PS1325 IDENTIFICATION AND TARGETING OF MOLECULAR SIGNATURES OF HYPOMETHYLATING AGENT RESISTANCE IN MYELODYSPLASTIC SYNDROME AND CHRONIC MYELOMONOCYTIC LEUKEMIA

Abstract

Background: Waldenström macroglobulinemia (WM) is a subset of lymphoplasmacytic lymphoma (LPL) with IgM paraprotein. Genetic hallmark of WM is MYD88 L265P mutation which is important for its diagnosis and pathophysiology of this disease. Other genetic changes including CXCR4 mutation, which is associated with WHIM syndrome, are reported to be common in WM. Whereas comprehensive information on genetic changes of LPL with other paraprotein like IgG is scarce. Formalin-fixed paraffin-embedded (FFPE) samples are available for genomic studies, but it contains abundant non-tumor cells. Aims: In this report, we aim to evaluate, first, genetic difference between WM and LPL with IgG paraprotein (IgG type) using targeted next-generation sequencing (NGS) from FFPE DNA and second, the usefulness of allele-specific oligonucleotide (ASO) PCR and peptide nucleic acid (PNA) PCR clamp method compared with next-generation sequencing. Methods: Twenty patients with pathologically confirmed LPL (10 of WMs and 10 of IgG type LPLs) were subjected to this study after informed consent. There were include 8 DNA extracted from bone marrow (BM) FFPE samples and 12 DNA extracted from BM cells. MYD88 L265P was detected by ASO-PCR. CXCR4 mutations were detected by PNA PCR clamp method with 18-mer PNA, which enables to detect the most frequently mutated amino acid: S338. Using the amplification products from that, detected mutations were confirmed by Sanger sequencing. We also tried detecting CXCR4 mutations by only Sanger sequencing. The mutations in 12 genes were detected by targeted sequencing on coding sequence with NGS. Mann-Whitney U test and Chi-squared test were used for statistical analysis. This study was approved by the research ethics committee of Gunma University Hospital. Results: Using NGS, we found the somatic mutations in WMs and the IgG type LPLs: MYD88 (4 vs. 5), CXCR4 (3 vs. 2), NOTCH2 (0 vs. 1), ARID1A (9 vs. 8), RAG2 (5 vs. 5), KMT2D (10 vs. 10), TP53 (0 vs. 0), MYBBP1A (0 vs. 1), CD79B (0 vs. 0), PRDM1 (0 vs. 1), CD274 (0 vs. 0), PDCD1LG2 (2 vs. 0); there were no statistical differences, suggesting genetic similarity between these two subsets of LPL, IgM type and IgG type. MYD88 L265P was found in 9/10 (90%) of WM cases and 6/10 (60%) of the IgG type by ASO-PCR (p = 0.40); However, it was found only in 4/10 (40%) of WM cases and 5/10 (50%) of the IgG type by NGS (p = 0.05). In addition, A260 V was found in 1 of IgG type LPL case. All MYD88 L265P mutations detected by NGS were also detected by ASO-PCR. CXCR4 mutations were found in 1/9 (11%) of WM cases (T318 frameshift) and 2/9 (22%) of IgG type (T318 frameshift and S338∗) by PNA PCR clamp method (p = 0.22). Whereas, these were found in 3/10 (30%) of WM cases and 2/10 (20%) of IgG type by NGS (p = 0.67). NGS enabled to detect the three CXCR4 mutations detected by PNA PCR clamp method and the mutations in outside of target area of PNA (E345 frameshift and L305F). Summary/Conclusion: Our NGS analysis suggest genetic similarity between two subsets of LPL, WM and IgG type. ASO-PCR is superior sensitive to NGS in detection of MYD88 L265P mutation, and PNA PCR clamp method enabled to detect the CXCR4 mutations in FFPE samples even if they were the frameshift mutations. Meanwhile, NGS analysis uncovered mutations other than ASO-PCR and PNA PCR clamp method targets.