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Background Use of exosomes as biomarkers in non‐small cell lung cancer (NSCLC) is an intriguing approach in the liquid‐biopsy era. Exosomes are nano‐sized vesicles with membrane‐bound proteins that reflect their originating cell. Prognostic biomarkers are needed to improve patient selection for optimal treatment. We here evaluate exosomes by protein phenotyping as a prognostic biomarker in NSCLC. Methods Exosomes from plasma of 276 NSCLC patients were phenotyped using the Extracellular Vesicle Array; 49 antibodies captured the proteins on the exosomes, and a cocktail of biotin‐conjugated antibodies binding the general exosome markers CD9, CD81 and CD63 was used to visualise the captured exosomes. For each individual membrane‐bound protein, results were analysed based on presence, in a concentration‐dependent manner, and correlated to overall survival (OS). Results The 49 proteins attached to the exosomal membrane were evaluated. NY‐ESO‐1, EGFR, PLAP, EpCam and Alix had a significant concentration‐dependent impact on inferior OS. Due to multiple testing, NY‐ESO‐1 was the only marker that maintained a significant impact on inferior survival (hazard rate (HR) 1.78 95% (1.78–2.44); p = 0.0001) after Bonferroni correction. Results were adjusted for clinico‐pathological characteristics, stage, histology, age, sex and performance status. Conclusion We illustrate the promising aspects associated with the use of exosomal membrane‐bound proteins as a biomarker and demonstrate that they are a strong prognostic biomarker in NSCLC., Highlights In NSCLC, exosomal membrane‐bound protein profiling is feasible in liquid biopsies.Nine exosomal proteins showed potential as prognostic markers in NSCLC.Exosomal NY‐ESO‐1 was a strong prognostic biomarkers in NSCLC.

Recent studies have suggested that mutations in the mitochondrial genome (mtDNA) may play a role in the development and response to treatment for human cancer. The aim of this study was to investigate whether mtDNA variations have any prognostic relevance, to clarify the spectra of mtDNA variation and to determine whether there was any correlation to known prognostic factors in acute myeloid leukemia (AML). To elucidate this, we sequenced the entire mtDNA in 56 AML patients and 14 control subjects. When analyzing the biologic impact of the non-synonymous variations in the mtDNA coding genes, we found an inferior disease-free survival for patients exhibiting variations in the two most important catalytic genes of the complex IV of the oxidative phosphorylation complexes (OXPHOS), that is, the cytochrome c oxidase subunit I and the cytochrome c oxidase subunit II (hazard ratio 2.6, P = 0.03; multivariate analysis). In addition, the most frequent variation was the T16311C in the control region, which was found in 11 (20%) of the 56 patients. This observation was confirmed in another cohort of 173 diagnostic AML samples. In this expanded group, the T16311C variation tended to be associated with chromosomal abnormalities.

This prospective Phase II study is the first to assess the feasibility and efficacy of maintenance 5-azacytidine for older patients with high-risk myelodysplastic syndrome (MDS), chronic myelomonocytic leukaemia and MDS-acute myeloid leukaemia syndromes in complete remission (CR) after induction chemotherapy. Sixty patients were enrolled and treated by standard induction chemotherapy. Patients that reached CR started maintenance therapy with subcutaneous azacytidine, 5/28 d until relapse. Promoter-methylation status of CDKN2B (P15 ink4b), CDH1 and HIC1 was examined pre-induction, in CR and 6, 12 and 24 months post CR. Twenty-four (40%) patients achieved CR after induction chemotherapy and 23 started maintenance treatment with azacytidine. Median CR duration was 13.5 months, >24 months in 17% of the patients, and 18-30.5 months in the four patients with trisomy 8. CR duration was not associated with CDKN2B methylation status or karyotype. Median overall survival was 20 months. Hypermethylation of CDH1 was significantly associated with low CR rate, early relapse, and short overall survival (P = 0.003). 5-azacytidine treatment, at a dose of 60 mg/m(2) was well tolerated. Grade III-IV thrombocytopenia and neutropenia occurred after 9.5 and 30% of the cycles, respectively, while haemoglobin levels increased during treatment. 5-azacytidine treatment is safe, feasible and may be of benefit in a subset of patients.

