Aberrant Isotype Class Switch Recombination Reactivation Leads to a Genome Wide Genomic Instability in Follicular Lymphoma

Introduction

Follicular lymphoma (FL) patients usually follow an indolent clinical course but suffer recurrent relapses, after few months or after long periods of remission. Many ultimately develop resistances to therapies and a subset, with an annual incidence of 2-3%, experience transformation to DLBCL. Understanding the molecular mechanisms responsible for the clonal evolution of these tumors thus appears essential to anticipate these events. As such, two B-cells specific genetic instability processes are suspected to drive lymphomagenesis: the aberrant somatic hypermutation (ASHM) and the isotype class switch recombination (CSR). ASHM occurs when AID loses its specificity and targets multiple oncogenes while CSR, also initiated by AID, initiates double strand breaks responsible for the acquisition of chromosomal translocations. However, while it is largely recognized that ASHM usually remains active in FL cells, little is known about a possible ongoing CSR activity in these tumors and its potential impact on progression.

Methods

We performed a detailed analysis of the structure of the IGH loci in 16 patients: 8 with FL at diagnosis, 4 with paired FL + DLBCL (tFL) biopsies and in 5 with de novo t(14;18) positive DLBCL using high throughput IGH RNA-sequencing. Sequencing libraries were prepared using a Template Switching 5′ RACE Protocol kit (New England Biolabs) starting from total RNA extracted from fresh-frozen biopsies. Single‐end 300 bp read sequencing was performed on a MiSeq or NextSeq 550Dx system.

Results

In agreement with previous studies, almost all FL showed genomic hallmarks of CSR activation at diagnosis (11/12). If 6 still expressed an IgM, the IGHCµ gene was deleted from the translocated allele in 9/9 t(14;18) positive cases while 2 negative biopsies had a t(3;14) and a t(8;14) involving IGH switch regions respectively. In one FL/DLBCL pair and one de novo DLBCL, we also discovered an as yet undocumented cryptic inversion on the der(18)t(14;18). This recurrent rearrangement, which is also present in the RL cell line, joins a DNA repeated region located upstream from BCL2 with an IGH switch region, resulting in a flip of the whole intervening BCL2-IGH fusion. The localization of these breakpoints and the detection of multiple mutations around the junctions, which remind the mutations introduced by AID at physiological CSR junctions, further argue for an aberrant CSR activation during lymphomagenesis.

Unexpectedly, we also observed that the two DLBCL biopsies which harbored this inversion, a second tFL and two independent FL expressed dozens of fusion transcripts where multiple ectopic sequences were fused to the first exons of an IGH constant gene (649, 398, 284, 112 and 87 unique fusions, respectively). Most of the fusions we tested were validated by RT-PCR and Sanger sequencing (12/15), ruling out the possibility that they could correspond to RNA-seq artefacts. Additionally, we observed that the majority of the 1261 unique ectopic sequences identified aligned upstream from canonical GT splicing sites on genomic DNA (91%), at the exact end of known exons (68.8%), with a strong bias toward the first exons of their corresponding genes (51.4%). Among these genes, we identified many AID targets, some of which are implicated in recurrent IGH translocation in B-cell lymphoma (CIITA, FOXP1, BCL11A, CCND3, RHOH, CXCR4, SOCS1, IL21R, NOTCH2...), suggesting that these fusions result from a genome wide instability linked to an ongoing aberrant CSR activation. Notably, four of these five patients experienced rapid and adverse clinical evolutions, dying less than 2 years after these clinical events, suggesting that this phenomenon may be correlated to tumor aggressiveness.

Conclusion

We present new molecular evidence thet CSR is activated during FL lymphomagenesis and propose that a reactivation of this process at the tumoral stage can lead to a genome-wide instability which results in the emergence of dozens of subclones harboring different IGH fusion genes. This aberrant CSR reactivation may thus, like ASHM, be a major contributor to FL clinical progression by providing these cells new important selective advantages.