{"doi":"10.1002/ajh.26184","title":"Cell‐free <scp>DNA</scp> analysis for detection of <scp><i>MYD88</i><sup>L265P</sup></scp> and <scp><i>CXCR4</i><sup>S338X</sup></scp> mutations in <scp>W</scp>aldenström macroglobulinemia","abstract":"Molecular testing for MYD88 and CXCR4 mutations is increasingly used in patients with Waldenström macroglobulinemia (WM). MYD88L265P is the most frequent variant (93–95%) in WM patients, while non-L265P MYD88 variants (1–2%) can rarely occur.1, 2 The presence of mutated MYD88 is associated with a decreased risk of histological transformation, longer overall survival, and sensitivity to ibrutinib.3, 4 Over 40 nonsense and frameshift CXCR4 variants have been identified in WM patients.5, 6 Nonsense CXCR4S338X variants are the most common, occurring in 50% of WM patients due to C > A or C > G nucleotide transversions.5, 6 Among CXCR4 mutations, CXCR4S338X has the largest clinical impact with higher serum IgM levels, symptomatic hyperviscosity, earlier time to initiation of frontline therapy, and shorter progression-free survival with ibrutinib.3 MYD88 and CXCR4 mutations can therefore provide important data regarding diagnosis, prognosis, and treatment response in WM.3 The use of bone marrow (BM) aspirate materials represents the current \"gold standard\" for molecular testing in WM.1-3, 5, 6 Although MYD88 and CXCR4 mutations can also be identified in peripheral blood (PB), the diagnostic yield in PB is inferior to BM, particularly for previously treated patients.7, 8 Tumor enrichment with B-cell selection can significantly improve testing sensitivity, but pre-sorting B cells is time-consuming and not feasible in most clinical laboratories.7, 9 Recent studies have demonstrated the feasibility of identifying MYD88 and CXCR4 mutations by using cell-free DNA (cfDNA) from WM patients.10-12 These findings prompted us to perform a comprehensive analysis comparing the use of cfDNA to matched BM and PB, with or without B-cell selection, for detection of the most common MYD88 (L265P) and CXCR4 (S338X) mutations in WM patients. We prospectively collected matched BM and PB samples from 28 consecutive WM patients. PB was collected in Streck Cell-Free DNA tubes (Streck, La Vista, Nevada) to preserve cfDNA. Tubes were centrifuged and plasma was isolated within 30 h of venipuncture. cfDNA was extracted using the QIAGEN Circulating Nucleic Acid Kit (Qiagen, Hilden, Germany) with all modifications employed by Kang et al.13 Both CD19-selected and unselected BM and PB mononuclear cells were isolated as before.2, 6, 7 Overall, five different tissue fractions were isolated for analysis: CD19-selected BM (BM19+), unselected BM (BMMC), CD19-selected PB (PB19+), unselected PB (PBMC), and cfDNA. Quantitative allele-specific polymerase chain reaction (AS-PCR) assays for MYD88L265P and CXCR4S338X mutations were performed for each tissue fraction using 2.5 ng of DNA as previously described.2, 6, 7 To avoid a potential batch effect, tissue fractions from the same patient were run on the same plate. Calculations were performed with R (R Foundation for Statistical Computing, Vienna, Austria). The p values < .05 were considered statistically significant. The Dana Farber/Harvard Cancer Center IRB approved this study, and all patients provided written consent for sample use. Baseline patient characteristics are shown in Table S1. Mutation testing for MYD88L265P and CXCR4S338X was performed in 28 and 23 patients, respectively; limited tumor DNA precluded evaluation of CXCR4S338X in five patients. Using BM19+ as the reference tissue,1-3, 5, 6 we benchmarked the test performance