Udgivelsesdato: 2009-Nov HOXA4 gene expression is a predictor for outcome in normal karyotypic acute myeloid leukaemia (AML) patients. Given that Meis1 is a co-factor for Hox genes, we hypothesized that the combined expression of HOXA4 and MEIS1 might add prognostic information in these patients. When diagnostic samples from 246 AML patients were divided into three main groups based on gene expression levels of HOXA4 combined with MEIS1 we found that within the group of patients exhibiting low levels of HOXA4, those with a high expression of MEIS1 had a significantly worse outcome than those exhibiting low MEIS1 expression (P = 0.025). Moreover, this prediction was independent of cytogenetics, mutational status of the NPM1 and FLT3 genes as well as upon WBC and age. To evaluate the possible contribution of regulatory events underlying these observations, 157 patient samples were subjected to promoter hypermethylation analysis. We observed that 77% were HOXA4- and 15%MEIS1 hypermethylated and that this epigenetic alteration was highly correlated to the gene expression level (MEIS1: P = 0.001; HOXA4: P = 0.007). Finally, we found a higher expression level and a higher frequency of hypermethylation of HOXA4 among patients with NPM1 mutations. In conclusion, our data show that the combination of low HOXA4 and low MEIS1 gene expression is a favourable predictor for outcome in all AML patients and that the expression levels are governed by the methylation state of these genes.

A novel transdermal formulation of fentanyl-containing dipropylene glycol droplets dispersed in a silicone matrix with a rate-controlling membrane was developed. Healthy male subjects (n = 24) received repeated 72-hour applications of fentanyl (50 mug/h) as the novel matrix and the conventional reservoir formulations in a randomized, 2-way crossover study. Blood samples were collected, and serum concentrations of fentanyl were assayed using liquid chromatography with mass spectrometry detection. The mean area under the curve (AUCtau) and peak concentrations (C(max)) of the matrix formulation were 84 838 pg.h/mL and 1680 pg/mL, respectively. Ratio and 90% confidence intervals of AUCtau and C(max) between the 2 formulations were within 80% to 125%. Adherence of the matrix formulation was higher than the reservoir formulation (62.5 vs 56.2%, P < .0001), without affecting skin irritation. Vital signs and adverse events of the 2 formulations were similar in nature and frequency. The novel matrix formulation displayed enhanced adherence and resulted in similar pharmacokinetics and tolerability as the reservoir formulation.

Although a number of molecular aberrations have been described in acute myeloid leukaemia (AML), no study has yet determined their relative prognostic importance. We have analysed blast cells from 250 adult patients treated at the same institution during a 15-year period. Balanced translocations were detected by multiplex polymerase chain reaction in 13% of the cases. Internal tandem duplication (ITD) of the FLT3 gene and partial tandem duplication of the MLL gene were found in 24% and 4%, respectively. Promoter hypermethylation of the P15, CDH1, ER and MDR1 genes was observed in 71%, 64%, 40% and 4%, respectively. Compared with normal bone marrow, the chemotherapy resistance protein MRP1 and apoptosis related genes BAX and CASPASE3 were found to be overexpressed in AML blasts. Univariate analysis revealed that the most important determinants of prognosis were presence of balanced translocations, age, white blood cell count and extramedullary disease, in order of statistical significance. In a multivariate analysis, balanced translocations retained their prognostic significance and FLT3 ITD as well as high gene expression of MDR1 were negative prognostic factors. From these data, which are the first to compare these molecular aberrations directly, we conclude that, when a battery of molecular changes is evaluated for upfront significance in AML, recurrent translocations are of prime importance for treatment outcome.

