Use of an integrated strip-free blood glucose monitoring system increases frequency of self-monitoring and improves glycemic control: Results from the ExAct study.

AIMS: We investigated the impact of using an integrated, strip-free system compared to the use of single-strip systems on testing frequency and glycemic control in individuals with insulin-treated diabetes. METHODS: This multinational, comparative, cluster-randomized, observational study included 311 patients with type 1 and insulin-treated type 2 diabetes who were performing SMBG at suboptimal frequencies. Sites were cluster-randomized to "integrated strip-free" system (EXP group) or any "single-strip" system (CNL group). Testing frequency and HbA1c were measured at baseline, 12 weeks and 24 weeks. RESULTS: At week 24, the EXP group showed an increase in SMBG frequency from baseline of 4.17 tests/week (95% CI 2.76, 5.58) compared with an increase of 0.53 tests/week (95% CI -0.73, 1.79) among CNL patients, resulting in a between-group difference of 3.63 tests/week (p < 0.0002). Mixed-effects models for repeated measurements (MMRM) controlling for baseline frequency of testing, country and clinical site confirmed a higher SMBG testing frequency in the EXP group compared to the CNL group, with a between-group difference of 2.70 tests/week (p < 0.01). Univariate analysis showed greater HbA1c reductions in the EXP group than CNL group: -0.44% (95% CI -0.59, -0.29) vs. -0.13% (95% CI -0.27, 0.01), respectively, p < 0.0002. MMRM analyses confirmed these HbA1c reductions. A greater percentage of EXP than CNL patients achieved HbA1c reductions of ≥0.5%: 45.1% vs. 29.1%, respectively, p < 0.01. CONCLUSIONS: The use of an integrated, strip-free SMBG system improved testing adherence and was associated with improvements in glycemic control.

ProAct study: new features of insulin pumps improve diabetes management and glycemic control in patients after transition of continuous subcutaneous insulin infusion systems.

BACKGROUND: Continuous subcutaneous insulin infusion (CSII) patients experience switches of pump systems on a regular basis. We investigated the impact of transition from older pumps to the Accu-Chek(®) Combo system (Roche Diagnostics Deutschland GmbH, Mannheim, Germany) on a patient's glycemic control and diabetes management. PATIENTS AND METHODS: In total, 299 patients (172 female, 127 male; mean±SD age, 39.4±15.2 years; CSII duration, 7.0±5.2 years) were enrolled by 61 European sites into this uncontrolled prospective trial. Glycemic control, safety, and diabetes management parameters were measured at baseline and after 3 and 6 months. Changes from baseline were analyzed. RESULTS: After transition to the new insulin pump, mean±SD hemoglobin A1c (HbA1c) values decreased from 7.8±1.1% (baseline) to 7.7±1.1% (end point). The proportion of patients with HbA1c <7.0% was slightly higher at the end of the study (29.6%) than at baseline (25.2%), whereas the proportion of patients with HbA1c >8.0% decreased (baseline, 36.2%; end point, 32.7%; P<0.05). The number of hypoglycemic episodes (blood glucose<70 mg/dL) improved slightly during the study (baseline, 40.4±34.0 events/quarter; end point, 39.2±33.9 events/quarter). Glycemic control improved significantly in the group with an initial HbA1c >8.0% (-0.46%; P<0.001) and remained solidly stable in the group with an initial HbA1c <7% (+0.04%; not significant). Short-term (<3 years) pump users (n=48) had a larger HbA1c decrease (-0.40%) than long-term (≥3 years) users (n=251) (-0.07%; P<0.05). The number of blood glucose measurements increased (3.7±1.9/day vs. 4.4±1.8/day; P<0.05), whereas the number of insulin boluses decreased (5.1±1.9/day vs. 4.6±1.5/day; P<0.05) during the study. CONCLUSIONS: Transition from older pump systems to the Accu-Chek Combo system in a large patient population resulted in stable glycemic control with significant improvements in HbA1c in patients with unsatisfactory baseline HbA1c and shorter pump use. Increased frequency of self-monitoring of blood glucose and decrease of bolus frequency could suggest a more confident diabetes management and a reduced need for correction boluses.

