Abstract: Classical Hairy Cell Leukemia (HCLc) is a rare B-cell malignancy characterized by hairy-like cell membrane projections. The cell of origin and pathway of malignant transformation in HCLc remain unknown. Early work using whole exome sequencing identified BRAFV600E as a genetic hallmark in HCLc (Tiacci et al 2011 and Weston-Bell et al 2016). However, no other recurrent coding mutations appeared, marking it an uncommon monogenic cancer. As BRAFV600E appears insufficient to drive tumorigenesis in other cancers (Dankort et al 2009; Rad et al 2013), it suggested that other driver alterations may be implicated in HCLc pathogenesis, perhaps in the non-coding genome, overlooked in exome studies alone. To probe this, we carried out whole genome sequencing (WGS) in HCLc. We established the genomic panorama, and importantly leveraged data to derive tumor phylogenies to determine the relative order of early driver mutational events that associate with the most recent common ancestor (MRCA) of all tumor cells. WGS was performed in 10 typical HCLc cases each paired with germline in purified cell sorted populations of disaggregated splenocytes. Sequencing involved 150 bp paired ends on Illumina HiSeq4000. Data analyses included identification of somatic single-nucleotide variants (SNVs) and small indels using CaVEMan and Pindel, respectively; copy-number aberrations (CNA) with Battenberg; and structural variants (SVs) with BRASS. The fitting of COSMIC mutational signatures was performed using mmsig. Mutational SNVs and CNA drivers were used for the analysis of tumor phylogenesis of individual tumors. We mapped the whole genome landscape in HCLc. Somatic coding variants emerged as previously reported. BRAFV600E was present as a clonal mutation in all HCLc tumors. Missense mutations in KLF2 (p.S275N and p.S275I) were identified in 2 tumors. Two somatic SNVs were detected in PRSS3 (p.D95G and p.R94S) and ANKRD30B (p.K388E and p.N392K) in 2 different cases respectively. In the noncoding genome, BCL6 and ZFP36L1 promoters were recurrently mutated in 5/10 cases. Driver mutations in BCL6 promoters have been reported in other B-cell malignancies to disrupt negative autoregulatory circuits (Pasqualucci et al 2003). Analysis of CNA showed a relatively stable genome (ploidy \textasciitilde 2). A large CNA chr5 gain was observed in a single tumor. Two additional focal CNA were spotlighted at chr17q25 (Gain) and chr20q11 (LOH) in two individual tumors. A recurrent interchromosomal translocation at chr12q14.3 locus was identified in 5 individual tumors, and an identical translocation at 14q21.3 with 15q11.2 observed in 2 tumors. Interestingly, novel kataegis-like mutations were observed at TRB loci across all HCLc cohort samples. We further identified somatic hypermutation (SHM) imprints in non-Ig loci, notably in BCL6. The mutational rate of AID targets at TRB and non-Ig loci were comparable to SHM occurring in Ig IGV genes. Deciphering the mutational signatures in HCLc genomes highlighted SBS9, a mutational signature associated with SHM in B-cell leukemia as one of the most relevant mechanisms, allied to the clock-like signatures of SBS1 and SBS5. Next, we utilized estimates of the cancer cell fraction to define which mutations are clonal and associate with the MRCA, and which mutations occured subclonally to derive individual tumor phylogenetic trees using methodology we have reported previously (Rabbie et al 2020; Ansari-Pour 2021). Here, analyses of clonal trajectories revealed that BRAFV600E occurred prior to subclonal diversification in MRCA in all cases. Significantly however, 1 or 2 additional driver mutations were conjointly detected with BRAFV600E in the MRCA in 6/10 HCLc cases prior to any subclonal evolution. Our data provide a comprehensive map of the whole genome in HCLc for the first time. Clonal evolutionary trajectories reveal heterogeneous genetic origins in HCLc, in which BRAFV600E is the sole driver mutation in one subset of disease, but requires 1 or 2 additional mutational drivers in the other subset to promote malignant transformation. Thus, unlike other cancers, BRAFV600E alone appears to suffice to induce malignant transformation in some HCLc. Additionally, SHM in non-Ig loci including BCL6 may implicate early traffic of the tumor cell of origin through the germinal center and early exit prior to acquisition of CD27, or mutational events at ectopic sites.