Giulia Fontanelli

The c.480C>G polymorphism of hOCT1 influences imatinib clearance in patients affected by chronic myeloid leukemia.

The aim of the study was to investigate any possible influence of polymorphisms of transmembrane transporters human organic cation transporter 1 (hOCT1), ABCB1, ABCG2 on imatinib pharmacokinetics in 33 men and 27 women (median age and range, 56 and 27–79 years, respectively) affected by chronic myeloid leukemia. A population pharmacokinetic analysis was performed to investigate imatinib disposition in every patient and the role of transporter polymorphisms. Results showed that the α1-acid glycoprotein and the c.480C>G genotype of hOCT1 had a significant effect on apparent drug clearance (CL/F) being responsible, respectively, for a 20% and 10% decrease in interindividual variability (IIV) of CL/F (from 50.1 up to 19.6%). Interestingly, 25 patients carrying at least one polymorphic c.480 G allele had a significant lower CL/F value with respect to the 35 c.480CC individuals (mean±s.d., 9.6±1.6 vs 12.1±2.3 l h−1, respectively; P<0.001). In conclusion, the hOCT1 c.480C>G SNP may significantly influence imatinib pharmacokinetics, supporting further analyses in larger groups of patients.

Real-Time PCR and Droplet Digital PCR: two techniques for detection of the JAK2(V617F) mutation in Philadelphia-negative chronic myeloproliferative neoplasms.

Philadelphia‐negative chronic myeloproliferative neoplasms (MPNs) are clonal disorders that present JAK2V617F mutation in 50–95% of cases. The main objective of this study was the comparison of two PCR methods, real‐time (qPCR) and droplet digital PCR (DD‐PCR) for detection of the JAK2V617F mutation, to assess analytic sensitivity, specificity, and feasibility of the two methods.

Antileukemic activity of sulforaphane in primary blasts from patients affected by myelo- and lympho-proliferative disorders and in hypoxic conditions.

Sulforaphane is a dietary isothiocyanate found in cruciferous vegetables showing antileukemic activity. With the purpose of extending the potential clinical impact of sulforaphane in the oncological field, we investigated the antileukemic effect of sulforaphane on blasts from patients affected by different types of leukemia and, taking into account the intrinsically hypoxic nature of bone marrow, on a leukemia cell line (REH) maintained in hypoxic conditions. In particular, we tested sulforaphane on patients with chronic lymphocytic leukemia, acute myeloid leukemia, T-cell acute lymphoblastic leukemia, B-cell acute lymphoblastic leukemia, and blastic NK cell leukemia. Sulforaphane caused a dose-dependent induction of apoptosis in blasts from patients diagnosed with acute lymphoblastic or myeloid leukemia. Moreover, it was able to cause apoptosis and to inhibit proliferation in hypoxic conditions on REH cells. As to its cytotoxic mechanism, we found that sulforaphane creates an oxidative cellular environment that induces DNA damage and Bax and p53 gene activation, which in turn helps trigger apoptosis. On the whole, our results raise hopes that sulforaphane might set the stage for a novel therapeutic principle complementing our growing armature against malignancies and advocate the exploration of sulforaphane in a broader population of leukemic patients.

Genetic predisposition and induced pro-inflammatory/pro-oxidative status may play a role in increased atherothrombotic events in nilotinib treated chronic myeloid leukemia patients

Several reports described an increased risk of cardiovascular (CV) events, mainly atherothrombotic, in Chronic Myeloid Leukemia (CML) patients receiving nilotinib. However, the underlying mechanism remains elusive. The objective of the current cross-sectional retrospective study is to address a potential correlation between Tyrosine Kinase Inhibitors (TKIs) treatment and CV events. One hundred and 10 chronic phase CML patients in complete cytogenetic response during nilotinib or imatinib, were screened for CV events and evaluated for: traditional CV risk factors, pro/anti-inflammatory biochemical parameters and detrimental ORL1 gene polymorphisms (encoding for altered oxidized LDL receptor-1). Multivariate analysis of the whole cohort showed that the cluster of co-existing nilotinib treatment, dyslipidaemia and G allele of LOX-1 polymorphism was the only significant finding associated with CV events. Furthermore, multivariate analysis according to TKI treatment confirmed IVS4-14 G/G LOX-1 polymorphism as the strongest predictive factor for a higher incidence of CV events in nilotinib patients. Biochemical assessment showed an unbalanced pro-inflammatory cytokines network in nilotinib vs imatinib patients. Surprisingly, pre-existing traditional CV risk factors were not always predictive of CV events. We believe that in nilotinib patients an induced “inflammatory/oxidative status”, together with a genetic pro-atherothrombotic predisposition, may favour the increased incidence of CV events. Prospective studies focused on this issue are ongoing.

Polycomb genes are associated with response to imatinib in chronic myeloid leukemia.

