First-line oxaliplatin-based chemotherapy and nivolumab for metastatic microsatellite-stable colorectal cancer-the randomised METIMMOX trial.

BACKGROUND: We evaluated first-line treatment of metastatic microsatellite-stable colorectal cancer with short-course oxaliplatin-based chemotherapy alternating with immune checkpoint blockade. METHODS: Patients were randomly assigned to chemotherapy (the FLOX regimen; control group) or alternating two cycles each of FLOX and nivolumab (experimental group). Radiographic response assessment was done every eight weeks with progression-free survival (PFS) as the primary endpoint. Cox proportional-hazards regression models estimated associations between PFS and relevant variables. A post hoc analysis explored C-reactive protein as signal of responsiveness to immune checkpoint blockade. RESULTS: Eighty patients were randomised and 38 in each group received treatment. PFS was comparable-control group: median 9.2 months (95% confidence interval (CI), 6.3-12.7); experimental group: median 9.2 months (95% CI, 4.5-15.0). The adjusted Cox model revealed that experimental-group subjects aged ≥60 had significantly lowered progression risk (p = 0.021) with hazard ratio 0.17 (95% CI, 0.04-0.76). Experimental-group patients with C-reactive protein <5.0 mg/L when starting nivolumab (n = 17) reached median PFS 15.8 months (95% CI, 7.8-23.7). One-sixth of experimental-group cases (all KRAS/BRAF-mutant) achieved complete response. CONCLUSIONS: The investigational regimen did not improve the primary outcome for the intention-to-treat population but might benefit small subgroups of patients with previously untreated, metastatic microsatellite-stable colorectal cancer. TRIAL REGISTRATION: ClinicalTrials.gov number, NCT03388190 (02/01/2018).

The association of urine markers of iodine intake with development and growth among children in rural Uganda: a secondary analysis of a randomised education trial.

OBJECTIVE: We examined associations of urine iodide excretion, proxy for iodine intake, with child development and growth. DESIGN: This is a secondary analysis of a 1:1 cluster-randomised trial with a 6-month nutrition/stimulation/hygiene education intervention among mothers of children aged 6-8 months to improve child development and growth. Development was assessed using Bayley Scales of Infant and Toddler Development-III (BSID-III) and Ages and Stages Questionnaire (ASQ), whereas anthropometry was used to assess growth. Urine iodide concentration (UIC) and urine iodide/creatinine ratio (ICR) were measured. SETTING: The current study was conducted in southern Uganda. PARTICIPANTS: We randomly selected 155 children from the 511 enrolled into the original trial and analysed data when they were aged 20-24 and 36 months. RESULTS: Median UIC for both study groups at 20-24 and 36 months were similar (P > 0·05) and within the normal range of 100-199 µg/l (0·79-1·60 µmol/l), whereas the intervention group had significantly higher ICR at 20-24 months. The BSID-III cognitive score was positively associated (P = 0·028) with ICR at 20-24 months in the intervention group. The ASQ gross motor score was negatively associated (P = 0·020) with ICR at 20-24 months among the controls. ICR was not significantly associated with anthropometry in the two study groups at either time-point. CONCLUSIONS: Following the intervention, a positive association was noted between ICR and child's cognitive score at 20-24 months, whereas no positive association with ICR and growth was detected. Iodine sufficiency may be important for child's cognitive development in this setting.

Longitudinal plasma metabolic profiles, infant feeding, and islet autoimmunity in the MIDIA study.

AIMS: The aim of this study was to investigate the longitudinal plasma metabolic profiles in healthy infants and the potential association with breastfeeding duration and islet autoantibodies predictive of type 1 diabetes. METHOD: Up to four longitudinal plasma samples from age 3 months from case children who developed islet autoimmunity (n = 29) and autoantibody-negative control children (n = 29) with the HLA DR4-DQ8/DR3-DQ2 genotype were analyzed using two-dimensional gas chromatography coupled to a time-of-flight mass spectrometer for detection of small polar metabolites. RESULTS: Plasma metabolite levels were found to depend strongly on age, with fold changes varying up to 50% from age 3 to 24 months (p < 0.001 after correction for multiple testing). Tyrosine levels tended to be lower in case children, but this was not significant after correction for multiple testing. Ornithine levels were lower in case children compared with the controls at the time of seroconversion, but the difference was not statistically significant after correcting for multiple testing. Breastfeeding for at least 3 months as compared with shorter duration was associated with higher plasma levels of isoleucine, and lower levels of methionine and 3,4-dihydroxybutyric acid at 3 months of age. CONCLUSIONS: Plasma levels of several small, polar metabolites changed with age during early childhood, independent of later islet autoimmunity status and sex. Breastfeeding was associated with higher levels of branched-chain amino acids, and lower levels of methionine and 3,4-dihydroxybutyric acid.

