EHA Library - The official digital education library of European Hematology Association (EHA)

MECHANISMS OF ADAPTATION TO IBRUTINIB IN HIGH RISK CHRONIC LYMPHOCYTIC LEUKEMIA
Author(s): ,
Gabriela Forestieri
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Lodovico Terzi di bergamo
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Jui Wan Loh
Affiliations:
Center for Systems and Computational Biology,Rutgers Cancer Institute of New Jersey, Rutgers University,New Brunswick,United States
,
Valeria Spina
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Antonella Zucchetto
Affiliations:
Clinical and Experimental Onco-Hematology Unit,Centro di Riferimento Oncologico di Aviano (CRO), IRCCS,Aviano,Italy
,
Adalgisa Condoluci
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland
,
Alessio Bruscaggin
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Wei Wu
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Martin Faderl
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Ferdinando Bonfiglio
Affiliations:
Institute of Oncology Research,Bellinzona,Switzerland
,
Elena Flospergher
Affiliations:
Department of Molecular Medicine,University of Pavia,Pavia,Italy
,
Tamara Bittolo
Affiliations:
Clinical and Experimental Onco-Hematology Unit,Centro di Riferimento Oncologico di Aviano (CRO), IRCCS,Aviano,Italy
,
Erika Tissino
Affiliations:
Clinical and Experimental Onco-Hematology Unit,Centro di Riferimento Oncologico di Aviano (CRO), IRCCS,Aviano,Italy
,
Amartya Singh
Affiliations:
Center for Systems and Computational Biology,Rutgers Cancer Institute of New Jersey, Rutgers University,New Brunswick,United States
,
Lorenzo De Paoli
Affiliations:
Division of Hematology, Department of Translational Medicine,University of Eastern Piedmont,Novara,Italy
,
Clara Deambrogi
Affiliations:
Division of Hematology, Department of Translational Medicine,University of Eastern Piedmont,Novara,Italy
,
Anna Maria Frustaci
Affiliations:
Department of Hematology,ASST Grande Ospedale Metropolitano Niguarda,Milan,Italy
,
Marina Deodato
Affiliations:
Department of Hematology,ASST Grande Ospedale Metropolitano Niguarda,Milan,Italy
,
Francesco Autore
Affiliations:
Fondazione Policlinico Universitario A Gemelli IRCCS,Rome,Italy
,
Michele Merli
Affiliations:
Division of Hematology,Ospedale di Circolo e Fondazione Macchi,Varese,Italy
,
Lydia Scarfò
Affiliations:
Division of Experimental Oncology,IRCCS San Raffaele Hospital,Milan,Italy
,
Silvia Rasi
Affiliations:
Division of Hematology, Department of Translational Medicine,University of Eastern Piedmont,Novara,Italy
,
Renzo Lucchini
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland
,
Jakob Passweg
Affiliations:
Department of Haematology,University Hospital Basel,Basel,Switzerland
,
Paolo Ghia
Affiliations:
Division of Experimental Oncology,IRCCS San Raffaele Hospital,Milan,Italy
,
Franco Cavalli
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland
,
Bernhard Gerber
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland
,
Georg Stuessi
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland
,
Francesco Passamonti
Affiliations:
Division of Hematology,Ospedale di Circolo e Fondazione Macchi,Varese,Italy
,
Michael Gregor
Affiliations:
Division of Haematology and Central Haematology Laboratory,Cantonal Hospital Lucerne,Lucerne,Switzerland
,
Luca Laurenti
Affiliations:
Fondazione Policlinico Universitario A Gemelli IRCCS,Rome,Italy
,
Dimitar Efremov
Affiliations:
Molecular Hematology,International Centre for Genetic Engineering and Biotechnology,Trieste,Italy
,
Alesandra Tedeschi
Affiliations:
Department of Hematology,ASST Grande Ospedale Metropolitano Niguarda,Milan,Italy
,
Gianluca Gaidano
Affiliations:
Division of Hematology, Department of Translational Medicine,University of Eastern Piedmont,Novara,Italy
,
Hossein Khiabanian
Affiliations:
Center for Systems and Computational Biology,Rutgers Cancer Institute of New Jersey, Rutgers University,New Brunswick,United States
,
Valter Gattei
Affiliations:
Clinical and Experimental Onco-Hematology Unit,Centro di Riferimento Oncologico di Aviano (CRO), IRCCS,Aviano,Italy
Davide Rossi
Affiliations:
Oncology Institute of Southern Switzerland,Bellinzona,Switzerland;Institute of Oncology Research,Bellinzona,Switzerland
(Abstract release date: 05/14/20) EHA Library. Forestieri G. 06/12/20; 294974; S154
Gabriela Forestieri
Gabriela Forestieri
Contributions
Abstract

