Increased proportion of mature NK cells is associated with successful imatinib discontinuation in chronic myeloid leukemia.

Recent studies suggest that a proportion of chronic myeloid leukemia (CML) patients in deep molecular remission can discontinue the tyrosine kinase inhibitor (TKI) treatment without disease relapse. In this multi-center, prospective clinical trial (EURO-SKI, NCT01596114) we analyzed the function and phenotype of T and NK cells and their relation to successful TKI cessation. Lymphocyte subclasses were measured from 100 imatinib-treated patients at baseline and 1 month after the discontinuation, and functional characterization of NK and T cells was done from 45 patients. The proportion of NK cells was associated with the molecular relapse-free survival as patients with higher than median NK-cell percentage at the time of drug discontinuation had better probability to stay in remission. Similar association was not found with T or B cells or their subsets. In non-relapsing patients the NK-cell phenotype was mature, whereas patients with more naïve CD56bright NK cells had decreased relapse-free survival. In addition, the TNF-α/IFN-γ cytokine secretion by NK cells correlated with the successful drug discontinuation. Our results highlight the role of NK cells in sustaining remission and strengthen the status of CML as an immunogenic tumor warranting novel clinical trials with immunomodulating agents.

PO-55 - Individual variation in hemostatic alterations caused by tyrosine kinase inhibitors - a way to improve personalized cancer therapy?

INTRODUCTION: During the last two decades, Bcr-Abl tyrosine kinase inhibitors (TKIs) have revolutionized the treatment of chronic myelogenous leukemia (CML), and are now considered standard treatment for this disease. However, TKIs can induce serious hemostatic side effects including cardiovascular disease and bleeding disorders. Blood platelet aggregation and formation of pro-coagulant platelets are important to allow a well-balanced hemostatic response. Therefore, a detailed understanding of what effect different TKIs exert on platelets and hemostasis could help to understand if there are differences of importance to minimize the risk of bleeding complications in treated patients. AIM: To investigate how TKIs used in CML (imatinib, dasatinib, nilotinib, bosutinib, and ponatinib) affect platelet activation and hemostasis. MATERIALS AND METHODS: We have developed a multi-parameter six color flow cytometry protocol to study different aspects of platelet function upon activation, e.g. formation of aggregatory (PAC-1-positive) and pro-coagulant (phosphatidylserine-exposing) platelets, exocytosis of alpha- and lysosomal granules and mitochondrial membrane potential.This protocol was performed in presence or absence of TKIs in blood from normal donors and in treated patients. Whole blood aggregometry (Multiplate®), thrombin generation in platelet-rich plasma and in vitro thrombus formation by free oscillation rheometry (ReoRox G2) was further evaluated in some situations. RESULTS: At clinically relevant concentrations, dasatinib significantly decreased the formation of procoagulant platelets. Ponatinib induced a slight decrease in formation of procoagulant platelets, whereas bosutinib and nilotinib showed opposite tendencies (n=7). Dasatinib also decreased platelet aggregation (n=4-6) and in vitro thrombus formation (n=3). Thrombin generation was not significantly affected by therapeutic levels of TKIs, whereas higher doses of dasatinib, bosutinib, ponatinib and imatinib significantly changed one or several of the thrombin generation parameters (n=7-8). Interestingly, large differences in response to the drugs were observed among the healthy donors, especially for dasatinib and bosutinib. Major inter-individual variations were also observed in dasatinib-treated patients, see Figure 1. CONCLUSIONS: Different TKIs show varying potency to affect platelet-based hemostasis. In addition, we found large inter-individual variations in how some drugs affected platelet function. Therefore, we suggest that development of a clinically useful protocol for platelet function testing could help to identify patients more susceptible to adverse drug reactions. Such a protocol could potentially help clinicians to gain insight into the risk of side effects, which could help to choose the most suitable drug for each individual patient.

Frequent and long-term follow-up of health-related quality of life following allogeneic haematopoietic stem cell transplantation.