HbA1c analysis by capillary electrophoresis - comparison with chromatography and an immunological method.

BACKGROUND: Haemoglobin A1c (HbA1c) has become an even more important analyte for clinical laboratories during recent years with the introduction of its diagnostic use for diabetes mellitus. Several different analytical principles can be used, each with their advantages and disadvantages. AIM: We wanted to compare Sebia Capillarys 2 Flex Piercing (Capillarys) with our routine HbA1c methods, which were an HPLC method (Tosoh G7) and an immunoassay (Tina-Quant on Roche Modular P) by analysing a large clinical material. Furthermore, we investigated sample stability. METHODS: HbA1c analysis was performed in parallel by all three methods for more than 600 patient samples including common and some rare haemoglobin variants, as well as for several controls, some with set target values. Sample stability at room temperature and refrigerated was assessed for up to seven days. RESULTS: Capillarys produced generally somewhat lower HbA1c values than both comparison methods, apparently due to positive bias for the comparison methods. Leaving out samples with haemoglobin variants, we found a mean bias (95% CI) for Capillarys compared to Tosoh G7 (without factorization) and Modular of -0.39 (-0.40 to -0.38) and -0.16 (-0.17 to -0.14) % HbA1c, respectively. HbA1c results were similar between instruments for samples from dialysis patients and for samples with heterozygous common haemoglobin variants, except that Tosoh G7 reported too low results in the presence of Hb E. For heterozygous Hb Raleigh, Capillarys and the immunoassay gave similar results. CONCLUSION: Capillarys is a convenient instrument for routine HbA1c analysis.

Analytical Bias Exceeding Desirable Quality Goal in 4 out of 5 Common Immunoassays: Results of a Native Single Serum Sample External Quality Assessment Program for Cobalamin, Folate, Ferritin, Thyroid-Stimulating Hormone, and Free T4 Analyses.

BACKGROUND: We undertook this study to evaluate method differences for 5 components analyzed by immunoassays, to explore whether the use of method-dependent reference intervals may compensate for method differences, and to investigate commutability of external quality assessment (EQA) materials. METHODS: Twenty fresh native single serum samples, a fresh native serum pool, Nordic Federation of Clinical Chemistry Reference Serum X (serum X) (serum pool), and 2 EQA materials were sent to 38 laboratories for measurement of cobalamin, folate, ferritin, free T4, and thyroid-stimulating hormone (TSH) by 5 different measurement procedures [Roche Cobas (n = 15), Roche Modular (n = 4), Abbott Architect (n = 8), Beckman Coulter Unicel (n = 2), and Siemens ADVIA Centaur (n = 9)]. The target value for each component was calculated based on the mean of method means or measured by a reference measurement procedure (free T4). Quality specifications were based on biological variation. Local reference intervals were reported from all laboratories. RESULTS: Method differences that exceeded acceptable bias were found for all components except folate. Free T4 differences from the uncommonly used reference measurement procedure were large. Reference intervals differed between measurement procedures but also within 1 measurement procedure. The serum X material was commutable for all components and measurement procedures, whereas the EQA materials were noncommutable in 13 of 50 occasions (5 components, 5 methods, 2 EQA materials). CONCLUSIONS: The bias between the measurement procedures was unacceptably large in 4/5 tested components. Traceability to reference materials as claimed by the manufacturers did not lead to acceptable harmonization. Adjustment of reference intervals in accordance with method differences and use of commutable EQA samples are not implemented commonly.

Urine NMR metabolomics analysis of breastfeeding biomarkers during and after pregnancy in a large prospective cohort study.

