PF344 HIGH FREQUENCY ASXL1 MUTATIONS AND MULTIPLE SUBCLONAL PIGA MUTATIONS ARE OBSERVED IN CELLULAR AND CELL‐FREE DNA IN PAROXYSMAL NOCTURNAL HAEMOGLOBINURIA
Authors: G. Ryland, L. Fox, M. Brown, M. Caruso, M. McBean, E. Thompson, J. Lickiss, J. Shortt, K. Mason, D. Westerman, D. Ritchie, J. Szer, P. Blombery
Journal: HemaSphere (2019)

Background: Paroxysmal nocturnal haemoglobinuria (PNH) is a life‐threatening clinical syndrome caused by acquired mutations in PIGA in the haemopoietic compartment. The clinicopathological features of PNH can overlap with aplastic anaemia (AA) and myelodysplastic syndrome (MDS) and these entities may represent different manifestations of a common underlying pathological process. To date, the landscape of acquired mutations in de novo PNH ( i.e. not preceded by a formal diagnosis of AA/MDS) remains incompletely characterised. Aims: To characterise the spectrum of acquired mutations in the cellular and cell‐free compartments in a cohort of patients with de novo PNH. Methods: DNA was extracted from peripheral blood leukocytes (n = 19) or bone marrow (n = 2) from 21 patients with a clinicopathological diagnosis of PNH (defined as a detectable PNH clone by flow cytometry, active haemolysis and not meeting formal diagnostic criteria for MDS or AA). Patients with a pre‐existing diagnosis of AA or MDS were excluded. Cell‐free DNA (cfDNA) was also collected from 19 patients. DNA sequencing libraries were prepared from cellular DNA using both the Peter MacCallum Cancer Centre PanHaem hybridisation and Myeloid amplicon panels (sensitivity 5%) which includes sequence variant detection for recurrently mutated genes associated with haematological malignancy and whole genome copy number assessment. A custom panel using Anchored Multiplex PCR target enrichment chemistry and unique molecular barcodes was used for cfDNA sequencing (sensitivity &lt;1%). Results: At time of sampling 18 of 21 patients were two or more years post diagnosis (median time since diagnosis of 8 years) with a median age of 41 years (range 23 ‐ 82). 16 patients were being treated with complement inhibitor therapy. PIGA mutations were detected in 19 patients, with multiple mutations detected in half the cohort (11 patients, range 2 ‐ 9 PIGA mutations per patient). 21 mutations were detected in the cellular DNA, while cfDNA sequencing revealed 30 additional PIGA mutations at low allelic burden (median allele frequency 2.19%, range 0.57% ‐ 4.86%) (20 frameshift, 15 missense, 10 splice site, 6 nonsense). Two patients did not have a detectable PIGA mutation but instead harboured focal copy number losses at the PIGA locus, which also involved ZRSR2 in the minimal deleted region. Remarkably, 7 of 21 patients had truncating ASXL1 mutations detectable in both the cellular and cfDNA (9 frameshift, 3 nonsense); in 4 patients multiple ASXL1 mutations were detected. Clonal pathogenic mutations were also detected in U2AF1 (2 patients) and BCOR (1 patient). Patients with ASXL1 and/or U2AF1 mutations had significantly lower neutrophil counts (mean 1.7 vs 1.08, p = 0.021) compared to those without. Haemoglobin and platelet counts were not statistically different between the two groups. Summary/Conclusion: Multiple subclonal PIGA mutations are detectable in patients with PNH. In addition acquired pathogenic variants in ASXL1 , U2AF1 and ZRSR2 were detected in a third of the cohort. Longitudinal studies are required to definitively determine the natural history of patients harbouring these pathogenic genomic variants. Furthermore cfDNA sequencing may represent a more sensitive approach for the detection of clinically relevant mutations in patients with PNH.