Revealing a Phenotypical Appearance of Ibrutinib Resistance in Patients With Chronic Lymphocytic Leukaemia by Flow Cytometry

Abstract

Background: Ibrutinib is widely known as an effective and well-tolerated therapeutical choice of the chronic lymphocytic leukaemia (CLL). However, acquired resistance may occur during the treatment, causing relapse. Early detection of ibrutinib resistance is an important issue, therefore we aimed to find phenotypic markers on CLL cells the expression of which may correlate with the appearance of ibrutinib resistance. Methods: We examined 28 patients' peripheral blood (PB) samples (treatment naïve, ibrutinib sensitive, clinically ibrutinib resistant). The surface markers' expression (CD27, CD69, CD86, CD184, CD185) were measured by flow cytometry. Furthermore, the BTK^C481S resistance mutation was assessed by digital droplet PCR. Moreover, the CLL cells' phenotype of a patient with acquired ibrutinib resistance was observed during the ibrutinib treatment. Results: The expression of CD27 (p = 0.030) and CD86 (p = 0.031) became higher in the clinically resistant cohort than in the ibrutinib sensitive cohort. Besides, we found that high CD86 and CD27 expressions were accompanied by BTK^C481S mutation. Our prospective study showed that the increase of the expression of CD27, CD69 and CD86 was noticed ahead of the clinical resistance with 3 months. Conclusion: Our study suggests that the changes of the expression of these markers could indicate ibrutinib resistance and the examination of these phenotypic changes may become a part of the patients' follow-up in the future.

Keywords: chronic lymphocytic leukaemia; drug resistance; flow cytometry; ibrutinib; targeted therapy.

FIGURE 1

The immunophenotype of CLL cells in different treatment cohorts. The expression level of five different surface markers (CD69, CD184, CD27, CD86, CD185) in three different cohorts (treatment naïve n = 10, ibrutinib sensitive n = 7, ibrutinib resistant n = 11) were measured by flow cytometry. Relative median fluorescent intensity (MFI) values were calculated as the difference between the MFI value of internal negative controls and B-cells. ANOVA or Kruskal-Wallis test with Holm-Sidak post hoc test were performed for statistical evaluation. *p < 0.05; **p < 0.01.

FIGURE 2

The change of CD27, CD69 and CD86 expression during ibrutinib treatment. Relative expression levels of three markers (CD27 (black line), CD69 (dotted line) and CD86 (dashed line)) and the CLL ratio (grey line) were analysed during ibrutinib treatment in a patient’s peripheral blood sample who ultimately became ibrutinib resistant (Clin. res.). We used the initial expression levels (day 0) of the CD27, CD69 and CD86 as a benchmark. The time point when the tendency has changed level marked by a vertical dotted line. When the markers’ expression exceeded the benchmark level marked by a vertical dashed line.

FIGURE 3

Phenotypic difference between BTK ^C481S mutated and wild type CLL samples. The relative expression of CD27, CD69 and CD86 surface markers and the BTK ^C481S mutation status in the clinically ibrutinib-resistant patients (n = 11) were compared. T-test or Mann-Whitney-test were performed for statistical evaluation. *p < 0.05; **p < 0.01.