Low‐dose total body irradiation (LTBI) is known for its antitumor immune modulatory effects. Moreover, there is theoretical ground suggesting that combining LTBI with interleukin‐2 (IL‐2) will have a synergistic immune‐mediated antitumor effect. However, the use of LTBI in combination with IL‐2 or other forms of immunotherapy has not been tested before. Aim of the work: To test the efficacy of combining LTBI and IL‐2 in controlling lung metastases in a murine model for malignant melanoma compared to IL‐2 alone. Material and methods: Ten‐week‐old female C57BL/6 mice were inoculated intravenously (on day 0) with 1 million B16F1 malignant melanoma cells. The mice received either no treatment (control group), LTBI alone (single fraction of 0.75 Gy), IL‐2 treatment alone (30,000 CU × 2 daily for 5 consecutive days), or a combination of LTBI and IL‐2. LTBI was given on day +7 and IL‐2 treatment started day +8. On day +14, the mice were sacrificed and the lungs were removed and analyzed for tumor burden. Lung sections were also tested for tumor‐infiltrating cells using immunohistochemical staining. Peripheral blood and splenic cells were collected and tested for the percentage of the various lymphocytic subsets using immunostaining and flow cytometry. Results: Tumor burden expressed as the percentage of lung area occupied with metastases (±1 SD), was the same in the control group (8.1 ± 4.9%), and in the group receiving LTBI alone (8.3 ± 4.5%). Tumor burden was reduced to 6.4%(±3.4%) in the IL‐2 alone group (P = 0.3) and further reduced to 3%(±1%) in the combined treatment group (P = 0.004). The difference in tumor burden between the IL‐2 alone group and the combined treatment group was statistically significant (P = 0.006). The combined treatment caused a significant increase in the number of natural killer (NK) cells and macrophages infiltrating the metastatic sites. This was associated with a significant increase in the percentage of CD122+(IL‐2Rβ) cells and NK cells in both peripheral blood and spleens. Conclusion: We conclude that combining LTBI and IL‐2 treatment is synergistic and therapeutically more effective than IL‐2 alone. The data points to NK cells and macrophages as likely major effectors of the synergistic outcome of the combined treatment. This observation may have important clinical implications in the treatment of patients with metastatic malignant melanoma.

Self-clustering of the five common nucleobases was investigated by electrospray ionization tandem mass spectrometry and shown to provide insight into the non-covalent interactions between identical bases. Alkali and ammonium cations significantly increase self-aggregation of the nucleobases and lead to the formation of uniquely stable magic number clusters. Sodium adducts of guanine, thymine and uracil preferentially take the form of tetrameric (quartet) clusters. This gas-phase result correlates with previously reported solution-phase data on sodium cation stabilized guanosine, thymine and uracil quartet structures believed to be responsible for telomere stabilization. In the presence of potassium, cesium or ammonium cations, pentameric magic number clusters are formed from thymine and uracil, while in solution the nucleoside isoguanosine yields clusters of this favored size. The formation of magic number metaclusters occurs for thymine and uracil in the presence of ammonium cations. These doubly charged 10- and 15-mers are tentatively attributed to the formation of pentamer/ammonium cation/ pentamer sandwich structures.

This paper describes the use of desorption chemical ionisation membrane inlet mass spectrometry (DCI-MIMS) for the detection of a broad range of common contaminants in water. In addition, we discuss the advantages/disadvantages of two different types of chemical ionisation reagent gases, i-butene (a Broensted acid reagent) and argon (a charge exchange reagent). We found that polyaromatic hydrocarbons was detectable at ppt levels, the estrogenic compounds diethyl phthalate and octylphenol at high ppt levels, steroid hormones at ppb levels, hydrophobic pesticides at low ppb levels, whereas hydrophilic pesticides and bisphenol A were not detectable at all. With the exception of the polyaromatic hydrocarbons and pentachlorophenol, none of the reported compounds have to our knowledge been detected previously by other MIMS systems. In most cases the Broensted acid reagent gave characteristic ions at high mass/charge ratio, whereas the charge exchange reagent gave less characteristic ions at low m/z ratio. However, the sensitivity was generally not as good with the Broensted acid reagent as with the charge exchange reagent, since the Broensted acid reagent, i-butene, gave a large chemical background. Copyright © 2001 John Wiley & Sons, Ltd.

The candidate tumour suppressor gene MMAC1/PTEN located at chromosome 10q23.3 has been reported to be frequently mutated in a number of solid tumours. Less is known about its status in leukaemia. In the present study we first analysed 13 leukaemia cell lines for mutations and homozygous deletions in MMAC1/PTEN using PCR and denaturing gradient gel electrophoresis (DGGE). We identified an intragenic deletion including MMAC1/PTEN exons 2-5 in an acute myelocytic leukaemia cell line, HL-60 blast, and an insertion of four nucleotides in exon 5 in an acute monocytic leukaemia cell line, U937. Analysis of 59 patients with acute myeloid leukaemia (AML), 26 patients with myelodysplastic syndromes (MDS) and 10 patients with chronic myeloid leukaemia (CML) only revealed a polymorphic base substitution in codon 44 in one AML patient, suggesting that mutations in the MMAC1/PTEN gene are infrequent genetic aberrations in myeloid leukaemia.