Whole Genome Sequencing of B-Cell Neoplasms with t(14;19)(q32;q13) Reveals Different Entities

Introduction: The t(14;19)(q32;q13) is a rare cytogenetic abnormality found in <0.1% of all B-cell neoplasms. The molecular features of this translocation are not well characterized. IGH-BCL3 rearrangement has been found in some tumors identified as "atypical" chronic lymphocytic leukemia (CLL) with aggressive clinical evolution. This translocation has also been observed in other B-cell neoplasms without clear evidence of the target gene. The mechanisms generating this translocation, the genomic profile of alterations of these cases, and whether different molecular features may be associated with specific entities are not known.

Aim: To elucidate the genomic features of B-cell neoplasms carrying the t(14;19) and their relationship to pathological characteristics of the tumors.

Materials and methods: We sequenced the whole-genome (WGS) of 13 cases in which the t(14;19) had been identified by conventional cytogenetics and/or FISH using a BCL3 break-apart probe. In six of these cases we performed RNA-seq. Pathological and clinical revision was conducted in all cases, 8 of them with tissue biopsies.

Results: The breakpoints of the t(14;19) were characterized at base-pair resolution using WGS. All breakpoints in chr14 were found within any of the class switch recombination (CSR) regions suggesting an aberrant CSR as the mechanism causing this alteration. The breakpoints on chr19 were found upstream (13 kb) the 5' untranslated region (UTR) of BCL3 in 8/13 (61.5%) cases. One additional case had the breakpoint further upstream (49 kb) of BCL3 truncating CEACAM16. The four remaining cases had breakpoints downstream of BCL3; two cases within CBLC, one in BCAM, and one after NECTIN2. Of note, the further upstream BCL3 case and the downstream BCL3 cases had mutated IGHV, while all upstream BCL3 cases had unmutated IGHV. Based on RNA-seq data, all upstream BCL3 cases (n=5) showed an upregulation of BCL3, while one downstream case with RNA-seq available showed upregulation of NECTIN2 and low levels of BCL3. The pathology review identified the four downstream BCL3 cases as marginal zone lymphomas whereas the cases with breakpoints upstream BCL3 (n=3 with tissue available) and the case further upstream BCL3 were classified as "atypical" CLL.

We next characterized the genomic landscape of these tumors based on the breakpoint on chr19 (upstream and downstream BCL3). The analysis of the WGS showed a lower number of mutations, copy number alterations (CNA), and structural variants (SV) in the upstream BCL3 group compared to the downstream BCL3 cases (mean of 2429.5 vs 6271.7 somatic mutations, 3.1 vs 11.7 CNA, and 4.4 vs 18 SV, respectively). In terms of specific driver mutations, the downstream BCL3 group carried mutations in genes previously described in MZL, such as KMT2D, NOTCH2, or KLF2 found in two cases. All but one case with the breakpoint upstream BCL3 carried trisomy 12 (tri12), which was absent in all cases with a downstream breakpoint.

Finally, we performed a differential expression analysis between 5 atypical CLL cases with BCL3 rearrangements vs 4 CLL without t(14;19) [all unmutated IGHV]. This analysis showed 578 genes upregulated and 720 genes downregulated in the BCL3-rearranged cases (q <0.05), including remarkable differences in the expression of previously described CLL hallmark genes, such as upregulation of EBF1 and downregulation of LEF1, FMOD, ADTRP, CLNK, IGSF3, TCF4. An analysis of the RNA-seq data of 294 CLL cases lacking the t(14;19) (Puente et al., Nature 2015) indicated that this transcriptional program was not related to IGHV mutational status nor to the presence of tri12. Nonetheless, we identified a small set of tri12 mutated IGHV CLL lacking the t(14;19) with a similar modulation of the expression of the above hallmark genes.

Conclusions: We have characterized the breakpoints of the t(14;19) at base-pair resolution and evidenced marked molecular and pathological differences of the tumors according to the location of the breakpoint. Tumors carrying the breakpoint downstream BCL3 exhibit a higher genomic complexity, driver alterations, and pathological features corresponding to MZL. Contrarily, tumors with the breakpoint upstream of BCL3 upregulate BCL3 and display lower genomic complexity as well as CLL-like features. Nonetheless, these cases have a different gene expression profile compared to conventional CLL characterized by LEF1 downregulation and EBF1 overexpression.