Circulating tumour DNA reflects treatment response and clonal evolution in chronic lymphocytic leukaemia

Abstract

Several novel therapeutics are poised to change the natural history of chronic lymphocytic leukaemia (CLL) and the increasing use of these therapies has highlighted limitations of traditional disease monitoring methods. Here we demonstrate that circulating tumour DNA (ctDNA) is readily detectable in patients with CLL. Importantly, ctDNA does not simply mirror the genomic information contained within circulating malignant lymphocytes but instead parallels changes across different disease compartments following treatment with novel therapies. Serial ctDNA analysis allows clonal dynamics to be monitored over time and identifies the emergence of genomic changes associated with Richter's syndrome (RS). In addition to conventional disease monitoring, ctDNA provides a unique opportunity for non-invasive serial analysis of CLL for molecular disease monitoring.

Figure 1. ctDNA dynamics reflect changes in disease burden across different tissue compartments.

(a) Case CLL020: plasma ctDNA levels by TP53 p.R209fs mutation (left) and IGH (right) decreased over the course of ofatumumab and venetoclax treatment, which paralleled the decline in mutant DNA levels in the MNL, the decrease in PBL count and the reduction in lymphadenopathy as assessed by imaging. (b) Case CLL018: the initial peripheral lymphocytosis observed post ibrutinib treatment coincided with a increase in the abundance of the NOTCH1 p.P2415fs mutation in the MNL. This contrasted with plasma ctDNA levels of both the NOTCH1 p.P2415fs mutation (left) and IGH (right), which instead reflected the reduction in radiological disease burden. (c) Case CLL001: following obinutuzumab therapy, rapid clearance of the circulating leukaemic cells was observed and the NOTCH1 p.P2415fs mutation became undetectable in the MNL. This coincided with a parallel decrease in plasma ctDNA as assessed by NOTCH1 p.P2415fs mutation (left) and IGH (right) levels, although ctDNA did not become undetectable. The patient then had compartmentalized disease progression with increasing lymphadenopathy that was matched by a plasma ctDNA rise. Subsequent ibrutinib treatment resulted in a reduction in lymphadenopathy and plasma ctDNA. These dynamic changes were not reflected in the PBL count or by the NOTCH1 p.P2415fs mutation in MNL DNA. (d) Correlation between the change in ctDNA/MNL MAF versus change in radiological disease burden (cm²) from the maximal value analysed for each individual patient across any time point. Of 38 matched serial time points from a total of 12 patients, the correlation with radiology was significantly better with ctDNA (r²=0.78, P<0.0001) than with MNL DNA (r²=0.24, P=0.0019). (e) Comparison of ctDNA and MNL detection across 111 matched time points. (f) Comparison of ctDNA with detection of CLL by multi-colour flow cytometry on PB or BM.

Figure 2. ctDNA detects clonal evolution in CLL patients with RS.

(a) Graphical representation of the linear pattern of evolution of CLL to RS in CLL004 revealed by LC-WGS of the plasma and WES of plasma (P) and BM/LN. (b) Depth of coverage (DOC) log₂ ratio plots from LC-WGS of plasma in CLL004 at baseline (top panel) and at RS (bottom panel) showing CNAs specific to the dominant CLL clone in yellow and new CNAs at the time of RS in blue. (c) Heat-map illustrating the distribution of predicted functional SNVs from WES at baseline (BM and P) and at progression to RS (LN and P shown in red) in CLL004. (d) Graphical representation of the linear pattern of evolution of CLL to RS in CLL054. (e) DOC log₂ ratio plots from LC-WGS of plasma in patient CLL054 at baseline (top) and at RS (bottom) showing CNAs specific to the dominant CLL clone (yellow) and new CNAs at the time of RS (blue). (f) Heat-map illustrating the distribution of predicted functional SNVs from WES at baseline (PB and P) and at progression to RS (BM, LN and P shown in red) in CLL054. (g) Graphical representation of the branched pattern of evolution of CLL to RS in CLL022. (h) DOC log₂ ratio plots from LC-WGS of plasma in CLL022 at baseline (top) and at RS (bottom) showing CNAs specific to the ancestral CLL clone (yellow), the dominant CLL clone (green) and new CNAs at the time of RS (blue). (i) ctDNA levels in CLL022 showed a decrease in the TP53 p.R248Q mutation post venetoclax treatment. In contrast, there was an increasing fractional abundance of TP53 p.C238S and SF3B1 p.K666M mutations observed in the plasma as the patient progressed to RS. These mutations were detected in plasma 64 days before the clinical diagnosis of RS. (j) Heat-map illustrating the distribution of predicted functional SNVs from WES at baseline (BM and P) and at progression to RS (BM and P shown in red) in CLL022.