Persistence of pre-leukemic clones during first remission and risk of relapse in acute myeloid leukemia.

Some patients with acute myeloid leukemia (AML) who are in complete remission after induction chemotherapy harbor persisting pre-leukemic clones, carrying a subset of leukemia-associated somatic mutations. There is conflicting evidence on the prognostic relevance of these clones for AML relapse. Here, we characterized paired pre-treatment and remission samples from 126 AML patients for mutations in 68 leukemia-associated genes. Fifty patients (40%) retained ⩾1 mutation during remission at a variant allele frequency of ⩾2%. Mutation persistence was most frequent in DNMT3A (65% of patients with mutations at diagnosis), SRSF2 (64%), TET2 (55%), and ASXL1 (46%), and significantly associated with older age (P<0.0001) and, in multivariate analyses adjusting for age, genetic risk, and allogeneic transplantation, with inferior relapse-free survival (hazard ratio, 2.34; P=0039) and overall survival (hazard ratio, 2.14; P=036). Patients with persisting mutations had a higher cumulative incidence of relapse before, but not after allogeneic stem cell transplantation. Our work underlines the relevance of mutation persistence during first remission as a novel risk factor in AML. Persistence of pre-leukemic clones may contribute to the inferior outcome of elderly AML patients. Allogeneic transplantation abrogated the increased relapse risk associated with persisting pre-leukemic clones, suggesting that mutation persistence may guide postremission treatment.Leukemia accepted article preview online, 18 December 2017. doi:10.1038/leu.2017.350.

Sequential therapy combining clofarabine and T-cell-replete HLA-haploidentical haematopoietic SCT is feasible and shows efficacy in the treatment of refractory or relapsed aggressive lymphoma.

Prognosis is poor for patients with biologically aggressive Non-Hodgkin lymphoma (NHL), refractory to chemotherapy or relapsed after autologous transplantation, especially when no disease control before allogeneic transplantation is achieved. In 16 patients (median age 53, median prior regimes 5) with relapsed or refractory non-remission NHL, we analysed retrospectively the efficacy of a sequential therapy comprising clofarabine re-induction followed by a reduced-intensity conditioning with fludarabine, CY and melphalan, and T-cell-replete HLA-haploidentical transplantation. High-dose CY was utilized post-transplantation. All patients engrafted. Early response (day +30) was achieved in 94%. Treatment-related grade III-IV toxicity occurred in 56%, most commonly transient elevation of transaminases (36%), while there was a low incidence of infections (19% CMV reactivation, 19% invasive fungal infection) and GVHD (GVHD: acute III-IV: 6%; mild chronic: 25%). One-year non-relapse mortality was 19%. After a median follow-up of 21 months, estimated 1- and 2-year PFS was 56 and 50%, respectively, with 11 patients (69%) still alive after 2 years. In summary, sequential therapy is feasible and effective and provides an acceptable toxicity profile in high-risk non-remission NHL. Presumably, cytotoxic reinduction with clofarabine provides enough remission time for the graft-versus lymphoma effect of HLA-haploidentical transplantation to kick in, even in lymphomas that are otherwise chemo-refractory.

Early assessment of minimal residual disease in AML by flow cytometry during aplasia identifies patients at increased risk of relapse.

In acute myeloid leukemia (AML), assessment of minimal residual disease (MRD) by flow cytometry (flow MRD) after induction and consolidation therapy has been shown to provide independent prognostic information. However, data on the value of earlier flow MRD assessment are lacking. Therefore, the value of flow MRD detection was determined during aplasia in 178 patients achieving complete remission after treatment according to AMLCG (AML Cooperative Group) induction protocols. Flow MRD positivity during aplasia predicted poor outcome (5-year relapse-free survival (RFS) 16% vs 43%, P<0.001) independently from age and cytogenetic risk group (hazard ratio for MRD positivity 1.71; P=0.009). Importantly, the prognosis of patients without detectable MRD was neither impacted by morphological blast count during aplasia nor by MRD status postinduction. Early flow MRD was also evaluated in the context of existing risk factors. Flow MRD was prognostic within the intermediate cytogenetic risk group (5-year RFS 15% vs 37%, P=0.016) as well as for patients with normal karyotype and NPM1 mutations (5-year RFS 13% vs 49%, P=0.02) or FLT3-ITD (3-year RFS rates 9% vs 44%, P=0.016). Early flow MRD assessment can improve current risk stratification approaches by prediction of RFS in AML and might facilitate adaptation of postremission therapy for patients at high risk of relapse.

