INTERFERON-ΑLPHA REVISITED: INDIVIDUALIZED IMMUNE-MODULATION EASED UP SELECTION PRESSURE OF TKI ON BCR-ABL MUTATED CLONES AND IMPROVES MOLECULAR RESPONSE IN HIGH-RISK CML PATIENTS

Background
CML patients in whom the tyrosine kinase inhibitor (TKI) therapy (imatinib, dasatinib, nilotinib) fails due to T315I BCR-ABL kinase domain mutation, should be switched to ponatinib or for bone marrow transplantation. However, in individual cases these treatment scenarios cannot be applied. We used treatment with interferon-α (IFN, given solo, sequentially or together with TKI) assuming that TKI-independent mechanism of action may lead to T315I mutant clone repression.  

Aims
To study an effect of IFN-based treatment on mutated BCR-ABL transcript level and overall response of CML patients who failed on TKIs. In addition to that to assess the role of the sensitive measurement of BCR-ABL mutant mRNA load dynamics and immune response induced by IFN in individualized treatment.

Methods
Six CML patients who had failed on TKIs due to T315I or other highly resistant mutational profile have been treated with individual IFN-based treatment strategies. Kinase domain mutations in cDNA of BCR-ABL were analyzed in total 54 samples (median 6 samples of each patient, range 5-15) of peripheral blood leukocytes using next-generation deep sequencing (GS Junior, Roche Applied Science). The T315I clone reactivity on lowering imatinib concentration (2.0, 1.0, 0.4 and 0 µM imatinib) was studied in vitro on KCL22-R cell line resistant to 4.0 µM imatinib with 45% of BCR-ABL T315I. KCL22-R were incubated in each imatinib concentration for 14 days and checked for mutation. The basic immune-profile was analyzed in 5/6 CML patients using flow-cytometry detecting populations of CD4+, CD8+, regulatory T-cells (Tregs) and  NK cells in specific time points (e.g. before IFN initiation,  during IFN-based treatment). 

Results

The switch of TKI to IFN-based treatment resulted in decreasing of mutated BCR-ABL mRNAs to undetectable levels in 4/6 patients. Three of those patients achieved major molecular response (MMR). IFN has been stopped, based on non-mutated BCR-ABL transcripts detection, continuing with solo nilotinib in 1/4 patient who achieved complete cytogenetic remission (CCgR). One case of MMR responding patient on solo IFN treatment showed decreased levels of CD3+ T-lymphocytes and elevated levels of NK cells. Other two patients who achieved MMR after IFN expressed elevated levels of NK cells representing probably an immune activation on dasatinib pre-treatment following with restoring immunological surveillance after application of combined treatment IFN with TKI. In 2/6 cases TKI cessation and IFN introduction did not contribute to the BCR-ABL mutations reduction and response improvement.

No changes in the BCR-ABL T315I transcript levels have been observed after 14 days of incubation of KCL22-R upon easement of selective pressure of imatinib. We assume that longer time of incubation, which is ongoing, would be necessary to follow-up any changes. 

Summary
In this work we showed that CML patients with TKI multi-resistant mutation T315I or other highly resistant BCR-ABL mutations may achieve CCgR or MMR thanks to IFN-based individualized treatment. These achievements may relate to immune-mediated anti-leukemic effects in this specific group of patients resulting in BCR-ABL clones repression. The sensitive measurement of mutated BCR-ABL transcript levels augments safety of this individualized treatment strategy.

 

Supported by IGA NT1389, by the project for conceptual development of research organization no. 00023736 of MZCR and IGA LF UP 01-2015.

Keyword(s): BCR-ABL, Chronic myeloid leukemia, Interferon alpha, Kinase domain mutant

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