Single-cell sequencing unveils extensive intratumoral heterogeneity of cancer/testis antigen expression in melanoma and lung cancer.

Cancer/testis antigens (CTAs) are widely expressed in melanoma and lung cancer, emerging as promising targets for vaccination strategies and T-cell-based therapies in these malignancies. Despite recognizing the essential impact of intratumoral heterogeneity on clinical responses to immunotherapy, our understanding of intratumoral heterogeneity in CTA expression has remained limited. We employed single-cell mRNA sequencing to delineate the CTA expression profiles of cancer cells in clinically derived melanoma and lung cancer samples. Our findings reveal a high degree of intratumoral transcriptional heterogeneity in CTA expression. In melanoma, every cell expressed at least one CTA. However, most individual CTAs, including the widely used therapeutic targets NY-ESO-1 and MAGE, were confined to subpopulations of cells and were uncoordinated in their expression, resulting in mosaics of cancer cells with diverse CTA profiles. Coordinated expression was observed, however, mainly among highly structurally and evolutionarily related CTA genes. Importantly, a minor subset of CTAs, including PRAME and several members of the GAGE and MAGE-A families, were homogenously expressed in melanomas, highlighting their potential as therapeutic targets. Extensive heterogeneity in CTA expression was also observed in lung cancer. However, the frequency of CTA-positive cancer cells was notably lower and homogenously expressed CTAs were only identified in one of five tumors in this cancer type. Our findings underscore the need for careful CTA target selection in immunotherapy development and clinical testing and offer a rational framework for identifying the most promising candidates.

Effect of dapagliflozin on collectins and complement activation in plasma from patients with type 2 diabetes and albuminuria: Data from the DapKid cohort.

BACKGROUND: Sodium-glucose cotransporter 2 (SGLT- 2) inhibitors exert cardiovascular and kidney-protective effects in people with diabetes. Attenuation of inflammation could be important for systemic protection. The lectin pathway of complement system activation is linked to diabetic nephropathy. We hypothesized that SGLT-2 inhibitors lower the circulating level of pattern-recognition molecules of the lectin cascade and attenuate systemic complement activation. METHODS: Analysis of paired plasma samples from the DapKid crossover intervention study where patients with type 2 diabetes mellitus (T2DM) and albuminuria were treated with dapagliflozin and placebo for 12 weeks (10 mg/day, n=36). ELISA was used to determine concentrations of collectin kidney 1 (CL-K1), collectin liver 1 (CL-L1), mannose-binding lectin (MBL), MBL-associated serine protease 2 (MASP-2), the anaphylatoxin complement factor 3a (C3a), the stable C3 split product C3dg and the membrane attack complex (sC5b-9). RESULTS: As published before, dapagliflozin treatment lowered Hba1C from 74 (14.9) mmol/mol to 66 (13.9) mmol/mol (p<0.0001), and the urine albumin/creatinine ratio from 167.8 mg/g to 122.5 mg/g (p<0.0001). Plasma concentrations of CL-K1, CL-L1, MBL, and MASP-2 did not change significantly after dapagliflozin treatment (P>0.05) compared to placebo treatment. The plasma levels of C3a (P<0.05) and C3dg (P<0.01) increased slightly but significantly, 0.6 [0.2] units/mL and 76 [52] units/mL respectively, after dapagliflozin treatment. The C9-associated neoepitope in C5b-9 did not change in plasma concentration by dapagliflozin (P>0.05). CONCLUSION: In patients with type 2 diabetes and albuminuria, SGLT-2 inhibition resulted in modest C3 activation in plasma, likely not driven by primary changes in circulating collectins and not resulting in changes in membrane attack complex. Based on systemic analyses, organ-specific local protective effects of gliflozins against complement activation cannot be excluded.

Randomized Trial of SGLT2 Inhibitor Identifies Target Proteins in Diabetic Kidney Disease.

