P777: runx1 variants with high variant allele frequency in myeloid neoplasms. germline or not?

Background:RUNX1 is a transcription factor frequently mutated in myeloid neoplasms e.g. 10% of acute myeloid leukemia (AML) patients, but RUNX1 variants detected in tumor tissue at a variant allele frequency (VAF) close to 50% can potentially be germline. As pathogenic germline RUNX1 variants are causative for RUNX1 Familial Platelet Disorder with Associated Myeloid Malignancies it is crucial to distinguish between somatic and germline variants. Previous studies in AML patients estimate the frequency of germline RUNX1 variants to be approximately 3%. The frequency of RUNX1 germline variants in lower risk myeloid neoplasms (MN) such as myelodysplastic syndrome (MDS), chronic myelomonocytic leukemia (CMML), clonal cytopenia of undetermined significance (CCUS) and idiopathic cytopenia of undetermined significance (ICUS) remains unknown. Aims: To estimate the frequency of RUNX1 germline variants in MN. Methods: Patients referred to a hematological department in Denmark with suspicion of MN (excluding myeloproliferative disorders) were included in the study. At first visit, a skin biopsy was drawn and frozen dry at -80 °C. RUNX1 variants were identified in either blood or bone marrow using 4 different amplicon based next generation sequencing panels. Variants detected with a VAF > 40 % were further classified as somatic or germline by bi-directional sanger sequencing of germline DNA purified from skin biopsies. Results: Among 595 patients, 85 (14%) had variants in RUNX1 and 28 patients (5%) had variants with a VAF above 40%. High VAF RUNX1 variants were either missense, frameshift, nonsense, or splice-site variants. A majority of the variants were located in the runt homology domain (RHD). We observed a higher proportion of patients with AML and a lower proportion of patients with CCUS in patients with a RUNX1 variant compared to patients without a RUNX1 variant, and this tendency was enhanced if the RUNX1 variants were present at VAF > 40%. Among 22 patients with a high VAF RUNX1 variant and available germline DNA two variants (9%) were classified as germline (NM_001754.5; c.668A>G, c.649G>A, minimal allel frequency reported in gnomAD; 0.012%, 0.003%). The c.668A>G variant was detected in a patient with a low platelet count and CCUS diagnosed at age 24. The c.649G>A variant was detected in a patient with a normal platelet count and MDS diagnosed at age 64. Both germline variants were missense variants located in the region between the RHD and the transactivating domain (TAD) and both were predicted to be pathogenic in-silico by several prediction tools. Unfortunately, family history wasn’t available for any of the two patients with germline variants. Pathogenic variants co-occurring at similar VAF as RUNX1 were detected in 17 out of 20 patients with a somatic RUNX1 variant, but not in any of the patients with germline variants (Figure 1). Pathogenic variants in SRSF2, TET2 and ASXL1 were frequently observed to co-occur with high VAF RUNX1 variants. Image:Summary/Conclusion:RUNX1 variants with a VAF above 40% were classified as germline in two out of 22 MN patients (9%). The variants were detected in patients with MDS and CCUS indicating that germline testing should be considered not only in AML, but also in other MN with high VAF of a RUNX1 variant. Furthermore, a high VAF RUNX1 variant without co-occurring variants with similar VAF in other genes should increase the consideration for germline testing.