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K. Van Roosbroeck et al.
xenografts, increasing the risk of DLBCL (Richter transfor- mation).53 The viral miRNA that correlate in our networks could be a marker of viral reactivation.
Several questions remain to be answered. First, what are the mechanisms that lead to the high level of correla- tion between miRNA during Richter transformation? Further investigation of the exact role of each of the sig- nature miRNA and their mechanisms of action in the Richter transformation process is warranted. For this, the development of in vitro and in vivo models of CLL to RS transformation is imperative. Second, it was surprising that, out of 15 deregulated miRNA identified by the Firefly assay in the extended cohort, only three could be confirmed by qRT-PCR in the same dataset. This is most likely due to a combination of factors, such as the poor quality of RNA from FFPE samples, sensitivity of the assays, and the difficulty in identifying good normalizers. In the Firefly assay, a combination of miR-15a, miR-191 and miR-26a were found to be the best for normalization, while for the qRT-PCR experiments, the geometric mean of U6 and RNU48 was found to have the least variability. In addition, the Firefly platform is not based on PCR amplification, while qRT-PCR is, which may also partial- ly explain the differences in results.
Some limitations need to be acknowledged with regard to this study. The number of patients included was small, so the statistical power of the analysis is limited. RS is a relatively rare disease and it is very difficult to obtain a large sample set. Furthermore, we analyzed only 40 human and viral miRNA using the Firefly assay and 25 miRNA using qRT-PCR, thus offering a limited view of the miRNA expression deregulation and miRNA network in RS. It is likely that other miRNA, which we overlooked by analyzing a restricted panel of miRNA, could be involved in the pathogenesis of RS. We tried to overcome this limitation by choosing the most suitable miRNA can- didates and using previous RNA-sequencing data,20 and
literature research of miRNA implicated in CLL.
Finally, the identification of SDE miR-21 (overex- pressed) and miR-150 (downregulated) and of HUS miR- 17, -29c and -191 demonstrates that RS is a genetically heterogeneous disease and offers new targets for the ther- apeutic development of anti-miRNA54 in this disease which currently does not have a confirmed treatment to
increase the poor life expectancy.
Acknowledgments
We thank Mike Tackett from FirePlex Service at Abcam (Cambridge, MA, USA) for the technical support.
Funding
Dr. Calin is the Felix L. Haas Endowed Professor in Basic Science. Work in his laboratory is supported by National Institutes of Health (NIH/NCATS) grant UH3TR00943-01 through the NIH Common Fund, Office of Strategic Coordination (OSC), National Cancer Institute (NCI) grants 1R01 CA182905-01 and 1R01CA222007-01A1, an NIGMS 1R01GM122775-01 grant, a U54 grant – UPR/MDACC Partnership for Excellence in Cancer Research 2016 Pilot Project, a Team DOD (CA160445P1) grant, a Ladies Leukemia League grant, a CLL Moonshot Flagship project, a SINF 2017 grant, and the Estate of C. G. Johnson, Jr. Work by Dr. Van Roosbroeck was supported in part by the Lauri Strauss Leukemia Foundation. Dr. Dragomir was supported by POC grant n.35/01.09.2016, ID 37_796. Dr. Bertoni was supported by grants from the Helmut Horten Foundation, the San Salvatore Foundation, and by Oncosuisse (OCS - 02296-08- 2008). Dr. Stilgenbauer and Dr. Bloehdorn were supported by the DFG through SFB1074 subprojects B1 and B2. Dr. You was supported in part by NIH/NCI R01 CA164346 and IRG of the University of Texas MD Anderson Cancer Center. Dr. Gaidano was supported by AIRC 5 x 1000 project 21198, AIRC, Milan, Italy. Dr. Zupo was supported by Italian Ministry of Health 5 × 1000 funds 2014 and 2015.
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