Abstract: S154

Type: Oral Presentation

Session title: New insights in the biology of chronic lymphocytic leukemia

Background
Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a partial remission (PR) with detectable minimal residual disease (MRD) which is maintained in high risk patients for ~3 years until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. 

Aims
The study aims at a multilayer characterization of the adaptation process that allows residual CLL cells to persist despite BTK inhibition. 

Methods
The study has enrolled 33 patients with CLL treated with ibrutinib. Longitudinal samples have been collected at pre-specified timepoints, including baseline, weeks 2, 24, 48, 72, 96, and 3 years of treatment. Samples were characterized by multilayer systematic approaches, including: i) profiling of proliferation, antigen expression and signalling by multiparametric and phospho-flow-cytometry; ii) profiling of genetic evolution on tumor gDNA and cellular free DNA (cfDNA); iii) profiling of transcriptomic evolution by RNA-seq. 

Results

During treatment, highly sensitive ultra-deep next generation sequencing did not disclose any non-synonymous somatic mutation of BTK and PLCG2 in MRD. Multicolor flow cytometry revealed an upregulation of B-cell receptor molecules (i.e. IgM, CD19) on MRD cells while B-cell receptor inhibitory molecules were downregulated (i.e. CD305, CD307b, FcμR). In addition, a number of homing and adhesion molecules were consistently upregulated on MRD cells (i.e CD29, CD49d). Collectively, these data indicate that MRD cells are not anergic B-cells, but instead adapts the surface machinery upon ibrutinib likely to increase the contact with the microenvironment. At transcriptomic level, most of the genes (N=1,235) were downregulated in MRD, while only a fraction was upregulated (N=114). Among downregulated genes, the switch-off of NF-kB signatures in MRD mirrored the lack of phospho-signaling through the BTK-PLCG2 axis, which was not restored neither after stimulation of the BCR, nor after stimulation of Toll-like receptor 9 (TLR9) or CD40. Transcriptomic evolution of MRD under ibrutinib also included the down modulation of proliferation and metabolism genes. Consistently, flow-cytometry analysis of MRD confirmed a reduction in Ki-67 expression, a decrease of the proliferative compartment, and an increase of the resting compartment, indicating that cell proliferation is not involved in refueling MRD. Among genes upregulated in MRD cells, a clear cluster of the MAPK signature (i.e. BRAF, MAP3K2, RAF1) was enriched. Consistently, upon stimulation of the B-cell receptor (but not of the TLR9) of MRD, we observed an increase of phosphosignaling in the MAPK pathway (i.e. pERK). At the genetic level, the clonal composition of MRD was maintained unchanged. Clonal evolution was limited to a fraction of cases, 6 of which acquired pathogenic non-synonymous somatic mutations in BRAF, NRAS, and KRAS.

Conclusion
MRD under ibrutinib adapts its phenotype, mainly in a non-genetic way by maintaining functional competence of B-cell receptor signaling through the MAPK pathway. Though rare, preferential acquisition of mutations in MAPK genes in MRD further suggests the relevance of this mechanism of adaptation. In terms of perspective, evidence pointing to the MAPK as a vulnerability of MRD under ibrutinib provides the rationale for combining MAPK pathway inhibitors to BTK inhibitors in order to achieve deeper and longer responses.

