A novel sequencing panel for somatic hypermutation (SHM) status determination

September 10, 2024

Written by: Debajeet Ghosh, PhD Student, Karsan Lab

Edited by: Alex Witt, PhD Candidate, Pryzdial Lab

A Next Generation Sequencing (NGS) panel developed by Dr. Aly Karsan’s group in the BC Cancer Research Centre and UBC Centre for Blood Research allows for simultaneous detection of somatic hypermutation status (SHM) and other key clinical variants to inform treatment of Chronic Lymphocytic Leukemia1.

B cells, a special class of white blood cells, rely on a phenomenon called somatic hypermutation (SHM) to produce functionally competent antibodies (i.e. immunoglobulins). SHM introduces variation to antigen binding sites of antibodies, particularly the immunoglobulin heavy variable chains (IGHV), to improve their affinity for antigens on the target cell. When SHM processes are disrupted, as in cases of B-cell malignancies like Chronic Lymphocytic Leukemia (CLL), patients’ B cells have minimal or no variability in the IGHV, contributing to more aggressive disease than their functionally competent counterparts2. Hence, IGHV somatic hypermutation status determination is a routine prognostic test for CLL patients. Patients with a negative SHM or unmutated (U-CLL) status tend to have increased disease progression and shorter overall survival, compared to mutated patients (M-CLL)2.

Figure: Sample selection and NGS data analysis pipeline.

Figure: Sample selection and NGS data analysis pipeline.

While Sanger Sequencing has been a ‘gold standard’ for SHM status determination, the low throughput nature and limited sensitivity in clonality determination leaves more to be desired. Dr. Aly Karsan’s group at the BC Cancer Research Centre has devised a targeted capture Next Generation Sequencing (NGS) panel that allows for simultaneous detection of SHM status along with other key clinical variants that dictate CLL treatment regimen3. The NGS panel designed by Grants et al. offers a powerful and versatile approach for comprehensive genomic profiling of CLL patients. The target capture technique with three different probe pools (described in detail below) allows for flexible customization of the panel, enabling the inclusion of virtually limitless additional variants – a marked improvement from its predecessor.

The published NGS pipeline featured 3 different probe pools:

  • An IGHV SHM pool: Sequences and identifies SHM status and IGH V(D)J rearrangement in the IGHV 1-7 and IGHJ 1-6 gene families.
  • An SNV/indel pool: Sequences and detects disruptive mutations in 11 different genes (such as TP53, MYD88 and NOTCH2) relevant to CLL in terms of outcome correlation, drug target determination, and diagnostic accuracy.
  • A del(17p) pool: Sequences genes in the 17p chromosomal arm to detect if the patient bears the chromosomal band deletion which is associated with worse outcomes.

The panel’s NGS capabilities also allows SHM determination of multiple clonal populations, or clonotype, in patients exhibiting tumor heterogeneity – something not possible with Sanger Sequencing.  

The NGS panel was validated using libraries constructed from 35 unique CLL patient samples. Compared to Sanger sequencing, the NGS panel results showed a very similar sequencing alignment for IGHV. For clonotype determination, the results were identical for all except two patients. For these samples, the NGS panel determined two different alleles of the IGHV gene that were not resolved by Sanger sequencing – highlighting its applicability in clonotype determination and accurate quantification. The panel showed high reproducibility in SHM determination, with a maximum variability of only 0.11% across technical replicates.

Increasing in SHM status detection sensitivity while simultaneously generating clues vital to CLL treatment regimens makes this targeted capture Next Generation Sequencing (NGS) panel from the Karsan lab a standout in diagnostic profiling tools.

REFERENCES:

  1. Grants, J. M. et al. Chronic Lymphocytic Leukemia IGHV Somatic Hypermutation Detection by Targeted Capture Next-Generation Sequencing. Clinical Chemistry 70, 273–284 (2024).
  2. Chronic Lymphocytic Leukemia: Disease Biology | Acta Haematologica | Karger Publishers. https://karger.com/aha/article/147/1/8/863029/Chronic-Lymphocytic-Leukemia-Disease-Biology.
  3. Stewart, J. P. et al. Validation of the EuroClonality-NGS DNA capture panel as an integrated genomic tool for lymphoproliferative disorders. Blood Adv 5, 3188–3198 (2021).