Introduction: Sodium-glucose cotransporter 2 inhibitors (SGLT2i) have emerged as novel therapeutics to treat diabetic kidney disease (DKD). Although the beneficial effects of SGLT2i have been demonstrated, their target mechanisms on kidney function are unknown. The current study aimed to elucidate these mechanisms by studying SGLT2i-induced changes in the urinary proteome of persons with type 2 diabetes (T2D) and DKD. Methods: A total of 40 participants with T2D were enrolled in a double-blinded randomized cross-over trial at the Steno Diabetes Center Copenhagen, Denmark. They were treated with 10 mg of dapagliflozin for 12 weeks. Thirty-two participants with complete urinary proteomics measures before and after the trial were included. All participants received renin-angiotensin system blockade and had albuminuria, (urine albumin-to-creatinine ratio [UACR] ≥30 mg/g). A type 1 diabetes (T1D) cohort consisting of healthy controls and persons with DKD was included for validation. Urinary proteome changes were analyzed using Wilcoxon signed-rank test. Functional enrichment analysis was conducted to discover affected biological processes. Results: Dapagliflozin treatment significantly (Padjusted < 0.05) affected 36 urinary peptide fragments derived from 19 proteins. Eighteen proteins were correspondingly reflected in the validation cohort. A multifold change in peptide abundance was observed in many proteins (A1BG, urinary albumin [ALB], Caldesmon 1, COLCRNN, heat shock protein 90-ß [HSP90AB1], IGLL5, peptidase inhibitor 16 [PI16], prostaglandin-H2-D-isomerase [PTGDS], SERPINA1). These also included urinary biomarkers of kidney fibrosis and function (type I and III collagens and albumin). Biological processes relating to inflammation, wound healing, and kidney fibrosis were enriched. Conclusion: The current study discovers the urinary proteome impacted by the SGLT2i, thereby providing new potential target sites and pathways, especially relating to wound healing and inflammation.

Effects of two- and twelve-weeks sodium-glucose cotransporter 2 inhibition on DNA and RNA oxidation: two randomized, placebo-controlled trials.

Animal studies have shown that SGLT2 inhibition decreases oxidative stress, which may explain the cardiovascular protective effects observed following SGLT2 inhibition treatment. Thus, we investigated the effects of two and twelve weeks SGLT2 inhibition on DNA and RNA oxidation. Individuals with type 2 diabetes (n = 31) were randomized to two weeks of treatment with the SGLT2 inhibitor empagliflozin treatment (25 mg once daily) or placebo. The primary outcome was changes in DNA and RNA oxidation measured as urinary excretion of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo), respectively. In another trial, individuals with type 2 diabetes (n = 35) were randomized to twelve weeks of dapagliflozin treatment (10 mg once daily) or placebo in a crossover study. Changes in urinary excretion of 8-oxodG and 8-oxoGuo were investigated as a posthoc analysis. Compared with placebo treatment, two weeks of empagliflozin treatment did not change urinary excretion of 8-oxodG (between-group difference: 0.3 nmol/24-hour (95% CI: -4.2 to 4.8)) or 8-oxoGuo (1.3 nmol/24-hour (95% CI: -4.7 to 7.3)). From a mean baseline 8-oxodG/creatinine urinary excretion of 1.34 nmol/mmol, dapagliflozin-treated individuals changed 8-oxodG/creatinine by -0.17 nmol/mmol (95% CI: -0.29 to -0.04) following twelve weeks of treatment, whereas placebo-treated individuals did not change 8-oxodG/creatinine (within-group effect: 0.10 nmol/mmol (95% CI: -0.02 to 0.22)) resulting in a significant between-group difference (p = 0.01). Urinary excretion of 8-oxoGuo was unaffected by dapagliflozin treatment. In conclusion, two weeks of empagliflozin treatment did not change DNA or RNA oxidation. However, a posthoc analysis revealed that longer-term dapagliflozin treatment decreased DNA oxidation. Clinicaltrials.gov: NCT02890745 and NCT02914691.HighlightsPlasma ferritin correlated with DNA and RNA oxidation in individuals with T2D.Twelve weeks dapagliflozin treatment decreased DNA oxidation.Dapagliflozin and empagliflozin treatment did not change RNA oxidation.Lipid peroxidation was unaffected by two weeks empagliflozin treatment.

Validity of Danish public criteria for providing flash glucose monitoring to participants with type 1 diabetes-An explorative cohort study.

INTRODUCTION: Flash glucose monitor (FGM) use is increasing. A set of Danish criteria for regulating the use has been released. We assessed their validity. METHODS: Patients with type 1 diabetes attending our clinic were offered Freestyle Libre Sensor for 12 months and stratified into fulfilling the Danish regional criteria (RC+) or not (RC-). Primary endpoint was achieving individualized target HbA1c. Secondary endpoints were HbA1c reduction ≥5%, time in range (TIR), time below range (TBR), daily scans, change in median HbA1c, and noted experiences. RESULTS: Two hundred seventy-eight participants were included. Forty-four participants met target HbA1c after 1 year. No difference between RC+ and RC- was observed (p = .136). Higher age was associated with probability to meet target HbA1c (RR = 3.15, [95% CI: 1.15, 8.62]) as was frequent scans (RR = 1.88, [95% CI: 0.99, 3.57]). One hundred twenty-three participants met an HbA1c reduction ≥5%, the majority represented in RC+ (p = .023). Higher baseline HbA1c was associated with a reduction of HbA1c ≥5% after 1 year (RR = 1.97, [95% CI: 1.40, 2.78]). There was no difference between RC+ and RC- in TIR, TBR, and daily scans. Positive experiences dominated from both participants and healthcare professionals. More positive experiences were noted from healthcare professionals in RC- (p = .003) but no difference in reported experiences among participants in RC+ and RC- (p = .880). CONCLUSION: The Danish Regional Criteria seems not a valid tool for regulation of FGM. Participants of older age and participants with more frequent daily scans might benefit more from FGM.

