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The metabolic response to an immune challenge in a viviparous snake, Sistrurus miliarius [RESEARCH ARTICLE]

C. M. Lind, J. Agugliaro, and T. M. Farrell

Mounting an immune response may be energetically costly and require the diversion of resources away from other physiological processes. Yet, both the metabolic cost of immune responses and the factors that impact investment priorities remain poorly described in many vertebrate groups. For example, although viviparity has evolved many times in vertebrates, the relationship between immune function and pregnancy has been disproportionately studied in placental mammals. To examine the energetic costs of immune activation and the modulation of immune function during pregnancy in a non-mammalian vertebrate, we elicited an immune response in pregnant and non-pregnant pygmy rattlesnakes, Sistrurus miliarius, using lipopolysaccharide (LPS). Resting metabolic rate (RMR) was measured using flow-through respirometry. Immune function was examined using bactericidal assays and leukocyte counts. The RMR of pygmy rattlesnakes increased significantly in response to LPS injection. There was no statistically significant difference in the metabolic response of non-reproductive and pregnant snakes to LPS. Mean metabolic increments for pregnant females, non-reproductive females, and males were 13%, 18%, and 26%, respectively. The ratio of heterophils to lymphocytes was elevated in response to LPS across reproductive categories; however, LPS did not impact plasma bactericidal ability in non-reproductive snakes. Although pregnant females had significantly higher plasma bactericidal ability compared to non-reproductive snakes prior to manipulation, their bactericidal ability declined in response to LPS. LPS administration also significantly reduced several litter characteristics, particularly when administrated relatively early in pregnancy. Our results indicate that immune performance is energetically costly, altered during pregnancy, and that immune activation during pregnancy may result in tradeoffs that affect offspring in a viviparous reptile.




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Thermo-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure of obese mice [RESEARCH ARTICLE]

Jing Wen, Tingbei Bo, Xueying Zhang, Zuoxin Wang, and Dehua Wang

Ambient temperature and food composition can affect energy metabolism of the host. Thermal transient receptor potential (thermo-TRPs) ion channels can detect temperature signals and are involved in the regulation of thermogenesis and energy homeostasis. Further, the gut microbiota has also been implicated in thermogenesis and obesity. In the present study, we tested the hypothesis that thermo-TRPs and gut microbiota are involved in reducing diet-induced obesity (DIO) during low temperature exposure. C57BL/6J mice in obese (body mass gain >45%), lean (body mass gain <15%), and control (body mass gain<1%) groups were exposed to high (23±1°C) or low (4±1°C) ambient temperature for 28 days. Our data showed that low temperature exposure attenuated DIO, but enhanced brown adipose tissue (BAT) thermogenesis. Low temperature exposure also resulted in increased norepinephrine (NE) concentrations in the hypothalamus, decreased TRP melastatin 8 (TRPM8) expression in the small intestine, and altered composition and diversity of gut microbiota. In DIO mice, there was a decrease in overall energy intake along with a reduction in TRP ankyrin 1 (TRPA1) expression and an increase in NE concentration in the small intestine. DIO mice also showed increases in Oscillospira, [Ruminococcus], Lactococcus, and Christensenella and decreases in Prevotella, Odoribacter, and Lactobacillus at the genus level in fecal samples. Together, our data suggest that thermos-TRPs and gut microbiota are involved in thermogenesis and energy metabolism during low temperature exposure in DIO mice.




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Fructose Production and Metabolism in the Kidney

Understanding fructose metabolism might provide insights to renal pathophysiology. To support systemic glucose concentration, the proximal tubular cells reabsorb fructose as a substrate for gluconeogenesis. However, in instances when fructose intake is excessive, fructose metabolism is costly, resulting in energy depletion, uric acid generation, inflammation, and fibrosis in the kidney. A recent scientific advance is the discovery that fructose can be endogenously produced from glucose under pathologic conditions, not only in kidney diseases, but also in diabetes, in cardiac hypertrophy, and with dehydration. Why humans have such a deleterious mechanism to produce fructose is unknown, but it may relate to an evolutionary benefit in the past. In this article, we aim to illuminate the roles of fructose as it relates to gluconeogenesis and fructoneogenesis in the kidney.