Second haematopoietic SCT using HLA-haploidentical donors in patients with relapse of acute leukaemia after a first allogeneic transplantation.

Haploidentical haematopoietic SCT (HSCT) using T-cell-replete grafts and post-transplant high-dose CY has found increasing acceptance. Our purpose was to evaluate the feasibility and outcome of this strategy as second HSCT incorporating donor change for acute leukaemia relapse after a first allogeneic transplantation. The courses of 20 consecutive adults (median age 37 years, 12 male) with AML (n=14), ALL (n=5) and acute bi-phenotypic leukaemia (n=1) were analysed retrospectively. Conditioning consisted of fludarabine, CY and either melphalan or TBI or tresosulfan+/-etoposide. Engraftment was achieved in 17 (85%), and a second remission was induced in 15 patients (75%) on day +30. The rate of grade II-IV acute GvHD was 35%, while chronic GvHD occurred in five patients. Most commonly observed grade III-IV toxicities were mucositis (30%), hyperbilirubinemia (20%), elevation of transaminases (20%) and creatinine (20%), while invasive fungal infection affected 30%. One-year non-relapse mortality (NRM) was 36%. At a median follow-up of 17 months, estimated 1-year OS was 45%, and 1-year relapse-free survival was 33%. This strategy was feasible and allowed for successful engraftment with a moderate rate of toxicity. Early outcome and NRM are at least comparable with results after a second HSCT from HLA-matched donors without donor change at HSCT2.

Comparative molecular analysis of therapy-related and de novo acute promyelocytic leukemia.

Therapy-related acute promyelocytic leukemia (t-APL) has been reported as a late complication of exposure to radiotherapy and/or chemotherapeutic agents targeting DNA topoisomerase II. We have analyzed in t-APL novel gene mutations recently associated with myeloid disorders. Unlike previous reports in acute myeloid leukemia (AML), our results showed neither IDHs nor TET2 mutations in t-APL. However we found an R882H mutation in the DNMT3A gene in a patient with t-APL suggesting a possible role of this alteration in the pathogenesis of t-APL.

Monitoring of WT1 expression in PB and CD34(+) donor chimerism of BM predicts early relapse in AML and MDS patients after hematopoietic cell transplantation with reduced-intensity conditioning.

Relapse of malignant disease remains the major complication in patients with acute myeloid leukemia (AML) or myelodysplastic syndrome (MDS) after hematopoietic cell transplantation (HCT) with reduced-intensity conditioning (RIC). In this study, we investigated the predictive value of disease-specific markers (DSMs), donor chimerism (DC) analysis of unsorted (UDC) or CD34(+) sorted cells and Wilms' tumor gene 1 (WT1) expression. Eighty-eight patients with AML or MDS were monitored after allogenic HCT following 2 Gy total-body irradiation with (n=84) or without (n=4) fludarabine 3 × 30 mg/m(2), followed by cyclosporin A and mycophenolate mofetil. DSMs were determined by fluorescence in situ hybridization (FISH) and WT1 expression by real-time polymerase chain reaction. Chimerism analysis was performed on unsorted or CD34(+) sorted cells, by FISH or short tandem repeat polymerase chain reaction. Twenty-one (24%) patients relapsed within 4 months after HCT. UDC, CD34(+) DC and WT1 expression were each significant predictors of relapse with sensitivities ranging from 53 to 79% and specificities of 82-91%. Relapse within 28 days was excluded almost entirely on the basis of WT1 expression combined with CD34(+) DC kinetics. Monitoring of WT1 expression and CD34(+) DC predict relapse of AML and MDS after RIC-HCT.

