The Genetic Landscape Of Mantle Cell Lymphoma and The Epigenetic Origins Of Lineage Specific Mutations

Mantle cell lymphoma is an uncommon form of non Hodgkin lymphoma that is characterized by poor responsiveness to chemotherapy and a high rate of mortality. While translocation of CCND1 is a defining feature of the disease, the role of collaborating somatic mutations that contribute to mantle cell lymphoma remains to be better defined.

In this study, we sought to better understand the patterns of mutations that occur in tumors derived from closely related stages of B cell differentiation. We began by performing exome sequencing in 55 cases of mantle cell lymphoma to broadly identify the mutational landscape of the disease. We then defined the chromatin structure of the normal counterpart B cells (naïve and germinal center B cells respectively) for mantle cell lymphoma and Burkitt lymphoma by profiling their epigenetic markers using chromatin immunoprecipitation followed by sequencing (ChIP-seq). We found that the somatic mutational profiles of mantle cell lymphoma and Burkitt lymphomas overlapped strongly with areas of open chromatin in their normal counterpart B cells, identifying B cell developmental lineage as a factor in the acquisition of somatic mutations.

We identified somatic mutations affecting 311 genes in at least one tumor/germline pair among MCL cases. These variants were not present in publicly available data from normal controls including dbSNP135, the 1000 Genomes Project and available exome sequencing data from healthy individuals without lymphoma. We further required each of the identified genes to have a minimum of two rare and predicted functional variants for genes already in COSMIC, and three such variants in novel genes. The most frequently mutated genes in mantle cell lymphoma were ATM (41.9%), CCND1 (14%) and TP53 (18.6%). Other frequently mutated genes included known oncogenes and tumor suppressor genes such as RB1, SMARCA4, and APC. Our data also implicated a number of genes not previously associated with mantle cell lymphoma, including POT1, FAT4 and ROBO2. Silencing mutations (frameshift and nonsense mutations) comprised a sizeable fraction of the genetic events in ATM, MLL3, RB1 and ROBO2, suggesting that those alterations result in a loss of function in mantle cell lymphoma.

To better understand the genetic differences between common non Hodgkin lymphomas, we examined exome sequencing data that we and others have generated from Burkitt lymphoma and diffuse large B cell lymphoma (DLBCL). We identified all genes that were mutated in roughly 10% of at least one lymphoma type and differentially mutated relative to at least one of the other lymphoma types (P<0.05, Fisher’s exact test). We found a number of genes that were predominantly mutated in each type. Mutations in ATM, CCND1, POT1 and RB1 occurred mostly in mantle cell lymphoma. Mutations in ID3 and MYC occurred predominantly in Burkitt lymphoma. Mutations in PIM1, FRMPD1 and CREBBP occurred mostly in DLBCLs. A number of genes had overlapping patterns of mutations between two or more of the diseases, including MLL3, TP53, ARID1A and SETD2.

We sought to define the relationship between the epigenetically determined chromatin state of normal B cells and the lymphomas that are thought to arise from them. We began by FACS-sorting normal naïve B cells, germinal center B cells and memory B cells from otherwise normal individuals undergoing tonsillectomy. We profiled the chromatin structure and epigenetic state of the normal B cells through chIP-seq on 6 different markers: H3K4me1, H3K4me3, H3Ac, H3K27me3, H4K36me3, and CTCF. Using these epigenetic markers, differences in open chromatin between naïve B cells and germinal center B cells were computed for genes that were differentially mutated in mantle cell lymphoma and Burkitt lymphoma. We found that difference in gene mutation frequency between mantle cell lymphoma and Burkitt lymphoma is highly associated with differences in open chromatin in their corresponding cells of origin (P=0.02, Fisher's exact test).

Our work demonstrates that the sequencing of relatively uncommon tumors such as mantle cell lymphoma and Burkitt lymphoma afford not only new insights into the genetics of these tumors, but also allow us to better examine the broader effects of lineage and epigenetic alterations in cancer. This work thus provides an important starting point for understanding the genetic diversity of mantle cell lymphomas and the interplay between genetic and epigenetic alterations in cancer.