scholarly journals Detection of emerging ibrutinib resistance by flow cytometry in patients with chronic lymphocytic leukemia

2021 ◽  
Author(s):  
Ferenc Takács ◽  
Lili Kotmayer ◽  
Ágnes Czeti ◽  
Gábor Szalóki ◽  
László Tamás ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Kinase Inhibitor ◽  
Acquired Resistance ◽  
Resistance Mutation ◽  
Lymphocytic Leukemia ◽  
Continuous Treatment ◽  
Clinical Resistance ◽  
Ibrutinib Resistance

Abstract Background: Bruton ’ s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, continuous treatment is required to maintain remission, which may lead to acquired ibrutinib resistance. Early detection of acquired resistance preceding clinical disease progression is an important issue. This is why our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment in sensitive and clinically resistant patients. Materials and methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples from CLL patients were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTK C481S resistance mutation was tested using digital droplet PCR (ddPCR) in samples from ibrutinib sensitive and resistant cases. In addition, we investigated the changes of CLL cells ’ phenotype during ibrutinib treatment in one patient with acquired ibrutinib resistance. Results: We found that the expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. Furthermore, the expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTK C481S mutation. In addition, our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance. Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required. Monitoring CD86 expression on peripheral blood CLL cells during ibrutinib treatment may become a potential new method to detect acquired ibrutinib resistance in the near future.

scholarly journals Detection of Emerging Ibrutinib Resistance by Flow Cytometry in Patients with Chronic Lymphocytic Leukemia

2021 ◽  
Author(s):  
Ferenc Takács ◽  
Lili Kotmayer ◽  
Ágnes Czeti ◽  
Gábor Szalóki ◽  
László Tamás ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Kinase Inhibitor ◽  
Resistance Mutation ◽  
Lymphocytic Leukemia ◽  
Flow Cytometric ◽  
Clinical Resistance ◽  
Treatment Naïve ◽  
Droplet Pcr ◽  
Ibrutinib Resistance

Abstract Purpose: Bruton’s tyrosine kinase inhibitor ibrutinib has revolutionized the treatment of chronic lymphocytic leukemia (CLL). Although ibrutinib is a highly effective drug, during the treatment acquired ibrutinib resistance may occur and its early detection is an important issue. Our aim was to investigate several phenotypic markers on CLL cells to reveal changes in their expression during ibrutinib treatment.Methods: In our study 28 (treatment naive, ibrutinib sensitive, clinically ibrutinib resistant) peripheral blood (PB), and 6 paired PB and bone marrow (BM) samples were examined. The expression of several surface markers (CD69, CD184, CD86, CD185, CD27) was assessed by flow cytometry in each sample. Furthermore, the presence of the BTKC481S resistance mutation was tested using digital droplet PCR. In addition, we investigated the changes of the phenotype of CLL cells during ibrutinib treatment in one patient with acquired ibrutinib resistance.Results: The expression of CD27 decreased during ibrutinib therapy but increased again at the onset of clinical resistance. Expressions of CD69 and CD86 were also elevated at the onset of clinical ibrutinib resistance. The expression of CD86 showed correlation between PB and BM samples. Relapsed cases with high CD86 expression were positive for BTKC481S mutation. Our prospective study showed that the increases in the expression of CD27, CD69 and CD86 were detectable up to several months before the onset of clinical resistance.Conclusion: Our research suggests that the flow cytometric measurements of certain markers, especially CD86, may predict development of ibrutinib resistance, however, confirmatory experiments are still required.

Receptor Tyrosine Kinase-Like Orphan Receptor 1 (ROR1) - a Putative Diagnostic Marker for Chronic Lymphocytic Leukemia (CLL).

