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4627 Reappearance of CXCR4dim/CD5bright Proliferative Fraction Associates with BTK Mutations and Anticipates Progression in Ibrutinib-Treated CLL

Program: Oral and Poster Abstracts
Session: 641. Chronic Lymphocytic Leukemias: Basic and Translational: Poster III
Hematology Disease Topics & Pathways:
Research, Lymphoid Leukemias, CLL, Translational Research, Diseases, Lymphoid Malignancies
Monday, December 11, 2023, 6:00 PM-8:00 PM

Federico Pozzo, PhD1*, Gabriela Forestieri2*, Filippo Vit, PhD3*, Erika Tissino, PhD4*, Tamara Bittolo5*, Robel Papotti6*, Annalisa Gaglio6*, Lodovico Terzi Di Bergamo7*, Agostino Steffan8*, Roberta Laureana9*, Agostino Tafuri, MD10, Annalisa Chiarenza, MD11*, Francesco Di Raimondo, MD12, Jacopo Olivieri13*, Francesco Zaja, MD14*, Luca Laurenti, MD15*, Maria Ilaria Del Principe, MD16*, Riccardo Bomben5*, Antonella Zucchetto, PhD17*, Davide Rossi18 and Valter Gattei, MD19

1Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico di Aviano, Aviano, Italy
2Laboratory of Experimental Hematology, Institute of Oncology Research, Bellinzona, Switzerland
3Clinical and Experimental Onco-Haematology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy, ITA
4Clinical and Experimental Onco-Hematology Unit, Centro Di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
5CRO Aviano National Cancer Institute,Clinical and Experimental Onco-Hematology Unit, Aviano, Italy
6Clinical and Experimental Onco-Haematology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
7Institute of Oncology Research, Bellinzona, CHE
8IRCCS National Cancer Institute, Aviano, PN, ITA
9Division of Haematology, University of Tor Vergata, Rome, Roma, ITA
10Department of Clinical and Molecular Medicine and Hematology, Sant'Andrea - University Hospital - Sapienza, University of Rome, Rome, Italy
11Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania, Catania, Italy
12Division of Hematology, Policlinico, Department of Surgery and Medical Specialties, University of Catania, Catania, ITA
13Clinica Ematologica, Centro Trapianti e Terapie Cellulari "Carlo Melzi", Azienda Sanitaria Universitaria Integrata di Udine, Udine, Italy
14DSM, University of Trieste, Trieste, Italy
15Istituto di Ematologia, Rome, ITA
16Hematology, Department of Biomedicine and Prevention, University Tor Vergata, Roma, Italy
17Clinical and Experimental Onco-Hematology Unit, Centro di Riferimento Oncologico di Aviano (CRO) IRCCS, Aviano, Italy
18Clinic of Hematology, Oncology Institute of Southern Switzerland, Ente Ospedaliero Cantonale, Bellinzona, Switzerland
19Centro Di Riferimento Oncologico, Aviano, Italy

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 the redistribution from nodal compartments to the blood stream. Nevertheless, little is known about the impact of long-term treatment of the BTK-inhibitor ibrutinib (IBR) on the PF/RF phenotype. Here, 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.

Longitudinal analysis of the PF under IBR in 31 CLL cases, enrolled in the IOSI-EMA-001 with sequential blood 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 in 100 IBR-treated CLL cases from the real world, referred for routine immunophenotyping (296 samples, median 3 samples/case). 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). 77 cases discontinued IBR due to: toxicity (n=13), progression (n=51), other/death (n=13). Mutations of BTK/PLCG2 were detected in 30 cases, 28 of which discontinued IBR due to progression and 2 for other/death.

To correlate reappearance of the PF with cause of discontinuation, we focused on 64 cases collected within 12 months before/after IBR discontinuation due to toxicity (n=10), progression (n=47) or other/death (n=7), and compared with 22 samples still on treatment for the next 12 months after testing (Panel A). ROC analysis of the PF suggested a criterion of PF>3% as the most discriminating to predict IBR discontinuation due to progression, with a sensitivity of 86.7% and a specificity of 71.1%. This cut-off could separate patients with a longer time-to-progression (median 56 vs. 45 months, p=0.031). 26/47 progressing cases bore BTK/PLCG2 mutations, with a PF higher than wild-type cases discontinuing for toxicity (8.4% vs. 2.2%; p<0.001) or still on treatment (1.8%, p<0.001). Notably, the 21 BTK/PLCG2- wild-type progressing cases also showed higher PF compared to cases discontinuing for toxicity (6.9% vs. 2.2%; p<0.001) or still on treatment (1.8%; p<0.001). Notably, PF levels showed no significant correlation with white blood cell counts. 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%).

Cell sorting of the PF/RF fractions from 10 cases showing PF reappearance after prolonged IBR treatment (median 50.7 months) revealed a mean of 1.7 BTK mutations per sample (27 total, range 1-4), a median VAF higher in the PF (23.0%, 0.3-92.3) on average 2.8 times larger than in the RF (5.2%, 0.1-84.8; p=0.003, paired rank test). A similar skewing 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).

Finally, mRNA-seq was performed in 20 cases, on matching PF/RF fractions both at pre-IBR and progression (9 cases with 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, indicating that PF at progression functionally resembles its pre-treatment counterpart. A strong enrichment of multiple proliferation-related signatures, present in pre-treatment samples, was significantly up-regulated also at progression, independently from BTK mutations (Panel B).

In summary, here we report that CLL progression under IBR associates with reappearance of the CD5bright/CXCR4dim PF which precedes clinical progression, increase of serum B2M and WBC counts. Transcriptomic/genetic profiling of the post-IBR PF suggests presence of clonal selection dynamics and active bypass signaling pathways, bona fide taking place within the lymph node microenvironment, including BTK/PLCG2 mutations. Clinically, longitudinal monitoring of the CXCR4/CD5 fractions by flow cytometry may provide a simple tool helping to intercept CLL progression under IBR therapy.

Disclosures: Zaja: Sobi: Honoraria, Research Funding; Grifols: Consultancy, Honoraria, Research Funding; Amgen: Honoraria, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Rossi: AbbVie, AstraZeneca, Gilead, BeiGene, BMS, Janssen, Lilly, Kyte: Honoraria, Research Funding.

*signifies non-member of ASH