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Probiotics for the Prevention of Ventilator-Associated Pneumonia: A Meta-Analysis of Randomized Controlled Trials

BACKGROUND:Ventilator-associated pneumonia (VAP) is a common and serious complication of mechanical ventilation. We conducted a meta-analysis of published randomized controlled trials to evaluate the efficacy and safety of probiotics for VAP prevention in patients who received mechanical ventilation.METHODS:We searched a number of medical literature databases to identify randomized controlled trials that compared probiotics with controls for VAP prevention. The results were expressed as odds ratios (OR) or mean differences with accompanying 95% CIs. Study-level data were pooled by using a random-effects model. Data syntheses were accomplished by using statistical software.RESULTS:Fourteen studies that involved 1,975 subjects met our inclusion criteria. Probiotic administration was associated with a reduction in VAP incidence among all 13 studies included in the meta-analysis (OR 0.62, 95% CI 0.45–0.85; P = .003; I2 = 43%) but not among the 6 double-blinded studies (OR 0.72, 95% CI 0.44–1.19; P = .20; I2 = 55%). We found a shorter duration of antibiotic use for VAP (mean difference −1.44, 95% CI −2.88 to −0.01; P = .048, I2 = 30%) in the probiotics group than in the control group, and the finding comes from just 2 studies. No statistically significant differences were found between the groups in terms of ICU mortality (OR 0.95, 95% CI 0.67–1.34; P = .77; I2 = 0%), ICU stay (mean difference –0.77, 95% CI –2.58 to 1.04; P = .40; I2 = 43%), duration of mechanical ventilation (mean difference –0.91, 95% CI –2.20 to 0.38; P = .17; I2 = 25%), or occurrence of diarrhea (OR 0.72, 95% CI 0.45–1.15; P = .17; I2 = 41%).CONCLUSIONS:The meta-analysis results indicated that the administration of probiotics significantly reduced the incidence of VAP. Furthermore, our findings need to be verified in large-scale, well-designed, randomized, multi-center trials.




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Human Metapneumovirus Infection in Hospitalized Children

BACKGROUND:Most children are exposed to human metapneumovirus (HMPV) by the age of 5 y. This study aimed to describe the morbidity associated with HMPV infections in a cohort of children in the Midwest of the United States.METHODS:This was a retrospective 2-center cohort study including children (0–17 y old) hospitalized with HMPV infections at 2 tertiary care pediatric hospitals from 2009 to 2013. Demographics, chronic medical conditions, viral coinfections, and hospitalization characteristics, including the need for respiratory support, high-flow nasal cannula, CPAP, bi-level positive airway pressure, invasive mechanical ventilation, pediatric ICU admission, acute kidney injury (AKI), use of extracorporeal membrane oxygenation, and length of stay, were collected.RESULTS:In total, 131 subjects were included. Those with one or more comorbidities were older than their otherwise healthy counterparts, with a median age of 2.8 y (interquartile range [IQR] 1.1–7.0) compared to 1.3 y (IQR 0.6–2.0, P < .001), respectively. Ninety-nine (75.6%) subjects required respiratory support; 72 (55.0%) subjects required nasal cannula, simple face mask, or tracheostomy mask as their maximum support. Additionally, 1 (0.8%) subject required high-flow nasal cannula, 1 (0.8%) subject required CPAP, 2 (1.5%) subjects required bi-level positive airway pressure, 15 (11.5%) subjects required invasive mechanical ventilation, 4 (3.1%) subjects required high-frequency oscillatory or jet ventilation, and 4 (3.1%) subjects required extracorporeal membrane oxygenation. Fifty-one (38.9%) subjects required pediatric ICU admission, and 16 (12.2%) subjects developed AKI. Subjects with AKI were significantly older than those without AKI at 5.4 y old (IQR 1.6–11.7) versus 1.9 y old (IQR 0.7–3.5, P = .003). After controlling for the presence of at least one comorbidity and cystic fibrosis, each year increase in age led to a 16% increase in the odds of AKI (P = .01). The median length of stay for the entire cohort was 4.0 d (IQR 2.7–7.0).CONCLUSIONS:Children hospitalized with HMPV may be at risk for AKI. Risk of HMPV-associated AKI appears to increase with age regardless of severity of respiratory illness or presence of comorbidities.




