Circulating tumour DNA analysis to detect clonal evolution in TP53 mutated CLL treated with sequential precision medicines

Circulating tumour DNA (ctDNA) is increasingly being investigated not only for the early detection of treatment failure but also for the assessment of clonal evolution during treatment, facilitating a more comprehensive assessment of tumour burden compared to existing techniques. Here, we describe the case of a male patient with TP53 mutated CLL, diagnosed in 2000,initially treated with 2 lines of immune-chemotherapy prior to relapse in 2012, when he received sequential treatment with the selective BTKi tiraburitinib followed by the selective BCL2 inhibitor, venetoclax. Using serial ctDNA analysis we studied the dynamics of clonal evolution during treatment with tirabrutinib and venetoclax prior to the development of Richter’s transformation(RT). To elucidate clonal dynamics leading to therapeutic resistance and subsequent RT, we carried out paired whole exome sequencing(WES), deep targeted sequencing using a customised 72 gene panel and digital droplet PCR (ddPCR) on sequential peripheral blood mononuclear cells (PBMCs) and ctDNA. ctDNA was derived from plasma samples obtained pre-, during and post-treatment with tirabrutinib and venetoclax. Tumour tissue was obtained at the point of diagnosis of RT. This patient initially responded for 31 months on a potentially non-saturating dose of 40mg OD of tirabrutinib prior to clinicalrelapse. Analysis of ctDNA did not detect either a BTK or PLCG2 mutation at initial progression and therefore the dose of tirabrutinib was increased to 600mg OD, the maximal dose permitted on study. A further clinical response was seen with a resultant secondary BTKi induced lymphocytosis. The patient remained in partial remission for an additional 12 months. Two months prior to further clinical relapse, the presence of a BTK p.L528W mutation was detected by ctDNA analysis, a mutation disrupts the BTKi binding site which has been previously described in refractory CLL (Sharma et al., 2016). On WES of PBMC, mutations were found in TP53p.V216M at 73% variant allele frequency (VAF) and MSH2 p.E572_Q574del at 29.6% VAF. Analysis of IGHV further demonstrated a single productive IGH clonotype of IGHV2-5*06.Following initiation of venetoclax, ctDNA levels of BTK p.L562W and TP53 p.V216M rapidly became undetectable, correlating with clinical response. The patient remained in remission for 13 months before developing rapidly progressive RT resistant to R-CHOP.WES of the RT tumour showed additional mutations, deletions and chromosomal translocations. However, deep-targeted sequencing uncovered a low level BCL2 p.A113G (1.8% VAF) undetected by WES. This mutation was also detected by ctDNA analysis at this timepoint demonstrating ctDNA offers a non-invasive rout to RT detection. The IGHV clonotype matched the pre-RT PBMC, indicating RT was clonally related to the CLL clone. A striking increase in the frequency of frameshift mutations in post-MSH2 aberration (119%, compared to sequenced samples pre-MSH2 acquisition) was observed, suggestive of chromosomal instability, which likely contributed to treatment resistance. The increase in VAF of MSH2 through sequential relapses in treatment, further suggests an association with RT development (tirabrutinib(16.7%), second relapse (29.6%), and RT (45.3%)).Here we have shown that mutational profiling of ctDNA is an effective tool for detecting clonal resistance to precision medicines prior to the development of clinical or radiological evidence of progression.