Session topic: 05. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research

Keyword(s): Chronic lymphocytic leukemia, Ibrutinib, Minimal residual disease (MRD)

Abstract: S154

Type: Oral Presentation

Session title: New insights in the biology of chronic lymphocytic leukemia

Background
Ibrutinib inhibits the BTK molecule downstream the B-cell receptor (BCR). Though highly active in high risk chronic lymphocytic leukemia (CLL), the most typical response achievable in patients is a partial remission (PR) with detectable minimal residual disease (MRD) which is maintained in high risk patients for ~3 years until the development of genetically driven resistance caused by the acquisition of mutations in the BTK or PLCG2 genes. 

Aims
The study aims at a multilayer characterization of the adaptation process that allows residual CLL cells to persist despite BTK inhibition. 

Methods
The study has enrolled 33 patients with CLL treated with ibrutinib. Longitudinal samples have been collected at pre-specified timepoints, including baseline, weeks 2, 24, 48, 72, 96, and 3 years of treatment. Samples were characterized by multilayer systematic approaches, including: i) profiling of proliferation, antigen expression and signalling by multiparametric and phospho-flow-cytometry; ii) profiling of genetic evolution on tumor gDNA and cellular free DNA (cfDNA); iii) profiling of transcriptomic evolution by RNA-seq. 

Results

During treatment, highly sensitive ultra-deep next generation sequencing did not disclose any non-synonymous somatic mutation of BTK and PLCG2 in MRD. Multicolor flow cytometry revealed an upregulation of B-cell receptor molecules (i.e. IgM, CD19) on MRD cells while B-cell receptor inhibitory molecules were downregulated (i.e. CD305, CD307b, FcμR). In addition, a number of homing and adhesion molecules were consistently upregulated on MRD cells (i.e CD29, CD49d). Collectively, these data indicate that MRD cells are not anergic B-cells, but instead adapts the surface machinery upon ibrutinib likely to increase the contact with the microenvironment. At transcriptomic level, most of the genes (N=1,235) were downregulated in MRD, while only a fraction was upregulated (N=114). Among downregulated genes, the switch-off of NF-kB signatures in MRD mirrored the lack of phospho-signaling through the BTK-PLCG2 axis, which was not restored neither after stimulation of the BCR, nor after stimulation of Toll-like receptor 9 (TLR9) or CD40. Transcriptomic evolution of MRD under ibrutinib also included the down modulation of proliferation and metabolism genes. Consistently, flow-cytometry analysis of MRD confirmed a reduction in Ki-67 expression, a decrease of the proliferative compartment, and an increase of the resting compartment, indicating that cell proliferation is not involved in refueling MRD. Among genes upregulated in MRD cells, a clear cluster of the MAPK signature (i.e. BRAF, MAP3K2, RAF1) was enriched. Consistently, upon stimulation of the B-cell receptor (but not of the TLR9) of MRD, we observed an increase of phosphosignaling in the MAPK pathway (i.e. pERK). At the genetic level, the clonal composition of MRD was maintained unchanged. Clonal evolution was limited to a fraction of cases, 6 of which acquired pathogenic non-synonymous somatic mutations in BRAF, NRAS, and KRAS.

Conclusion
MRD under ibrutinib adapts its phenotype, mainly in a non-genetic way by maintaining functional competence of B-cell receptor signaling through the MAPK pathway. Though rare, preferential acquisition of mutations in MAPK genes in MRD further suggests the relevance of this mechanism of adaptation. In terms of perspective, evidence pointing to the MAPK as a vulnerability of MRD under ibrutinib provides the rationale for combining MAPK pathway inhibitors to BTK inhibitors in order to achieve deeper and longer responses.

Session topic: 05. Chronic lymphocytic leukemia and related disorders - Biology & Translational Research

Keyword(s): Chronic lymphocytic leukemia, Ibrutinib, Minimal residual disease (MRD)

By clicking “Accept Terms & all Cookies” or by continuing to browse, you agree to the storing of third-party cookies on your device to enhance your user experience and agree to the user terms and conditions of this learning management system (LMS).

Cookie Settings
Accept Terms & all Cookies