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Correction to "Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine" [Errata]




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Circadian Clock-Controlled Drug Metabolism: Implications for Chronotherapeutics [Minireview]

Dependence of drug metabolism on dosing time has long been recognized. However, only recently are the underlying mechanisms for circadian drug metabolism being clarified. Diurnal rhythmicity in expression of drug-metabolizing enzymes is believed to be a key factor determining circadian metabolism. Supporting the notion that biological rhythms are generated and maintained by the circadian clock, a number of diurnal enzymes are under the control of the circadian clock. In general, circadian clock genes generate and regulate diurnal rhythmicity in drug-metabolizing enzymes via transcriptional actions on one or two of three cis-elements (i.e., E-box, D-box, and Rev-erb response element or RAR-related orphan receptor response element). Additionally, cycling or clock-controlled nuclear receptors such as hepatocyte nuclear factor 4α and peroxisome proliferator–activated receptor are contributors to diurnal enzyme expression. These newly discovered mechanisms for each of the rhythmic enzymes are reviewed in this article. We also discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics. Our discussion is also extended to two diurnal transporters (P-glycoprotein and multidrug resistance-associated protein 2) that have an important role in drug absorption. Although the experimental evidence is lacking in metabolism-based chronoefficacy, circadian genes (e.g., Rev-erbα) as drug targets are shown to account for diurnal variability in drug efficacy.

SIGNIFICANCE STATEMENT

Significant progress has been made in understanding the molecular mechanisms for generation of diurnal rhythmicity in drug-metabolizing enzymes. In this article, we review the newly discovered mechanisms for each of the rhythmic enzymes and discuss how the rhythms of enzymes are translated to circadian pharmacokinetics and drug chronotoxicity, which has direct implications for chronotherapeutics.




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Ontogeny and Cross-species Comparison of Pathways Involved in Drug Absorption, Distribution, Metabolism, and Excretion in Neonates (Review): Kidney [Minireview]

The kidneys play an important role in many processes, including urine formation, water conservation, acid-base equilibrium, and elimination of waste. The anatomic and functional development of the kidney has different maturation time points in humans versus animals, with critical differences between species in maturation before and after birth. Absorption, distribution, metabolism, and excretion (ADME) of drugs vary depending on age and maturation, which will lead to differences in toxicity and efficacy. When neonate/juvenile laboratory animal studies are designed, a thorough knowledge of the differences in kidney development between newborns/children and laboratory animals is essential. The human and laboratory animal data must be combined to obtain a more complete picture of the development in the kidneys around the neonatal period and the complexity of ADME in newborns and children. This review examines the ontogeny and cross-species differences in ADME processes in the developing kidney in preterm and term laboratory animals and children. It provides an overview of insights into ADME functionality in the kidney by identifying what is currently known and which gaps still exist. Currently important renal function properties such as glomerular filtration rate, renal blood flow, and ability to concentrate are generally well known, while detailed knowledge about transporter and metabolism maturation is growing but is still lacking. Preclinical data in those properties is limited to rodents and generally covers only the expression levels of transporter or enzyme-encoding genes. More knowledge on a functional level is needed to predict the kinetics and toxicity in neonate/juvenile toxicity and efficacy studies.

SIGNIFICANCE STATEMENT

This review provides insight in cross-species developmental differences of absorption, distribution, metabolism, and excretion properties in the kidney, which should be considered in neonate/juvenile study interpretation, hypotheses generation, and experimental design.




