In-Depth miRNA Profiling Of Germinal Center Derived B-Cell Lymphomas By Next Generation Sequencing: A Report From The German Icgc-Mmml-Seq Project

The discovery of microRNAs (miRNAs), a group of small non-coding regulatory RNAs, revolutionized the field of posttranscriptional gene regulation. Mature miRNAs are single-stranded RNA molecules of 20- to 23-nucleotide length that control gene expression. They typically reduce the translation and stability of mRNAs important in tumorigenesis mediating processes such as inflammation, cell cycle regulation, stress response, differentiation, apoptosis, and invasion.

Germinal center (GC) derived B-cell lymphomas, including Burkitt lymphoma (BL), diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), are the most frequent malignant lymphomas. Although clear distinctions on histologic and genetic grounds exist, there is also a large number of cases with intermediate features, not unequivocally attributable to one of these entities.

Recently, a signature of 38 miRNAs containing MYC regulated and nuclear factor-kB pathway-associated miRNAs was published, which differentiated BL from DLBCL. However, available data is preliminary as published profiles are not derived from large sample collections and also originate mostly from PCR-based approaches and microarrays. Yet, only sequencing-based approaches allow for an unbiased analysis and the discovery of novel miRNAs and small RNA classes.

The ICGC (International Cancer Genome Consortium)-MMML-Seq (Molecular Mechanisms in Malignant Lymphoma by Sequencing) Consortium aims at fully characterizing a total of 250 GC derived B-cell lymphomas over a period of five years. We here report the miRNA analysis of the first 49 patient samples including BL, DLBCL and FL using Illumina technology. We are recording the classical miRNA fraction (18-35 nucleotides) as well as a larger size fraction (35-90 nucleotides).

Initial differential expression analyses comparing BL against non-BL showed eight miRNAs to be differentially expressed. This comprises hsa-miR-150, which has been ascribed a role in the adaptive immune response and which was shown to be upregulated in cutaneous marginal zone B-cell lymphomas. Further differentially expressed miRNAs include e.g. hsa-miR-211, hsa-miR-548ac and hsa-miR-1244-1/3, which represent novel targets in lymphomagenesis.

Among the many bioinformatically predicted novel miRNAs, we have been able to validate four candidates by Northern Blot experiments and are currently performing functional studies.

Mutational analysis is ongoing, but so far has yielded two miRNAs with mutations in the mature sequences (hsa-miR-4537, hsa-miR-758).

In conclusion, our initial series of 49 cases from the ICGC-MMML-Seq cohort has already given exciting insights into the role of miRNAs in GC derived B-cell lymphomas. (Supported by BMBF through 01KU1002A-J)