Background: Mutations (mut) in the TP53 gene are found in distinct mature B-cell neoplasms (B-NHL) and are frequently associated with poor outcome. Risk stratification relies heavily on TP53mut status and is crucial to guide treatment. In addition, TP53mut are also observed in clonal hematopoiesis of indeterminate potential (CHIP) which is associated with an increased risk to develop myeloid neoplasm, mainly post cytotoxic therapy (pCT). Molecular analysis for TP53mut is mostly performed from native peripheral blood (PB) or bone marrow (BM) samples and thus agnostic to the cellular origin of TP53mut.

Aim: To investigate the cellular origin of TP53mut in patients with B-NHL in samples with 1) low tumor cell content or 2) a discrepancy between variant allele fraction (VAF) and clone size as determined by flow cytometry (FC).

Methods: 121 patients (PB/BM: 97/24; median age: 72 years [36-91]; female: 36%) with B-NHL were included: 105 samples with clone sizes <5% (median: 2% [0.2-5]) and 16 samples with discrepant TP53mut VAF and FC result (median difference: 29% [7-73]). Discrepancy was assessed using twice the VAF (%), adjusted for CNV, and compared to the FC (%) value. In 105 cases B-NHL cells were enriched to a median of 22% [5-100] within a lymphoid background (>95% B- and T-cells) while in 15 of further 16 samples B-NHL cells were purely sorted (median enrichment 99% [94-100]) besides T-cells and granulocytes as other sorted fractions. Sorted samples were analyzed by NGS. Cryopreserved cells of 2/15 patients and 1 additional patient were processed with the Single-Cell DNA (Takahashi, MissionBio) and Protein Sequencing Protocol (TotalSeq-D Heme Oncology Panel, Biolegends).

Results: Parallel analysis of both lymphoma-enriched and native sample material of 105 B-NHL patients revealed in total 166 mut. 109 mut were found in the lymphoid fraction, 57 in native sample material and 27 mut were detected in both fractions. We identified in total 9 patients with TP53mut: 5 only in lymphoid, 2 both in lymphoid/native and 2 only in native material indicating TP53mut as representing CHIP. Due to high clinical relevance of lineage assignment for TP53mut, we further investigated fresh PB samples from 16patients with discordant TP53mut VAF (%) and lymphoma clone size (%) as previously determined in these patients. Sorted B-NHL cells, T-cells and granulocytes of 15/16 patients showed 10 samples with TP53mut solely B-NHL-associated and 2 patients with TP53mut in B-NHL cells and additionally in T-cells or granulocytes. Importantly, 3 patients showed no TP53mut in B-NHL but either TP53mut in T-cells and granulocytes, only in granulocytes, or have been assigned to co-occurring multiple myeloma (MM) in BM only. In the first single cell analysed sample 3 distinct subclonal TP53mut in B-NHL cells were confirmed, whereof 1 harbored an additional del(17p), which was also present in T- and NK-cells at lower level. The other 2 TP53mut were also present in granulocytes. Second single cell analysed sample confirmed TP53mut and del(17p) in B-NHL cells which were additionally identified in granulocytes but not in other compartments. Third sample, not sort-analyzed, excluded TP53mut in B-NHL or any other cell type except plasma cells of a co-occurring MM present in BM only. Overall, single-cell analysis confirmed and refined distinct lineage involvement due to increased sensitivity and specificity. In summary, we detected TP53mut in myeloid cells in 4/16 patients, suggesting myeloid CHIP, whereof in 2 patients TP53mut would initially have been falsely attributed to B-NHL. In 2 other patients we confirmed that TP53mut was solely attributable to co-occuring MM.

Conclusion: Molecular analysis of unprocessed (native) samples often cannot determine the clonal origin of TP53mut or clearly link them to either B-NHL or CHIP. In 10 out of 25 patient samples, we found TP53mut lineage assignment beyond B-NHL association. While B-cell enrichment can improve sensitivity and reduce false negatives, it does not help detect false positives caused by CHIP in native samples. Although our study focused on cases with low B-NHL infiltration or discrepant TP53mut VAF and FC results, combining genetic and FC data is essential to identify such discrepancies. Accurately determining whether a TP53mut comes from B-NHL or co-existing CHIP is critical for prognosis, assessing the risk of developing a myeloid neoplasm, and choosing the right treatment for B-NHL patients.

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2025
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