More Somatic Mutations Including Those in the Aid-Motif with High-Risk, Histologically Non-Transformed Follicular Lymphomas

Follicular lymphoma (FL) is the most prevalent among indolent subtypes of non-Hodgkin lymphomas. While the histologic transformation associates with disease aggressiveness in variant FL, a minor population of non-transformed FL patients also shows a shorter period for disease control despite the use of rituximab (Rit) immunochemotherapy. In this study, we investigated the clinical and genetic features of high-risk non-transformed FL. With the median follow-up period of 61 months for 103 consecutively treated patients with histologically non-transformed FL including 85 patients treated with the first-line Rit-containing therapies and 18 with watch and wait, the 5-year overall survival (OS) and progression-free survival (PFS) of the entire cohort were 93.3% and 54.4%, respectively. Chemotherapy was initiated according to the GELF criteria. Although neither FLIPI nor FLIPI2 sufficiently predicted the risks for either OS or PFS, the presence of two extranodal involvements, including peripheral blood (PB) (N=7, p=0.006) and/or bone (N=6, p=0.007), at diagnosis were found to be significantly associated with poor PFS, but not OS, in our cohort. Indeed, 13 high-risk patients with PB and/or bone involvements at diagnosis showed a significantly shorter median PFS of 27.2 months compared with that of the 90 standard-risk patients, who have not yet reached the median (p<0.001). Other factor, including age, performance status, disease stage, histologic grade, laboratory tests etc., showed no prognostic impact in our cohort. Next, to determine the genetic characteristics of the high-risk subset of FLs, we randomly selected 14 FLs, including 5 high- and 9 standard-risk FLs according to our disease-risk criteria, and performed whole exome sequencing analysis of 14 tumor samples at diagnosis with matched germline samples. The mean numbers of total mutated genes in the high-risk and in the standard-risk groups were 190 and 138 (p=0.04), respectively, and the mean numbers of genes with non-synonymous mutations were 52 and 39 (p=0.06), respectively. To investigate the consequence of multiple mutational processes, we analyzed the profiles of substitution subtypes including flanking bases in the mutated genes and identified a CG>TG substitution to be the most common mutational signature, while there was no difference with regard to the composition of the signature between the high- and standard-risk FLs. "Kataegis", a pattern of clustered hypermutations, was observed mostly in the target motif of activation-induced cytidine deaminase (AID), recurrently on chromosome 2p12 (involving the IGK locus), 14q32 (IGH), 18q21 (BCL2), 21p11, and 22q11 (IGLL). The mean number of C>T mutations in the AID-motif was significantly increased in the high-risk FLs over that in the standard-risk FLs (17 vs. 11, p=0.02), whereas there was no difference in the mean number of mutations in the target motif of APOBEC, which is considered to be the major driving agent responsible for kataegis in various solid cancers, between the two risk groups (8 vs. 7, p=0.49). For functional assessment of mutated genes, CREBBP and TNFRSF14 mutations were identified as the driver mutations of FL regardless of the disease risk using background mutation rate analysis. Hypergeometric test on all of the non-synonymous mutated genes from 14 FLs resulted in 71 significantly enriched gene ontologies (GOs), including the terms "cell development and differentiation", "regulation of cell death", "immunity mediated by lymphocytes, including B cells", "hematopoiesis", and "differentiation of leukocytes, including lymphocytes". In addition, pathway analysis demonstrated that mutated genes were enriched in the Notch and B cell receptor signaling pathways. However, we were not able to identify GOs or signaling pathways specific to the high-risk FL patients that could explain the different outcomes of the disease. Non-synonymous mutations in the high-risk FLs were found in genes, such as MEF2B and FUBP1, and were suggested to be associated with cancer aggressiveness, but not with the AID pathway. Overall, our results suggested that genomic instability, which allows for the emergence of distinct mutations not only by kataegis due to AID activity but also by additional mechanisms that increase mutations, underlies development of the high-risk phenotype in FLs.