{"doi":"10.1002/hon.70096_471","title":"471 | LONGITUDINAL scRNA‐seq CASE STUDY IDENTIFIES THREE STABLE AND DISTINCT B‐CELL SUBTYPES IN SERIAL SAMPLES OF A DIFFUSE B‐CELL LYMPHOMA FROM A SINGLE DOG","abstract":"G. Walker and A. Treeful equally contributing author. Introduction: Diffuse B-cell lymphomas are the most common hematologic malignancies in dogs, with low long-term remission rates (5%–10% surviving ≥ 2 years) and unclear mechanisms of therapy resistance and relapse. In a veterinary clinical trial combining oncolytic virotherapy (VSV) and immune checkpoint blockade, we aim to identify mechanisms of immunotherapy resistance. Here we describe a preliminary longitudinal scRNAseq analysis to evaluate intratumoral cellular composition and transcriptional dynamics in relation to treatment response. Methods: A 6-year-old neutered male goldendoodle with stage 5a B cell lymphoma was randomized into the VSV-only arm. Serial lymph node biopsies (n = 7) were collected at screening, day 0 (VSV treatment), and days 3, 10 (FNA and resection), 28, 49, and 70. CHOP chemotherapy was initiated on day 30. The day 70 sample did not pass quality control for further processing. Single-cell sequencing was performed using the 10X Chromium 3′ (v3.1) platform and reads were aligned to the canFam4_Y genome with CellRanger. Seurat (v5.2.1) in R (v4.4.0) was used for sample merging and subsequent quality control, feature selection, clustering, and cell annotation alongside GCESS analysis. Results: We obtained data for 182,356 high-quality cells across all analyzed samples (n = 6), with B cells comprising ∼92%, T cells ≤ 6%, and other immune cells ∼2%. The resected tumor yielded the highest quality data. GCESS and Seurat analysis identified three transcriptionally distinct B-cell subpopulations, characterized by either reduced ribosomal protein expression (RPS19, RPS26, RPLP1, RPS14, RPL9), a cell proliferation signature (TOP2A, MKI-67, CENPF, STMN1, HMGB2, TPX2), or a hybrid +ribosomal/-proliferation state. Intriguingly the three subpopulations were stable across all time points, other than the disappearance of the proliferative population post-chemotherapy. Conclusions: Despite any technical variability (e.g., biopsy strategy, intra-tumoral heterogeneity) our key findings were consistent across all time points. The persistence of three B-cell subtypes suggests preservation of lineage specific signals, although implications remain unclear. Plausible biological explanations vary from three independently evolved malignant clones, to a transformed monoclonal cluster with residual or reactive non-malignant populations. VSV treatment had minimal impact on B-cell transcriptional profiles in this dog, but an evident loss of proliferation signals post-chemotherapy was consistent with biological expectations. Ongoing analyses will assess potential effects of VSV on the T-cell compartment and compare these subtypes to circulating B cells and normal lymph node tissues for further validation. Research funding declaration: Research funding provided by the National Institutes of Health (NIH). Keywords: genomics, epigenomics, and other -omics; canine lymphoma as a surrogate; immunotherapy Potential sources of conflict of interest: S. Naik Stock ownership: Ownership in stock of Vryiad which makes virotherapy products, including the VSV used in the present study.","journal":"Hematological Oncology","year":2025,"id":568515,"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":0.9496,"is_data_producer":false,"deposit_databanks":null,"is_oa":true,"file_count":0,"downloads":0,"has_version_chain":false,"published_date":"2025-01-01","fair_score":null,"fair_percentile":null,"algorithm_id":"datarank_citation_only_1hop_v6","ranking_scope":"data_only","authors":[{"id":923758,"name":"Amy E. Treeful","orcid":"0000-0002-7270-0538","position":1,"is_corresponding":false},{"id":497571,"name":"Marissa Macchietto","orcid":"0000-0002-9877-6020","position":2,"is_corresponding":false},{"id":1321298,"name":"Kelly Reid","orcid":null,"position":3,"is_corresponding":false},{"id":337450,"name":"Davis Seelig","orcid":"0000-0002-1733-8177","position":4,"is_corresponding":false},{"id":630063,"name":"Kimberly Demos-Davies","orcid":"0000-0001-6901-9963","position":5,"is_corresponding":false},{"id":1472779,"name":"Andreas Winter","orcid":"0000-0003-0757-8721","position":6,"is_corresponding":false},{"id":1472778,"name":"Manuela Büettner","orcid":"0000-0003-4351-6333","position":7,"is_corresponding":false},{"id":325073,"name":"Lauren A. Burt","orcid":"0000-0002-0348-5643","position":8,"is_corresponding":false},{"id":1050378,"name":"Sara Pracht","orcid":null,"position":9,"is_corresponding":false},{"id":1472780,"name":"Veronica Penza","orcid":"0000-0003-1096-560X","position":10,"is_corresponding":false},{"id":1473340,"name":"C. Yung","orcid":null,"position":11,"is_corresponding":false},{"id":1473339,"name":"R. Luethcke","orcid":null,"position":12,"is_corresponding":false},{"id":262888,"name":"Ralf H. Adams","orcid":"0000-0003-3031-7677","position":13,"is_corresponding":false},{"id":1473346,"name":"R. Ditcovsky","orcid":null,"position":14,"is_corresponding":false},{"id":1473347,"name":"Alana R. Kuzmik","orcid":null,"position":15,"is_corresponding":false},{"id":302949,"name":"Daniel A. Vallera","orcid":"0000-0003-4587-3238","position":16,"is_corresponding":false},{"id":539026,"name":"A. Borgatti","orcid":null,"position":17,"is_corresponding":false},{"id":390707,"name":"Morgan Henson","orcid":"0000-0001-7882-426X","position":18,"is_corresponding":false},{"id":1321299,"name":"Caitlin Feiock","orcid":null,"position":19,"is_corresponding":false},{"id":393152,"name":"Jaime F. Modiano","orcid":"0000-0001-6398-7648","position":20,"is_corresponding":false},{"id":44525,"name":"Shalin H. Naik","orcid":"0000-0003-0299-3301","position":21,"is_corresponding":false},{"id":539028,"name":"A. L. Sarver","orcid":null,"position":22,"is_corresponding":false},{"id":1472787,"name":"Gordon Walker","orcid":"0000-0002-5278-6792","position":0,"is_corresponding":true}],"reference_count":0,"raw_metadata":null,"created_at":"2026-07-19T02:56:52.212268Z","pmid":null,"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":[]}