Cardiovascular function of workers exposed to carbon disulphide.

OBJECTIVES: The aim of the study was to verify that exposure to carbon disulphide (CS(2)) up to 10 ppm results in a negative inotropic effect on cardiovascular function. METHODS: In a cross-sectional study a total of 325 workers exposed to CS(2) in the rayon-producing industry and 179 controls from the same plants were examined. The exposure assessment was based on personal air sampling and biological monitoring (2-thiothiazolidine-4-carboxylic acid (TTCA) in urine) for all persons. The examination consisted of a standardised questionnaire, physical examination, assessment of body fat mass, ergometric test with the determination of work capacity at heart rates of 100, 130, 150 and 170 beats/min, and electrocardiography. RESULTS: The mean external exposure was 6.04 ppm CS(2) (range: 0.03-91.08); the mean internal exposure was 1.14 mg TTCA/g creatinine (range: 0.02-11.50). The workers exposed to CS(2) showed better physical fitness. The diameters of the left heart chamber of the exposed persons were not significantly different when compared with occupationally non-exposed workers, but there was a tendency of increasing diameters for the exposed employees. In the multiple linear regression the diameters showed physiologically plausible correlations with the body mass index, body fat mass, alcohol consumption, and physical fitness, but not, however, with the exposure, neither with the exposure group in all persons nor with the internal or external exposure within the exposed workers. CONCLUSIONS: In this study, differences in the heart chamber diameters between exposed persons and controls could not be confirmed. Differences in physical fitness and constitution can explain differences in heart chamber diameters.

Simultaneous quantification of citalopram, clozapine, fluoxetine, norfluoxetine, maprotiline, desmethylmaprotiline and trazodone in human serum by HPLC analysis.

We describe an analytical procedure for the simultaneous quantification of citalopram (seropram), clozapine (leponex), fluoxetine (fluctine), norfluoxetine, maprotiline (ludiomil), desmethylmaprotiline and trazodone (trittico) in human serum within a period of 11.5 minutes using reversed phase HPLC. After 2 liquid/liquid extractions in the sample preparation phase, the drugs and metabolites were separated on a C18 column using a mobile phase consisting of acetonitrile/buffer (30/70, v:v) at 70 degrees C, a flow rate of 1.5 m/min and haloperidol as internal standard. Absorption and native fluorescence signals of the eluted compounds were detected simultaneously at 260 nm and 227/300 nm (excitation/emission), respectively. The calibration ranges for citalopram, clozapine, fluoxetine, norfluoxetine, maprotiline, and desmethylmaprotiline ranged from 50-400 microg/l and for trazodone from 50-3,200 microg/l. The CVs varied between 0.6% and 5.5% (within-run) and between 3.2% and 7.1% (between-run). Recoveries were > 90% for all pharmaceuticals. We noticed no interferences from several commonly used drugs.

[Practical usage of a quality management system according to ISO 9001 : 2000 focusing on the double contrast (barium) enema]. / Praktische Anwendung eines Qualitätsmanagementsystems nach ISO 9001:2000 am Beispiel von Qualitätsverbesserungsmassnahmen beim Kolon-Kontrastmitteleinlauf, Germany.

PURPOSE: We describe the implementation of quality improvement measures in a quality management system. METHODS: With questionnaires for radiologists and patients, we investigated the relations between patient preparation and diagnostic quality serving the main purpose, to improve diagnostic quality with the help of more detailed patient information (e.g., changing preparation sheets and handling out "peri-med" information sheets to the patients). Furthermore, a comparative data ascertainment at other institutes was integrated. RESULTS: For the group of outpatients, increasing process quality (patient information) and outcome (diagnostic quality) could be achieved. Taking aspects of quality costs into consideration, a decrease in costs due to failures was achieved. CONCLUSION: More detailed patient information has positive effects on diagnostic quality of the double contrast (barium) enema.

Lack of association between childhood stroke after varicella and human leukocyte antigen (HLA)-B51.