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1587-1587
Author(s):  
Sabrina Uhrmacher ◽  
Magdalena Hertweck ◽  
Julian Paesler ◽  
Felix Erdfelder ◽  
Alexandra Filipovich ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Chronic Lymphocytic Leukemia ◽  
Tyrosine Kinase ◽  
Healthy Volunteers ◽  
Receptor Tyrosine Kinase ◽  
Peripheral Blood ◽  
Lymphocytic Leukemia ◽  
Orphan Receptor ◽  
Flow Cytometric

Abstract Abstract 1587 Poster Board I-613 In chronic lymphocytic leukemia (CLL) WNT signaling is constitutively active and several members of this signaling pathway are uniformely upregulated in these cells. Apart from classical WNT receptors like FZD and LRP6, receptor tyrosine kinase-like orphan receptor 1 (ROR1) has been shown to function as a receptor for WNT proteins, too. Furthermore, it could recently be demonstrated that ROR1 is frequently expressed on the surface of CLL cells and might therefore serve as a therapeutic target in this disease. However, so far only little is known about the expression status of this protein in different patients. Moreover, a diagnostic antibody for flow cytometric investigations is lacking. Thus, the aim of our study was to i) establish a directly labelled anti-ROR1 antibody for flow cytometry, ii) to confirm previous results on ROR1 expression in CLL, iii) to investigate ROR1 expression in different cell compartments and iv) correlate our findings to known markers of risk and disease progression. Peripheral blood of CLL patients as well as healthy volunteers was subjected to flow cytometric analysis. Besides standard determination of leukocyte subpopulations ZAP70 and CD38 status was assessed according to current diagnostic recommendations. In addition, ROR1 surface expression was first detected by flow cytometry using a specific primary antibody directed against ROR1 and a fluorescent labelled secondary antibody. Using this experimental setting we found that ROR1 is expressed on 63.4% of all neoplastic CLL cells and also on 30.5% of T cells in the peripheral CLL blood. In contrast, no ROR1 expression could be detected on NK cells, B cells, CD8+- or CD4+-T cells of healthy individuals. To improve the analytical technique the ROR1 antibody was directly conjugated with Phycoerythrin (PE) and the experiments were repeated. With the conjugated antibody we detected ROR1 expression on 97.1% of neoplastic CLL cells and virtually on no T lymphocytes. ROR1 expression levels correlated neither with the expression of ZAP70 nor with CD38. Again, we could not detect ROR1 expression on peripheral blood cells of our healthy volunteers. Taken together, ROR1 expression appears to be highly restricted to CLL cells. If in addition to CD5 and CD19 ROR1 detection is included into diagnostic flow cytometric panels the specificity and sensitivity of immunophenotypic CLL diagnostics may be greatly enhanced. Disclosures Hallek: Roche: Consultancy, Honoraria, Research Funding.

Phase IIa Study of the Anti-CXCL12/SDF-1 Spiegelmer® Nox-A12 Alone and Combined with Bendamustine/Rituximab in Patients with Relapsed Chronic Lymphocytic Leukemia (CLL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4593-4593
Author(s):  
Marco Gobbi ◽  
Federico Caligaris-Cappio ◽  
Marco Montillo ◽  
Stephanie Vauléon ◽  
Stefan Zöllner ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Plasma Concentration ◽  
Chronic Lymphocytic Leukemia ◽  
Healthy Volunteers ◽  
Peripheral Blood ◽  
Combined Treatment ◽  
Single Agent ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Cd34 Cells

