Retrospective Analysis Sheds Light on Mutations Linked With BTK Inhibitor Resistance in CLL


Patients with chronic lymphocytic leukemia who experience disease progression during treatment with either covalent or noncovalent BTK inhibitors displayed a higher frequency of BTK mutations in L528W as well as RAS/RAF/MAPK pathway alterations, indicating that these alterations may play a role in the development of BTK inhibitor resistance.

Kiyomi Mashima, MD, PhD

Kiyomi Mashima, MD, PhD

Patients with chronic lymphocytic leukemia (CLL) who experience disease progression during treatment with either covalent or noncovalent BTK inhibitors displayed a higher frequency of BTK mutations in L528W as well as RAS/RAF/MAPK pathway alterations, indicating that these alterations may play a role in the development of BTK inhibitor resistance, according to findings from a retrospective report presented at the2023 International Workshop on CLL .1

In a summary of mutations detected during BTK inhibitor treatment,mutations that were new or had increasing variant allele frequency (VAF) were significantly more common in patients with progressive disease (PD) vs those with nonprogressive (NP) disease (P < .001).

Additionally, L528W mutations were identified in 2 patients who received first-line ibrutinib (Imbruvica) and 1 patient who received pirtobrutinib (Jaypirca). Among the 6 patients in the PD group who received treatment with pirtobrutinib, 4 displayed T474 mutations. Activating mutations in the RAS/RAF/MAPK pathway were also enriched in the PD group vs the NP group; total mutations in MAPK-related genes also had higher VAFs (P = .0596).

“Our results suggest that MAPK pathway mutations are also related to BTK inhibitor resistance,” lead study author Kiyomi Mashima, MD, PhD, a postdoctoral researcher at Dana-Farber Cancer Institute, said in an oral presentation of the data. “L528W may also be related to BTK inhibitor resistance since it can occur during both ibrutinib and pirtobrutinib treatment.”

A total of 31 patients had paired clinical sequencing results before and during BTK inhibitor treatment. Comparison of mutational clonal evolution based on responses to BTK inhibitors revealed that all reported T474 mutations were observed during pirtobrutinib treatment.

Further analysis of these patients demonstrated significant enrichment of mutations in BTK (P < .01), PLCG2 (P < .05), TP53 (P < .01), SF3B1 (P < .01), and NOTCH2 (P < .05) in the PD group. A trend toward an increasing proportion of XPO1 mutations in the PD group vs the NP group was observed, although this was not statistically significant. (P = .079), 

“Importantly, all the patients in the PD group exhibited at least 1 or more increased or new BTK PLCG2 or TP53 mutation. In contrast, no BTK, PLCG2, or TP53 mutations newly appeared or increased in the NP group,” Mashima added. “Pre-existing 4 TP53 mutations and 6 NOTCH1 mutations among the NP group decreased or completely disappeared during BTK inhibitor treatment.”

Prior research has shown that the most common cause of covalent BTK inhibitor resistance in patients with CLL are BTK mutations occurring at the cysteine 481 residue, which are known to prevent the binding of covalent BTK inhibitors. Alternative site BTK mutations, such as T474 and L528, have been shown to confer resistance to noncovalent BTK inhibitors, with some of these mutations correlating with diminished or absent kinase activity. Further research is needed to improve understanding of the pattern of mutations during BTK inhibitor treatment and to identify alternative mechanisms of resistance, which are estimated to affect between one-third to one-half of patients being treated with this drug class.

“BTK inhibitors have dramatically improved the prognosis of CLL in the past few years,” Mashima emphasized. “However, acquired BTK inhibitor resistance remains a major problem.”

To obtain this information, investigators conducted a retrospective analysis of clinical sequencing results from patients at Dana-Farber Cancer Institute who were receiving BTK inhibitor treatment between 2014 and 2022. Sequencing results were obtained using a comprehensive clinical-grade, 88-gene next-generation sequencing panel.

A total of 765 treatment periods of 609 patients with CLL were initially identified; this was narrowed down to 470 clinical sequencing reports from 301 patients. Of these, 133 clinical sequencing reports were identified as occurring during the treatment period (80%) or at patients’ next office visit within 3 months after BTK inhibitor regimen completion (20%). Fifteen cases were excluded because they lacked treatment history and/or treatment effect assessment (n = 13), ibrutinib was used for graft-vs-host disease (n = 1), or acute myeloid leukemia had developed (n = 1).

Investigators ultimately obtained a total of 118 clinical sequencing reports from 85 patients. Patients were divided based on their disease status at the time of sequencing. A total of 36 patients who were experiencing disease progression were assigned to the PD group, which had a median observation time of 37.0 months from BTK inhibition. The remaining 49 patients without disease progression were designated as the NP group, and had a median observation period of 26.3 months. Of the 118 sequencing results, 60 came from the PD group and 58 were obtained from the NP group.

Patients in the PD group were heavily pretreated, according to Mashima, with a higher percentage of patients having previously received newer-generation BTK inhibitors vs those in the ND group. In the PD group, 69% of patients received ibrutinib, 11% received acalabrutinib (Calquence), and 17% were treated with pirtobrutinib; corresponding percentages in group NP were 82%, 18%, and 0%, respectively.

A total of 216 distinct mutations were identified across 57 genes, comprising 158 variants in the PD group and 121 variants in the NP group. BTK mutations were exclusively detected in 15 patients within the PD group; 7 of these patients received ibrutinib, 2 were treated with acalabrutinib, and 6 received pirtobrutinib. These BTK mutations included C481S (c.1442G>C), C481S (c.1441T>A), C481S (c.1442_1443delGCinsCT), C481R, C481Y, C481F, L528W, T474I, and T474L.

“Our cohort is small, and our study is based on limited targeted sequencing data,” Mashima concluded. “Further work is required and is currently ongoing to elucidate other mechanisms of BTK inhibitor resistance in CLL.”

Dr Mashima did not declare any personal or financial interests.


Mashima K, Fernandes SM, Shupe S, et al. Mutations detected in real world clinical sequencing during BTK inhibitor treatment including novel BTK inhibitors in chronic lymphocytic leukemia (CLL). Presented at: 2023 International Workshop on CLL; October 6-9, 2023; Boston, Massachusetts. Abstract 1554200.

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