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Theophylline Acetaldehyde as the Initial Product in Doxophylline Metabolism in Human Liver [Articles]

Doxophylline (DOXO) and theophylline are widely used as bronchodilators for treating asthma and chronic obstructive pulmonary disease, and DOXO has a better safety profile than theophylline. How DOXO’s metabolism and disposition affect its antiasthmatic efficacy and safety remains to be explored. In this study, the metabolites of DOXO were characterized. A total of nine metabolites of DOXO were identified in vitro using liver microsomes from human and four other animal species. Among them, six metabolites were reported for the first time. The top three metabolites were theophylline acetaldehyde (M1), theophylline-7-acetic acid (M2), and etophylline (M4). A comparative analysis of DOXO metabolism in human using liver microsomes, S9 fraction, and plasma samples demonstrated the following: 1) The metabolism of DOXO began with a cytochrome P450 (P450)–mediated, rate-limiting step at the C ring and produced M1, the most abundant metabolite in human liver microsomes. However, in human plasma, the M1 production was rather low. 2) M1 was further converted to M2 and M4, the end products of DOXO metabolism in vivo, by non-P450 dismutase in the cytosol. This dismutation process also relied on the ratio of NADP+/NADPH in the cell. These findings for the first time elucidated the metabolic sites and routes of DOXO metabolism in human.

SIGNIFICANCE STATEMENT

We systematically characterized doxophylline metabolism using in vitro and in vivo assays. Our findings evolved the understandings of metabolic sites and pathways for methylxanthine derivatives with the aldehyde functional group.




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Drug Metabolism and Disposition




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Perturbation of phosphoglycerate kinase 1 (PGK1) only marginally affects glycolysis in cancer cells [Metabolism]

Phosphoglycerate kinase 1 (PGK1) plays important roles in glycolysis, yet its forward reaction kinetics are unknown, and its role especially in regulating cancer cell glycolysis is unclear. Here, we developed an enzyme assay to measure the kinetic parameters of the PGK1-catalyzed forward reaction. The Km values for 1,3-bisphosphoglyceric acid (1,3-BPG, the forward reaction substrate) were 4.36 μm (yeast PGK1) and 6.86 μm (human PKG1). The Km values for 3-phosphoglycerate (3-PG, the reverse reaction substrate and a serine precursor) were 146 μm (yeast PGK1) and 186 μm (human PGK1). The Vmax of the forward reaction was about 3.5- and 5.8-fold higher than that of the reverse reaction for the human and yeast enzymes, respectively. Consistently, the intracellular steady-state concentrations of 3-PG were between 180 and 550 μm in cancer cells, providing a basis for glycolysis to shuttle 3-PG to the serine synthesis pathway. Using siRNA-mediated PGK1-specific knockdown in five cancer cell lines derived from different tissues, along with titration of PGK1 in a cell-free glycolysis system, we found that the perturbation of PGK1 had no effect or only marginal effects on the glucose consumption and lactate generation. The PGK1 knockdown increased the concentrations of fructose 1,6-bisphosphate, dihydroxyacetone phosphate, glyceraldehyde 3-phosphate, and 1,3-BPG in nearly equal proportions, controlled by the kinetic and thermodynamic states of glycolysis. We conclude that perturbation of PGK1 in cancer cells insignificantly affects the conversion of glucose to lactate in glycolysis.




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Transcriptome reconstruction and functional analysis of eukaryotic marine plankton communities via high-throughput metagenomics and metatranscriptomics [METHOD]

Large-scale metagenomic and metatranscriptomic data analyses are often restricted by their gene-centric approach, limiting the ability to understand organismal and community biology. De novo assembly of large and mosaic eukaryotic genomes from complex meta-omics data remains a challenging task, especially in comparison with more straightforward bacterial and archaeal systems. Here, we use a transcriptome reconstruction method based on clustering co-abundant genes across a series of metagenomic samples. We investigated the co-abundance patterns of ~37 million eukaryotic unigenes across 365 metagenomic samples collected during the Tara Oceans expeditions to assess the diversity and functional profiles of marine plankton. We identified ~12,000 co-abundant gene groups (CAGs), encompassing ~7 million unigenes, including 924 metagenomics-based transcriptomes (MGTs, CAGs larger than 500 unigenes). We demonstrated the biological validity of the MGT collection by comparing individual MGTs with available references. We identified several key eukaryotic organisms involved in dimethylsulfoniopropionate (DMSP) biosynthesis and catabolism in different oceanic provinces, thus demonstrating the potential of the MGT collection to provide functional insights on eukaryotic plankton. We established the ability of the MGT approach to capture interspecies associations through the analysis of a nitrogen-fixing haptophyte-cyanobacterial symbiotic association. This MGT collection provides a valuable resource for analyses of eukaryotic plankton in the open ocean by giving access to the genomic content and functional potential of many ecologically relevant eukaryotic species.




