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rence of invasive fungal infections, in particular invasive aspergillosis with an unusually high number of cases with central nervous system involvement.3-5 This associ- ation was somewhat unexpected since invasive fungal infections do not belong to the spectrum of infections observed in patients with XLA and patients with chronic lymphocytic leukemia are considered to be at low-risk of invasive aspergillosis.6
It has been known for decades that neutrophils repre- sent the major effectors in the host’s defense mecha- nisms against Aspergillus.7,8 Quantitative and qualitative (e.g. NADPH oxidase component deficiency in chronic granulomatous disease) defects affecting neutrophils are among the most important risk factors for developing invasive aspergillosis.7,9 BTK has been extensively stud- ied in B cells but data on its role in neutrophils are limit- ed. Although previous work indicates that BTK is expressed by neutrophils and plays a role in neutrophil development and function,10 very little is known about the potential effect of BTK or its inhibition on antifungal immunity.11
This prompted us to test the hypothesis that ibrutinib administration might impair neutrophil responses against Aspergillus. We performed in vitro experiments on neutrophils from healthy donors and analyzed neu- trophil functions in patients receiving ibrutinib at differ- ent time points during the course of treatment. Here we show that ibrutinib treatment is associated with multiple defects in neutrophil functions, in particular decreased CD11b surface expression, reduction of reactive oxygen species (ROS) production and impairment of the cells’ capacity to kill Aspergillus fumigatus.
Methods
Patients
Thirty-three patients from three centers treated with ibrutinib for a lymphoid malignancy were enrolled in the study (Table 1). The main indications for ibrutinib treatment were chronic lym- phocytic leukemia (n=23; 72% of patients) and mantle-cell lym- phoma (n=4; 12.5% of patients). Patients receiving corticoid ther- apy or granulocyte colony-stimulating factors were not consid- ered. Neutrophils were collected from patients just before ibruti- nib treatment was initiated (M0; n=23) then approximately 1 month (M1; n=22) and 3 months later (M3; n=18). No patient was neutropenic. Because a complete longitudinal (M0, M1 and M3) follow up could not be obtained in all patients, the sample size varies between experiments and time points. No available data have been subtracted. No cases of invasive aspergillosis were diagnosed throughout the study.
Neutrophil isolation
Neutrophils were isolated using the dextran-Ficoll method, as detailed in the Online Supplement.
Stimulation conditions
Whole-blood samples (500 μL) were stimulated for 2 h at 37°C with either 106 germinating conidia from an Aspergillus fumigatus sensu stricto clinical isolate or 5 ng/mL bacterial lipopolysaccharide (LPS) (Sigma Aldrich) or phosphate-buffered saline (PBS) as a con- trol. Details are provided in the Online Supplement.
Surface molecule expression of neutrophils
For cytometry analysis anti-human antibodies directed against the following human antigens were used: CD11b, CD14, CD15,
Table 1. Characteristics of patients and blood samples included in the study.
Characteristics
Numbers of patients
Mean age in years [range] Male/female sex
Disease: n (%)
Chronic lymphocytic leukemia Mantle-cell lymphoma Other lymphoid malignanciesa
Before ibrutinib therapy
23
69.9 [54-85] 13/10
19
2
2
Month 1
22
66.9 [41-86] 14/8
18
3
1
Month 3
18
66.1 [41-84] 11/7
14
2
2
Dosage of ibrutinib (mg/day)
Number of therapeutic lines before ibrutinib: mean [range]
1.8 [1-6] 1.8 [1-6] 1.8 [1-6] 765
280 12 420 not applicable 17 13 560 43
Last therapeutic line before ibrutinib administration
Delay between the last therapeutic line and the start of ibrutinib therapy
Time to ibrutinib initiation: mean / median [range] (days)
Neutrophil count: mean (cells/μL) [range] Lymphocytes count: mean (cells/μL) [range]
RFCb
R BENDAc
R CHOPd RMTXARA-Ce Others
<6months
6 months - 12 months 12 months - 60 months
8 1 2 5
6 3 1 6
5 1 9 7
34.2 / 34 [18-62] 4 945 [120-13 800]
45 426 [400-148 226]
3 1 1 8
4 1 5 8
105 / 97 [76-161] 3 145 [390-6 470]
37 195 [1 200-122 930]
4 0 12 >60months 7
not applicable
5 388 [1 180-15 200]
22 890 [590-57 700]
Ibrutinib impairs anti-Aspergillus responses
aOtherlymphoidmalignanciesincludedprimarycentralnervoussystemlymphoma(n=2),diffuselargeB-celllymphoma(n=1), intraocularlymphoma(n=1)andWaldenström macroglobulinemia; bRFC: rituximab-fludarabine-cyclophosphamide; cR BENDA: rituximab plus bendamustine; dR CHOP: rituximab plus cyclophosphamide, doxorubicin, vin- cristine, and prednisone or prednisolone; eR MTX ARA-C: rituximab plus methotrexate and cytarabine.
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