Somatic alterations in the melanoma genome: a high-resolution array-based comparative genomic hybridization study.

We performed DNA microarray-based comparative genomic hybridization to identify somatic alterations specific to melanoma genome in 60 human cell lines from metastasized melanoma and from 44 corresponding peripheral blood mononuclear cells. Our data showed gross but nonrandom somatic changes specific to the tumor genome. Although the CDKN2A (78%) and PTEN (70%) loci were the major targets of mono-allelic and bi-allelic deletions, amplifications affected loci with BRAF (53%) and NRAS (12%) as well as EGFR (52%), MITF (40%), NOTCH2 (35%), CCND1 (18%), MDM2 (18%), CCNE1 (10%), and CDK4 (8%). The amplified loci carried additional genes, many of which could potentially play a role in melanoma. Distinct patterns of copy number changes showed that alterations in CDKN2A tended to be more clustered in cell lines with mutations in the BRAF and NRAS genes; the PTEN locus was targeted mainly in conjunction with BRAF mutations. Amplification of CCND1, CDK4, and other loci was significantly increased in cell lines without BRAF-NRAS mutations and so was the loss of chromosome arms 13q and 16q. Our data suggest involvement of distinct genetic pathways that are driven either through oncogenic BRAF and NRAS mutations complemented by aberrations in the CDKN2A and PTEN genes or involve amplification of oncogenic genomic loci and loss of 13q and 16q. It also emerges that each tumor besides being affected by major and most common somatic genetic alterations also acquires additional genetic alterations that could be crucial in determining response to small molecular inhibitors that are being currently pursued.

Identification of ARHGEF17, DENND2D, FGFR3, and RB1 mutations in melanoma by inhibition of nonsense-mediated mRNA decay.

Gene identification by nonsense-mediated mRNA decay inhibition (GINI) has proven to be a strategy for genome-wide discovery of genes containing inactivating mutations in colon and prostate cancers. Here, we present the first study of inhibition of the nonsense-mediated mRNA decay (NMD) pathway in melanoma. We used a combination of emetine and actinomycin D treatment to stabilize mRNAs containing premature termination codons (PTCs), followed by microarray analysis and sequencing to identify novel tumor suppressor genes (TSGs) in a panel of 12 melanoma cell lines. Stringent analysis of the array data was used to select 35 candidate genes for sequencing. Of these, 4 (11%) were found to carry PTCs, including ARHGEF17, DENND2D, FGFR3, and RB1. While RB1 mutations have previously been described in melanoma, the other three genes represent potentially novel melanoma; TSGs. ARHGEF17 showed a G1865A mutation leading to W622X in a cell line derived from a mucosal melanoma; in RB1 a C1411T base change resulting in Q471X was discovered in a cell line derived from an acral melanoma; and the FGFR3 and DENND2D genes had intronic insertions leading to PTCs in cell lines derived from superficially spreading melanomas. We conclude that although the false positive rate is high, most likely due to the lack of DNA mismatch repair gene defects, the GINI protocol is one approach to discover novel TSGs in melanoma.

Differential gene expression in melanocytic nevi with the V600E BRAF mutation.

We studied gene expression in 18 melanocytic nevi with and four nevi without the V600E mutation in the BRAF gene using HG-U133A 2.0 microarray with 22,277 transcripts. Data analysis revealed 92 genes up-regulated and 105 genes down-regulated in nevi with the mutation compared to nevi without mutation. Pathway analysis showed that differentially regulated genes mapped to 10 genetic networks. The major network included genes involved in cell death, cell cycle, and cellular growth and proliferation. Up-regulated genes in nevi with the mutation included CDKN2A, CDKN1C, and MITF; whereas down-regulated genes included those involved in apoptotic and other pathways. Principal component analysis identified 22 probe sets (20 genes) that caused separate segregation of nevi with and without mutations. In conclusion, our data showed differences in gene expression between nevi with and without the V600E BRAF mutation. Moreover, nevi with mutations showed over-expression of genes involved in melanocytic senescence and cell cycle inhibition.

