Key Points CH may be associated with broader ill health (worse performance status, increased and potentially novel comorbidities). Serum interleukin-6 is elevated in people with CH and genetic subtypes, providing a view of the human systemic inflammatory landscape of CH.

Acquired, inactivating mutations in Tet methylcytosine dioxygenase 2 (TET2) are detected in peripheral blood cells of a remarkable 5%–10% of adults greater than 65 years of age. They impart a hematopoietic stem cell advantage and resultant clonal hematopoiesis of indeterminate potential (CHIP) with skewed myelomonocytic differentiation. CHIP is associated with an overall increased risk of transformation to a hematological malignancy, especially myeloproliferative and myelodysplastic neoplasms (MPN, MDS) and acute myeloid leukemia (AML), of approximately 0.5% to 1% per year. However, it is becoming increasingly possible to identify individuals at greatest risk, based on CHIP mutational characteristics. CHIP, and particularly TET2-mutant CHIP, is also a novel, significant risk factor for cardiovascular diseases, related in part to hyper-inflammatory, progeny macrophages carrying TET2 mutations. Therefore, somatic TET2 mutations contribute to myeloid expansion and innate immune dysregulation with age and contribute to prevalent diseases in the developed world—cancer and cardiovascular disease. Herein, we describe the impact of detecting TET2 mutations in the clinical setting. We also present the rationale and promise for targeting TET2-mutant and other CHIP clones, and their inflammatory environment, as potential means of lessening risk of myeloid cancer development and dampening CHIP-comorbid inflammatory diseases.

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Introduction: The expanding diagnostic and clinical significance of genes associated with myeloid malignancies necessitates higher throughput testing. Targeted next-generation sequencing (NGS) of DNA and RNA may be an ideal approach to capture these variants and eventually streamline, or replace, conventional cytogenetic testing. We present our clinical validation of the OncomineTM Myeloid NGS assay and early experience regarding the clinical impact in patients with suspected or known myeloid malignancies. Methods: OncomineTM Myeloid (Thermo Fisher Scientific) panel (OMP) validation was conducted at Kingston Health Sciences Centre (KHSC) on anonymized and commercial samples according to IQMH and ACMG standards for NGS (PMID: 23887774). As of April 2018 the test has been formally validated and offered at KHSC. Prior to this, patients were consented at the time of blood (PB) or bone marrow (BM) collection for research-based testing. BM and PB samples were collected, gDNA and RNA extracted, and libraries prepared using the Ion ChefTM System. Sequencing was completed using the OMP on Ion PGM (research phase) and S5XL (clinical) sequencers. The OMP uses amplicon-based semiconductor sequencing technology to cover 40 DNA genes recurrently mutated in myeloid malignancies (23 hotspots and 17 full coding), 29 RNA fusion driver genes and 10 gene expression targets. Sequencing data were analyzed using Torrent Suite, Torrent Variant Caller, Ion Reporter (v.5.2-5.6) and Alamut software. The OMP includes a Torrent Suite FLT3-ITD "plug-in" and Ion Reporter "Oncomine filter". Orthogonal clinical assays were performed where necessary for additional confirmation. Variants were interpreted based on a 4-tier classification system developed by Li et al (PMID: 27993330). Variants in the top 3 tiers were reported after assessment by at least one clinician and one certified medical geneticist. Actionability was assessed in the context of electronic diagnostic and clinical information and molecular board review. Results: Validation involved 24 paired DNA-RNA samples previously run at KHSC on an early access OMP version and also at an external site (kind exchange by Dr. N. Carson, Saint John, NB), along with SeraCareTM DNA and RNA controls. Validation run metrics were as follows: DNA means (mapped reads 1306216, read length 226bp, uniformity 98%, depth 2518), RNA means (mapped reads 260350, read length 114bp). Complete concordance was found for RNA variants while the DNA filter chain was only modified to better capture a JAK2 exon 12 variant in a SeraCare control. The minimum depth of coverage was subsequently set to 400X for somatic DNA variants and sensitivity set at 5% variant allele fraction (VAF) for clinical reporting. To date, we have performed sequencing for 38 samples from 35 unique patients (avg age = 64.5y). Of these, 12/35 (34%) involved suspected or known myelodysplastic syndromes (MDS), 12/35 (34%) with acute myeloid leukemia (AML), 4/35 (12%) with query myeloproliferative neoplasms (MPNs), 2/35 (5%) with MDS/MPN, and 5/35 (14%) other (2 B-ALL, 1 aplastic, 1 hairy cell + ?MDS, 1 ?CHIP). OMP detected 56 variants (in 80% of cases), including 43% tier 1, 48% tier 2, and 9% tier 3 variants. The most frequently mutated genes included TET2 (21%), SRSF2 (14%), SF3B1 (9%), TP53 (5%), and RUNX1 (5%). The average VAF was 36% for all samples, with an average of 1.5 variants per sample. Of detected variants, 73% were potentially actionable. Specifically, 51% of actionable variants facilitated or clarified diagnosis (38% of total variants), 32% (23%) affected prognosis, and 17% (13%) had the potential to affect treatment decisions. Among 10 patients with suspected (but subthreshold for) MDS or MPN, clonality was detected in 5, while 5 had no variants, prompting further investigation of reversible cytopenia or cytosis. We are rapidly accruing patients and expect to have OMP profiles for at least 100 patients at the time of the ASH 2018 meeting. Conclusions: Oncomine Myeloid DNA and RNA molecular profiling has found greatest clinical use at our centre in the realm of MDS and AML. In the majority of patients, OMP permits the identification of actionable variants that may aid clinicians in achieving or clarifying diagnoses, inform follow-up and prognosis, and guide treatment decisions. The OMP represents a promising strategy to address the challenge of expanding, important molecular features of myeloid malignancies. Disclosures No relevant conflicts of interest to declare.