Abstract Abstract 4593 Background NOX-A12 is a novel, potent, L-aptamer inhibitor of CXCL12/SDF-1, a chemokine which attracts and activates immune- and non-immune cells. The signaling of CXCL12 has been shown to play an important role in the pathophysiology of chronic lymphocytic leukemia (CLL), especially in the interaction of leukemic cells with their tissue microenvironment. The therapeutic concept of NOX-A12 is to inhibit such tumor-supporting pathways and thereby sensitizing the CLL cells towards chemotherapy. Methods The purpose of this phase IIa study is to evaluate the safety and efficacy of NOX-A12 in combination with background chemo-immunotherapy of bendamustine and rituximab (BR) in patients with relapsed CLL. The described study is being performed in compliance with ethical principles based on the Declaration of Helsinki and ICH-GCP guidelines. The study population was split into a pilot and expansion group. In the pilot group, 3 cohorts of 3 patients each received escalating doses of single agent NOX-A12 two weeks prior to the combined treatment of NOX-A12 and BR. Interim data from these patients are reported. Based on previous Phase I studies in healthy volunteers, pilot patients received a dose of 1, 2 or 4 mg/kg body weight (BW) single agent NOX-A12 on day -14, followed by a 2-weeks period of safety, PK and PD assessments prior to the combined treatment with NOX-A12 and BR. To date, the first cohort of the pilot group already progressed to the 2nd cycle of combined treatment. Evaluation criteria included adverse events according to CTCAE V4, flow cytometry of peripheral blood CD34+ cells and CLL cells, pharmacokinetics of NOX-A12, plasma concentration of CXCL12 and tumor response (NCI-WG 1996 criteria, updated 2008). Results To date 3 patients (age range: 58 – 65 years) have been enrolled in the pilot group of this study. They had received 1 or 2 prior therapies, but no bendamustine. Single i.v. doses of 1 mg/kg BW NOX-A12 had no clinically relevant effects on vital signs, 12-lead ECG parameters and laboratory parameters. One patient reported grade 1 pain in the lower limbs two days after treatment with NOX-A12. This event was not dose-limiting and resolved spontaneously on the same day. Flow cytometry of CD34+ cells and CLL cells (CD19+/CD5+high) showed a rapid mobilization of these cells into the peripheral blood on day 1. Interestingly, return to baseline was not complete at the last assessment on day 3 (for details see Figure 1). The NOX-A12 pharmacokinetics in these 3 patients (for concentration-time profile see Figure 2) is very comparable to healthy volunteers receiving i.v. NOX-A12, with a maximum plasma concentration of 1.52 ± 0.14 μM after 1 h (tmax) and a plasma elimination half-life of about 50 h. As seen in healthy volunteers the plasma concentration of CXCL12 increased upon NOX-A12 treatment and reached a maximum of 0.434 ± 0.076 μM at 24 to 72 h p.a. without ever approaching the plasma concentration of NOX-A12 (Figure 2). Conclusion Single i.v. doses of NOX-A12 at 1 mg/kg BW were safe and well tolerated; the maximum tolerated dose was not reached. NOX-A12 induced a long-lasting mobilization of CD34+ cells and leukemic cells in patients with relapsed CLL, consistent with a mechanism of action based on CXCL12 inhibition. Patient accrual and identification of an optimal chemosensitization regimen of NOX-A12 combined with BR is being continued. Disclosures: Vauléon: NOXXON Pharma AG: Employment. Zöllner:NOXXON Pharma AG: Employment. Dümmler:NOXXON Pharma AG: Employment. Kruschinski:NOXXON Pharma AG: Employment. Fliegert:NOXXON Pharma AG: Employment.

scholarly journals Novel Agents in Chronic Lymphocytic Leukemia: New Combination Therapies and Strategies to Overcome Resistance

Cancers ◽  
2021 ◽  
Vol 13 (6) ◽  
pp. 1336
Author(s):  
Moritz Fürstenau ◽  
Barbara Eichhorst
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Acquired Resistance ◽  
Resistance Mechanisms ◽  
Lymphocytic Leukemia ◽  
Novel Agents ◽  
New Combination ◽  
Special Focus ◽  
Combination Therapies ◽  
Clinical Resistance ◽  
Btk Inhibitors

The approval of Bruton’s tyrosine kinase (BTK) inhibitors such as ibrutinib and acalabrutinib and the Bcl-2 inhibitor venetoclax have revolutionized the treatment of chronic lymphocytic leukemia (CLL). While these novel agents alone or in combination induce long lasting and deep remissions in most patients with CLL, their use may be associated with the development of clinical resistance. In this review, we elucidate the genetic basis of acquired resistance to BTK and Bcl-2 inhibition and present evidence on resistance mechanisms that are not linked to single genomic alterations affecting these target proteins. Strategies to prevent resistance to novel agents are discussed in this review with a special focus on new combination therapies.