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Endogenous PAD4 in Breast Cancer Cells Mediates Cancer Extracellular Chromatin Network Formation and Promotes Lung Metastasis

Peptidyl arginine deiminase 4 (PAD4/PADI4) is a posttranslational modification enzyme that converts protein arginine or mono-methylarginine to citrulline. The PAD4-mediated hypercitrullination reaction in neutrophils causes the release of nuclear chromatin to form a chromatin network termed neutrophil extracellular traps (NET). NETs were first described as antimicrobial fibers that bind and kill bacteria. However, it is not known whether PAD4 can mediate the release of chromatin DNA into the extracellular space of cancer cells. Here, we report that murine breast cancer 4T1 cells expressing high levels of PADI4 can release cancer extracellular chromatin networks (CECN) in vitro and in vivo. Deletion of Padi4 using CRISPR/Cas9 abolished CECN formation in 4T1 cells. Padi4 deletion from 4T1 cells also reduced the rate of tumor growth in an allograft model, and decreased lung metastasis by 4T1 breast cancers. DNase I treatment, which degrades extracellular DNA including CECNs, also reduced breast to lung metastasis of Padi4 wild-type 4T1 cells in allograft experiments in the Padi4-knockout mice. We further demonstrated that DNase I treatment in this mouse model did not alter circulating tumor cells but decreased metastasis through steps after intravasation. Taken together, our genetic studies show that PAD4 plays a cell autonomous role in cancer metastasis, thus revealing a novel strategy for preventing cancer metastasis by inhibiting cancer cell endogenous PAD4.

Implications:

This study shows that PADI4 can mediate the formation of CECNs in 4T1 cells, and that endogenous PADI4 plays an essential role in breast cancer lung metastasis.

Visual Overview:

http://mcr.aacrjournals.org/content/molcanres/18/5/735/F1.large.jpg.




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OLR1 Promotes Pancreatic Cancer Metastasis via Increased c-Myc Expression and Transcription of HMGA2

Pancreatic cancer is one of the most lethal human malignancies, partly because of its propensity for metastasis. However, the mechanisms of metastasis in pancreatic cancer remain unclear. Oxidized low-density lipoprotein receptor 1 (OLR1), a lectin-like scavenger receptor that recognizes several ligands, such as oxidized low-density lipoprotein, was previously reported in cardiovascular and metabolic diseases. The role and mechanism of OLR1 in pancreatic cancer is unclear. In this study, we found that OLR1 expression was significantly higher in pancreatic cancer tissues than that in adjacent normal tissues and closely associated with reduced overall survival. OLR1 promoted proliferation and metastasis of pancreatic cancer cells in vitro and in vivo. Mechanistically, OLR1 increased HMGA2 transcription by upregulating c-Myc expression to promote the metastasis of pancreatic cancer cells. In addition, patients with pancreatic cancer with high expression of OLR1–c-Myc–HMGA2 axis showed worse prognosis compared with patients with low expression of OLR1–c-Myc–HMGA2 axis.

Implications:

Our findings suggested that the OLR1–c-Myc–HMGA2 axis promotes metastasis of pancreatic cancer cells and may serve as potential therapeutic targets and prognosis markers for patients with pancreatic cancer.




