“Snapshotting” Somatic Hypermutation in Single Follicular Lymphoma Cells

• Published in: Blood 2021-11, Vol.138 (Supplement 1), p.1151-1151
• Main Authors: Sepulveda-Yanez, Juleta H., Alvarez-Saravia, Diego, Quinten, Edwin, Menafra, Roberta, Kloet, Susan L., Monajemi, Ramin, Kielbasa, Szymon M., Navarrete Signorile, Marcelo, van Bergen, Cornelis A.M., Veelken, Hendrik
• Format: Article
• Language: English
• ISSN: 0006-4971; 1528-0020
• DOI: 10.1182/blood-2021-150475

Introduction Follicular Lymphoma (FL) is one of the most prevalent B-cell neoplasms and despite recent advances remains incurable in most cases. FL cells are malignant counterparts of normal germinal center B-cells. At molecular level FL are characterized by the t(14;18) which results in the overexpression of the BCL-2. These apoptosis resistant cells accumulate somatic mutations associated with tumorigenesis and progression. Whereas several mutagenic mechanisms shape the FL genomic landscape, up to 22% of the mutations may be attributed to activation-induced cytidine deaminase (AID) activity. Under physiological conditions AID is responsible for somatic hypermutation (SHM) in immunoglobulin genes (IG) of germinal center B-cells. In fact, it is accepted that ongoing SHM still occurs at relatively high rates in FL cells as compared with other germinal center lymphoid neoplasms. Moreover, we recently demonstrated the direct effect of AID overexpression inducing somatic mutations driving murine and human B-cell neoplasm progression in vivo. Therefore, unveiling the molecular basis of AID activity in lymphoma cells remains essential to understand lymphomagenesis and to develop novel targeted therapies. The advent of single-cell high-throughput sequencing has enabled the analysis of molecular events at an unprecedented resolution. Therefore, we designed a study to capture AID-induced somatic hypermutation at the single cell level in FL. Methods Tumor samples derived from 14 FL patients were analyzed, as controls, we chose a B-cell malignancy with lower SHM rates and included 5 samples derived from chronic lymphocytic leukemia (CLL) and 2 from monoclonal B lymphocytosis (MBL). Single cell whole cDNA libraries were obtained by 10X Genomics. Immunoglobulin gene single cell libraries were prepared by enrichment with seminested amplification using 3 ′ constant domain primers followed by 10X Genomics prep. Both single-cell libraries were sequenced in paired-end mode (2 × 150 bp) on an Illumina Hiseq platform. We developed an immunoglobulin alignment tool to enable the analysis of highly mutated IG sequences derived from FL. A consensus sequence was obtained by transcript associated with a unique molecular identifier (UMI) for every cell. Then, the presence of variants in the IG by position in a particular cell was annotated. These observations were filtered using quality parameters (read depth => 25 , frequency of the event => 20%, UMIs supporting every variant => 5)