AIM Imatinib is a tyrosine kinase inhibitor that has revolutionized the treatment of chronic myeloid leukemia (CML). Despite its efficacy, about a third of patients discontinue the treatment due to therapy failure or intolerance. The rational identification of patients less likely to respond to imatinib would be of paramount clinical relevance. We have shown that transmembrane transporter hOCT1 genotyping predicts imatinib activity. In parallel, Polycomb group genes (PcGs) are epigenetic repressors implicated in CML progression and in therapy resistance. PATIENTS & METHODS We measured the expression of eight PcGs in paired pre- and post-imatinib bone marrow samples from 30 CML patients. RESULTS BMI1, PHC3, CBX6 and CBX7 expression was significantly increased during imatinib treatment. Post-treatment levels of CBX6 and CBX7 predicted 3-month response rate. Measurement of post-treatment BMI1 levels improved the predictive power of hOCT1 genotyping. CONCLUSION These results suggest that the expression levels of PcGs might be useful for a more accurate risk stratification of CML patients.

Association of the hOCT1/ABCB1 genotype with efficacy and tolerability of imatinib in patients affected by chronic myeloid leukemia

PurposeThe present study was aimed at investigating whether imatinib pharmacogenetics is related to its pharmacodynamics in patients affected by chronic myeloid leukemia.MethodsThrough a procedure based on a sequence of classical statistics methods, we investigated the possible relationships between treatment efficacy/tolerability and combinations of time-independent variables as gender and genetic covariates in the form of single nucleotide polymorphisms (SNPs) or combinations thereof. Moreover, since the drug tolerability has a strong incidence on the discontinuation of the therapy, we investigated whether the time of manifestation of the most frequent toxic effects can be related to time-independent patients’ characteristics or not.ResultsWe found that a combination of two polymorphisms, namely hOCT1 c.480C>G (rs683369) and ABCB1 c.3435C>T (rs1045642), seems to play the role of predictor for imatinib in both efficacy and toxicity. Furthermore, the time of manifestation of edema toxicity is found to be associated to a combination of gender and ABCB1 c.3435C>T, whereas the time of manifestation of cramp toxicity appears related to gender.ConclusionsThe novelty of this study is dual: the achievement of results that potentially have a significant clinical interest and the demonstration that the adoption of composed covariates may represent a unique tool to study different aspects of the treatment with imatinib.

The hOCT1 and ABCB1 polymorphisms do not influence the pharmacodynamics of nilotinib in chronic myeloid leukemia

First-line nilotinib in chronic myeloid leukemia is more effective than imatinib to achieve early and deep molecular responses, despite poor tolerability or failure observed in one-third of patients. The toxicity and efficacy of tyrosine kinase inhibitors might depend on the activity of transmembrane transporters. However, the impact of transporters genes polymorphisms in nilotinib setting is still debated. We investigated the possible correlation between single nucleotide polymorphisms of hOCT1 (rs683369 [c.480C>G]) and ABCB1 (rs1128503 [c.1236C>T], rs2032582 [c.2677G>T/A], rs1045642 [c.3435C>T]) and nilotinib efficacy and toxicity in a cohort of 78 patients affected by chronic myeloid leukemia in the context of current clinical practice. The early molecular response was achieved by 81% of patients while 64% of them attained deep molecular response (median time, 26 months). The 36-month event-free survival was 86%, whereas 58% of patients experienced toxicities. Interestingly, hOCT1 and ABCB1 polymorphisms alone or in combination did not influence event-free survival or the adverse events rate. Therefore, in contrast to data obtained in patients treated with imatinib, hOCT1 and ABCB1 polymorphisms do not impact on nilotinib efficacy or toxicity. This could be relevant in the choice of the first-line therapy: patients with polymorphisms that negatively condition imatinib efficacy might thus receive nilotinib as first-line therapy.

Reduced circulating B-lymphocytes and altered B-cell compartments in patients suffering from chronic myeloid leukaemia undergoing therapy with Imatinib