Acute and Long-Term Treatment With Dapagliflozin and Association With Serum Soluble Urokinase Plasminogen Activator Receptor.

Background: Elevated soluble urokinase plasminogen activator receptor (suPAR) is highly associated with increased risk of diabetic complications. Dapagliflozin is a drug inhibiting the sodium-glucose co-transporter 2 in the kidney to decrease blood glucose, while also decreasing risk of kidney disease, heart failure, and death. Therefore, we have investigated suPAR as a monitor for treatment effect with dapagliflozin in diabetes. Methods: suPAR was measured in two double-blinded randomized clinical cross-over trials. The first trial investigated the effect of a single dose dapagliflozin 50 mg or placebo 12 h after intake, in individuals with type 1 diabetes and albuminuria. The second trial investigated the effect of a daily dose dapagliflozin 10 mg or placebo for 12 weeks, in individuals with type 2 diabetes and albuminuria. suPAR was measured in serum samples taken, in the acute trial, after treatment with dapagliflozin and placebo, and in the long-term trial, before and after treatment with dapagliflozin and placebo. Effect of dapagliflozin on suPAR levels were assessed using paired t-test. Results: 15 participants completed the acute trial and 35 completed the long-term trial. Mean difference in suPAR between dapagliflozin and placebo in the acute trial after 12 h was 0.70 ng/ml (95% CI: 0.66; 1.33, p = 0.49). In the long-term trial the mean difference was 0.06 ng/ml (95% CI -0.15; 0.27, p = 0.57). Conclusion: Based on our findings we conclude that suPAR is not a feasible marker to monitor the effect of treatment with dapagliflozin. Thus, a further search of suitable markers must continue.

Spectrum of germline AIRE mutations causing APS-1 and familial hypoparathyroidism.

Objective: The autoimmune polyendocrine syndrome type 1 (APS-1) is an autosomal recessive disorder characterised by immune dysregulation and autoimmune endocrine gland destruction. APS-1 is caused by biallelic mutations affecting the autoimmune regulator (AIRE) gene on chromosome 21q22.3, which facilitates immunological self-tolerance. The objective was to investigate >300 probands with suspected APS-1 or isolated hypoparathyroidism for AIRE abnormalities. Methods: Probands were assessed by DNA sequence analysis. Novel variants were characterised using 3D modelling of the AIRE protein. Restriction enzyme and microsatellite analysis were used to investigate for uniparental isodisomy. Results: Biallelic AIRE mutations were identified in 35 probands with APS-1 and 5 probands with isolated hypoparathyroidism. These included a novel homozygous p.(His14Pro) mutation, predicted to disrupt the N-terminal caspase activation recruitment domain of the AIRE protein. Furthermore, an apparently homozygous AIRE mutation, p.Leu323fs, was identified in an APS-1 proband, who is the child of non-consanguineous asymptomatic parents. Microsatellite analysis revealed that the proband inherited two copies of the paternal mutant AIRE allele due to uniparental isodisomy. Hypoparathyroidism was the most common endocrine manifestation in AIRE mutation-positive probands and >45% of those harbouring AIRE mutations had at least two diseases out of the triad of candidiasis, hypoparathyroidism, and hypoadrenalism. In contrast, type 1 diabetes and hypothyroidism occurred more frequently in AIRE mutation-negative probands with suspected APS-1. Around 30% of AIRE mutation-negative probands with isolated hypoparathyroidism harboured mutations in other hypoparathyroid genes. Conclusions: This study of a large cohort referred for AIRE mutational analysis expands the spectrum of genetic abnormalities causing APS-1.

Calcilytic NPSP795 Increases Plasma Calcium and PTH in an Autosomal Dominant Hypocalcemia Type 1 Mouse Model.