B-RAF and N-RAS mutations are preserved during short time in vitro propagation and differentially impact prognosis.

In melanoma, the RAS/RAF/MEK/ERK signalling pathway is an area of great interest, because it regulates tumor cell proliferation and survival. A varying mutation rate has been reported for B-RAF and N-RAS, which has been largely attributed to the differential source of tumor DNA analyzed, e.g., fixed tumor tissues or in vitro propagated melanoma cells. Notably, this variation also interfered with interpreting the impact of these mutations on the clinical course of the disease. Consequently, we investigated the mutational profile of B-RAF and N-RAS in biopsies and corresponding cell lines from metastatic tumor lesions of 109 melanoma patients (AJCC stage III/IV), and its respective impact on survival. 97 tissue biopsies and 105 biopsy-derived cell lines were screened for B-RAF and N-RAS mutations by PCR single strand conformation polymorphism and DNA sequencing. Mutations were correlated with patient survival data obtained within a median follow-up time of 31 months. B-RAF mutations were detected in 55% tissues and 51% cell lines, N-RAS mutations in 23% tissues and 25% cell lines, respectively. There was strong concordance between the mutational status of tissues and corresponding cell lines, showing a differing status for B-RAF in only 5% and N-RAS in only 6%, respectively. Patients with tumors carrying mutated B-RAF showed an impaired median survival (8.0 versus 11.8 months, p = 0.055, tissues; 7.1 versus 9.3 months, p = 0.068, cell lines), whereas patients with N-RAS-mutated tumors presented with a favorable prognosis (median survival 12.5 versus 7.9 months, p = 0.084, tissues; 15.4 versus 6.8 months, p = 0.0008, cell lines), each in comparison with wildtype gene status. Multivariate analysis qualified N-RAS (p = 0.006) but not B-RAF mutation status as an independent prognostic factor of overall survival. Our findings demonstrate that B-RAF and N-RAS mutations are well preserved during short term in vitro propagation and, most importantly, differentially impact the outcome of melanoma patients.

Gene expression profiling of human endometrial-trophoblast interaction in a coculture model.

Investigating the interaction of human endometrium and trophoblast during implantation is difficult in vitro and impossible in vivo. This study was designed to analyze the effect of trophoblast on endometrial stromal cells during implantation by comprehensive gene profiling. An in vitro coculture system of endometrial stromal cells with first-trimester trophoblast explants was established. Trophoblast and endometrial stromal cells were separated after 24 h. Gene expression of endometrial stromal cells after coculture was compared with the gene expression of endometrial stromal cells cultured alone by microarray analysis. We confirmed the expression of distinct genes using real-time PCR. Genes up-regulated included those for inflammatory response, immune response, and chemotaxis (pentraxin-related gene 3, chemokine ligands, IL-8, IL-1 receptors, IL-18 receptor, IL-15, IL-15 receptor, TNF-alpha-induced protein 6, and IL-6 signal transducer), regulators of cell growth (IGF-binding proteins 1 and 2) and signal transduction. Also up-regulated were genes for growth and development, glucose metabolism, and lipid metabolism: DKK-1, WISP, IGF-II, hydroxysteroid 11beta-dehydrogenase 1, hydroxyprostaglandin dehydrogenase 15, prostaglandin E synthase, prostaglandin F receptor, aldehyde dehydrogenase 1 family, member A3 and phosphatidic acid phosphatase type 2B. Other genes included genes for cell-cell signaling (pre-B-cell colony-enhancing factor 1), proteolysis, calcium ion binding, regulation of transcription, and others. Down-regulated genes included genes for proteolysis (MMP-11 and mitochondrial intermediate peptidase), genes for cell death (caspase 6, death-associated protein kinase 1, and histone deacetylase 5), transcription factors (sex determining region Y-box 4, dachshund homolog 1, ets variant gene 1, and zinc finger protein 84 and 435), and genes for humoral immune response (CD24 antigen). Trophoblast has a significant impact on endometrial stromal cell gene expression. Some of the genes regulated by trophoblast in endometrial stromal cells are already known to be regulated by progesterone and show the endocrine function of trophoblast during pregnancy. Others are genes so far unknown to play a role in endometrial-trophoblast interaction and open a wide field of investigation.

