CLL Progression Is Associated with Increased Clonal Diversity and Replication Stress
Authors: Agathanggelou Angelo , Skowronska Anna , Davies Nick , Kwok Marwan , Clockie Samuel , Griffiths Mike , Taylor Alexander , Stankovic Tatjana 
Published: October 2019

Abstract
There is increasing evidence to suggest that genetic lesions accumulate during CLL progression, often involving mutations in DNA damage response genes ATM and TP53, or SF3B1. However, little is known about the mechanisms which contribute towards genomic instability and clonal diversification during CLL progression. Genomic instability is a major cause of subclonal diversification and can be driven by replication stress (RS). It has been shown that in solid tumours RS drives tumourigenesis through the following steps: presence of unreplicated DNA, accumulation of DNA damage, activation and functional loss of ATM/p53 and genomic instability.
In this study we addressed whether high proliferation rates during CLL progression are associated with increased subclonal diversification and with RS. We compared samples from the indolent and progressive stages of CLL, both in individual patients and in patient cohorts. We determined clonal diversification by two complementary methods: multiplexed-FISH and next generation sequencing (NGS). We measured RS by an assay that recognizes a region of unreplicated DNA.
Using multiplexed-FISH that identifies the location of multiple genetic alterations at the single cell level, we identified in 3/11 relapsed samples the occurrence of novel subclones with ATM (11q) or TP53 (17p) deletions that were not observed in the pre-treatment tumours. These findings were corroborated by targeted NGS of recurrently mutated genes in CLL which revealed novel subclones carrying ATM, TP53, NOTCH1, SF3B1 or BIRC3 mutations at the time of relapse in 9 out of 22 paired samples. Of particular interest was the acquisition of multiple mutations in the TP53 gene observed in four patients. These data demonstrate an intrinsic propensity for clonal diversification during CLL progression.
Having demonstrated an association between disease progression and subclonal diversification in CLL, we next sought to identify a mechanism behind this phenomenon. RS results in under-replicated DNA which is sequestered into nuclear compartments marked by 53BP1 protein. Therefore, the appearance of 53BP1 bodies in G1-cells represents a marker of RS. We observed that 53BP1 bodies were not present in PBMCs from healthy individuals, and that 20 CLL samples from an indolent stage of disease contained a low level of this RS marker. This was in stark contrast to the elevated proportion of CLL cells with 53BP1 bodies observed in 13 progressive tumours (p<0.0001). Finally to confirm that replication stress increases during disease progression we analysed a cohort of individual patients at different stages during the evolution of their disease. Intriguingly, and consistent with our hypothesis, we found that the proportion of 53BP1 body positive cells was stable during the indolent phase of disease and only increased significantly when the tumour made the transition from indolent to the progressive stage (p=0.0009).
Collectively, our data indicate that replication stress correlates with the stage of CLL progression and may provide a mechanism behind the observed subclonal diversification.

Disclosures
No relevant conflicts of interest to declare.