BACKGROUND: Modern metabolomic profiling has not yet been applied to human breastfeeding research. A common reason for breastfeeding cessation is perceived insufficient milk production. We investigated broad biochemical profiles in maternal urine collected during and after pregnancy to identify biomarkers related to reduced reported breastfeeding. METHODS: Fasting urine was collected at three consultations (visit V1: gestational week 8-20; V2: week 28 ± 2; V3: 10-16 weeks postpartum) in the STORK Groruddalen program, a prospective, multiethnic cohort study of gestational diabetes involving healthy, pregnant women in Oslo, Norway, and analyzed using NMR spectroscopy. Breastfeeding at V3 was recorded in three categories: Exclusively breastfeeding (n = 326), partially breastfeeding (n = 156) and formula feeding (n = 67). RESULTS: Five metabolites were relevant to breastfeeding. Lactose was detected at V1 and increased to 0.1 mM/mM creatinine at V2. Postpartum excretion at V3 was significantly higher in exclusively breastfeeding women than partially or non-breastfeeding (median = 0.29, 0.23 and 0.04 mM/mM creatine, respectively; ANOVA p-value = 2e-70). Glycine excretion at V3 (0.12, 0.10 and 0.06, respectively; p = 2e-5) and at V2 were associated with breastfeeding (0.34, 0.33 and 0.26, respectively; p = 4e-5). Creatine and two unidentified substances also correlated with breastfeeding. NMR metabolomics found no other metabolites differing between categories during pregnancy (V1, V2), and did not predict individual breastfeeding postpartum (V3). CONCLUSION: Decreased glycine excretion at V2 may indicate difficulties meeting the metabolic demands of the growing fetus, but urine profiles contained otherwise little indication of early adaptations during pregnancy towards reduced biological potential to breastfeed.

Glyoxalase 1 enzyme activity in erythrocytes and Ala111Glu polymorphism in type 1-diabetes patients.

AIMS/HYPOTHESIS: The enzyme glyoxalase 1 (GLO1) can inactivate the glycoxidation product methylglyoxal that is thought to be an important contributor to the pathogenesis of vascular complications in diabetes. We aimed to study erythrocyte GLO1 activity and whether the Ala111Glu GLO1 gene polymorphism affected GLO1 activity. METHODS: Fasting erythrocyte GLO1 activity was measured spectrophotometrically. The A111G gene polymorphism, assessed in DNA from leucocytes was analyzed in patients with type 1-diabetes and normal kidney function and compared with a control group. RESULTS: Sixty-one patients with type 1-diabetes duration of 26.1 (10.7) years, mean (SD) with a HbA1c of 7.8 (0.9)%, 61.7 (9.9) mmol/mol and normal glomerular filtration rate were compared with 62 age- and sex-matched healthy controls. GLO1 activity was 0.206 (0.183-0.231) median (25-75% percentiles) U/mg Hb in the control group vs. 0.192 (0.165-0.224) in the diabetes group, (p = 0.149). In the diabetes group GLO1 correlated with HbA1c (r = 0.33, p < 0.01) and oxidized glutathione (GSSG) (r = - 0.34, p < 0.01) and in the control group with GSH (r = 0.37, p < 0.005) and fasting glucose (r = 0.26, p < 0.04). In a multiple regression analysis with GLO1 activity as the dependent variable, including the Ala111Glu polymorphism, the significant independent variables were log GSSG (ß - 0.318, p = 0.02) and HbA1c (ß 0.285, p = 0.041) in the diabetes group and log GSH, (ß 0.407, p = 0.004) in the control group. CONCLUSIONS/INTERPRETATION: Erythrocyte glyoxalase 1 activity did not differ between patients with type 1-diabetes and controls. The Ala111Glu glyoxalase gene polymorphism did not have an effect on glyoxalase 1 activity in either group.

Do changes in persistent organic pollutants after bariatric surgery cause endocrine disruption?

BACKGROUND: Bariatric surgery results in weight loss, marked endocrine changes and the release of persistent organic pollutants (POPs). The release of POPs might cause endocrine disruption. The study aimed to explore associations between POPs and adiponectin, leptin and ghrelin in subjects undergoing bariatric surgery. METHODS: The study included 63 subjects with severe obesity (men/women: 13/50), age (years): 45.0 (8.5), and BMI (kg/m2) 39.1 (3.4). Analyses of adiponectin, leptin and ghrelin and POPs (hexachlorobenzene (HCB), dichlorodiphenyldichloroethylene (p,p'-DDE), polychlorinated biphenyl (PCB) 118 (dioxin-like compound; dl), and sum 6 PCB (PCB 28, -52, -101, -138, -153, and -180) were performed before and 12 months after bariatric surgery. RESULTS: There were significant increases in adiponectin and all POPs and a fall in leptin after surgery. The main finding was the highly significant associations between adiponectin and all POPs. The increase in HCB explained 38% of the variation in adiponectin. CONCLUSIONS: If the POP-associated increase in adiponectin is a causal effect, the release of POPs might have important clinical consequences. Adiponectin has both positive and negative clinical effects exerted by essentially unknown mechanisms. The effects of released POPs on the metabolic functions in subjects undergoing bariatric surgery deserve further evaluation.