Human leukocyte antigen (HLA)-B51 has been suggested as an immunogenetic marker for a genetic predisposition to vascular occlusion in response to an immunological stimulus. Varicella has been reported to be a possible risk factor for stroke. We performed DNA-based HLA typing in 11 young patients (mean age: 5.2 years) with unexplained ischaemic stroke. In eight of them varicella had occurred before their stroke. HLA-B51 was negative in all 11 patients and we did not find any significant accumulation of other HLA-subgroups. Our study does not support an association between susceptibility to stroke after varicella and HLA-B51.

Comparison between three transmucosal routes of administration of midazolam in children.

Midazolam was applied transmucosally in 47 children randomly assigned to three different groups. Group N received 0.2 mg.kg-1 nasally, group R 0.5 mg.kg-1 rectally, and group S 0.2 mg.kg-1 sublingually. All groups were treated 60 min prior to a planned i.v. puncture with EMLA. Reliable and valid psychological parameters (such as emotional situation, shivering, awareness, respiratory rate and facial colour) were scored after premedication and before and after i.v. puncture, 20 min after premedication and until induction. A blood sample was drawn 10, 30 and 60 min after premedication and the levels of midazolam, alpha-hydroxy-midazolam, ACTH, glucose and cortisol were measured. In all three groups the plasma levels of midazolam 10 min after premedication were higher than 70 ng.ml-1 (accepted as a sedative level). 30 min after premedication the midazolam level in the sublingual group was statistically significantly higher than in the nasal group and the psychological parameters in all three groups were significantly changed (10 min after premedication). The psychological parameters were not significantly different between the three groups over the whole study. Sublingual premedication has some advantages (most readily accepted, highest plasma levels and lowest deviations) and could be the first choice in premedication of children. All three transmucosal applications are safe and well accepted, although nasal application was rejected by two of the children.

Carbon disulphide. IV. Cardiovascular function in workers in the viscose industry.

OBJECTIVE: The aim of the study was to examine whether an increase can be detected in the prevalence of coronary heart disease or a higher prevalence of unusual cardiological findings in workers with occupational exposure to carbon disulphide (CS2) at the level of the threshold limit value of 10 ppm currently valid in occupational medicine. METHODS: In a cross-sectional study we investigated 247 men occupationally exposed to CS2 and a comparable control group (n = 222). The current exposure to CS2 was measured using personal air monitoring and biological monitoring of all test persons. A cumulative exposure index (median of CS2 exposure in the past multiplied by the duration of employment) was calculated. In addition to collecting comprehensive anamnestic data on all persons, we carried out a physical examination, an ultrasound examination of the large arteries, a resting and exercise ECG and an echocardiographic examination. RESULTS: No increase could be found in the prevalence of coronary heart disease or of arteriosclerotic findings in the exposed subjects. There was no difference in the distribution of the performance of the two groups in the ergometric tests. The echocardiogram showed a median increase in the diameter of the left atrium and left ventricle of 1-2 mm in the exposed subjects. These differences could also be confirmed statistically after multiple linear regression analysis. The left ventricular, telesystolic diameter was positively associated (P < 0.05) with internal exposure (CS2 metabolite in urine), and fractional shortening revealed a plausible negative trend (P = 0.0755). Current external exposure (CS2 in air) and cumulative exposure did not influence any of the parameters investigated. CONCLUSION: The findings may indicate a negatively inotropic effect of CS2 so far unknown in man. However, no clinical relevance for this effect was apparent.

Carbon disulphide. III. Risk factors for coronary heart diseases in workers in the viscose industry.

