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J. Delgado et al.
rently selected in small cohorts of patients after CIT.63,64 Clonal evolution plays an important role not only in resistance to CIT, but also to novel agents. Indeed, differ- ent point mutations have been identified in the BTK and PLCG2 genes in patients previously treated with the BTK inhibitor ibrutinib,65 and in the BCL2 gene in patients relapsing after treatment with the BCL2 antagonist vene- toclax.66 Resistance to these agents has been associated with these mutations in around 70% of cases, although they are usually subclonal and their specific role causing resistance needs to be proven.67 Other resistance mecha- nisms involve upregulation of BCL-XL and MEK/ERK, and cell reprogramming and transdifferentiation to cell subtypes that do not require BCR signaling.65,67–70
Epigenomic landscape
The genome of CLL features widespread hypomethyla- tion, and a large fraction of the differences between U- CLL and M-CLL can be attributed to their different cell of origin in germinal center-independent or -experienced B cells, respectively.5 Major hypomethylation changes occur at transcription factor binding sites such as TCF3, PU.1/SPIB, NFAT and EGR, and enhancers that modulate genes relevant for CLL pathogenesis involved in B-cell function, BCR signaling, and NF-κB activation among others. This methylation profile is already acquired at the MBL stage3 and remains relatively stable over time. However, some CLL have intratumor variability in cer- tain regions, which may alter the expression of several genes and facilitate tumor evolution.71 Of note, this vari- ability is greater in U-CLL than in M-CLL and is associat- ed with increasing number of subclones.7,71
Several groups have evaluated the full reference
epigenome of CLL, providing a genome-wide map of his-
tone marks and three-dimensional chromatin architec-
ture.6,72–74 Surprisingly, there was a significant variability in
active regulatory regions among individual patients. This
variability, and also the total number of active sites, was
larger in U-CLL than in M-CLL. Around 80% of these
active sites were also present in normal naïve, germinal
center, memory, or plasma cells.6 Some of these active
regions are seen in all CLL cases but in none of the normal
B-cell subtypes, and may therefore be crucial for CLL
pathogenesis. Most of these de novo active regions target
regulatory loci and super-enhancers enriched in transcrip-
tion binding motifs of NFAT, FOX, TCL/LEF, and PAX5,
which have been shown to play a role in CLL pathogen-
esis and could potentially be targeted pharmacological- ly.26,72
Somatic mutations in chromatin remodeler genes could modify the epigenomic landscape of CLL, but they are uncommon in this malignancy compared to other lym- phoid neoplasms. CHD2 is mutated in 5% of CLL and 7% of MBL.75 The histone methyltransferase SETD2 and ARID1A are also mutated in a small proportion of patients. Of note, MYD88 mutations and trisomy 12 are associated with specific remodeling of chromatin activa- tion and accessibility regions. More specifically, the epigenomic profile induced by MYD88 mutations targets regulatory regions related to NF-κB signaling,6 whereas the epigenetic configuration of trisomy 12 CLL is charac- terized by a subtype-specific hypomethylation signature associated with increased H3K27 acetylation, which leads to the overexpression of 25 target genes including RUNX3.76
Pathogenic mechanisms in the evolution of the disease
CLL is always preceded by an often unnoticed prema- lignant state known as high-count MBL.77 Low-count MBL may persist for a long time but the risk of progres- sion is negligible.78 Yearly, 1% of cases of high-count MBL evolve into CLL requiring therapy,79 and 2-10% of patients with symptomatic CLL eventually develop Richter transformation.80 At the other end of the spec- trum, around 30% of patients with CLL never require any CLL-specific therapy and die of other causes, and 1-2% of them even experience spontaneous regression of their disease.81 It is therefore evident that the rate and pattern of growth (or even decline) of the disease can vary greatly among patients.
Patients with high-count MBL carry mutations in driver genes which may be detected at a median of 41 months prior to progression to CLL.3,82 The mutation rates for the most common drivers (e.g., SF3B1, DDX3X, BIRC3, ATM) are comparable between MBL and CLL, with only a few genes being more commonly mutated in CLL (NOTCH1, TP53, XPO1).82,83 Patients with MBL with mutated drivers have a shorter time to first treatment compared to cases without mutations. Once CLL is established, the growth dynamics of tumor cells is heterogeneous. Some patients exhibit a logistic-like behavior in which the clone stabi- lizes over time, whereas some others show an exponen- tial-like growth pattern.84 This exponential growth, clini- cally defined as “short lymphocyte doubling time” is still considered an adverse prognostic parameter in CLL.85,86 As expected, the median number of driver mutations (both clonal and subclonal) is higher in patients with exponen- tial growth, and this patient population also displays unmutated IGHV genes more frequently. In addition, the rate of clonal evolution after therapy (i.e., with a signifi- cant shift in at least one subclone) is also higher in patients with exponential growth (Figure 4).
Transformation of CLL into an aggressive lymphoma occurs in 2-10% of patients and in most of them (>90%) corresponds to a DLBCL, but Hodgkin lymphoma may also occur.87 The DLBCL usually emerges as a linear evo- lution of the same CLL clone with only rare cases deriv- ing from a branching divergent subclone.88 CLL carrying stereotyped subset 8 (IGHV4-39), NOTCH1 or TP53 mutations and complex karyotypes are at higher risk of transformation after CIT. Transformed DLBCL frequently add CDKN2A deletions and MYC translocations or amplifications on top of the genomic alterations already present in the original CLL, but lack the common muta- tions observed in primary DLBCL indicating that they may correspond to a different biological category.80 Richter transformation also occurs in patients treated with BTK inhibitors. These tumors do not usually acquire BTK or PLCG2 mutations but, if these were present in the original CLL, subclones may emerge with additional independent mutations.89,90
In the rare instances in which the disease regresses spon- taneously, the patients uniformly have mutated IGHV genes, no stereotypes, low proliferative activity and poor migration to proliferation centers as exemplified by a high CXCR4 expression. Interestingly, patients with sponta- neous regression show reduced T-cell exhaustion and increased T-cell proliferation, confirming the important role of the immune system in CLL progression.91 Moreover, driver mutations are also present in these patients but they always remain stable without subclonal shifts.91
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