Calcilytics are calcium-sensing receptor (CaSR) antagonists that reduce the sensitivity of the CaSR to extracellular calcium. Calcilytics have the potential to treat autosomal dominant hypocalcemia type 1 (ADH1), which is caused by germline gain-of-function CaSR mutations and leads to symptomatic hypocalcemia, inappropriately low PTH concentrations, and hypercalciuria. To date, only one calcilytic compound, NPSP795, has been evaluated in patients with ADH1: Doses of up to 30 mg per patient have been shown to increase PTH concentrations, but did not significantly alter ionized blood calcium concentrations. The aim of this study was to further investigate NPSP795 for the treatment of ADH1 by undertaking in vitro and in vivo studies involving Nuf mice, which have hypocalcemia in association with a gain-of-function CaSR mutation, Leu723Gln. Treatment of HEK293 cells stably expressing the mutant Nuf (Gln723) CaSR with 20nM NPSP795 decreased extracellular Ca2+-mediated intracellular calcium and phosphorylated ERK responses. An in vivo dose-ranging study was undertaken by administering a s.c. bolus of NPSP795 at doses ranging from 0 to 30 mg/kg to heterozygous (Casr +/Nuf ) and to homozygous (Casr Nuf/Nuf ) mice, and measuring plasma PTH responses at 30 min postdose. NPSP795 significantly increased plasma PTH concentrations in a dose-dependent manner with the 30 mg/kg dose causing a maximal (≥10-fold) rise in PTH. To determine whether NPSP795 can rectify the hypocalcemia of Casr +/Nuf and Casr Nuf/Nuf mice, a submaximal dose (25 mg/kg) was administered, and plasma adjusted-calcium concentrations measured over a 6-hour period. NPSP795 significantly increased plasma adjusted-calcium in Casr +/Nuf mice from 1.87 ± 0.03 mmol/L to 2.16 ± 0.06 mmol/L, and in Casr Nuf/Nuf mice from 1.70 ± 0.03 mmol/L to 1.89 ± 0.05 mmol/L. Our findings show that NPSP795 elicits dose-dependent increases in PTH and ameliorates the hypocalcemia in an ADH1 mouse model. Thus, calcilytics such as NPSP795 represent a potential targeted therapy for ADH1. © 2020 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.

Effect of dapagliflozin on cardiac function in people with type 2 diabetes and albuminuria - A double blind randomized placebo-controlled crossover trial.

AIMS: Sodium glucose transport inhibitors (SGLT2i) can reduce risk of heart failure (HF) and cardiovascular death in people with type 2 diabetes (T2D) and existing cardiovascular disease. Our aim was to examine the effect of the SGLT2i dapagliflozin on cardiac function in people with T2D and albuminuria. METHODS: A secondary analysis of a double-blind, randomized, cross-over study of 12 weeks treatment with dapagliflozin 10 mg versus placebo. Myocardial function was assessed by echocardiography and biomarkers of cardiac risk were measured. An exploratory diastolic composite of echocardiographic variables was computed. RESULTS: Of the 36 participants completing the study 89% were male, mean age 64 ±â€¯8 years, diabetes duration 16.4 ±â€¯4.7 years and HbA1c 73 ±â€¯15 mmol/mol (8.9 ±â€¯1.4%), 30.6% had former cardiovascular events and 32% had macroalbuminuria. Mean left ventricular ejection fraction (LVEF) was 55.4% after placebo and 54.3% after dapagliflozin (p = 0.15), global longitudinal strain -16.1 vs. -15.9, (p = 0.64), E/e' 7.6 vs. 7.6 (p = 0.082), and tissue Doppler velocity e' 10.0 vs. 10.6 (p = 0.05). The composite score showed diastolic function improvement of 19.8% (p = 0.021). No other significant changes were observed. CONCLUSIONS: Dapagliflozin may have minor effects on diastolic function in people with T2D, albuminuria and preserved LVEF.

Neonatal Hypocalcemic Seizures in Offspring of a Mother With Familial Hypocalciuric Hypercalcemia Type 1 (FHH1).

