Sorter enrichment of small B-NHL clones for NGS analyses

In addition to histology and immunophenotyping, molecular genetics plays an important role in the diagnosis of B-cell lymphomas (WHO 2022). With the introduction of modern high-throughput sequencing methods (Next Generation Sequencing, NGS), hundreds of thousands of genome regions can now be analyzed in parallel within a very short time. The so-called panel testing allows the analysis of several lymphoma-relevant genes (approx. 66) in one test run in less than one week and thus plays an important role in:

• confirming the diagnosis (e.g. MYD88, BRAF, NOTCH2, KLF6)

• the assessment of prognosis (e.g. TP53, CXCR4)

of B-cell lymphomas.

However, low-infiltrating or washout lymphomas (<5% clone size) cannot currently be investigated molecularly by panel NGS due to lack of sensitivity. Therefore, for hairy cell leukemia and lymphoplasmocytic lymphoma, sensitive quantitative PCR analyses at the mRNA level have been established at MLL, allowing reliable detection of mutations in BRAF and MYD88 even for clone sizes <5%. However, in other B-NHL entities, a diagnostic challenge may arise under the following circumstances:

• In the presence of clinical symptoms and necessary therapy

• No clear immunophenotype (e.g., CD5-negative splenic B-NHL).

• histological tissue is not available (e.g. intraperitoneal lymph nodes).

Fluorescence-activated cell sorting is a method in which cells are labeled using fluorescently labeled antibodies and can be specifically deflected and captured by applying a voltage to an electric field. This makes it possible to identify lymphoma cells and isolate and enrich them from the cell suspension.

In a preliminary study, we sorter-enriched 30 different patient samples with small B-NHL clones. After enrichment of malignant B cells to 17% of leukocytes (median) in a total cell count of 700,000 cells (median), we were able to reliably perform NGS (lymphoid panel). Lymphoma-relevant mutations were found in 50% of the samples examined. For reference, NGS analyses were also performed on the respective unsorted patient sample. Without prior enrichment of the lymphoma cells, mutations were found in only 13% of the cases, with only one of the unsorted samples (3%) completely revealing all mutations.

These data demonstrate the need for malignant B cell enrichment in B-NHL samples with small clone size. Sorter-based enrichment represents an ideal method to address this diagnostic challenge. We are currently expanding the number of test samples (n>30) in order to validate this method in a timely manner and, in diagnostically relevant individual cases, to reliably offer NGS analyses for B-cell lymphomas with small clone size. For this purpose, we currently have two sorter devices (CytoFlex SRT, Beckman  Coulter) are available.