Nilotinib and Dasatinib Evoked Distinct Immune Effects during Tfr Phase of Each Discontinuation Trial
Authors: Fujioka Yuki , Takahashi Naoto , Ohtake Shigeki , Atsuta Yoshiko , Minami Yosuke , Iriyama Noriyoshi , Kiyoi Hitoshi , Miyazaki Yasushi , Matsumura Itaru 
Published: November 2021

Abstract
Introduction: Tyrosine kinase inhibitor (TKI) has dramatically improved the prognosis of chronic myeloid leukemia (CML) patients. Recently, many trials of TKI discontinuation revealed that approximately 40% to 60% of CML patients who received long time TKI therapy reached the treatment-free remission (TFR), thus now TFR is proposed as one of the goals in CML treatment. Achieving deep molecular response (DMR) by TKI therapy is a minimum requirement of a challenge to TKI discontinuation in CML patient, actually CML patients with molecular residual disease (MRD) showed worse consequence than undetectable MRD (IJH, 2017). On the other hand, it has been known that some patients sustain a molecular response for a long time despite BCR-ABL fusion gene positivity in their peripheral blood. We hypothesized that the residual malignant cells were eliminated by host immune systems in the patients with continuous TFR. Practically, we obtained the data that the immune responses of the patients with continuous TFR were more activated than relapsed patients in the discontinuation trial of imatinib (ASH, 2019). Here, we reported immune effects before and after TFR phase in two Japanese discontinuation trials of second-generation TKIs (JALSG N-STOP216 and D-STOP216).
Methods: Japanese patients with CML-CP treated with nilotinib or dasatinib as the first line for at least three years and confirmed in DMR for at least two years were eligible. Patients who received other TKI or stem cell transplantations were excluded. Patients were re-confirmed in MR4.5 before discontinuing TKI and they were sampled peripheral blood at pre- and 1, 3 months after stopping TKIs and after retreatment (figure 1). Peripheral blood mononuclear cells (PBMCs) were subjected to staining with immune markers and analyzed by flow cytometry.
Results: 51 patients treated with nilotinib and 49 patients treated with dasatinib were assessed clinical outcomes. At 12 months, 39/51 patients (76.5%) and 27/49 patients (55.1%) remained TFR in N-STOP216 and D-STOP216 respectively (EHA, 2021). For immunological analysis, 48 patients and 43 patients were analyzed by flowcytometry. We classified the patient of each trial into two groups (TFR group and Retreatment (RET) group) (figure 1). The frequency of CD4 + T cells and CD8 + T cells in CD3 + T cells was not different between both groups in each trial. The frequency of FoxP3 +CD4 + regulatory T (Treg) cells were not different between both groups in each trial. However, the kinetics of Treg cells, especially effector Treg (eTreg; FoxP3 hiCD45RA -) cells from Pre-stopping dasatinib to 1 month after stopping dasatinib, significantly increased in TFR groups but not in RET group (figure 2). This difference in kinetics of eTreg cells was not observed in nilotinib discontinuation trial (figure 2). In N-STOP216 trial, there were no differences between TFR and RET groups in the immuno-phenotype of CD8 + T cells, NK cells, or B cells, while granulocytic myeloid-derived suppressor cells (G-MDSCs) increased at 1 month after stopping nilotinib in RET group (0.27% in RET group vs. 0.08% in TFR group, P=0.011) (figure 3). On the other hand, in D-STOP216, CD8 + T cells from the patients with continuous TFR showed less exhausted phenotype (PD-1 +CD8 +T cells 37.4% in RET group vs. 49.8% in TFR group, p=0.018) and more proliferative activity (Ki67+CD8 cells 5.03% in RET group vs. 8.91% in TFR group, p=0.043) compared with RET group at 1 month after stopping dasatinib (figure 4). There were no differences in NK cells, B cells, and MDSCs between TFR and RET groups in D-STOP216.
Conclusion: We found that each TKI evoked different immune responses after stopping TKI. Nilotinib has been developed as higher specific for BCR-ABL, therefore immune cells were not affected because of its narrower off-target effect. MDSCs developed by hematopoietic stem cells with BCR-ABL were decreased by the direct effect of nilotinib in the patients with continuous TFR. Antitumor immune responses might be diminished by residual MDSCs in RET group consequently it would be a molecular relapse. On the other hand, dasatinib has different immune effects probably due to its broad-spectrum kinase inhibitory effects. T cell immune responses might be exhausted by long term exposure to dasatinib in RET group. This study showed the possibility of different mechanisms of relapse caused by distinct immune effects.
Figure 1 Figure 1.

Disclosures
Takahashi: Kyowahakko-Kirin: Research Funding; Ono: Research Funding; Asahikasei: Research Funding; Toyamakagaku: Research Funding; Eizai: Research Funding; Chugai: Research Funding; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau. Atsuta: Astellas Pharma Inc.: Speakers Bureau; Mochida Pharmaceutical Co., Ltd.: Speakers Bureau; Meiji Seika Pharma Co, Ltd.: Honoraria; AbbVie GK: Speakers Bureau; Kyowa Kirin Co., Ltd: Honoraria. Minami: Bristol-Myers Squibb Company: Honoraria; Novartis Pharma KK: Honoraria; Pfizer Japan Inc.: Honoraria; Takeda: Honoraria; Astellas: Honoraria; Ono: Research Funding; CMIC: Research Funding. Iriyama: Bristol Myers Squibb: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Pfizer: Honoraria, Speakers Bureau; Otsuka: Honoraria, Speakers Bureau. Kiyoi: Kyowa Kirin: Honoraria; Fijifilm: Honoraria; Eisai: Honoraria; Dainippon Sumitomo: Honoraria; Daiichi Sankyo: Honoraria; celgene: Honoraria; Astellas: Honoraria; Otsuka: Honoraria; Perseus Proteomics: Honoraria; Pfizer: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Takeda: Honoraria; Zenyaku Kogyo: Honoraria. Miyazaki: Novartis: Honoraria; Abbvie: Honoraria; Kyowa-Kirin: Honoraria; Nippon-Shinyaku: Honoraria; Chugai: Honoraria; Bristol-Myers Squibb: Honoraria; Pfizer: Honoraria; Sumitomo-Dainippon: Honoraria, Research Funding; Janssen: Honoraria; Eisai: Honoraria; Daiichi-Sankyo: Honoraria; Astellas: Honoraria; Takeda: Honoraria; Sanofi: Honoraria. Matsumura: Ono: Research Funding; Otsuka: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Shionogi: Research Funding; Taiho: Research Funding; Takeda: Research Funding; Sumitomo Dainippon: Research Funding; Nihon Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Japan Blood Products Organization: Research Funding; Mundipharma: Research Funding; AYUMI Pharmaceutical: Research Funding; Eli Lilly Japan: Research Funding; Novartis: Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding; MSD: Research Funding; Mitsubishi Tanabe: Research Funding; Amgen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Kyowa Kirin: Research Funding; Eisai: Research Funding; Chugai: Research Funding; Astellas: Speakers Bureau; Asahi Kasei: Research Funding; Addvie: Research Funding.