of MYD88L265P and CXCR4S338X detection for BMMC, PB19+, PBMC, and cfDNA (Table 1). The rates of concordance between the different tissue fractions and BM19+ were consistent with previous studies.7, 9-12 We then compared the clinicopathological characteristics between patients with concordant and discordant results for MYD88L266P by cfDNA. Discordant patients had a lower median BM involvement (9% vs. 45%; p = .04) and serum IgM level (985 vs. 1597 mg/dL; p = .02), as well as a higher hemoglobin level (12.3 vs. 10.7 g/dL; p = .03) versus concordant patients. The BM involvement significantly corr","journal":"American Journal of Hematology","year":2021,"id":197516,"datarank":0.0,"base_score":0.0,"endowment":0.0,"self_citation_contribution":0.0,"citation_network_contribution":0.0,"self_endowment_contribution":0.0,"citer_contribution":0.0,"corpus_percentile":null,"corpus_rank":null,"citation_count":12,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9568,"is_data_producer":false,"deposit_databanks":null,"is_oa":false,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2021-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":356236,"name":"Zachary R. Hunter","orcid":"0000-0002-1689-1691","position":1,"is_corresponding":false},{"id":480823,"name":"Lian Xu","orcid":"0000-0002-0714-6446","position":2,"is_corresponding":false},{"id":645220,"name":"Nicholas Tsakmaklis","orcid":null,"position":3,"is_corresponding":false},{"id":357515,"name":"Amanda Kofides","orcid":null,"position":4,"is_corresponding":false},{"id":357511,"name":"Manit Munshi","orcid":null,"position":5,"is_corresponding":false},{"id":771545,"name":"Xia Liu","orcid":"0000-0001-7599-6285","position":6,"is_corresponding":false},{"id":232466,"name":"Maria Luisa Guerrera","orcid":"0000-0002-0730-4678","position":7,"is_corresponding":false},{"id":644060,"name":"Carly Leventoff","orcid":"0000-0002-3703-3901","position":8,"is_corresponding":false},{"id":645219,"name":"Timothy P. White","orcid":null,"position":9,"is_corresponding":false},{"id":644061,"name":"Catherine Flynn","orcid":"0000-0001-7645-3469","position":10,"is_corresponding":false},{"id":234432,"name":"Kirsten Meid","orcid":null,"position":11,"is_corresponding":false},{"id":356238,"name":"Christopher J. Patterson","orcid":"0000-0002-9652-2203","position":12,"is_corresponding":false},{"id":232467,"name":"Guang Yang","orcid":"0000-0003-3049-4200","position":13,"is_corresponding":false},{"id":644062,"name":"Andrew R. Branagan","orcid":"0000-0002-3868-9267","position":14,"is_corresponding":false},{"id":405104,"name":"Shayna Sarosiek","orcid":"0000-0002-0075-6735","position":15,"is_corresponding":false},{"id":232463,"name":"Jorge J. Castillo","orcid":"0000-0001-9490-7532","position":16,"is_corresponding":false},{"id":232462,"name":"Steven P. Treon","orcid":"0000-0001-6393-6154","position":17,"is_corresponding":false},{"id":356237,"name":"Joshua Gustine","orcid":"0000-0001-6717-7669","position":18,"is_corresponding":false},{"id":357514,"name":"Maria Demos","orcid":null,"position":0,"is_corresponding":true}],"reference_count":15,"raw_metadata":null,"created_at":"2026-07-18T23:50:23.532351Z","pmid":"33819355","pmcid":null,"fwci":null,"citation_percentile":null,"influential_citations":0,"oa_status":null,"license":null,"views":0,"total_file_size_bytes":0,"version_count":0,"fair_f":null,"fair_a":null,"fair_i":null,"fair_r":null,"fair_zscore":null,"fair_rationale":null,"fair_model":null,"fair_agent_version":null,"fair_fulltext_source":null,"fair_has_llm":null,"fair_computed_at":null,"clinical_trials":[],"software_tools":[],"db_accessions":[],"linked_datasets":[],"topics":[]}