Validation of Minimal Residual Disease Flow Cytometry Method for Residual Disease Monitoring in Chronic Lymphocytic Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4196-4196
Author(s):  
Shabnam Tangri ◽  
Annalee Estrellado ◽  
Julie Ranuio ◽  
Elisa Romeo ◽  
Jonathan Lawson ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Light Chain ◽  
Peripheral Blood ◽  
Residual Disease ◽  
Lymphocytic Leukemia ◽  
Normal Cells ◽  
Chain Analysis ◽  
Minimal Residual

Abstract Monitoring Minimal Residual Disease (MRD) of Chronic Lymphocytic Leukemia (CLL) patients who achieved complete remission has been difficult and challenging using flow cytometry detection methods in peripheral blood Earlier flow cytometry methods described for detection of MRD relied on the assessment of CD20 expression that maybe compromised during rituximab therapy (CD5/19/20/79B and light chain analysis) or were done using the classic CLL staining panel (CD5/19/23 and light chain analysis) that had a low sensitivity. To facilitate the development of methods that would be suitable for rituximab containing regimens and have better sensitivity, a panel of antibody combinations was identified for use in an internationally standardized flow cytometric approach for MRD detection in CLL-treated patients (Rawstron et al., 2007). This panel consists of 5 combinations of antibodies specific for 1-sIgKappa/sIgLambda/CD19/CD5, 2-CD45/CD14/CD19/CD5, 3-CD43/CD79b/CD19/CD5, 4-CD22/CD81/CD19/CD5 and 5-CD20/CD38/CD19/CD5 antigens. The first two combinations are utilized for confirmation of clonality and assessment of Tcell contamination rate within the B cell gate, whereas combinations 3, 4 and 5 are specific for MRD detection. This standardized 5-combination panel was technically validated by Genoptix Medical Laboratory for use in BiogenIdec’s clinical trials in CLL with Lumiliximab. To validate this panel assay, selected CLL samples were mixed with normal donor blood or “disease-free” bone marrow specimens, to achieve 1%, 0.1%, 0.05% and 0.01% of CLL cells in “non-CLL” leukocytes. Our results show that it is possible to identify up to 1 CLL cell in 10,000 normal cells in some but not all cases, and that on a consistent basis our analysis is able to identify 1 CLL cell in 1000 normal cells. In comparison, the classic screening panel commonly used to diagnose CLL has a limit of detection (LOD) of about 1% (1 leukemic cell in 100 normal cells); thus this new method provides a 10 to 100 fold improvement in sensitivity. The enhanced sensitivity and LOD of the new assay is mainly due to the reduction of the non-CLL cell background in peripheral blood and bone marrow Furthermore, to make analysis guidelines consistent among analysts, an internal semi-quantitative clustering scoring analysis system was developed for this assay. The cluster scoring scheme assigns a negative score to scattered events with no clearly visible clone; well grouped events with tight visible clones are assigned positive scores and those gated events are considered for MRD final call. To reliably define the presence of MRD, at least 2 of the 3 MRD specific antibody combinations must have a positive clustering score. Due to the small number of cells to be analyzed, collection of 500,000 events is recommended. Finally, the results of additional studies showed that the anticoagulant employed may impact the stability of the antigens over a period of 7 days, and for accurate MRD determination blood specimens should be drawn into Heparin vacutainer tubes when long shipping times are expected.

scholarly journals BTKC481S-Mediated Resistance to Ibrutinib in Chronic Lymphocytic Leukemia

2017 ◽  
Vol 35 (13) ◽  
pp. 1437-1443 ◽  
Author(s):  
Jennifer A. Woyach ◽  
Amy S. Ruppert ◽  
Daphne Guinn ◽  
Amy Lehman ◽  
James S. Blachly ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
Acquired Resistance ◽  
Resistance Mutation ◽  
Lymphocytic Leukemia ◽  
Clinical Relapse ◽  
Resistance Mutations ◽  
Screening Population ◽  
History Of ◽  
Sequential Studies

