Abstract - Text

Abstract-Text

Introduction: Knowledge of the mechanisms that lead to translocations in hematological malignancies provides essential insights into the development of these diseases. In IGH-associated translocations, a common pathomechanism for the generation of Double-Strand-Breaks (DSBs) seems to be mediated by the RAG1/RAG2 complex at the IGH-locus. At the IGH-translocation partners, a second mechanism based on the activity of the AID, causing a T:G mismatch that is recognized by RAG1/RAG2, has been proposed. Further non-IGH-associated translocations as the t(12;21)/ETV6-RUNX1 are suggested to be caused by a RAG1/RAG2-mediated mechanism. For the rare B-lymphoblastic leukemia/lymphoma with t(5;14)(q31.1;q32.3)/IL3-IGH-fusion that is recognized as full entity in the WHO-Classification, the mechanisms are unknown so far. We report a molecular genetic analysis of two new cases of B-lymphoblastic leukemia (B-ALL) with t(5;14)(q31;q32)/IL3-IGH-fusion.

Methods: DNA from two cases with the diagnosis of a B-ALL was available for molecular analyses. The presence of the t(5;14) was confirmed by FISH with specific probes for the IL3 gene and IGH locus. Whole genome sequencing (WGS) was performed with NovaSeq6000. Confirmation of the WGS results by amplification and Sanger-sequencing of the direct IL3-JH IGH segments was possible in one case.

Results: WGS and direct amplification of the IL3-IGH fusion in case 1 and WGS in case 2 allowed mapping of the breaks on chromosome 5 to the 5`-non-coding region of IL3. The breakpoint region of both cases spanned about 1200 nucleotides and showed an increased GC-content and a high number of CpG dinucleotides, which were significantly clustered to the breakpoint region. This pointed to an involvement of the AID and RAG1/RAG2 activity in the generation of the DSBs. The breaks on chromosome 14 in case 1 showed all features of an illegitimate V(D)J recombination, like the localization in the JH4 and DH1-26 segments and the presence of recombination signal sequences (RSS). In case 2, the localization of the breakpoints in the constant region of IGH elicited the mediation of an illegitimate V(D)J recombination as the pathomechanism involved. However, the flanking sequences at the breakpoints showed features of cryptic RSS (cRSS) that might likely function as a target for RAG1/RAG2.

Conclusions: We present here the first characterization of the pathomechanisms leading to the t(5;14)/IL3-IGH-fusion in B-ALL. Our observations indicate that the DSBs at the IL3 locus on chromosome 5 are probably mediated by the activity of AID, leading to a T:G mismatch and followed by RAG1/RAG2 activity. The DSBs on chromosome 14 are suspected to be caused by RAG1/RAG2 activity either, as expected, by an illegitimate V(D)J recombination or at cRSS. Therefore, the activity of RAG1/RAG2 is likely the major driver for the development of the translocation in t(5;14)/IL3-IGH-fusion cases analyzed here.