Additive Value of High-Throughput Sequencing of Myeloid-Associated Gene Mutations in Diagnosing Myeloproliferative Neoplasms
Yongbao Wang, Albert K Ho, Shere Billouin-Fraser, Qiulu Pan, Dan Jones. Quest Diagnostics Nichols Institute, Chantilly, VA
Background: Myeloproliferative neoplasms (MPN) are clonal hematopoietic disorders that must be distinguished from more common reactive hematologic expansions. Mutation of JAK2, particularly V617F, is found in almost all cases of polycythemia vera and 40-50% of essential thrombocythemia and primary myelofibrosis serving as a clonal marker for diagnosis. However, there remain a significant number of patients with hematologic findings highly suspicious for MPN with no JAK2 mutation and an uninformative karyotype. Using an advanced sequencing panel containing 7 genes commonly mutated in MPNs, we investigated the additive diagnostic benefit of high-throughput sequencing in establishing clonal markers of disease.
Design: Thirteen blood samples with suspected MPN that were for negative for JAK2 V617F mutation were analyzed. BCR-ABL1-positive chronic myelogenous leukemia cases were excluded. JAK2 V617F mutation was assessed by a quantitative pyrosequencing method. Genomic DNA was subjected to PCR-based DNA sequencing using a custom-designed 161-amplicon panel. The panel included regions of ASXL1, EZH2, IDH1, IDH2, KRAS, NRAS and TET2 that have mutations associated with myeloid neoplasms. The amplicons were sequenced using the Ion Torrent PGM platform (Life Technologies) and analyzed using Sequence Pilot (JSI Medical). All mutations identified were confirmed by bidirectional Sanger sequencing or pyrosequencing. Control blood samples from unaffected individuals and from JAK2-mutated MPNs were used for comparison.
Results: Five of thirteen (38.5%) JAK2-unmutated samples submitted for MPN evaluation showed one or more mutations in the 7 other genes. These included TET2 alterations (4 different indels and G429R), NRAS point mutations (G12V, G12D), EZH2 mutation (R690H, and an indel) and IDH2 (R140Q, 2 cases). Two of the samples had 4 different mutations. The level of mutations ranged from 7% to 57% of the sequence reads with a target read depth of >200 per amplicon. Normal blood samples (n = 10) showed no mutations except for one non-synonymous change in TET2, representing a possible SNP. The MPN samples with high levels of JAK2 V617F mutation did not show mutations in the other seven genes.
Conclusions: High-throughput sequencing in samples submitted for MPN evaluation can identify myeloid-associated mutations as clonal markers in more than 30% of additional cases that are negative for JAK2 V617F mutation. Sequencing panels are likely to be a cost-effective routine diagnostic assay, providing more informative clonality data than routine karyotyping in this group of MPNs.
Tuesday, March 5, 2013 9:30 AM
Poster Session III # 173, Tuesday Morning