To evaluate risk factors for coronary heart disease and factors which can influence the course of acute myocardial infarction in workers exposed to CS2 we performed a cross-sectional study of 247 workers in the viscose industry. The control group of 222 men from the same plant was comparable for age, social status and physical work. The CS2 exposure determined by personal air sampling ranged from < 0.2 ppm to 65.7 ppm (median: 4.0 ppm) and the duration of exposure ranged from 4 to 220 (median: 66) months. Using a multiple linear regression model we found neither higher blood pressure at rest or after exercise, nor hyperlipoproteinaemia in a higher degree, nor lower high-density lipoprotein (HDL) or lower apolipoprotein A-I levels, nor higher blood glucose values, nor indicators of direct cardiotoxic effects or signs of disturbances in blood coagulation in the exposed group in comparison to controls. Regarding the influence of chronic exposure on the investigated parameters, we found an inverse correlation of the cumulative exposure (mean CS2 exposure in the department multiplied by the duration of work in this department) with the HDL concentration. The HDL levels correlated with the duration but not with the intensity of exposure. In the same way the apolipoprotein A-I levels showed a negative association with the duration of exposure in the exposed group as well as in the control group. The HDL concentrations showed the same trend for the controls. It therefore seems that this finding is more likely due to confounding factors than to the CS2 exposure. As all subjects (exposed and controls) have done shift work, in some cases for a long time, this kind of work could be responsible for the negative relationship between the duration of employment as a shift worker and the apolipoprotein A-I and HDL levels. At the current air-borne levels no significant differences were found between the exposed persons and the controls in the distribution frequency for blood pressure values, lipoproteins, blood glucose, blood coagulation and indicators of direct cardiotoxic effects.

[Analysis of myocardial evoked potentials in relation to ergometric load and heart rate for use in frequency-adaptive cardiac pacemakers]. / Analyse evozierter Myokardpotentiale in Abhängigkeit von ergometrischer Belastung und Schlagfrequenz für die Anwendung in frequenzadaptiven Herzschrittmachern.

The aim of this study was to evaluate the ventricular evoked response (VER) measured with unipolar fractally coated pacing leads for use with rate-responsive pacemakers. To this end, the morphology of the VER, its variation with pacing rate and various levels of physical loading, and the long-term stability of the signal were studied using the telemetric features of implantable pacemakers. Fractally coated electrodes were used in order to minimize the stimulation artefact, thus enabling a reliable unipolar measurement of the VER. The VER shows uniform basic morphology, and remains virtually unchanged after 3 months; moreover, frequency and load-dependent changes of VER morphology were identical for all patients. A special programming device has been developed to evaluate a rate adaptive algorithm for optimizing the pacing rate to the current loading situation, and was successfully tested in two patients. The algorithm was shown to be capable of calculating a heart rate adequate for haemodynamic demand.

Interaction of N-methyl-anthraniloyl-labelled porcine apolipoprotein A-I with porcine lecithin: cholesterol acyltransferase: an energy transfer study.

1. To investigate whether a direct protein-protein interaction between apoA-I and lecithin:cholesterol acyltransferase (LCAT) is necessary for the activation of the enzyme, apoA-I was labelled with N-methylisatoic anhydride at lysine residues. The intermolecular resonance energy transfer from tryptophan residues of LCAT (donor) to N-methyl-anthraniloyl (NMA)-labelled apoA-I (NMA-apoA-I) (acceptor) was used as a sensitive fluorescence method for studying molecular interactions. 2. In the absence of lipids no fluorescence energy transfer was measurable. 3. Fluorescence energy transfer occurred from LCAT to NMA-apoA-I in the presence of liposomes with phospholipid/cholesterol ratios ranging from 5:1 to 18:1 and regardless whether only 1 or up to 5 NMA-apoA-I molecules resided at the liposome surface. 4. This indicates a preferred binding of the enzyme directly to or in spatial proximity to the activator protein NMA-apoA-I even if enough space at the liposome surface is available to allow LCAT binding at a distance, where no energy transfer is measurable.

Low polarization pacing lead for detecting the ventricular-evoked response.