Health-related quality of life (HRQL) was evaluated in 94 patients undergoing allogeneic haematopoietic stem cell transplantation (HSCT) after myeloablative (MAC, n = 18) or reduced intensity conditioning (RIC, n = 76). HRQL was assessed with the EORTC QLQ C-30 during the inpatient period as well as during the following 3 years, i.e., at baseline and 12 times thereafter. Functional status and global quality of life decreased from baseline to weeks 2 and 3, especially role and social functions. Symptoms increased significantly during the first 3 weeks, particularly appetite loss, nausea and vomiting, diarrhoea and fatigue. It took at least 1 year for HRQL to return to the baseline level. The only function that improved significantly 3 years after HSCT was role function. Patients treated with MAC experienced significantly worse HRQL at baseline than patients treated with RIC, as well as more pain, sleep disturbance and appetite loss in weeks 3 and 4. Patients with extensive chronic graft-versus-host disease experienced reduced HRQL. These results provide a clinically useful overview of patients' HRQL during and after HSCT and indicate when they require increased support. The results demonstrate the importance of close follow-ups during the first year after HSCT to improve preventive or supportive interventions.

A potential role of fetal hemoglobin in the development of multidrug resistance.

Our previous data from a human leukemic cell line made resistant to the nucleoside analog (NA) 9-ß-D-arabinofuranosylguanine (AraG) revealed a massive upregulation of fetal hemoglobin (HbF) genes and the ABCB1 gene coding for the multidrug resistance P-glycoprotein (P-gp). The expression of these genes is regulated through the same mechanisms, with activation of the p38-MAPK pathway and inhibition of methylation making transcription factors more accessible to activate these genes. We could show that AraG, as well as other NAs, and P-gp substrates could induce global DNA demethylation and induction of Hbγ and P-gp both at the mRNA and protein expression level. We speculate that the expression of HbF prior to drug exposure or in drug-resistant cell lines is a strategy of the cancer to gain more oxygen, and thereby survival benefits. We also believe that P-gp may be induced in order to excrete Hb degradation products from the cells that would otherwise be toxic. By using Hbγ siRNA and pharmacological inhibitors of HbF production we here present a possible relationship between HbF induction and multi-drug resistance in a human leukemia cell line model.

Association of ABCB1 polymorphisms with survival and in vitro cytotoxicty in de novo acute myeloid leukemia with normal karyotype.

Overexpression of the multi-drug transporter P-glycoprotein, encoded by the ABCB1 gene, is a clinically relevant problem in acute myeloid leukemia (AML). Polymorphisms in ABCB1 might contribute to cancer risk and therapeutic response. We therefore investigated the influence of polymorphisms G1199A, C1236T, G2677T/A and C3435T on cancer susceptibility, in vitro cytotoxicity and overall survival in 100 de novo AML patients with normal karyotype. Patients with 1236C/C or 2677G/G genotypes showed poorer survival than patients with other genotypes (P=0.03 and P=0.02, respectively). Both these genotypes were significant factors for survival in multivariate analysis, along with age, NPM1 and FLT3 mutation status. In vitro cytotoxicity studies demonstrated that leukemic cells from 1236T/T and 2677T/T patients were significantly more susceptible to mitoxantrone (P=0.02), and tended to be more susceptible to etoposide and daunorubicin (P=0.07-0.09), but not to cytarabine. No significant difference in allele frequencies was found between patients and healthy volunteers (n=400).

A prospective evaluation of patients' health-related quality of life during auto-SCT: a 3-year follow-up.

Few studies have evaluated long-term health-related quality of life (HRQL) in patients during auto-SCT. This prospective study examined HRQL in 96 eligible patients before, during and up to 3 years after auto-SCT. The aim of the study was to make a comprehensive assessment of the frequency and severity of different symptoms in patients undergoing auto-SCT. The European Organization for Treatment and Research of Cancer Quality of Life Questionnaire (EORTC QLQ C-30) was administered 13 times. The second week during treatment was the period when patients had the lowest HRQL regarding both total quality of life and function and symptom scales. The patients recovered quickly and just two months after transplantation the baseline values were restored. Three years after transplantation most of the items in the questionnaire had stabilized, except role function and dyspnea, which had improved. There were significant differences between multiple myeloma (MM) and lymphoma patients' physical function, quality of life, fatigue and pain during week 2. At the 3-year follow-up, lymphoma patients indicated a better HRQL than MM patients. The quick recovery of patients after transplantation suggests that treatment is well tolerated; however, the supportive care could be improved at week 2, especially for the lymphoma patients.

Cell cycle dependent regulation of deoxycytidine kinase, deoxyguanosine kinase, and cytosolic 5'-nucleotidase I activity in MOLT-4 cells.

