Ultra-Deep Sequencing of De Novo IGHV Mutations in Activated CLL Cells: Evidence for Activation-Induced Deaminase Function.

Abstract 2545

B-cell chronic lymphocytic leukemia (CLL) clones often acquire new mutations and cytogenetic aberrations over time. In other human cancers, including lymphomas and solid tumors, activation-induced deaminase (AID), which normally causes immunoglobulin (Ig) somatic hypermutation (SHM) and isotype class switch recombination (CSR) in germinal center B cells, is expressed and functions abnormally to cause mutations promoting aggressiveness. In CLL, AID mRNA expression in the leukemic cells correlates with increased adverse cytogenetic aberrations and worse clinical outcomes. Furthermore, CLL cells activated by culture with CD32-transfected murine L cells, anti-CD40 and interleukin-4, produce AID protein with associated functions: DNA breaks, Ig CSR, and Ig heavy chain (IGH) variable (IGHV) gene SHM. To evaluate AID-mediated SHM in CLL more accurately, ultra-deep sequencing was performed on CLL clone's IGH cDNA prior to and after in vitro activation in one unmutated CLL (U-CLL) case (CLL1278, 0.0% mutated IGHV3–30) and one mutated CLL (M-CLL) case (CLL1299, 4.9% mutated IGHV3–23). Additionally, to examine activation of CLL IGH mutation in vivo, ultra-deep sequencing was performed on cells from one U-CLL case (CLL1083, 0.0% mutated IGHV4-b) prior to and after adoptive transfer into the NOD/SCID/γc^null (NSG) mouse, a xenograft model of CLL, where upregulation of AID protein occurs in CD5⁺CD19⁺ human CLL cells.

Libraries were created for ultra-deep sequencing using the 454 FLX system (Roche) by PCR amplification with IGHV family-specific framework1 (Lprimer) and IGH constant region Cμ (IGHM) (Rprimer) primers on cDNA obtained from CLL cells prior to (day 0) or after in vitro culture for 7 (CLL1278) or 14 days (CLL1278; CLL1299) or from NSG spleen CLL cells collected 35 days after transfer (CLL1083). The resulting 461,153 sequence reads were processed to generate separate datasets with fixed sequence block lengths for each primer. The Lprimer sequence blocks included only 5'IGHV sequence, while the Rprimer sequence blocks encompassed 3'IGHV, IGH diversity, and IGH joining genes (IGHVDJ) as well as 5'IGHM sequence. Individual subclone sequences that occurred at least twice were extracted from each of the datasets and the unique de novo subclones not shared between day 0 and activation were analyzed for mutations.

All three CLL cases showed increases in 5'IGHV and 3'IGHVDJ subclones with activation. After in vitro activation, for CLL1278, 123,518 total sequence reads produced 68 unique subclones as compared to 33 at day 0; and for CLL1299, 163,358 total sequence reads produced 78 unique subclones as compared to 61 at day 0. Likewise, after in vivo activation in the NSG mouse, for CLL1083, 174,472 total sequence reads produced 91 unique subclones as compared to 56 at day 0. In contrast, all three CLL cases showed decreases in 5'IGHM subclones after activation. After in vitro activation, CLL1278 and CLL1299 decreased from 22 and 20 unique day 0 subclones to 13 and 16 unique subclones. Similarly, CLL1083 showed a decrease from 20 unique day 0 subclones to 11 unique subclones after transfer into the NSG mouse. After normalization for read number and block sequence length, all three CLL cases showed an increase in 5'IGHV mutation with CLL cell activation (fold change relative to 5'IGHM = 3.4, 2.2, and 4.6 for CLL1278, CLL1299, and CLL1083, respectively). This increase in IGHV mutation relative to IGHM following activation is consistent with AID activity. Furthermore, examination of mutation sites in these subclones revealed an increase in mutations in AID hotspot motifs (GYW or WRC) in the 5'IGHV of all three CLL cases with activation (fold change = 2.0, 1.9, and 2.5 for CLL1278, CLL1299, and CLL1083, respectively), which was not observed further downstream in 3'IGHVDJ and 5'IGHM.

Thus, by analyzing a very large number of IGH sequences in CLL cells after activation in vitro or in vivo, a pattern of de novo mutations consistent with AID activity is found. Furthermore, since both U-CLL and M-CLL clones exhibited AID activity, these findings indicate that AID-mediated SHM is not limited by CLL IGHV mutation status. Finally, these data support a model of AID-promoted mistargeted mutations, which may lead to adverse cytogenetic aberrations and unfavorable outcomes in CLL.