TKI rotation-induced persistent deep molecular response in multi-resistant blast crisis of Ph+ CML

Abstract

In chronic myeloid leukemia (CML) resistance against one or more BCR-ABL1 tyrosine kinase inhibitors (TKI) remains a clinical challenge. Preclinical data suggest that TKI combinations may overcome resistance. We report on a heavily pre-treated 78 year-old female patient with CML who developed multi-resistant blast crisis with bone marrow fibrosis and a Ph- clone. Treatment with ponatinib resulted in blast cell clearance, decrease in fibrosis, and disappearance of BCR-ABL1, but also in severe thrombocytopenia with bleedings requiring platelet transfusions. We therefore switched from ponatinib to bosutinib. During bosutinib, platelet counts recovered. However, after 6 months, BCR-ABL1 mRNA levels increased to > 1%. Therefore, we ´switched back´ to ponatinib, and this was again followed by disappearance of BCR-ABL1 and a decrease in platelets. During the next 2 years, we applied ponatinib and bosutinib in continuous rotation-cycles and added hydroxyurea in order to suppress all sub-clones and to balance between efficacy and potential side effects following the principle of personalized medicine. With this approach the patient remained in complete molecular response and reached normal blood counts and a normal quality of life without vascular or other side effects. In conclusion, TKI rotation is a novel potent approach to suppress multiple resistant sub-clones and to balance between clinical efficacy and side effects in patients with advanced CML. Clinical trials are now warranted to show that TKI-rotation is in general safe and effective in these patients.

Keywords: BCR-ABL1 mutations; CML; drug resistance; nilotinib; ponatinib.

Conflict of interest statement

CONFLICTS OF INTEREST

PV received honoraria from Novartis, Pfizer, BMS, and Ariad, and research grants from Novartis and Ariad. The authors declare no other conflicts of interest.

Figures

Figure 1. Overview of clinical course and response to TKI rotation therapy

(A) Response to treatment with nilotinib and ponatinib, and influence of TKI therapy on blood counts and BCR-ABL1 mRNA levels: The patient was treated with nilotinib after resistance against dasatinib had been documented. During nilotinib, BCR-ABL1 decreased but did not disappear. After several months, BCR-ABL1 increased again and cytopenia developed. Finally, the patient developed blast crisis and treatment with ponatinib was initiated. In response to ponatinib, BCR-ABL1 mRNA levels decreased, but the patient developed severe thrombocytopenia. (B) Response to treatment with ponatinib/bosutinib-rotation and hydroxyurea (HU): Because of thrombocytopenia, the patient was switched to bosutinib (after a short phase of imatinib-bridging). During bosutinib, platelet counts recovered slowly, but BCR-ABL1 increased again. Therefore, we switched back to ponatinib, and finally decided to apply ponatinib and bosutinib in continuous rotation-cycles together with low-dose HU. Under this therapy, blood counts normalized, the patient entered a stable continuous complete response, and has a normal quality of life without major side effects.

Figure 2. Clonal evolution during TKI therapy as assessed by HUMARA

The HUMARA assay was performed with peripheral blood mononuclear cells in certain time intervals (from 2005 = month 0) as indicated. As assessed by HUMARA, a monoclonal pattern was seen in 2005 (month 0) and at the time of relapse with a Ph-negative clone when bone marrow (BM) fibrosis was detected (months 95–97). However, after successful treatment with dasatinib and nilotinib (months 9–22), a polyclonal pattern was obtained by HUMARA testing.

Figure 3. Drug combination effects in CML cells

(A) KU812 cells (upper panel) and K562 cells (lower panel) were incubated in control medium (Co), with various concentrations of ponatinib or bosutinib, or with a combination of both drugs at a fixed ratio (KU812 at 1:16; K562 at 1:100) at 37°C for 48 hours. Thereafter, 3H-thymidine was added for 16 hours, and uptake of 3H-thymidine was measured in a β-counter. Results are expressed as percent of medium control and represent the mean ± S.D. of quadruplicates. In case of KU812 cells, the drug combination was found to be highly synergistic, whereas in K562 cells, mostly additive effects were obtained. (B) Primary patient-derived blast cells were incubated in control medium (Co) or with increasing concentrations of hydroxyurea (HU), ponatinib, or bosutinib at 37°C and 5% CO2 for 48 hours as indicated. Then, 3H-thymidine uptake was measured. Results are expressed as percent of control and represent the mean ± S.D. of triplicates. (C) KU812 cells were incubated with ponatinib (0.1 nM: ■-■, 0.5 nM: ▲−▲, 1.0 nM: ▼-▼, or control medium: •-•) at 37°C for 4 hours. Then, cells were washed and incubated in control medium (Co) or bosutinib at various concentrations as indicated for another 48 hours. Thereafter, 3H-thymidine uptake was measured. Results are expressed as percent of control and represent the mean ± S.D. of triplicates. (D) KU812 cells were incubated in control medium (Co), ponatinib (0.2 nM), bosutinib (10 nM), or a combination of both drugs at 37°C for 48 hours. Thereafter, the percentage of AnnexinV/PI-positive cells was determined by flow cytometry. Results represent the mean ± S.D. of 3 independent experiments. Asterisk (*): p < 0.05 compared to control.

Figure 4. High capacity drug testing using patient-derived CML blast crisis cells

(A) Nanoliter amounts of BCR-ABL1 TKI and other drugs were pre-seeded at 4 defined concentrations into 384-well microtiter plates using acoustic compound transfer. CML cells (10,000 per well) were automatically plated and incubated with drugs at 37°C for 72 hours. Thereafter, cell viability was determined using Cell-Titer Glo assay. Responses were determined by measuring the percentage of viable cell (relative to control) at the 4 drug concentrations applied. (B) Cells were exposed to the drug combinations ´bosutinib+hydroxyurea´ (upper panel), ´ponatinib+hydroxyurea´ (middle panel), and ´ponatinib+bosutinib´ (lower panel) in the robotic drug testing assay.