Bcl-2 Family (Bcl-2, Mcl-1, Bim and Bax) and Btk Expression in B-Cell Chronic Lymphocytic Leukemia (B-CLL) Lymphocytes in Different Lymphoid Compartments and Changes during Btk Inhibitor Therapies (ibrutinib or acalabrutinib)

B-cell chronic lymphocytic leukemia (B-CLL) is characterized with variable infiltration by B -CLL lymphocytes of various lymphoid compartments, i.e peripheral blood (PB), bone marrow (BM) and lymph nodes (LN) and lymphoid organs. Interactions with various microenvironments may result in different disease activity and resistance to apoptosis. Novel agents targeting Btk and Bcl-2 show remarkable activity in B-CLL. However, they may have different activity in different lymphoid compartments while Btk inhibitors also may cause significant B-CLL lymphocyte redistribution between compartments.

Aim of this study was to evaluate: 1) is there a different expression of Bcl-2 family of anti- and pro-apoptotic proteins (Bcl-2, Bax, Bim, mcl-1) and Btk in B-CLL lymphocytes from different lymphoid compartments; 2) relationship of observed expressions to disease parameters (TTM, TD, stage, B2MG, LDH); 3) changes during treatment with Btk inhibitors.

We have included 28 B-CLL patients (18/10 M/F, median age 71 yr, range 48-85) treated with Btk inhibitors (ibrutinib 24, acalabrutinib 4). There were 15 previously treated patients and 13 treatment naïve patients. All evaluated patients had samples from PB and BM before treatment while 19 patients had also LN samples. At least one follow-up sample from PB had 20 patients. Median TTM score was 10.2 (range 3.4-23.9) and TD score was 0.68. Rai stage III/IV had 8 patients. Expression of Bcl-2, Mcl-1, Bim, Bax and Btk was determined by flow cytometry in CD19+CD5+ lymphocytes from fresh samples taken simultaneously by venipuncture (PB) or fine needle aspiration (BM and LN).

There is higher expression of Bcl-2, Mcl-1 in LN compared to PB and BM (p<0.05), while there is no change in follow-up PB during ibrutinib/acalbrutinib treatment. There is no significant difference in Bim expression between compartments, while there is trend for higher expression in PB follow-up sample (p=0.054). There is no significant difference in Bax expression between PB, BM and LN compartments, while there is significant drop in Bax expression in PB follow-up sample during treatment (p<0.05). p-Btk has higher expression in LN compared to PB and BM (p<0.05). Follow-up PB p-Btk expression is comparable to pretreatment expression in PB (despite redistribution). We haven't found significant correlation of Bcl-2 family expression and p-Btk expression to the parameters of tumor mass (TTM) and tumor distribution (TD).

In conclusion, there is a different pattern of expression of anti-apoptotic and pro-apoptotic Bcl-2 family members and Btk between lymphoid compartments. Our results may indicate higher BCR signaling and higher antiapoptotic activity in B-CLL lymphocytes in lymph node microenvironment. With redistribution during Btk inhibitor treatment B-CLL lymphocytes assume PB phenotype with downregulation of Btk and anti-apoptotic Bcl-2 and Mcl-1 proteins. Downregulation of pro-apoptotic Bax and possible upregulation of Bim protein indicate that there may be more complex interplay between various proteins resulting in different disease course. These results again emphasize the role of LN microenvironment in pathogenesis of B-CLL and may provide insights for treatment strategies combining targeted agents (Btk inhibitors and Bcl-2 antagonists).

Jaksic:Astra Zeneca: Consultancy, Speakers Bureau; Abbvie: Speakers Bureau; Johnson and Johnson: Speakers Bureau. Ivic:Johnson and johnson: Speakers Bureau; Abbvie: Speakers Bureau. Mitrovic:Abbvie: Speakers Bureau; Johnson ąnd Johnson: Speakers Bureau. Pirsic:Johnson and Johnson: Speakers Bureau. Pejsa:Johnson and Johnson: Speakers Bureau; Abbvie: Speakers Bureau; Astra Zeneca: Speakers Bureau. Kusec:Abbvie: Consultancy, Speakers Bureau.