Activation of nucleoside analogues is dependent on kinases and 5'-nucleotidases and the balance between the activity of these enzymes. The purpose of this study was to analyze deoxycytidine kinase, deoxyguanosine kinase, and 4 different 5'-nucleotidases during cell cycle progression in MOLT-4 cells. The activity of both kinases was cell cycle dependent and increased during proliferation while the activity of cytosolic 5'-nucleotidase I decreased. We could show that the kinase activity was higher than the total nucleotidase activity, which was unchanged or decreased during cell cycle progression. These data may be important in designing modern combination therapy with nucleoside analogues.

Pharmacological basis for cladribine resistance in a human acute T lymphoblastic leukaemia cell line selected for resistance to etoposide.

Cross-resistance between different classes of anti-neoplastic agents can jeopardize successful combination cancer chemotherapy. In this study, we observed an unexpected cross-resistance between the podophyllotoxine derivative etoposide (VP) and the nucleoside analogue cladribine (CdA) in CCRF-CEM cells developed for resistance to VP. The resistant cells also displayed 14- and twofold resistance to cytarabine (ara-C) and gemcitabine respectively. Closer analysis of these cells showed that they contained lower amounts of topoisomerase (topo) IIalpha (P < 0.001) and beta protein (P < 0.026), formed substantially lower amounts of the topo II-DNA complex, and had a markedly decreased level of Fas (CD95/APO-1)-ligand mRNA expression. Interestingly, Fas expression in the resistant cells did not differ from that in the parental cell line. No differences were observed in the accumulation/efflux of daunorubicin or in the gene expressions of P-glycoprotein, multidrug resistance-associated protein and the lung resistance-related protein. The activity of deoxycytidine kinase (dCK), responsible for activation of CdA and ara-C, was the same for resistant and wild-type cells. However, there was an increase in the activity of the cytosolic 5'-nucleotidases (5'-NT), responsible for deactivation of nucleotides, amounting to 206% (P < 0.001) for the high Km and 134% (P < 0.331) for the low Km 5'-NT in resistant cells. The high Km 5'-NT is probably responsible for the decreased amount of the active metabolite CdA 5'-triphosphate [40% decreased (P < 0.045)], as well as for other purine ribonucleosides and deoxyribonucleosides triphosphates in the resistant cells. In contrast, a significantly higher deoxycytidine triphosphate (dCTP) level (167%, P < 0.001) was observed in the resistant cells. Thus, this study suggests that the major cause of resistance to the nucleoside analogues CdA and ara-C in cells selected for resistance to VP is a result of metabolic alterations producing increased activity of 5'-NT and higher dCTP levels. Furthermore, these results indicate that there is a common factor in the regulation of nucleotide-degrading enzymes and DNA topoisomerases, which may be altered in cross-resistant cells.

Biochemical pharmacology and resistance to 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine, a novel analogue of cladribine in human leukemic cells.

The objective of the present study was to investigate the biochemical pharmacology of 2-chloro-2'-arabino-fluoro-2'-deoxyadenosine (CAFdA)--a fluorinated analogue of cladribine [2-chloro-2'-deoxyadenosine, Leustatin (CdA)] with improved acid and metabolic stability--in human leukemic cell lines and in mononuclear cells isolated from patients with chronic lymphocytic leukemia (CLL) and acute myelocytic leukemia (AML). We have also made and characterized two cell lines that are not sensitive to the growth inhibitory and cytotoxic effects of CAFdA. Incubation of cells isolated from the blood of CLL and AML patients with various concentrations of CdA or of CAFdA accumulated CdA and CAFdA nucleotides in a dose-dependent manner. A significantly higher rate of phosphorylation to monophosphates was observed for CAFdA than for CdA in cells from CLL patients (n = 14; P = 0.04). The differences in the phosphorylation were even more pronounced for the respective triphosphates in both CLL (n = 14; P = 0.001) and AML (n = 4; P = 0.04) cells. Retention of CAFdA 5'-triphosphate (CAFdATP) was also longer than that for CdA 5'-triphosphate (CdATP) in cells from leukemic patients. The relative efficacy of CAFdA as a substrate for purified recombinant deoxycytidine kinase (dCK), the key enzyme in the activation of nucleoside analogues, was very high and exceeded that of CdA as well as the natural substrate, deoxycytidine, by a factor of 2 and 8, respectively. The Km for CAFdA with dCK was also lower than that for CdA, as measured in crude extracts from the human acute lymphoblastic leukemia cell line CCRF-CEM and the promyelocytic leukemia cell line HL60. Acquired resistance to CAFdA in HL60 and in CCRF-CEM cell lines was directly correlated to the decreased activity of the nucleoside phosphorylating enzyme, dCK. Resistant cells also showed a considerable degree of cross-resistance to analogues that were activated by dCK. These observations demonstrated that dCK phosphorylates CAFdA more efficiently than CdA. Furthermore, CAFdATP is apparently more stable than CdATP and the mechanisms of resistance to CAFdA are similar to those leading to CdA resistance. These results encourage studies on the clinical effect of CAFdA in lymphoproliferative diseases.