CONTEXT: Familial hypocalciuric hypercalcemia type 1 (FHH1) is caused by loss-of-function mutations of the calcium-sensing receptor (CaSR) and is considered a benign condition associated with mild-to-moderate hypercalcemia. However, the children of parents with FHH1 can develop a variety of disorders of calcium homeostasis in infancy. OBJECTIVE: The objective of this work is to characterize the range of calcitropic phenotypes in the children of a mother with FHH1. METHODS: A 3-generation FHH kindred was assessed by clinical, biochemical, and mutational analysis following informed consent. RESULTS: The FHH kindred comprised a hypercalcemic man and his daughter who had hypercalcemia and hypocalciuria, and her 4 children, 2 of whom had asymptomatic hypercalcemia, 1 was normocalcemic, and 1 suffered from transient neonatal hypocalcemia and seizures. The hypocalcemic infant had a serum calcium of 1.57 mmol/L (6.28 mg/dL); normal, 2.0 to 2.8 mmol/L (8.0-11.2 mg/dL) and parathyroid hormone of 2.2 pmol/L; normal 1.0 to 9.3 pmol/L, and required treatment with intravenous calcium gluconate infusions. A novel heterozygous p.Ser448Pro CaSR variant was identified in the hypercalcemic individuals, but not the children with hypocalcemia or normocalcemia. Three-dimensional modeling predicted the p.Ser448Pro variant to disrupt a hydrogen bond interaction within the CaSR extracellular domain. The variant Pro448 CaSR, when expressed in HEK293 cells, significantly impaired CaSR-mediated intracellular calcium mobilization and mitogen-activated protein kinase responses following stimulation with extracellular calcium, thereby demonstrating it to represent a loss-of-function mutation. CONCLUSIONS: Thus, children of a mother with FHH1 can develop hypercalcemia or transient neonatal hypocalcemia, depending on the underlying inherited CaSR mutation, and require investigations for serum calcium and CaSR mutations in early childhood.

Gestational diabetes and the human salivary microbiota: a longitudinal study during pregnancy and postpartum.

BACKGROUND: An aberrant composition of the salivary microbiota has been found in individuals with type 2 diabetes, and in pregnant women salivary microbiota composition has been associated with preeclampsia and pre-term birth. Pregnant women, who develop gestational diabetes (GDM), have a high risk of developing type 2 diabetes after pregnancy. In the present study we assessed whether GDM is linked to variation in the oral microbial community by examining the diversity and composition of the salivary microbiota. METHOD: In this observational study the salivary microbiota of pregnant women with GDM (n = 50) and normal glucose regulation (n = 160) in third trimester and 9 months postpartum was assessed by 16S rRNA gene amplicon sequencing of the V1-V3 region. GDM was diagnosed in accordance with the International Association of the Diabetes and Pregnancy Study Groups (IADPSG) criteria. Cross-sectional difference in alpha diversity was assessed using Student's t-test and longitudinal changes were assessed by mixed linear regression. Cross-sectional and longitudinal difference in beta diversity was assessed by permutational multivariate analyses of variance. Differentially abundant genera and OTUs were identified by negative binomial regression. RESULTS: In the third trimester, two species-level operational taxonomic units (OTUs), while eight OTUs postpartum were differentially abundant in women with GDM compared with normoglycaemic women. OTU richness, Shannon diversity and Pielou evenness decreased from late pregnancy to 9 months after delivery regardless of glycaemic status. CONCLUSION: GDM is associated with a minor aberration of the salivary microbiota during late pregnancy and postpartum. For unknown reasons richness of the salivary microbiota decreased from late pregnancy to postpartum, which might be explained by the physiological changes of the immune system during human pregnancy.

The Cancer/Testis Antigen Gene VCX2 Is Rarely Expressed in Malignancies but Can Be Epigenetically Activated Using DNA Methyltransferase and Histone Deacetylase Inhibitors.

Identification of novel tumor-specific targets is important for the future development of immunotherapeutic strategies using genetically engineered T cells or vaccines. In this study, we characterized the expression of VCX2, a member of the VCX/Y cancer/testis antigen family, in a large panel of normal tissues and tumors from multiple cancer types using immunohistochemical staining and RNA expression data. In normal tissues, VCX2 was detected in the germ cells of the testis at all stages of maturation but not in any somatic tissues. Among malignancies, VCX2 was only found in tumors of a small subset of melanoma patients and thus rarely expressed compared to other cancer/testis antigens such as GAGE and MAGE-A. The expression of VCX2 correlated with that of other VCX/Y genes. Importantly, we found that expression of VCX2 was inversely correlated with promoter methylation and could be activated by treatment with a DNA methyltransferase inhibitor in multiple breast cancer and melanoma cell lines and a breast cancer patient-derived xenograft. The effect could be further potentiated by combining the DNA methyltransferase inhibitor with a histone deacetylase inhibitor. Our results show that the expression of VCX2 can be epigenetically induced in cancer cells and therefore could be an attractive target for immunotherapy of cancer.

Effects of Dapagliflozin on Volume Status When Added to Renin-Angiotensin System Inhibitors.

