Lymphoma Cell Sorting to Enrich Low Degree Infiltration Clonal B-Cells for Diagnostic Delineation of Molecular Genetic Background By NGS
Authors: Ecker Veronika , Mueller Martha-Lena , Meggendorfer Manja , Eder Christiane , Baer Constance Regina , Haferlach Claudia , Haferlach Torsten , Kern Wolfgang 
Published: December 2023

Introduction: According to WHO 2022 classificationthe vast majority of B-cell lymphoma entities continue to be defined histologically and by their immunophenotype. However, genetics help to define these entities and provide prognostic information. NGS targeted panel sequencing with short turn-around time has emerged as powerful diagnostic tool to uncover relevant genes frequently mutated in B-cell lymphoma. Yet its sensitivity limits its application to lymphoma cases with clone sizes &gt;5%. In case of clinical symptoms and need for therapy small B-cell clones (&lt;5%) can be diagnostically challenging if tissue for histopathology is difficult to obtain and immunophenotype is not characteristic for a specific lymphoma entitiy such as splenic B-cell lymphoma.
Aim: To enrich small B-cell clones (&lt;5%) by cell sorting to &gt;15% to enable lymphoma NGS panel sequencing for potential genetic characterization and risk stratification.
Methods: Peripheral blood or bone marrow samples (n=43) that containedsmall B-cells clones (&lt;5%) as identified by flow cytometric routine diagnostics were stained for cell sorting by a combination of monoclonal antibodies against 9 B-cell antigens, including CD5, CD19 and CD45. The gating strategy excludes debris and doublets and defines B- and T-cells out of CD45-positive events. By application of cell sorting (SRT Cytoflex, Beckman Coulter) we aimed at &gt;500,000 cells consisting of B- and T-cells, containing &gt;15% clonal B-cells to ensure sufficient NGS sensitivity but minimize sort time. DNA was extracted from sorted samples and lymphoma NGS panel sequencing (66 genes ~ 1,500x coverage, min. 400x) was performed (NovaSeq 6000, Illumina). For technical and biological validation, native sample material was sequenced in parallel for a subset of patient samples (n=30).
Results: In total43samples with &lt;5% clonal B-cells were applied for cell sorting and clonal B-cells were enriched to a median of 17% (range, 3-97%) in a median of 800,000 cells (range, 127,400-3,780,000). In a pilot study to optimize the sorting procedure 8 of 13 sorted samples (62%) revealed quantitatively sufficient and good quality DNA and could be successfully analyzed by NGS. In 4 of these 8 samples (50%) mutations were identified.
Optimized processing regarding cell sorting and DNA extraction was applied to further 30 samples all of which could be successfully sequenced. Overall, in 50% of all samples with NGS analysis after cell sorting (19/38) one or more mutations (total number of mutations: n=30) associated with B-cell lymphoma were detected. Mutations comprised genes frequently affected in B-cell lymphoma: MYD88 (30% of detected mutations, 9/30), CXCR4 (20%, 6/30), NOTCH1 (7%, 2/30), NOTCH2 (3%, 1/30), KMT2D (7%, 2/30), TP53 (13%, 4/30), BRAF (7%, 2/30), KLF2 (10%, 3/30) and DNMT3A (3%, 1/30).
All of the cases negative for NGS after sorting (n=15) were negative also by NGS on matched native sample material without prior enrichment of clonal B-cells. Out of the 15 cases with positive NGS results after sorting NGS on native sample material revealed mutations in only 4 (27%) cases (only 1 case with detection of all mutations) while in 11 (73%) cases no mutations were detected. In the 15 cases positive after sorting median variant allele frequency (VAF) was 8.8% (range 3.4-38.3%). As anticipated, in the 4 cases with mutations detected also without sorting VAF was lower in native sample material (median 3.1%, range 1.98-4.9%) as compared to sorted sample material (median 8.9%, range 6.8-23.2%, p=0.0412).
Conclusion: Cell sorting can overcome the diagnostic challenge of low degree infiltration B-cell lymphoma that require molecular genetics for further classification and risk prediction prior to therapy. Analysis of 30 samples and comparison with matched native material revealed that enrichment targeting 15% of clonal B-cells in a total of &gt;500.000 cells are desirable for successful and sensitive NGS panel sequencing. In 50% of such cases mutations are detectable after sorting. Cell sorting to enrich clonal B-cells thus is a powerful tool to enable NGS panel sequencing for low percentage mature B-cell lymphoma and should be considered for routine diagnostics of B-cell lymphoma.