Nilotinib-induced vasculopathy: identification of vascular endothelial cells as a primary target site.

The BCR/ABL1 inhibitor Nilotinib is increasingly used to treat patients with chronic myeloid leukemia (CML). Although otherwise well-tolerated, Nilotinib has been associated with the occurrence of progressive arterial occlusive disease (AOD). Our objective was to determine the exact frequency of AOD and examine in vitro and in vivo effects of Nilotinib and Imatinib on endothelial cells to explain AOD-development. In contrast to Imatinib, Nilotinib was found to upregulate pro-atherogenic adhesion-proteins (ICAM-1, E-selectin, VCAM-1) on human endothelial cells. Nilotinib also suppressed endothelial cell proliferation, migration and tube-formation and bound to a distinct set of target-kinases, relevant to angiogenesis and atherosclerosis, including angiopoietin receptor-1 TEK, ABL-2, JAK1 and MAP-kinases. Nilotinib and siRNA against ABL-2 also suppressed KDR expression. In addition, Nilotinib augmented atherosclerosis in ApoE-/- mice and blocked reperfusion and angiogenesis in a hindlimb-ischemia model of arterial occlusion, whereas Imatinib showed no comparable effects. Clinically overt AOD-events were found to accumulate over time in Nilotinib-treated patients. After a median observation-time of 2.0 years, the AOD-frequency was higher in these patients (29.4%) compared to risk factor- and age-matched controls (<5%). Together, Nilotinib exerts direct pro-atherogenic and anti-angiogenic effects on vascular endothelial cells, which may contribute to development of AOD in patients with CML.

Next-generation sequencing identifies major DNA methylation changes during progression of Ph+ chronic myeloid leukemia.

Little is known about the impact of DNA methylation on the evolution/progression of Ph+ chronic myeloid leukemia (CML). We investigated the methylome of CML patients in chronic phase (CP-CML), accelerated phase (AP-CML) and blast crisis (BC-CML) as well as in controls by reduced representation bisulfite sequencing. Although only ~600 differentially methylated CpG sites were identified in samples obtained from CP-CML patients compared with controls, ~6500 differentially methylated CpG sites were found in samples from BC-CML patients. In the majority of affected CpG sites, methylation was increased. In CP-CML patients who progressed to AP-CML/BC-CML, we identified up to 897 genes that were methylated at the time of progression but not at the time of diagnosis. Using RNA-sequencing, we observed downregulated expression of many of these genes in BC-CML compared with CP-CML samples. Several of them are well-known tumor-suppressor genes or regulators of cell proliferation, and gene re-expression was observed by the use of epigenetic active drugs. Together, our results demonstrate that CpG site methylation clearly increases during CML progression and that it may provide a useful basis for revealing new targets of therapy in advanced CML.

Quantitative monitoring of BCR/ABL1 mutants for surveillance of subclone-evolution, -expansion, and -depletion in chronic myeloid leukaemia.

BACKGROUND: In chronic myeloid leukaemia (CML), clonal evolution with resistance to tyrosine kinase inhibitors (TKIs) is often triggered by BCR/ABL1 mutations. However, in the context of the complex pro-oncogenic signalling networks which ultimately lead to clonal expansion and disease progression, the exact contribution of BCR/ABL1 mutants remains uncertain. Recent data indicate that detection of BCR/ABL1 mutant subclones does not permit prediction of their expansion dynamics and their potential to become drivers of resistant disease. METHODS: To determine the patterns of clonal evolution and the distinct proliferation kinetics of individual BCR/ABL1 mutants during treatment, we employed ligase-dependent polymerase chain reaction (LD-PCR) analysis for quantitative surveillance of CML subclones with various tyrosine kinase domain (TKD) mutations including M244V, L248V, G250E, E255K, T315I, F317L-A/G, M351T and F359V. FINDINGS: Inadequate treatment responses were observed in 27 of 100 patients investigated and 16 were found to bear one or more BCR/ABL1 TKD mutations in separate subclones. Rapid subclone expansion upon onset or switch of TKI treatment was common and sometimes preceded corresponding changes in BCR/ABL1 transcript levels. Mutant subclones were found to respond differentially and sometimes unexpectedly to various treatment modalities. Decline and persistent depletion of specific mutation-bearing subclones in response to treatment could be documented by LD-PCR surveillance. INTERPRETATION: The observations show that quantitative monitoring of mutant BCR/ABL1 subclones by LD-PCR is a powerful tool for detection of clonal evolution, subclone-expansion and subclone-depletion and can contribute to optimised management of patients with CML.

