 versus IDH2 mutations in myelodysplastic syndromes: a Mayo Clinic study of 277 patients. Leukemia 2012;26:101-105.
8. Thota S, et al. Genetic alterations of the cohesin complex genes in myeloid malignancies. Blood 2014;124:1790-1798.
9. Malcovati L, et al. SF3B1 mutation identifies a distinct subset of myelodysplastic syndrome with ring sideroblasts. Blood 2015;126:233-241.
10. Bejar R, et al. TET2 mutations predict response to hypomethylating agents in myelodysplastic syndrome patients. Blood 2014;124:2705-2712.
11. Jadersten M, et al. TP53 mutations in low-risk myelodysplastic syndromes with del(5q) predict disease progression. J Clin Oncol 2011;29:1971-1979.
12. Arber DA, et al. The 2016 revision to the World Health Organization classification of myeloid neoplasms and acute leukemia. Blood 2016; 127:2391-405.
13. Bejar R, et al. Somatic mutations predict poor outcome in patients with myelodysplastic syndrome after hematopoietic stem-cell transplantation. J Clin Oncol 2014;32:2691-2698.
14. Kwok B, et al. MDS-associated somatic mutations and clonal hematopoiesis are common in idiopathic cytopenias of undetermined significance. Blood 2015;126:2355-2361.
15. Cargo C, et al. Targeted sequencing identifies patients with preclinical MDS at high risk of disease progression. Blood 2015;126:2362-2365.
16. Vannucchi AM, et al. Mutations and prognosis in primary myelofibrosis. Leukemia 2013;27:1861-1869.
17. Elena C, et al. Integrating clinical features and genetic lesions in the risk assessment of patients with chronic myelomonocytic leukemia. Blood 2016;doi:10.1182/blood-2016-05-714030.
18. Papaemmanuil E, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med 2016;374:2209-2221.
19. Lindsley RC, et al. Acute myeloid leukemia ontogeny is defined by distinct somatic mutations. Blood 2015;125:1367-1376.

methodology/product platform
Capture-based, next-generation sequencing (NGS)

specimen type and requirements
Preferred:
Bone marrow: 2-5 mL in EDTA (purple-top) tube.
Peripheral blood: 2-5 mL in EDTA (purple-top) tube.
Acceptable alternative:
Bone marrow: 2-3 mL in sodium heparin (green-top) tube.
Peripheral blood: 2-3 mL in Sodium Heparin (green-top) tube.
Unacceptable: Specimens received fixed in alternative fixation methods. Decalcified, frozen or fresh tissue.
Note: Use refrigerated cold pack for transport. Make sure cold pack is not in direct contact with specimen.
DO NOT FREEZE.

turnaround time
Global (TC & PC): 10-12 days

cpt code(s)
81450 (x1)

medicare moldx cpt code
Not applicable

regulatory classification
Laboratory developed test (LDT)

ordering option
Global (TC & PC)