Purpose Therapeutic targeting of Bruton tyrosine kinase (BTK) with ibrutinib in chronic lymphocytic leukemia has led to a paradigm shift in therapy, and relapse has been uncommon with current follow-up. Acquired mutations in BTK and PLCG2 can cause relapse, but data regarding the prevalence and natural history of these mutations are limited. Patients and Methods Patients accrued to four sequential studies of ibrutinib were included in these analyses. Deep sequencing for BTK and PLCG2 was performed retrospectively on patients who experienced relapse and prospectively on a screening population. Results With a median follow-up time of 3.4 years, the estimated cumulative incidence of progression at 4 years is 19% (95% CI, 14% to 24%). Baseline karyotypic complexity, presence of del(17)(p13.1), and age less than 65 years were risk factors for progression. Among patients who experienced relapse, acquired mutations of BTK or PLCG2 were found in 85% (95% CI, 71% to 94%), and these mutations were detected an estimated median of 9.3 months (95% CI, 7.6 to 11.7 months) before relapse. Of a group of 112 patients examined prospectively, eight patients have experienced relapse, and all of these patients had acquired resistance mutations before relapse. A resistance mutation was detected in an additional eight patients who have not yet met criteria for clinical relapse. Conclusion Relapse of chronic lymphocytic leukemia after ibrutinib is an issue of increasing clinical significance. We show that mutations in BTK and PLCG2 appear early and have the potential to be used as a biomarker for future relapse, suggesting an opportunity for intervention.

scholarly journals Contribution of Flow Cytometry Immunophenotyping in Diagnostic of Acute and Chronic Leukemias

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 5198-5198
Author(s):  
Aldair Sousa Paiva ◽  
Hugo Diogenes De Oliveira Paiva ◽  
Geraldo Barroso Cavalcanti ◽  
Lenilton Silva Silveira ◽  
Lorena KF Silva ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Chronic Lymphocytic Leukemia ◽  
Peripheral Blood ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
Leukemic Cells ◽  
Acute Leukemias ◽  
Cell Chronic Lymphocytic Leukemia ◽  
Bone Morrow

Abstract BACKGROUND: Today immunophenotyping by flow cytometry is an useful adjunct to cytomorphologyc analysis to correct diagnostic of leukemias. It provides objective and quantitative data allowing for a high level of sensitivity detection and better characterization of acute and chronic leukemias. The purpose of this study was to demonstrate the contribution of the immunophenotyping by monoclonal antibodies (Mo.Ab.) in leukemic cells from patients with acute and chronic leukemias. METHODS: Analyzed 76 patients with leukemias before the treatment. The diagnostic of leukemias was based on the morphological and immunophenotyping analysis of leukemic cells from peripheral blood and bone morrow. The cytomorphologycal analysis was based on French - American - Britsh criteria (FAB classification) in blood and bone marrow films stain by leishmann and the immunophenotyping by flow cytometry with a panel of Mo.Ab. specific to acute leukemias as: CD1a, CD2, CD3, CD4, CD5, CD8, CD7, CD10, CD13, CD19, CD20, CD103, CD22, CD33, CD34, CD117, CD38, HLADR, TdT, anti-mieloperoxidase, anti-IgM and anti-kappa and lambda light chain. Further clinical and laboratory information as age, sex, presence of tumoral mass, lymphadenopathy, hepatosplenomegaly, hemoglobin determination, total and specific white cell count and cytomorphological analysis of blood film and bone morrow smear. RESULTS: Thirty four patients presented acute myeloid leukemia (AML), twenty acute lymphoblastic leukemia (ALL), nineteen B-cell chronic lymphocytic leukemia (B-CLL), two T-cell chronic lymphocytic leukemia and one case was hairy cell leukemia (HCL). Males were more frequently found in all types of leukemias. ALL were more observed in children (age < 15 years old) and in AML however were more frequently in adults patients. The chronic leukemias were presented in patients with 50 years old or more in all cases. The correlation between the immunophenotyping and clinical pathological profile of these leukemias demonstrated that ALL were more associated to lymphadenopathy, AML to hepatosplenomegaly, and CLL to lymphadenopathy and high count of white cells in peripheral blood. The thrombocytopenia and anemia were found in more cases of acute leukemia. CONCLUSION: This date suggest that today the immunophenotyping by flow cytometry is an important methodology to diagnostic and classification of leukemias. Disclosures No relevant conflicts of interest to declare.

scholarly journals An Unusual Case of Prolymphocytic Leukemia Transformation in a Patient With Chronic Lymphocytic Leukemia