Immunosuppression and atypical infections in CML patients treated with dasatinib at 140 mg daily.

BACKGROUND: The multikinase inhibitor dasatinib exerts growth-inhibitory effects in patients with imatinib-resistant chronic myeloid leukaemia (CML). In first clinical trials, side effects of dasatinib, 140 mg daily, were reported to be mild and tolerable. PATIENTS AND METHODS: We examined the side effect profile in 16 patients with imatinib-resistant CML who received 140 mg dasatinib daily in our center. RESULTS: Dasatinib produced substantial and sometimes severe or even life-threatening side effects with > or = 10% body weight loss (6/16 patients), pleural effusions grade II or higher (12/16) and infectious complications (12/16), including atypical infections not seen in imatinib-treated patients. One patient developed Epstein-Barr-Virus-positive mucosal leucoplakia, one died from pneumonia caused by pneumocystis carinii and three patients developed a skin-cancer. Most events were recorded within the first 2 years of therapy, only skin tumours developed after the second year. In ex vivo experiments performed in dasatinib-treated patients, transient suppression of IgE-dependent activation of blood basophils and TcR-dependent activation of T-lymphocytes was found. Moreover, in drug-binding studies, dasatinib was found to bind to several key kinase-targets of the immune system including Lyn and Btk, in mast cell, basophil, B-cell and T-cell lines. CONCLUSION: Dasatinib acts not only anti-neoplastic in CML but may also act as an immunosuppressive agent when applied at 140 mg daily, and produces frequent pleural effusions and weight loss in advanced CML.

Clinical and prognostic significance of histamine monitoring in patients with CML during treatment with imatinib (STI571).

BACKGROUND: Although imatinib is highly effective in chronic myeloid leukemia (CML), drug-resistance may occur. Therefore, monitoring of minimal residual disease (MRD) during treatment with imatinib is important. However, most MRD-parameters are expensive and require special technology. We determined the value of histamine as MRD-marker in CML. PATIENTS AND METHODS: Histamine levels were measured serially in whole blood samples before and during imatinib therapy in 80 CML patients by radioimmunoassay. RESULTS: Histamine levels were highly upregulated in CML at diagnosis compared to healthy controls, and correlated with the presence of basophils. During treatment with imatinib, histamine levels decreased and returned to normal levels in those achieving a complete cytogenetic response (CCR). Loss of CCR during therapy was invariably accompanied by an increase in histamine. Moreover, a histamine level of >100 ng/ml three or six months after start of imatinib was associated with a significantly reduced probability of survival (p<0.05). Whereas basophils were found to correlate well with histamine during imatinib, no correlations were found between histamine and Ph+ metaphases or histamine and BCR/ABL. CONCLUSION: Histamine-monitoring during treatment with imatinib is of prognostic significance.

Liposomal cytarabine for treatment of myeloid central nervous system relapse in chronic myeloid leukaemia occurring during imatinib therapy.

BACKGROUND: Central nervous system (CNS) relapse in chronic myeloid leukaemia (CML) is rare and if recorded is usually found to occur in patients with lymphoblastic transformation. The BCR/ABL tyrosine kinase inhibitor imatinib is highly effective in patients with CML, but hardly crosses the blood-brain barrier. PATIENTS AND METHODS: We report on two CML patients who developed a myeloid CNS relapse during treatment with imatinib. One patient was in major cytogenetic response at the time of CNS relapse. In both cases, the myeloid origin of neoplastic cells in the cerebrospinal fluid (CSF) was demonstrable by immunophenotyping, and their leukaemic origin by detection of the BCR/ABL oncoprotein. No BCR/ABL kinase domain mutations were found. Both patients received intrathecal liposomal cytarabine (50 mg each cycle; 6 cycles). In one patient, additional CNS radiation was performed, whereas in the other, consecutive treatment with dasatinib (70 mg per os twice daily) was started. RESULTS: In response to therapy, the clinical symptoms resolved, and the leukaemic cells in the CSF disappeared in both cases. After three months of observation, both patients are in complete cytogenetic and major molecular response, without evidence for a systemic or a CNS relapse. CONCLUSIONS: 'Anatomic' resistance against imatinib in the CNS can lead to a myeloid CNS relapse. Liposomal cytarabine with or without radiation is effective as local therapy in these patients. For systemic treatment and prophylaxis, BCR/ABL kinase inhibitors crossing the blood-brain barrier such as dasatinib should be considered in patients with CNS relapse.