Differences in global gene expression in melanoma cell lines with and without homozygous deletion of the CDKN2A locus genes.

We studied differential global gene expression in four melanoma cell lines with three cell lines without homozygous deletion of the CDKN2A locus using HG-U133A microarrays with 22 277 transcripts. None of the cell lines carried mutations in the B-RAF and N-RAS genes. Data analysis using stringent criteria showed specific upregulation of 70 genes and downregulation of 86 genes in cell lines with homozygous deletion of the CDKN2A gene. A comparison with previous expression data showed overlapping of upregulation and downregulation of seven and 23 genes, respectively, in melanoma cell lines with homozygous deletion of the CDKN2A locus or mutations in the B-RAF and N-RAS genes. Microarray data for eight selected genes were validated with an extended number of cell lines using quantitative real-time polymerase chain reaction. The upregulated genes in cell lines with the deletion besides others included MAGE A2 [fold change 128, 95% confidence interval (CI) 82.8-172.2; t-test P=0.004], MAGE A6 (fold change 623, 95% CI 473.4-772.1; t-test P=0.001), MAGE A12 (fold change 90, 95% CI 65.1-115.5; t-test P=0.001) and dopachrome tautomerase (fold change 42, 95% CI 32.5-51.8; t-test P=0.001). Downregulated genes included interleukin 18 (fold change 489, 95% CI 146.4-831.2; t-test P=0.04), ID2 (fold change 3, 95% CI 2.2-4.9; t-test P=0.001), KLF4 (fold change 9, 95% CI 4.3-14.7; P=0.01) and CD24 antigen (fold change 1308, 95% CI 766.0-1850.8; t-test P=0.01). The upregulated genes common to cell lines with homozygous deletion of the CDKN2A gene and mutations in B-RAF and N-RAS gene included those that are involved in RAS/RAF/MEK/ERK pathways. Our results highlight effects of homozygous deletion of the CDKN2A locus on global gene expression.

Effect of common B-RAF and N-RAS mutations on global gene expression in melanoma cell lines.

We studied global gene expression in three melanoma cell lines with the most common and potent V600E mutation in the B-RAF gene-four cell lines with a common Q61R mutation in the N-RAS gene and three cell lines with no mutations using human HG-U133A 2.0 micro-arrays with 22 277 transcripts. Data analysis using stringent criteria revealed several upregulated and downregulated genes in cell lines with B-RAF and N-RAS mutations compared with cell lines without mutations. We found 29 genes specifically upregulated and 32 genes downregulated in cell lines with B-RAF mutations, whereas 70 genes were upregulated and 39 downregulated in cell lines with N-RAS mutations; 11 genes showed overlapping upregulation and 45 downregulation. The micro-array data for nine selected genes were validated by the real-time PCR technique. Expression of a large number of genes, that encode members or regulators of the RAS/RAF/MEK/ERK pathways or are involved in metastasis or invasion, was affected in cell lines with mutations in B-RAF and N-RAS. Upregulated genes in cell lines with mutations included dual-specificity phosphatase 6 (DUSP6), sprouty 2 (SPRY2), v-akt murine thymoma viral oncogene homolog 3 (AKT3) and matrix metalloproteinase 14 (MMP14); downregulated genes included interleukin 18 (IL18), Krüppel-like factor 5 (KLF5) and inhibitor of DNA binding 2 (ID2). Our results, though carried on cell lines, provide a novel insight into the effect of mutations in the B-RAF and N-RAS genes on global gene expression in melanoma and highlight the complexity of mechanisms involved in tumour initiation and maintenance.