Multi-Omics Profiling of Longitudinal Samples Reveals Early Genomic Changes in Follicular Lymphoma

Introduction: Follicular lymphoma (FL) is an indolent malignancy, characterized by multiple relapses during the disease course. Annually around 2-3% of patients experience transformation to aggressive disease (tFL), most commonly to diffuse large B-cell lymphoma (DLBCL). Transformation and progression of disease within 2 years (POD24) are associated with poor prognosis, yet the molecular events underlying these processes are not well understood. The existence of common progenitor cells (CPCs) has been inferred from genetic analyses of longitudinal biopsies. Improved characterization of genetic alterations associated with CPCs in cases with transformation and POD24 may improve our understanding of disease progression, and reveal molecular markers for high-risk disease. Methods: To identify early genetic events during FL pathogenesis and molecular events underlying progression and transformation, we performed whole-exome sequencing of 97 serial tumor biopsies and matched normal samples purified from peripheral blood from 44 FL patients; 22 who experienced transformation (tFL) and 22 with progression of FL (nFL). Nineteen patients(both groups) experienced POD24. SNP6.0 data was available for 93 sequenced tumors and was used to infer allele-specific copy number alterations. Bulk RNA sequencing data was available for 64 sequenced tumors. Several computational tools were applied to identify potential cancer driver genes (IntOGen, MutSig2CV, 2020plus), mutational signatures, and to study clonal evolution (PyClone, ClonEvol). CIBERSORTx was used to infer B-cell specific transcriptomes from bulk RNAseq data. Results: Deep whole exome sequencing confirmed recurrent mutations in epigenetic regulators (CREBBP, KMT2D, EZH2, and EP300) and known copy number alterations. The mutational landscape was similar in nFL and tFL patients, except for higher recurrence of EZH2 mutations in tFL. We used CIBERSORTx to impute B-cell transcriptomes from bulk RNA-sequencing data to identify differentially expressed genes associated with common mutations. We discovered that numerous histone genes were downregulated in biopsies with EZH2 mutations, and in pretreatment samples from tFL patients, potentially contributing to epigenetic deregulation. Based on the mutational pattern in each biopsy, we calculated the genomic distances between longitudinal samples. This revealed great variability in nFL and tFL patients, compatible with complex evolutionary patterns independent of transformation. Tumor clonal evolution analysis identified CREBBP and KMT2D mutations as early genetic events in FL pathogenesis, likely occurring prior to the early CNAs that remained stable throughout disease progression such as gain in 12p and 18p and loss in 6p. Conclusions: Identification of such early and potentially essential genetic events may therefore provide opportunities for early disease detection and monitoring of progression.