Sodium glucose co-transporter 2 (SGLT2) inhibitors reduce the risk of heart and kidney failure in patients with type 2 diabetes, possibly due to diuretic effects. Previous non-placebo-controlled studies with SGLT2 inhibitors observed changes in volume markers in healthy individuals and in patients with type 2 diabetes with preserved kidney function. It is unclear whether patients with type 2 diabetes and signs of kidney damage show similar changes. Therefore, a post hoc analysis was performed on two randomized controlled trials (n = 69), assessing effects of dapagliflozin 10 mg/day when added to renin-angiotensin system inhibition in patients with type 2 diabetes and urinary albumin-to-creatinine ratio ≥30 mg/g. Blood and 24-h urine was collected at the start and the end of treatment periods lasting six and 12 weeks. Effects of dapagliflozin compared to placebo on various markers of volume status were determined. Fractional lithium excretion, a marker of proximal tubular sodium reabsorption, was assessed in 33 patients. Dapagliflozin increased urinary glucose excretion by 217.2 mmol/24 h (95% confidence interval (CI): from 155.7 to 278.7, p < 0.01) and urinary osmolality by 60.4 mOsmol/kg (from 30.0 to 90.9, p < 0.01), compared to placebo. Fractional lithium excretion increased by 19.6% (from 6.7 to 34.2; p < 0.01), suggesting inhibition of sodium reabsorption in the proximal tubule. Renin and copeptin increased by 46.9% (from 21.6 to 77.4, p < 0.01) and 33.0% (from 23.9 to 42.7, p < 0.01), respectively. Free water clearance (FWC) decreased by -885.3 mL/24 h (from -1156.2 to -614.3, p < 0.01). These changes in markers of volume status suggest that dapagliflozin exerts both osmotic and natriuretic diuretic effects in patients with type 2 diabetes and kidney damage, as reflected by increased urinary osmolality and fractional lithium excretion. As a result, compensating mechanisms are activated to retain sodium and water.

Up-front F18-FDG PET/CT in suspected salivary gland carcinoma.

OBJECTIVE: To investigate whether a 18F-FDG PET/CT (PET/CT)-based diagnostic strategy adds decisive new information compared to conventional imaging in the evaluation of salivary gland tumours and the detection of cervical lymph node metastases, distant metastases, and synchronous cancer in patients with salivary gland carcinoma. METHODS: The study was a blinded prospective cohort study. Data were collected consecutively through almost 3 years. All patients underwent conventional imaging-magnetic resonance imaging (MRI) and chest X-ray (CXR)-in addition to PET/CT prior to surgery. Final diagnosis was obtained by histopathology. MRI/CXR and PET/CT were interpreted separately by experienced radiologists and nuclear medicine physicians. Interpretation included evaluation of tumour site, cervical lymph node metastases, distant metastases, and synchronous cancer. RESULTS: Ninety-one patients were included in the study. Thirty-three patients had primary salivary gland carcinoma and eight had cervical lymph node metastases. With PET/CT, the sensitivity was 92% and specificity 29% regarding tumour site. With MRI/CXR, the sensitivity and specificity were 90% and 26%, respectively. Regarding cervical lymph node metastases in patients with salivary gland carcinoma, the sensitivity with PET/CT was 100% and with MRI/CXR 50%. PET/CT diagnosed distant metastases in five patients, while MRI/CXR detected these in two patients. Finally, PET/CT diagnosed two synchronous cancers, whereas MRI/CXR did not detect any synchronous cancers. CONCLUSIONS: Compared with MRI/CXR PET/CT did not improve discrimination of benign from malignant salivary gland lesions. However, PET/CT may be advantageous in primary staging and in the detection of distant metastases and synchronous cancers.

PET/CT Versus Standard Imaging for Prediction of Survival in Patients with Recurrent Head and Neck Squamous Cell Carcinoma.