A novel 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) splice variant with an alternative exon 1 potentially encoding an extended N-terminus.

BACKGROUND: The major rate-limiting enzyme for de novo cholesterol synthesis is 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). HMGCR is sterically inhibited by statins, the most commonly prescribed drugs for the prevention of cardiovascular events. Alternative splicing of HMGCR has been implicated in the control of cholesterol homeostasis. The aim of this study was to identify novel alternatively spliced variants of HMGCR with potential physiological importance. RESULTS: Bioinformatic analyses predicted three novel HMGCR transcripts containing an alternative exon 1 (HMGCR-1b, -1c, -1d) compared with the canonical transcript (HMGCR-1a). The open reading frame of the HMGCR-1b transcript potentially encodes 20 additional amino acids at the N-terminus, compared with HMGCR-1a. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was used to examine the mRNA levels of HMGCR in different tissues; HMGCR-1a was the most highly expressed variant in most tissues, with the exception of the skin, esophagus, and uterine cervix, in which HMGCR-1b was the most highly expressed transcript. Atorvastatin treatment of HepG2 cells resulted in increased HMGCR-1b mRNA levels, but unaltered proximal promoter activity compared to untreated cells. In contrast, HMGCR-1c showed a more restricted transcription pattern, but was also induced by atorvastatin treatment. CONCLUSIONS: The gene encoding HMGCR uses alternative, mutually exclusive exon 1 sequences. This contributes to an increased complexity of HMGCR transcripts. Further studies are needed to investigate whether HMGCR splice variants identified in this study are physiologically functional.

Gene expression results in lipopolysaccharide-stimulated monocytes depend significantly on the choice of reference genes.

BACKGROUND: Gene expression in lipopolysaccharide (LPS)-stimulated monocytes is mainly studied by quantitative real-time reverse transcription PCR (RT-qPCR) using GAPDH (glyceraldehyde 3-phosphate dehydrogenase) or ACTB (beta-actin) as reference gene for normalization. Expression of traditional reference genes has been shown to vary substantially under certain conditions leading to invalid results. To investigate whether traditional reference genes are stably expressed in LPS-stimulated monocytes or if RT-qPCR results are dependent on the choice of reference genes, we have assessed and evaluated gene expression stability of twelve candidate reference genes in this model system. RESULTS: Twelve candidate reference genes were quantified by RT-qPCR in LPS-stimulated, human monocytes and evaluated using the programs geNorm, Normfinder and BestKeeper. geNorm ranked PPIB (cyclophilin B), B2M (beta-2-microglobulin) and PPIA (cyclophilin A) as the best combination for gene expression normalization in LPS-stimulated monocytes. Normfinder suggested TBP (TATA-box binding protein) and B2M as the best combination. Compared to these combinations, normalization using GAPDH alone resulted in significantly higher changes of TNF-alpha (tumor necrosis factor-alpha) and IL10 (interleukin 10) expression. Moreover, a significant difference in TNF-alpha expression between monocytes stimulated with equimolar concentrations of LPS from N. meningitides and E. coli, respectively, was identified when using the suggested combinations of reference genes for normalization, but stayed unrecognized when employing a single reference gene, ACTB or GAPDH. CONCLUSIONS: Gene expression levels in LPS-stimulated monocytes based on RT-qPCR results differ significantly when normalized to a single gene or a combination of stably expressed reference genes. Proper evaluation of reference gene stabiliy is therefore mandatory before reporting RT-qPCR results in LPS-stimulated monocytes.

HDAC2 expression and variable number of repeats in exon 1 of the HDAC2 gene in corticotroph adenomas.

