Single-cell sequencing demonstrates complex resistance landscape in CLL and MCL treated with BTK and BCL2 inhibitors

Abstract

The genomic landscape of resistance to targeted agents (TAs) used as monotherapy in chronic lymphocytic leukemia (CLL) is complex and often heterogeneous at the patient level. To gain insight into the clonal architecture of acquired genomic resistance to Bruton tyrosine kinase (BTK) inhibitors and B-cell lymphoma 2 (BCL2) inhibitors in CLL, particularly in patients carrying multiple resistance mutations, we performed targeted single-cell DNA sequencing of 8 patients who developed progressive disease (PD) on TAs (either class). In all cases, analysis of single-cell architecture revealed mutual exclusivity between multiple resistance mutations to the same TA class, variable clonal co-occurrence of multiple mutations affecting different TAs in patients exposed to both classes, and a phenomenon of multiple independent emergences of identical nucleotide changes leading to canonical resistance mutations. We also report the first observation of established BCL2 resistance mutations in a patient with mantle cell lymphoma (MCL) following PD on sequential monotherapy, implicating BCL2 as a venetoclax resistance mechanism in MCL. Taken together, these data reveal the significant clonal complexity of CLL and MCL progression on TAs at the nucleotide level and confirm the presence of multiple, clonally independent, mechanisms of TA resistance within each individual disease context.

Figure 1.

Clonal relationships between resistance mutations in CLL (n = 8 patients) and MCL (n = 1 patient) inferred from variant-based analysis of scDNAseq data. Clones are shown with their defining variants (ie, all detected nonsynonymous coding or splice variants, excluding germ line polymorphisms), clone size (percentage of analyzed cells), and number of cells. Clones with size <1.5% are indicated with a dashed circle. Zygosity of each variant is indicated (heterozygous [HET], homozygous [HOM], or hemizygous [HEM]), with the exception of BCL2, for which zygosity was not determined because of poor sequence quality (supplemental Figure 1). The BCL2 Val156Asp mutation detected in bulk sequencing data in patients CLL-G (at progression during venetoclax treatment) and MCL-A was not assessable on the scDNAseq panel, and MCL1 amplification was not assessable by bulk sequencing (patient CLL-G). Arrows indicate inferred clonal relationships (with the dashed arrow indicating a possible clonal relationship). Each clone was assessed for MCL1 copy number gain and TP53 copy number loss (CNL), and these are indicated when detected (supplemental Table 4; supplemental Figure 2). Clones harboring established or putative BCL2i and BTKi resistance mutations or both are indicated in red, blue, or purple, respectively. Myeloid clones (inferred by variant allele frequency analysis of samples taken at different time points containing little or no CLL disease) are indicated with gray text (patients CLL-B and CLL-F). BTK C481S mutations are followed by a suffix (a-d) to denote which nucleotide change was observed: a indicates NM_000061.2:c. 1441T>A; b, NM_000061.2:c.1442G>C; c, NM_000061.2:c.1442_1443delinsCT; and d, NM_000061.2:c.1440_1441delinsGA. Targeted agent exposure for each patient is shown (venetoclax [VEN], zanubrutinib [ZANU], or ibrutinib [IBR]).

Figure 1.

Clonal relationships between resistance mutations in CLL (n = 8 patients) and MCL (n = 1 patient) inferred from variant-based analysis of scDNAseq data. Clones are shown with their defining variants (ie, all detected nonsynonymous coding or splice variants, excluding germ line polymorphisms), clone size (percentage of analyzed cells), and number of cells. Clones with size <1.5% are indicated with a dashed circle. Zygosity of each variant is indicated (heterozygous [HET], homozygous [HOM], or hemizygous [HEM]), with the exception of BCL2, for which zygosity was not determined because of poor sequence quality (supplemental Figure 1). The BCL2 Val156Asp mutation detected in bulk sequencing data in patients CLL-G (at progression during venetoclax treatment) and MCL-A was not assessable on the scDNAseq panel, and MCL1 amplification was not assessable by bulk sequencing (patient CLL-G). Arrows indicate inferred clonal relationships (with the dashed arrow indicating a possible clonal relationship). Each clone was assessed for MCL1 copy number gain and TP53 copy number loss (CNL), and these are indicated when detected (supplemental Table 4; supplemental Figure 2). Clones harboring established or putative BCL2i and BTKi resistance mutations or both are indicated in red, blue, or purple, respectively. Myeloid clones (inferred by variant allele frequency analysis of samples taken at different time points containing little or no CLL disease) are indicated with gray text (patients CLL-B and CLL-F). BTK C481S mutations are followed by a suffix (a-d) to denote which nucleotide change was observed: a indicates NM_000061.2:c. 1441T>A; b, NM_000061.2:c.1442G>C; c, NM_000061.2:c.1442_1443delinsCT; and d, NM_000061.2:c.1440_1441delinsGA. Targeted agent exposure for each patient is shown (venetoclax [VEN], zanubrutinib [ZANU], or ibrutinib [IBR]).