2021 ◽  
Vol 9 ◽  
pp. 232470962199076 ◽  
Author(s):  
Stephen Bell ◽  
Natalia Lattanzio ◽  
Julaine Braham ◽  
Victoria Campdesuner ◽  
Qassem Abdelal ◽  
...  
Keyword(s):  
Chronic Lymphocytic Leukemia ◽  
White Blood Cells ◽  
Acquired Resistance ◽  
Treatment Strategies ◽  
Surface Protein ◽  
Lymphocytic Leukemia ◽  
Prolymphocytic Leukemia ◽  
First Line Therapy ◽  
Ibrutinib Resistance ◽  
Mature Lymphocyte

B-cell prolymphocytic leukemia (B-PLL) is a rare leukemia characterized by rapidly increasing leukocytosis with splenomegaly and lymphadenopathy. Treatment strategies are largely based on studies of chronic lymphocytic leukemia (CLL). Antibodies against the cell surface protein CD20 are considered to be first-line therapy. A 76-year-old male with known CLL presented 2 weeks after starting chemoimmunotherapy for newly refractory CLL after failing ibrutinib therapy. White blood cell count was elevated at 226.7 × 103/µL. Fluorescent in situ hybridization analysis of a bone marrow specimen showed new development of complex cytogenetics. Flow cytometry revealed B cells appearing slightly dimmer on CD45 and brighter on CD20 compared with typical B-CLL suggestive of less mature lymphocyte forms. The patient was diagnosed with B-PLL and started on obinutuzumab and venetoclax with rapid normalization of white blood cells. This case recapitulates the challenges in diagnosing and treating B-PLL. Ibrutinib resistance is a growing area of study with several proposed mechanisms of acquired resistance. The pathogenesis of B-PLL is not completely understood, although mutations in MYC are presumed to play a role.

scholarly journals Single-Cell RNA-seq Reveals LGALS1 and LAG3 as Novel Drivers of Ibrutinib Resistance in Chronic Lymphocytic Leukemia

2021 ◽  
Author(s):  
Hui Jin ◽  
Bin Huang ◽  
Zijuan Wu ◽  
Huayuan Zhu ◽  
Hanning Tang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Chronic Lymphocytic Leukemia ◽  
Single Cell ◽  
Mononuclear Cells ◽  
Kinase Inhibitor ◽  
Expression Profiles ◽  
Marker Gene ◽  
Lymphocytic Leukemia ◽  
Primary Cells ◽  
Ibrutinib Resistance

Abstract BackgroundChronic lymphocytic leukemia (CLL) is a highly heterogeneous malignant lymphoproliferative B-cell disorder that can be treated using ibrutinib, a Bruton’s tyrosine kinase inhibitor. However, the ibrutinib resistance of CLL patients has caused widespread concerns, necessitating the development of novel treatment strategies. MethodsHere, we identified lectin galactoside-binding soluble 1 (LGALS1) and lymphocyte-activating gene 3 (LAG3) as potential markers for ibrutinib-resistant CLL using single-cell RNA sequencing (scRNA-seq), and the results were validated in an ibrutinib-resistant CLL cell line (MEC1-IR) and primary cells from CLL patients. Marker-gene expression was detected while functional analyses were conducted with or without OTX008, a selective Galectin-1 inhibitor. ScRNA-seq revealed that the biological features, gene expression profiles, and clonal signatures of peripheral blood mononuclear cells (PBMCs) from patients with ibrutinib-resistant CLL were distinct from those displayed by PBMCs from ibrutinib-sensitive patients.ResultsA close correlation between LGALS1 and LAG3 expression was observed and these factors were found to be highly expressed in ibrutinib-resistant CLL, with diagnostic and prognostic stratification, indicating that they may serve as drivers of ibrutinib-resistant CLL. Concordantly, LGALS1 and LAG3 expression was higher in ibrutinib-resistant CLL cells and primary cells, and OTX008 suppressed proliferation and induced apoptosis in both cells.ConclusionLGALS1 and LAG3 gene panel is promising indicator of ibrutinib-sensitivity and prognosis marker of CLL. LGALS1 inhibitor OTX008 is effective in CLL patients, both those naïve to and those resistant to ibrutinib.

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