PhD Scientific Days 2023

Budapest, 22-23 June 2023

Pathology - Posters C

Clonal characterization and somatic hypermutation assessment by next-generation sequencing in chronic lymphocytic leukemia

Tamás László1, Erik Zajta1, Róbert Horváth1, Lili Kotmayer1, Lajos Hegyi1, Csaba Bödör1

1 HCEMM-SE Molecular Oncohematology Research Group, Pathology and Experimental Cancer Research, Semmelweis University, Budapest

Text of the abstract

Introduction: The somatic hypermutation (SHM) status of the immunoglobulin heavy chain gene (IGHV) variable region is one of the most important prognostic biomarkers of chronic lymphocytic leukemia (CLL). Currently IGHV status is determined by conventional Sanger sequencing, but this method has several drawbacks, including high laboratory requirements or low efficiency in cases with multiple clonal rearrangements.
Aims: In our study, we aimed to set up a next-generation sequencing (NGS)-based assay for the assessment of SHM status and to compare the results obtained by NGS and the gold-standard Sanger sequencing.
Methods: In our study, we examined peripheral blood samples from 61 patients in a retrospective cohort of CLL cases with predominantly complex gene rearrangements. Sanger sequencing was performed using so-called “Leader” and “FR1” primers according to the recommendations of the European Research Initiative on CLL. NGS analysis was performed using the LymphoTrack Dx IGHV Leader Somatic Hypermutation Assay on a Miseq instrument. Spearman’s correlation was used to assess the correlation between the results obtained by NGS and Sanger sequencing.
Results: IGHV status could be determined by both methods in 66% (40/61) of the samples tested. The NGS-based SHM determination showed a positive correlation with Sanger sequencing (r=0.938, p<0.0001) in terms of the exact SHM values. Conventional Sanger sequencing failed in 21% (13/61) of the cases, while assessment of the SHM status was successful in these cases using NGS methodology. These patients were associated with multiple IGH rearrangmenets in 38% of samples tested (5/13), whereas we could not find any specific explanation for this phenomenon in 62% (8/13) of the cases. In 9.8% of the samples tested we couldn’t assess the SHM status using neither Sanger sequencing nor NGS due to policlonality (2/6) or the lack of productive clonal rearrangement (4/6). The NGS-based methodology was significantly more successful in determining IGHV status than conventional Sanger sequencing (p=0.028).
Conclusion: The results obtained from NGS-based IGHV SHM analysis correlate reliably with the results obtained from conventional Sanger sequencing. NGS can determine IGHV status with higher efficiency compared to the gold-standard Sanger sequencing.
Funding: K21_137948, H2020-739593, TKP2021-EGA-24 TKP2021-NVA-15, Elixir Hungary