A Multiomics Approach for Longitudinal Treatment Response Evaluation of Chronic Lymphocytic Leukemia Patients Receiving BTK Inhibitor Monotherapy
Authors: Hansen Marcus Høy , Veyhe Sólja Remisdóttir , Cédile Oriane , Dahlmann Sara Kamuk , Juul-Jensen Karen , Frederiksen Henrik , Thomassen Mads , Dybkaer Karen , Abildgaard Niels , Nyvold Charlotte Guldborg 
Published: December 2024

The approach to managing chronic lymphocytic leukemia (CLL) has changed considerably. Treatment strategies vary, ranging from watchful waiting in the initial stages to Bruton tyrosine kinase (BTK) inhibitor monotherapy, chemo-immunotherapy, or stem cell transplantation in advanced stages of the disease. A notable aspect of BTK inhibitors, such as ibrutinib, is the phenomenon of prolonged lymphocytosis, a condition shown not to impact survival rates.
Despite a high response rate, complete molecular remission is infrequent with ibrutinib monotherapy (Strati & al. 2020), unlike cases treated with venetoclax, rituximab, or combinations thereof (Roberts & al. 2015). This persistent residual lymphocytosis during ibrutinib treatment does not indicate a lack of response or disease progression (Herman & al. 2014; Barrientos & al. 2019). Because the extent of this clonal inertia remains to be thoroughly investigated at the molecular level, this methodological study investigated a multi-omics approach to profile the clonal, molecular CLL architecture from treatment initiation and response follow-up.
Methods
In this proof-of-concept study, we characterized purified B cells at diagnosis and follow-up (8 and 16 months) from two patients (pt.) with CLL, unmutated IGHV, receiving ibrutinib as first-line treatment. Blood samples showed a lymphocyte count of 127 and 20.9·109/L. Clonotyping using rearranged IGH locus was done at all three time points, averaging 0.6 million (M) reads. Whole-exome (90 M reads), full-transcript mRNA (63 M reads), and single-cell sequencing (~56000 cells) were performed at the time of diagnosis and last follow-up.
Results
The mono-therapeutic intervention reduced peripheral lymphocytes to 3.67-6.86·109/L at 8 months and 3.66-8.87·109/L at 16 months follow-up after ibrutinib initiation. Only minor changes of the clonal CLL burden within the B-cell compartment were found from 54.80% clonotypic rearrangements (pt. 1, IGHV3-23*04 J4*02) at diagnosis and 60.12 to 66.81% at follow-up. For pt. 2 (IGHV1-69*13 J6*02), this was 86.41% at diagnosis, followed by 85.96 to 88.62% at the time of follow-up.
Genomic lesions were confirmed transcriptionally, showing archetypical NOTCH1, SF3B1 point mutations, and ASXL1frameshift in C-terminal exon 12/13. Also, allelic and chromosomal imbalances from 13q14 deletions were observed in both samples at the DNA level, in agreement with cytogenetics. Additionally, mutated TP53, loss of 10q23, monoallelic deletion of ATM (11q21-q24), and the IGH locus from 14q32 were found for pt. 2. At the molecular level, pt. 2, several factors contribute to a high-risk profile. However, both patients were molecularly stable nearly 1.5 years after initiating ibrutinib treatment.
In summary, a direct correlation between putative somatic variants was found between diagnosis and relapse (ρPearson=0.91, Pt-statistics=0.0001) with a variant overlap of 82% (132/162, &gt;0.05 variant allele frequency, VAF). No significant change in VAF from diagnosis to follow-up was observed for DNA or RNA (PU-test=0.92 and PU-test=0.79). As no clonal progression was indicated by at the DNA level, we turned to single-cell resolution of the B cells compartment, showing an expressional shift from baseline to follow-up in biological pathways related to apoptosis, cellular response to stress, and canonical NFKB signaling transduction specific with significantly increased expression profiles at follow-up. Bulk RNA sequencing of the coding transcriptome quantitatively confirmed this increased expression of apoptotic markers.
Conclusion
We show that the CLL burden in the B-cell compartments remains high in either case, with no indications of clonal evolution, progression, or clearance of lymphocytosis at the molecular level. Collectively, striking clonal concordance and stability are evident, genomically and transcriptionally. Thus, we extend the early findings of Byrd & al., Woyach & al., and others, showing that lymphocytosis is a feature of ibrutinib. However, the results also show a molecular persistency of this lymphocytosis with precision for follow-up evaluation, with relevance for high-risk profiles. We are currently applying this methodology to profile a cohort to 3.5 years of follow-up.