*p versus normals and versus CML-1= 0.001. Data are expressed as means ± SD. To the Editor Imatinib mesylate, an inhibitor of several tyrosine kinases, such as Abelson gene (ABL), Breakpoint Cluster RegionAbelson gene (BCR-ABL1), c-KIT and platelet-derived growth factor receptors [1] is used to treat chronic myeloid leukaemia (CML) in chronic phase and is able to interfere with some immunologic pathways. Imatinib modifies some biological aspects of B-lymphocyte, being able to induce hypogammaglobulinemia and to alter the phenotype of bone marrow plasma cells [2,3]. A recent report by De Lavallade et al. [4] has shed new light on the mechanisms by which Imatinib and other tyrosine kinase inhibitors can affect B-lymphocyte immune response. We report our experience on 34 patients with CML undergoing Imatinib therapy. We evaluated the circulating B-lymphocyte compartments by means of multiparameter flow cytometry. Samples from 34 CML patients (23 men, 11 women, age 37–83 years), undergoing Imatinib treatment (Glivec, Novartis Pharmaceuticals Corporation, East Hanover, NJ, USA), 400mg/daily) as the first line therapy, were studied at the time of periodical (every 3months) monitoring of response to therapy. At the time of the study, the patients were not undergoing therapy with other drugs. None of the patients had a clinical history of recurrent infections. Twenty-three normal subjects (age 19–65 years) were evaluated as control group. The following blood parameters were registered: white blood cells (WBC), total lymphocyte percentage and absolute number, γ-globulin levels. The disease was monitored by conventional chromosome analysis (quinacrine and Giemsa banding), FISH analysis (LSI BCR-ABLdual-colour dual-fusion translocation probe), polymerase chain reaction (PCR) for the BCR/ABL1 translocation using the standardized PCR protocol of the European BIOMED-1 project and the standard operating procedures of LabNet guidelines [5]. Responders were defined as patients who obtained at least a three-log reduction in the BCR-ABL1messenger RNA level (MMR> 3). Major response was defined as MMR> 4 and complete molecular response as MMR> 5. Immunophenotyping was carried out by a FacsCanto II cytometer equipped with two lasers (488 and 633 nm), using a five-colour method with the following monoclonal antibodies: CD20/PerCP-Cy5.5, CD19/PE-Cy.7, CD27/APC and CD45/APC-Cy7.7 (Becton Dickinson, Franklin Lakes, NJ, USA). The antibody panel was completed by a F(ab′)2

Sorafenib As Monotherapy or in Association With Cytarabine and Clofarabine for the Treatment of Relapsed/Refractory FLT3 ITD-Positive Advanced Acute Myeloid Leukemia

Clinical Practice Points � Acute myeloid leukemia (AML) associated with fmsrelated tyrosine kinase 3 (FLT3) internal tandem duplication mutation has an increased relapse rate and a reduced overall survival. � Here we report 3 cases of advanced refractory AML treated using sorafenib alone or in association with chemotherapy, at a standard dose of 400 mg twice daily. All the patients had been previously treated with several chemotherapy lines and their status was complicated by severe infectious disease. � After sorafenib treatment, clinical complete remission but not molecular remission was achieved in all cases. Median duration of the response of these patients was relatively short (40 days), but in 1 patient it was possible to continue the therapy at the reduced dose of 600 mg daily as a bridge to allogeneic stem cell transplantation. � We confirm that sorafenib is active in FLT3 mutated AML and we suggest that this drug could be used in more precocious stages of this disease to achieve a deeper hematological response as a bridge to bone marrow transplant.

Increased values of the circulating PDGFβ sustains the "withdrawal syndrome" after tyrosine kinase inhibitor discontinuation in patients affected by chronic myeloid leukemia.

A Swedish group recently reported that up to 30% of patients affected by chronic myeloid leukemia (CML) who stopped imatinib in the context of the EURO-SKI trial presented for the first two months with musculoskeletal pains, more frequently localized in the shoulders and hips or extremities, with the symptoms often miming rheumatic polymyalgia. In that series, the C-reactive protein was increased in two patients only, and serum protein electrophoresis showed inflammatory signs in one-third of the assessed cases. Some of those patients have been described as sensitive to paracetamol, whereas other patients needed long-term treatment with corticosteroids [1]. With these premises,we decided to evaluate a panel of some inflammatory and anti-inflammatory cytokines (IL2, IL6, IL10, TNFα, IFNγ, VEGF, and PDGFβ) in four patients who showed the same symptoms already described by Richter et al. [1] in order to try to better understand the mechanism underlying this “withdrawal syndrome”. Seven patients affected by CML in chronic phase discontinued imatinib and one patient dasatinib during 2013/2014 at the Hematology Unit of the Pisa University, all for severe toxicities: cramps, ocular or intestinal bleeding, edema, diarrhea, skin toxicity, PAOD. Three subjects were male and five female; their median age was 64 years (range 54–78); all patients received a Tyrosine Kinase Inhibitor (TKI) for more than 5 years, with a median time from CML diagnosis of 10 years. At the moment of discontinuation, all patients were in deep molecular response (MR or MR) for at least 24 months. The initial Sokal risk score was low in 3 patients, intermediate in 4, and high in one patient; Hasford was low in 3 cases, and intermediate in the remaining 5; EUTOS score was low in all cases, except for one. The clinical characteristics of our patients are reported in Table 1. From the discontinuation of treatment, the BCR-ABL1/ABL1 International Scale (IS) ratio was monitored by quantitative PCR every month for the first 3 months, then every 2 months. Four of the above reported 8 patients developed the “TKIwithdrawal syndrome” after 2–3weeks from the TKI discontinuation, 3 after imatinib and one after dasatinib interruption. All these 4 patients presented with muscolo-skeletal pains, initially localized in the shoulders, and then even to the arms and hips, persistent during night also, with consequent relevant sleep disorders. These symptoms were graded as 2 according to the Common Criteria for Adverse Events scale (version 4.0, 2010).