DP132 | A NOVEL APPROACH FOR HIGHLY SENSITIVE AND RAPID IDENTIFICATION OF HMGA2 SUBMICROSCOPIC DELETIONS IN MYELOPROLIFERATIVE NEOPLASMS

Recently, Handa S. et al. (PMID:39715853) identified a novel submicroscopic deletion involving HMGA2 3’UTR (Δ3’HMGA2) in Primary Myelofibrosis (PMF) and Accelerated (AP) or Blast Phase (BP) patients (14/169, 8.3%). The Δ3’HMGA2 detected by authors involved ≤3.1kbp (12/14 cases) or ≥87kbp (2/14 cases) and was often associated to ASXL1 and CALR mutations and higher risk of disease evolution. Given its clinical relevance, we developed a streamlined method to rapidly detect Δ3’HMGA2 via targeted long-read sequencing with ultra-high coverage and a tailored automated analysis (<u>https://github.com/blackSquare225/RdD</u>). Custom primers were designed to generate a 4,735bp amplicon, spanning HMGA2 exon5 and 3’UTR, and further sequenced through ONT-PromethION. The analytical pipeline enables the calling of the most represented Δ3’HMGA2 isoforms. The method was first tested in a Training Cohort (TC) of 187 samples (108 MF, 79 MPN-BP) and further validated in a Validation Cohort (VC) of 136 samples (96 MF, 40 MPN-BP). All patients provided written informed consent (IRB #14560). In TC (median on-target read depth: 91,284x; 95%CI:15,691–291,322), we detected Δ3’HMGA2 in 2 PMF (Variant Allele Frequency, VAF: 8% and 0.1%), 1 CALRmut and 1 JAK2mut, respectively; 1 post-ET MF (VAF:21.3%) with NF1mut and 1 MPN-BP (VAF:0.6%) with JAK2, ASXL1, GATA2, SH2B3 mutations. In VC (median on-target read depth:72,864x; 95%CI:12,544–164,352) we found Δ3’HMGA2 in 3 PMF: 1 (VAF:0.4%) associated with JAK2, SH2B3, TP53, NRAS, 1 (VAF:70.5%) with CALR, ASXL1 and 1 (VAF:0.5%) with CALR, IDH1, CBL, NRAS and 1 (VAF:28.6%) post-ET MF associated with JAK2mut. Results were confirmed by Sanger method. All the Δ3’HMGA2-mutated patients (8/323, 2.5%) had normal karyotype at standard cytogenetics. None showed disease progression (median follow-up: 38 months) except for the JAK2mut PMF who progressed to MF-AP. Handa et al. suggested Δ3’HMGA2 as potential biomarkers of advanced MF and our findings confirm its presence in MF/MPN-BP. Given the known role of HMGA2 in disease progression and differences in Δ3’HMGA2 frequency from Handa et al., likely due to unique cohort characteristics, larger studies are needed to define its prevalence in MPNs and other blood cancers. At this regard, the assay we developed enables rapid (≤12h per run), simultaneous (up to 96 samples per run), and sensitive (limit of detection: 0.05%VAF) screening of Δ3’HMGA2, which standard cytogenetics or short-read sequencing might miss.