{"doi":"10.1002/hem3.30","title":"Robust identification of conventional and leukemic nonnodal mantle cell lymphomas using epigenetic biomarkers","abstract":"Mantle cell lymphoma (MCL) is a B-cell lymphoma whose heterogeneous clinical presentation and evolution seem to be mediated by differences in cell of origin as well as in genetic, epigenetic, and transcriptional profiles.1-4 MCL is currently categorized into two distinct molecular subtypes. Conventional MCLs (cMCLs), usually clinically aggressive, are derived from germinal center-inexperienced B cells and show a high burden of molecular alterations. In contrast, leukemic nonnodal MCLs (nnMCLs) frequently present with a more indolent disease originating from germinal center-experienced cells and show an overall lower genetic complexity.1 Current methods to discern the two MCL molecular subtypes include expression of SOX11, IGHV mutational status, and gene expression profiling (16 gene panel L-MCL162). However, the differential diagnosis of cMCL and nnMCL poses several challenges as no method can unequivocally differentiate the two subtypes in fresh, frozen, or paraffin-embedded formalin-fixed (FFPE) material in the most affected tissues, for example, lymph node (LN), peripheral blood (PB), and bone marrow (BM). For instance, SOX11 expression determined by immunohistochemistry might be only partial with unclear significance or difficult to stain in BM samples, and the L-MCL16 gene expression signature has been designed for PB samples. In this context, the use of DNA methylation biomarkers may represent an alternative strategy, as DNA methylation is comparably more stable than gene or protein expression levels and can be measured in DNA obtained from different sample types. In MCL, a comprehensive analysis of the DNA methylome revealed a signature differentiating two epigenetic subtypes, termed C1 and C2 MCL. This signature was mostly related to imprints of B cells at different maturation stages, and the C1 subtype was enriched for cases with SOX11 expression, unmutated IGHV status, and showed more genetic alterations and shorter overall survival than C2 MCLs.3 Based on these findings, C1 showed a strong overlap with cMCL, and C2 with nnMCLs. The DNA methylation signature differentiating C1 and C2 MCLs3 can be reduced to only three CpG sites while maintaining the detection accuracy,5 paving the way to generate locus-specific assays for clinical use. In the present study, we designed DNA methylation assays for these three CpG sites (Illumina CpG cluster IDs: cg23892310, cg07769421, and cg03425785) using bisulfite pyrosequencing (BisPyroSeq),6 a cost-effective method that shows high performance in a benchmarking study comparing techniques for epigenetic biomarkers.7 Additionally, as DNA methylation is affected by the tumor cell content of the sample, we also mined previously reported signatures and obtained two CpGs to estimate B-cell purity (Illumina CpG cluster IDs: cg00226923 and cg03860768).5 See Supporting Information for detailed technical information regarding the assays. We initially optimized the BisPyroSeq assays for the three MCL-stratifying CpGs using in vitro methylated DNA as well as DNA extracted from two MCL cell lines (Granta-519 and Z-138, both cMCL models) and 30 primary MCLs with available Illumina arrays from previous studies1, 3 and therefore known methylation status, suitable as a control. Methylation values from BisPyroSeq and methylation arrays were highly concordant (cg23892310: R = 0.91, p = 2.6 × 10−12; cg07769421: R = 0.92, p = 6.4 × 10−13; cg03425785: R = 0.85, p = 3.8 × 10−9; Figure 1A and Supporting Information S1: Table 1). Next, we quantified DNA methylation levels in 17 cases of which different sample types were available (three frozen and PB material, eight frozen and FFPE, two with FFPE and PB, one with two different FFPE samples, and three with frozen, PB, and FFPE material). We observed that our DNA methylation assays were very highly reproducible in different tissues and sample types, in all three measured CpGs, leading to identical classification in all cases (Figure 1B and Supporting Inform","journal":"HemaSphere","year":2024,"id":493715,"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":0,"citer_count":0,"citers_with_citation_signal":0,"citers_with_endowment":0,"datacite_reuse_total":0,"is_dataset":false,"is_dataset_confidence":0.9695,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2024-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":244344,"name":"Marta Kulis","orcid":"0000-0001-8104-9620","position":1,"is_corresponding":false},{"id":244341,"name":"Martí Duran‐Ferrer","orcid":"0000-0003-1666-5819","position":2,"is_corresponding":false},{"id":13762,"name":"Cristina López","orcid":"0000-0001-6644-1659","position":3,"is_corresponding":false},{"id":244339,"name":"Guillem Clot","orcid":"0000-0003-2588-7413","position":4,"is_corresponding":false},{"id":244337,"name":"Ferran Nadeu","orcid":"0000-0003-2910-9440","position":5,"is_corresponding":false},{"id":1340377,"name":"Mónica Romo","orcid":null,"position":6,"is_corresponding":false},{"id":69884,"name":"Eva Giné","orcid":"0000-0002-0532-1204","position":7,"is_corresponding":false},{"id":14394,"name":"Armando López‐Guillermo","orcid":"0000-0002-8588-8381","position":8,"is_corresponding":false},{"id":244356,"name":"Sı́lvia Beà","orcid":"0000-0001-7192-2385","position":9,"is_corresponding":false},{"id":14337,"name":"Elı́as Campo","orcid":"0000-0001-9850-9793","position":10,"is_corresponding":false},{"id":14406,"name":"José Ignacio Martin-Subero","orcid":"0000-0001-8809-5195","position":11,"is_corresponding":false},{"id":1134974,"name":"Marco M. Bühler","orcid":"0000-0003-0850-3326","position":0,"is_corresponding":true}],"reference_count":8,"raw_metadata":null,"created_at":"2026-07-19T02:09:03.883685Z","pmid":"38434527","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":[]}