2018 - Over-Time Monitoring of CXCR4dim/CD5bright Proliferative Fraction Anticipates Relapse In Ibrutinib-Treated CLL : a reservoir for BTK mutations?

Reciprocal expression of CXCR4 and CD5 on the surface of chronic lymphocytic leukemia (CLL) cells can discriminate the proliferative fraction (CD5high/CXCR4dim, PF), recently egressed from the lymph node, from the resting fraction (CXCR4high/CD5dim, RF) of older, quiescent cells. BTK inhibitors affect the proliferative capacity of CLL cells and cause their redistribution from nodal compartments to the blood stream. Little is known about the impact of long-term ibrutinib (IBR) treatment on the PF/RF phenotype. Therefore, we monitored the PF dynamics in two cohorts of IBR-treated CLL cases with long-term follow-up, and correlated with the emergence of IBR resistance.
Peripheral blood samples were collected from CLL patients who either entered the IOSI-EMA-001 study (NCT02827617) or were referred to our institution for routine immunogenetic analyses. Immunophenotypic analyses were performed using a FACSCanto II flow cytometer (BD Bioscience). DNA sequencing was performed with targeted amplicons, while RNA-seq was performed with the mRNA Library prep Kit on a Nextseq instrument (Illumina) and analyzed with R package DESeq2.
Longitudinal analysis of the PF under IBR in 31 CLL cases (IOSI-EMA-001) with sequential samplings (156 samples, median 6 samples/case) showed a depletion of the PF over time, from 17.7% at pre-treatment to 4.75, 0.4, 1.5, 0.6, 1.5% at later timepoints (0.5, 6, 12, 18, 24 months; p < 0.001, U test), along with loss of proliferative potential by Ki67 expression.
We then retrospectively analyzed the PF from a wider cohort of 100 IBR-treated CLL cases from the real world, referred for routine immunophenotyping (296 samples, median 3 samples/case; Fig. A-B). Median PF of pre-IBR samples was 12.0% (range 0.7-50), with a significant drop to 2.7% within one year of treatment and to 1.0% in the second year (p < 0.001, U test). Out of 100 cases, 71 discontinued IBR within 5 years (17 toxicity, 46 progression, 8 other/death). Mutations of BTK/PLCG2 were detected in 29/100 cases (29%); among these, 24 (64.2%) discontinued IBR due to progression/relapse (23/24) or toxicity (1/24).
To correlate the reappearance of the PF with cause of IBR discontinuation and with presence of BTK/PLCG2 mutations, we focused on 59 samples collected within 12 months before/after IBR discontinuation due to toxicity (n=11), progression (n=43) or other/death (n=5), and compared with 26 samples still on treatment for the next 12 months after testing. Mutations were present in 22/43 progressing cases, with a PF higher than wild-type (WT) cases discontinuing for toxicity (8.9% vs. 2.4%; p=0.012) or still on treatment (2.8%, p=0.015; Fig.E upper comparisons). Notably, the 21 WT progressing cases also showed higher PF compared to cases discontinuing for toxicity (6.9% vs. 2.4%; p=0.004) or to cases still on treatment (6.9% vs. 2.8%; p=0.024; Fig. C lower comparisons). Sequential dosages of plasmatic B2M in progressing cases (n=5) revealed steady levels below the 3.5 ug/mL threshold, despite the PF increase (median PF 18%; Fig.D).
We then isolated the PF/RF fractions from 10 cases, who developed BTK mutations and with PF reappearance after prolonged IBR treatment (median 50.7 months). Sequencing revealed 27 BTK mutations (mean 1.7 mutations per sample, range 1-4), with a median VAF higher in the PF (23.0%, 0.3-92.3) than in the RF (5.2%, 0.1-84.8; p=0.003, paired rank test); overall, the PF VAF was on average 2.8 times larger than the RF (0.01-38). A similar trend was present for PLCG2 mutations (median PF VAF: 2.0%, 0.1-6.2; RF VAF: 1.35%, 0.4-2.8; p=0.014) (Fig. E).
We performed mRNA-seq on 20 cases, with matching PF/RF fractions from samples at pre-IBR and progression, the latter enriched in BTK mutations. Differential expression signature of pre-IBR PF vs. RF, concordant with published gene sets, drove co-clustering of post-IBR PF/RF with their respective counterparts (Fig. F), indicating that PF at progression functionally resembles its pre-treatment counterpart.
To conclude, here we report: i) that IBR treatment abrogates the PF fraction within 1 year of treatment, expanding previous short-term observations; ii) its reconstitution after long-term treatment often predates clinical progression; iii) the enrichment of BTK/PLCG2 mutations in the reconstituted PF suggests the presence of active clonal selection dynamics, bona fide taking place within the lymph node microenvironment. Clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under IBR therapy.