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A. Vogelsberg et al.
The mutational spectrum identified in our study is con- sistent with published data obtained in FL and t(14;18)+ DLBCL. Both lymphomas were shown to be particularly enriched for mutations in the epigenetic regulators CREBBP, EZH2, KMT2D, and EP300, as well as for alter- ations of TNFRSF14, a gene encoding for a receptor of the tumor necrosis factor family.23,30,31,37,38 CREBBP mutations have consistently been described as drivers of lymphoma- genesis and as early genetic events in FL.22,44-47 In agree- ment with this and in line with a recent report, we found CREBBP to be the most commonly affected gene in ISFN samples, with mutations being shared with the clonally related manifest lymphomas in three cases.15 Moreover, ISFN and DLBCL of case 4, which lacked CREBBP alter- ations, exhibited a shared mutation of the closely related acetyltransferase EP300. CREBBP and EP300 mutations have been suggested to play similar roles in the pathogen- esis of FL and DLBCL and are therefore usually mutually exclusive.23,47,48 Alterations of EZH2, KMT2D, and TNFRSF14 have been described as both early driver and as accelerator mutations.37,38,44-46,49 We confirm that these mutations can occur during the presumably earliest stages of the disease, evidently years before the diagnosis of malignant lymphoma.14,15 Alterations of KMT2D and EZH2 were, however, detected only in the manifest lym- phomas of three of and five of seven cases respectively, suggesting they were often acquired later, possibly driv- ing the clone towards the malignant transformation. Notably, BCL2 mutations frequently occur at the earliest stages as well and are likely primarily an indicator of AID activity, rather than heralding aggressive behavior, as sug- gested by other authors.50 PIM1 and IGLL5, two addition- al genes known to be affected by aberrant SHM, were also mutated in one and two ISFN samples, respectively.31 Re-arrangements of MYC and alterations of TP53 are common drivers of FL transformation.38 Accordingly, five of our cases carried these genetic alterations and as expected, they were only detected in the aggressive BCL and not in the clonally related ISFN lesions. However, due to technical limitations, the presence of these mutations in minor ISFN subclones cannot be excluded completely.
Although we were able to investigate the evolution of ISFN at multiple levels, this study has some limitations, in particular, the small sample size due to the rarity of iden- tifiable ISFN lesions in patients with aggressive BCL,
which warrants validation in further studies. The neces- sary restriction to FFPE tissue also narrowed the scope of feasible analyses and raised the detection threshold in our targeted NGS analysis because of low level sequencing artifacts. Nevertheless, systematic validation allowed us to delineate the clonal and genetic evolution of aggressive BCL starting from an early progenitor lesion.
In summary, our data extend previous studies and provide first evidence that t(14;18)+ DLBCL and HGBL can arise from clonally related ISFN without FL as an intermediate step. Moreover, during this progression, similar to the clonal evolution and transformation of FL, branched evolution with both private and shared alterations is common. Our results further confirm that ISFN is subject to persistent AID activity and frequently acquires secondary genetic alter- ations, in addition to the defining t(14;18) translocation.
Disclosures
No conflicts of interest to disclose.
Contributions
FF, IB and AV wrote the manuscript; FF conceived and designed the study, selected the cases, and supervised the exper- imental work and data analysis; LQ-M helped designing the study, reviewed the cases and helped writing the manuscript; AV performed the experimental work and analyzed the data; JuS, JaS, and IB supervised the experimental work and data analy- sis; BM performed FISH analysis; BF and IAM-M helped with case selection; PB and SN provided bioinformatics support and constructed the phylogenetic trees; MR-P, MAP, KH and GO contributed with cases and provided clinical information. All con- tributing authors revised the manuscript.
Acknowledgements
The authors would like to thank Julia Bein, Sylvia Hartmann and Martin-Leo Hansmann for their cooperation and are grate- ful to Franziska Mihalik, Rebecca Braun, Inga Müller, Dennis Thiele, Isabell Haußmann, Gerd Janke, and Sema Colak for their excellent technical assistance. We acknowledge support by Open Access Publishing Fund of University of Tübingen.
Funding
This work was supported by grants from the Deutsche Forschungsgemeinschaft (DFG) to LQ-M (QU144/1-1) and FF (FE597/4-1) and the IZKF Promotionskolleg (E0500520).
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