The purpose of this study was to examine whether staging with 18F-FDG PET/CT better predicts survival in patients with recurrent head and neck squamous cell carcinoma (HNSCC) than chest x-ray (CXR) plus head and neck MRI or chest CT (CCT) plus head and neck MRI. Methods: This was a prospective cohort study based on paired data. Consecutive patients with histologically verified HNSCC recurrence were enrolled from September 2013 to March 2016. All patients underwent CXR/MRI, CCT/MRI, and PET/CT on the same day and before biopsy. All imaging studies underwent masked interpretation by separate teams of experienced nuclear physicians or radiologists. Recurrent carcinomas were categorized as localized (equivalent to primary stages I-II), locally advanced (equivalent to primary stages III-IVB), or metastatic (equivalent to primary stage IVC). Discriminative abilities for each imaging strategy with respect to cancer-specific and stage-based survival were compared using Kaplan-Meier analysis, Cox proportional-hazards regression with the Harrell concordance index (C-index), and net reclassification improvement. Results: In total, 110 patients (90 men and 20 women; median age, 66 y; range, 40-87 y) were included. PET/CT significantly changed the assigned tumor stage when compared with imaging strategies based on CXR/MRI or CCT/MRI (P < 0.001 for both). Kaplan-Meier analysis of PET/CT-based staging showed progressively worsened prognosis with localized, locally advanced, or metastatic disease (log-rank test, P < 0.001), whereas CXR/MRI and CCT/MRI were unable to distinguish between these groups in terms of survival (log-rank test, P = 0.18 and P = 0.58, respectively). Overall discriminative ability in predicting cancer-specific mortality was significantly greater for PET/CT (C-index, 0.72) than for CXR/MRI (C-index, 0.55) (P = 0.001) and CCT/MRI (C-index, 0.55)(P < 0.001). The addition of PET/CT to either CXR/MRI or CCT/MRI was associated with a significantly positive net reclassification improvement (P < 0.001 for both). Conclusion: Contrary to standard imaging strategies, PET/CT-based staging in recurrent HNSCC was able to significantly discriminate among the survival courses of patients with local, locally advanced, or metastatic disease and predict their respective survival probability.

Urinary tubular biomarkers as predictors of kidney function decline, cardiovascular events and mortality in microalbuminuric type 2 diabetic patients.

AIMS: Urinary levels of kidney injury molecule 1 (u-KIM-1) and neutrophil gelatinase-associated lipocalin (u-NGAL) reflect proximal tubular pathophysiology and have been proposed as risk markers for development of complications in patients with type 2 diabetes (T2D). We clarify the predictive value of u-KIM-1 and u-NGAL for decline in eGFR, cardiovascular events (CVE) and all-cause mortality in patients with T2D and persistent microalbuminuria without clinical cardiovascular disease. METHODS: This is a prospective study that included 200 patients. u-KIM-1 and u-NGAL were measured at baseline and were available in 192 patients. Endpoints comprised: decline in eGFR > 30%, a composite of fatal and nonfatal CVE consisting of: cardiovascular mortality, myocardial infarction, stroke, ischemic heart disease and heart failure based on national hospital discharge registries, and all-cause mortality. Adjusted Cox models included traditional risk factors, including eGFR. Hazard ratios (HR) are provided per 1 standard deviation (SD) increment of log2-transformed values. Relative integrated discrimination improvement (rIDI) was calculated. RESULTS: During the 6.1 years' follow-up, higher u-KIM-1 was a predictor of eGFR decline (n = 29), CVE (n = 34) and all-cause mortality (n = 29) in adjusted models: HR (95% CI) 1.68 (1.04-2.71), p = 0.034; 2.26 (1.24-4.15), p = 0.008; and 1.52 (1.00-2.31), p = 0.049. u-KIM-1 contributed significantly to risk prediction for all-cause mortality evaluated by rIDI (63.1%, p = 0.001). u-NGAL was not a predictor of any of the outcomes after adjustment. CONCLUSIONS: In patients with T2D and persistent microalbuminuria, u-KIM-1, but not u-NGAL, was an independent risk factor for decline in eGFR, CVE and all-cause mortality, and contributed significant discrimination for all-cause mortality, beyond traditional risk factors.

A PET/CT-Based Strategy Is a Stronger Predictor of Survival Than a Standard Imaging Strategy in Patients with Head and Neck Squamous Cell Carcinoma.