OBJECTIVES: Alterations in protein expression of histone deacetylase 2 (HDAC2) have been demonstrated in various neoplasms, and lack of nuclear expression of HDAC2 has previously been shown in some human and canine corticotroph adenomas. This study aimed to examine HDAC2 expression in a Norwegian cohort of corticotroph adenomas, screen for exonic HDAC2 gene variants in the adenomas and correlate the results with clinical data. PATIENTS AND DESIGN: Forty-four patients with verified Cushing's disease or Nelson's syndrome, positive ACTH staining and tissue available for immunohistochemistry and/or DNA sequencing were included. Ninety-four controls were chosen from the Norwegian Bone Marrow Registry. RESULTS: Histone deacetylase 2 expression examined by immunohistochemistry was strongly reduced in 3/30 adenomas. There were no association between HDAC2 expression and clinical variables. A previously unidentified insertion of three bases in a region coding for a polyserine cluster in exon 1 of the HDAC2 gene was identified in 6/32 adenomas. No other mutations in HDAC2 exons were found. Examination of DNA extracted from peripheral blood confirmed germ-line origin of the exon 1 insertion. The same insertion was also found in 28/94 of the controls (i.e., not significantly different from the patients). CONCLUSIONS: Strongly reduced HDAC2 protein expression was confirmed in a small portion of corticotroph tumours. Mutations in HDAC2 exons are unlikely to play an important role in the development of corticotroph adenomas.

Distinct Pattern of Endoplasmic Reticulum Protein Processing and Extracellular Matrix Proteins in Functioning and Silent Corticotroph Pituitary Adenomas.

Functioning (FCA) and silent corticotroph (SCA) pituitary adenomas act differently from a clinical perspective, despite both subtypes showing positive TBX19 (TPIT) and/or adrenocorticotropic hormone (ACTH) staining by immunohistochemistry. They are challenging to treat, the former due to functional ACTH production and consequently hypercortisolemia, and the latter due to invasive and recurrent behavior. Moreover, the molecular mechanisms behind their distinct behavior are not clear. We investigated global transcriptome and proteome changes in order to identify signaling pathways that can explain FCA and SCA differences (e.g., hormone production vs. aggressive growth). In the transcriptomic study, cluster analyses of differentially expressed genes revealed two distinct groups in accordance with clinical and histological classification. However, in the proteomic study, a greater degree of heterogeneity within the SCA group was found. Genes and proteins related to protein synthesis and vesicular transport were expressed by both adenoma groups, although different types and a distinct pattern of collagen/extracellular matrix proteins were presented by each group. Moreover, several genes related to endoplasmic reticulum protein processing were overexpressed in the FCA group. Together, our findings shed light on the different repertoires of activated signaling pathways in corticotroph adenomas, namely, the increased protein processing capacity of FCA and a specific pattern of adhesion molecules that may play a role in the aggressiveness of SCA.

Plasma immunological markers in pregnancy and cord blood: A possible link between macrophage chemo-attractants and risk of childhood type 1 diabetes.

PROBLEM: Previous studies have suggested that immune perturbations during pregnancy can affect offspring type 1 diabetes (T1D) risk. We aimed to identify immunological markers that could predict offspring T1D or that were linked to T1D risk factors. METHOD OF STUDY: We quantified selected circulating immunological markers in mid-pregnancy (interleukin [IL]-1ß, IL-1ra, IL-2Rα, IL-2, -4, -5, -6, -10, -12p70, 13, -17A, GM-CSF, IFN-γ, CXCL10, CCL 2, CCL3, CCL4, TNF) and cord blood plasma (neopterin and kynurenine/tryptophan ratio) in a case-control study with 175 mother/child T1D cases (median age 5.8, range 0.7-13.0 years) and 552 controls. RESULTS: Pre-pregnancy obesity was positively associated with CCL4, CXCL10, kynurenine/tryptophan ratio and neopterin (P < .01). The established T1D SNPs rs1159465 (near IL2RA) and rs75352297 (near CCR2 and CCR3) were positively associated with IL-2Rα and CCL4, respectively (P < .01). There was a borderline association of CCL4 and offspring T1D risk, independent of maternal obesity and genotype. When grouping the immunological markers, there was a borderline association (P = .05) with M1 phenotype and no association between M2-, Th1-, Th2- or Th17 phenotypes and offspring T1D risk. CONCLUSION: Increased mid-pregnancy CCL4 levels showed borderline associations with increased offspring T1D risk, which may indicate a link between environmental factors in pregnancy and offspring T1D risk.