Certified DNA Reference Materials to Compare HER2 Gene Amplification Measurements Using Next-Generation Sequencing Methods.

The National Institute of Standards and Technology (NIST) Standard Reference Materials 2373 is a set of genomic DNA samples prepared from five breast cancer cell lines with certified values for the ratio of the HER2 gene copy number to the copy numbers of reference genes determined by real-time quantitative PCR and digital PCR. Targeted-amplicon, whole-exome, and whole-genome sequencing measurements were used with the reference material to compare the performance of both the laboratory steps and the bioinformatic approaches of the different methods using a range of amplification ratios. Although good reproducibility was observed in each next-generation sequencing method, slightly different HER2 copy numbers associated with platform-specific biases were obtained. This study clearly demonstrates the value of Standard Reference Materials 2373 as reference material and as a calibrator for evaluating assay performance as well as for increasing confidence in reporting HER2 amplification for clinical applications.

Genetic analysis of patients with leukemic transformation of myeloproliferative neoplasms shows recurrent SRSF2 mutations that are associated with adverse outcome.

Leukemic transformation (LT) of myeloproliferative neoplasms (MPNs) is associated with a poor prognosis and resistance to therapy. Although previous candidate genetic studies have identified mutations in MPN patients who develop acute leukemia, the complement of genetic abnormalities in MPN patients who undergo LT is not known nor have specific molecular abnormalities been shown to have clinical relevance in this setting. We performed high-throughput resequencing of 22 genes in 53 patients with LT after MPN to characterize the frequency of known myeloid mutations in this entity. In addition to JAK2 and TET2 mutations, which occur commonly in LT after MPN, we identified recurrent mutations in the serine/arginine-rich splicing factor 2 (SRSF2) gene (18.9%) in acute myeloid leukemia (AML) transformed from MPNs. SRSF2 mutations are more common in AML derived from MPNs compared with LT after myelodysplasia (4.8%) or de novo AML (5.6%), respectively (P=.05). Importantly, SRSF2 mutations are associated with worsened overall survival in MPN patients who undergo LT in univariate (P=.03; HR, 2.77; 95% CI, 1.10-7.00) and multivariate analysis (P<.05; HR, 2.11; 95% CI, 1.01-4.42). These data suggest that SRSF2 mutations contribute to the pathogenesis of LT and may guide novel therapeutic approaches for MPN patients who undergo LT.