The beta cell glucokinase promoter variant is an unlikely risk factor for diabetes mellitus. Diabetes Incidence Study in Sweden (DISS).

Glucokinase plays an important role in the regulation of insulin secretion and is therefore an attractive candidate gene for both insulin dependent (IDDM) and non-insulin-dependent (NIDDM) diabetes mellitus. A single G-A nucleotide polymorphism at the -30 position of the beta-cell specific promoter region of the glucokinase gene was previously associated with reduced beta-cell function. In the present study we analysed 268 consecutive newly diagnosed Swedish patients classified with either IDDM (n = 205), NIDDM (n = 31) or unclassifiable (n = 32) diabetes between the ages of 15 and 35 years along with a group of 158 age- and sex-matched control subjects. The beta-cell promoter region was amplified by the polymerase chain reaction and the G-A variant identified by single strand conformational polymorphism. There was no significant difference in allele frequencies of G and A between any of the subject groups and likewise, no significant difference in the frequencies of the G/G, G/A, or A/A genotypes. Eight subjects were homozygous for the less common A allele, five had IDDM and three were control subjects. Our results suggest that the -30 beta-cell glucokinase promoter variant is not associated with IDDM.

Bleeding Meckel's diverticulum presenting as focal extravasation on pertechnetate scintigraphy.

The evaluation of gastrointestinal hemorrhage may include a Meckel's scan, particularly in children. Typically, a Meckel's diverticulum shows discrete, focal uptake in the right lower abdomen. The authors describe an adult whose bleeding Meckel's diverticulum manifested as brisk, intermittent intraluminal tracer extravasation.

Determination of meropenem in plasma by high-performance liquid chromatography and a microbiological method.

A rapid and selective high-performance liquid chromatographic (HPLC) method for the quantitative determination of meropenem in plasma is described. The drug was separated from plasma after plasma protein precipitation with 15% of trichloroacetic acid. The mobile phase consisted of acetonitrile-water-glacial acetic acid (21.2, 78 and 0.8% v/v, respectively) delivered at a flow rate of 1.2 ml/min. Meropenem was quantified using ultraviolet detection at 296 nm. Meropenem and the internal standard (pheniramine) were well separated from plasma components. The drug could be assayed by the HPLC method in the presence of its analogue, imipenem. The detection limit in plasma was 25 ng/ml of meropenem. The results were compared with those of agar for a microbiological diffusion method using Escherichia coli ATCC 25922 as the test organism. The sensitivity of the microbiological assay was less than 5 ng/ml, but this decreased at higher concentrations. Both methods were applied to the determination of the drug in aqueous solutions and in plasma.

The effect of caffeine on the human macular circulation.

The acute effect of caffeine on the retinal circulation was studied in 14 healthy volunteers using the blue field simulation technique, which provides measurements of the velocity of leukocytes flowing within the macular capillaries. Subjects adjusted the mean velocity (Vm) of computer-simulated leukocytes moving on a cathode ray tube screen to match that of their own entoptically perceived leukocytes before and 1 hr after a double-masked, randomized administration of 200 mg caffeine or placebo. Caffeine produced an average 13% +/- 5% (SEM) decrease in Vm (P less than 0.05) and a 9% +/- 3% increase in diastolic blood pressure (P less than 0.05). The decrease in Vm and, presumably, blood flow occurring despite the increased diastolic blood pressure probably is attributable to retinal vasoconstriction. This effect may result from caffeine's known inhibitory effect on adenosine, a potent vasodilator of the retinal vasculature.