The cardiac response to a pacing pulse is potentially useful for rate adaptive pacemakers and threshold tracking systems. However, until now capture recognition of the ventricular-evoked response by the use of a single electrode for stimulation as well as detection was limited by the electrode polarization. Electronic measures against the stimulus polarization artifact have not been successful due to the variability of the after potential or the requirement of additional battery power. Following the idea of Lewin, Myers and Parsonnet, who introduced the idea of a non-polarizable porous electrode for physiological stimulation, titanium nitride (TiN) and iridium (Ir) coatings with fractal surface structure have been developed with high electro-chemical active surface areas and Helmholtz double-layer capacities of up to 50,000 microF/cm2, thus reducing the polarization artifact significantly. Two types of endocardial leads (10 with a fractal TiN coating and 5 with a fractal Ir coating) were implanted in the apex of the right ventricle and the polarization artifact, as well as the evoked response, was measured. Both types of pacing leads show a 90% reduction in the polarization artifact in comparison to conventional leads. If an autoshort of approximately 20 to 50 ms is applied after the pacing pulse, the polarization artifact of these leads is negligible, thus enabling reliable detection of at least the repolarization phase of the ventricular-evoked response, which is fully sufficient for capture recognition. Additionally, due to their low polarization losses, TiN- or Ir-coated electrodes with fractal surface structure have a unique stimulation and detection performance.

Nonimmunological assay of urinary albumin based on laser-induced fluorescence.

We describe the first nonimmunological assay of albumin in urine with a detection limit of 1 mg/L. The method is simple, rapid, and accurate. It is based on the probe Albumin Blue 670, which becomes highly fluorescent on binding to albumin. An inexpensive diode laser was used as the light source for measurement of laser-induced fluorescence. The assay was coupled to a flow-injection analysis system capable of running 20 samples per hour. The working range was 1-100 mg/L, which covered albumin concentrations found in nonpathological urine and in urine with slightly increased albumin. This range makes prediction of nephropathy possible at an early stage. Other serum proteins and hemoglobin do not interfere. The coefficients of variation were < 4% and < 7% within one day and from day to day, respectively. A correlation coefficient of 0.990 (n = 100) was obtained for comparison with the Behring nephelometric assay.

Sputter-deposited TiN electrode coatings for superior sensing and pacing performance.

The sensing and pacing performance of pacemaker electrodes is characterized by the electrochemical properties of the electrode/tissue interface affecting tissue reactions and the kinetics of the ionic exchange. The usually smooth metallic electrode surface results in a high pass filter characteristic. To better match the electrode's filter characteristic to the spectral content of the depolarization signal, various combinations of electrode shape, material and surface structure have been researched. The electrode with sputter-deposited TiN coating presented in this report has been designed to meet the demand for low acute as well as chronic thresholds and superior sensing performance not only with respect to spontaneous activity but also regarding the detection of the evoked response. The clinical results obtained with this electrode prove the excellent pacing and sensing properties resulting from minimized polarization losses and optimized filtering of the signal to be detected, respectively. The acute and chronic clinical advantages over previous concepts are attributed mainly to the biocompatibility of the material used and the microcrystalline surface structure achieved by the coating process. The design concept of the new electrode is presented together with the clinical results obtained. While the advancements in microelectronics and battery technology have certainly formed the basis for the development of pulse generators featuring an ever increasing versatility of functions at the same or even smaller pacemaker dimensions, from a point of view of pacing system performance the development of improved electrode concepts as the one presented must be regarded as equally indispensable.

Ultraviolet fluorescence of human sera: I. Sources of characteristic differences in the ultraviolet fluorescence spectra of sera from normal and cancer-bearing humans.

The ultraviolet fluorescence emission spectra of sera from apparently healthy persons and of sera from cancer patients frequently show significantly different curve shapes. A biparametric fluorescence method has been developed to use these deviations to detect patients with malignant diseases (Clin Chem 1986;32:1974-8). However, the pathobiochemical reasons for these differences of diagnostic importance have thus far been unclear. To find a relevant explanation for the described effect, we focused our interest on human serum proteins, the materials mainly responsible for the intrinsic fluorescence of sera in this spectral region. Human sera of various protein compositions were selected according to their protein pattern, determined by cellulose acetate electrophoresis, and their intrinsic fluorescence properties were investigated. We found that the emission spectra of human sera were correlated with their relative protein compositions, albumin and alpha-2 globulins being the most significantly correlated with the fluorescence intensity ratios. Because increased percentages of alpha-2 globulins and decreased percentages of albumin frequently accompany malignancies, we suggest that this condition accounts for the differences between the emission spectra of sera from normal and cancer-bearing humans.