Our purpose was to examine whether staging of head and neck squamous cell carcinoma (HNSCC) by upfront 18F-FDG PET/CT (i.e., on the day of biopsy and before the biopsy) discriminates survival better than the traditional imaging strategies based on chest x-ray plus head and neck MRI (CXR/MRI) or chest CT plus head and neck MRI (CCT/MRI). Methods: We performed a masked prospective cohort study based on paired data. Consecutive patients with histologically verified primary HNSCC were recruited from Odense University Hospital from September 2013 to March 2016. All patients underwent CXR/MRI, CCT/MRI, and PET/CT on the same day. Tumors were categorized as localized (stages I and II), locally advanced (stages III and IVB), or metastatic (stage IVC). Discriminative ability for each imaging modality with respect to HNSCC staging were compared using Kaplan-Meier analysis, Cox proportional hazards regression with the Harrell C-index, and net reclassification improvement. Results: In total, 307 patients with histologically verified HNSCC were included. Use of PET/CT significantly altered the stratification of tumor stage when compared with either CXR/MRI or CCT/MRI (χ2, P < 0.001 for both). Cancer stages based on PET/CT, but not CXR/MRI or CCT/MRI, were associated with significant differences in mortality risk on Kaplan-Meier analyses (P ≤ 0.002 for all PET/CT-based comparisons). Furthermore, overall discriminative ability was significantly greater for PET/CT (C-index, 0.712) than for CXR/MRI (C-index, 0.675; P = 0.04) or CCT/MRI (C-index, 0.657; P = 0.02). Finally, PET/CT was significantly associated with a positive net reclassification improvement when compared with CXR/MRI (0.184, P = 0.03) but not CCT/MRI (0.094%, P = 0.31). Conclusion: Tumor stages determined by PET/CT were associated with more distinct prognostic properties in terms of survival than those determined by standard imaging strategies.

Calcimimetic and calcilytic therapies for inherited disorders of the calcium-sensing receptor signalling pathway.

The calcium-sensing receptor (CaS receptor) plays a pivotal role in extracellular calcium homeostasis, and germline loss-of-function and gain-of-function mutations cause familial hypocalciuric hypercalcaemia (FHH) and autosomal dominant hypocalcaemia (ADH), respectively. CaS receptor signal transduction in the parathyroid glands is probably regulated by G-protein subunit α11 (Gα11 ) and adaptor-related protein complex-2 σ-subunit (AP2σ), and recent studies have identified germline mutations of these proteins as a cause of FHH and/or ADH. Calcimimetics and calcilytics are positive and negative allosteric modulators of the CaS receptor that have potential efficacy for symptomatic forms of FHH and ADH. Cellular studies have demonstrated that these compounds correct signalling and/or trafficking defects caused by mutant CaS receptor, Gα11 or AP2σ proteins. Moreover, mouse model studies indicate that calcilytics can rectify the hypocalcaemia and hypercalciuria associated with ADH, and patient-based studies reveal calcimimetics to ameliorate symptomatic hypercalcaemia caused by FHH. Thus, calcimimetics and calcilytics represent targeted therapies for inherited disorders of the CaS receptor signalling pathway. LINKED ARTICLES: This article is part of a themed section on Molecular Pharmacology of GPCRs. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.21/issuetoc.

Head-to-Head Comparison of Chest X-Ray/Head and Neck MRI, Chest CT/Head and Neck MRI, and 18F-FDG PET/CT for Detection of Distant Metastases and Synchronous Cancer in Oral, Pharyngeal, and Laryngeal Cancer.

The purpose of this study was to determine the detection rate of distant metastasis and synchronous cancer, comparing clinically used imaging strategies based on chest x-ray + head and neck MRI (CXR/MRI) and chest CT + head and neck MRI (CHCT/MRI) with 18F-FDG PET/CT upfront in the diagnostic workup of patients with oral, pharyngeal, or laryngeal cancer. Methods: This was a prospective cohort study based on paired data. Consecutive patients with histologically verified primary head and squamous cell carcinoma at Odense University Hospital from September 2013 to March 2016 were considered for the study. Included patients underwent CXR/MRI and CHCT/MRI as well as PET/CT on the same day and before biopsy. Scans were read masked by separate teams of experienced nuclear physicians or radiologists. The true detection rate of distant metastasis and synchronous cancer was assessed for CXR/MRI, CHCT/MRI, and PET/CT. Results: A total of 307 patients were included. CXR/MRI correctly detected 3 (1%) patients with distant metastasis, CHCT/MRI detected 11 (4%) patients, and PET/CT detected 18 (6%) patients. The absolute differences of 5% and 2%, respectively, were statistically significant in favor of PET/CT. Also, PET/CT correctly detected 25 (8%) synchronous cancers, which was significantly more than CXR/MRI (3 patients, 1%) and CHCT/MRI (6 patients, 2%). The true detection rate of distant metastasis or synchronous cancer with PET/CT was 13% (40 patients), which was significantly higher than 2% (6 patients) for CXR/MRI and 6% (17 patients) for CHCT/MRI. Conclusion: A clinical imaging strategy based on PET/CT demonstrated a significantly higher detection rate of distant metastasis or synchronous cancer than strategies in current clinical imaging guidelines, of which European ones primarily recommend CXR/MRI, whereas U.S. guidelines preferably point to CHCT/MRI in patients with head and neck squamous cell carcinoma.