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Hepatic Transporter Alterations by Nuclear Receptor Agonist T0901317 in Sandwich-Cultured Human Hepatocytes: Proteomic Analysis and PBPK Modeling to Evaluate Drug-Drug Interaction Risk [Metabolism, Transport, and Pharmacogenomics]

In vitro approaches for predicting drug-drug interactions (DDIs) caused by alterations in transporter protein regulation are not well established. However, reports of transporter regulation via nuclear receptor (NR) modulation by drugs are increasing. This study examined alterations in transporter protein levels in sandwich-cultured human hepatocytes (SCHH; n = 3 donors) measured by liquid chromatography–tandem mass spectrometry–based proteomic analysis after treatment with N-[4-(1,1,1,3,3,3-hexafluoro-2-hydroxypropan-2-yl)phenyl]-N-(2,2,2-trifluoroethyl)benzenesulfonamide (T0901317), the first described synthetic liver X receptor agonist. T0901317 treatment (10 μM, 48 hours) decreased the levels of organic cation transporter (OCT) 1 (0.22-, 0.43-, and 0.71-fold of control) and organic anion transporter (OAT) 2 (0.38-, 0.38-, and 0.53-fold of control) and increased multidrug resistance protein (MDR) 1 (1.37-, 1.48-, and 1.59-fold of control). The induction of NR downstream gene expression supports the hypothesis that T0901317 off-target effects on farnesoid X receptor and pregnane X receptor activation are responsible for the unexpected changes in OCT1, OAT2, and MDR1. Uptake of the OCT1 substrate metformin in SCHH was decreased by T0901317 treatment. Effects of decreased OCT1 levels on metformin were simulated using a physiologically-based pharmacokinetic (PBPK) model. Simulations showed a clear decrease in metformin hepatic exposure resulting in a decreased pharmacodynamic effect. This DDI would not be predicted by the modest changes in simulated metformin plasma concentrations. Altogether, the current study demonstrated that an approach combining SCHH, proteomic analysis, and PBPK modeling is useful for revealing tissue concentration–based DDIs caused by unexpected regulation of hepatic transporters by NR modulators.

SIGNIFICANCE STATEMENT

This study utilized an approach combining sandwich-cultured human hepatocytes, proteomic analysis, and physiologically based pharmacokinetic modeling to evaluate alterations in pharmacokinetics (PK) and pharmacodynamics (PD) caused by transporter regulation by nuclear receptor modulators. The importance of this approach from a mechanistic and clinically relevant perspective is that it can reveal drug-drug interactions (DDIs) caused by unexpected regulation of hepatic transporters and enable prediction of altered PK and PD changes, especially for tissue concentration–based DDIs.




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Additional Local Therapy for Liver Metastases in Patients with Metastatic Castration-Resistant Prostate Cancer Receiving Systemic PSMA-Targeted Therapy

The aim of this study was to evaluate the efficacy of 177Lu-prostate-specific membrane antigen (PSMA)-617 (177Lu-PSMA) and selective internal radiation therapy (SIRT) for the treatment of liver metastases of castration-resistant prostate cancer. Methods: Safety and survival of patients with metastatic castration-resistant prostate cancer and liver metastases assigned to 177Lu-PSMA alone (n = 31) or in combination with SIRT (n = 5) were retrospectively analyzed. Additionally, a subgroup (n = 10) was analyzed using morphologic and molecular response criteria. Results: Median estimated survival was 5.7 mo for 177Lu-PSMA alone and 8.4 mo for combined sequential 177Lu-PSMA and SIRT. 177Lu-PSMA achieved discordant therapy responses with both regressive and progressive liver metastases in the same patient (best vs. worst responding metastases per patient: –35% vs. +63% diameter change; P < 0.05). SIRT was superior to 177Lu-PSMA for the treatment of liver metastases (0% vs. 56% progression). Conclusion: The combination of 177Lu-PSMA and SIRT is efficient and feasible for the treatment of advanced prostate cancer. 177Lu-PSMA alone seems to have limited response rates in the treatment of liver metastases.




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Assessing Radiographic Response to 223Ra with an Automated Bone Scan Index in Metastatic Castration-Resistant Prostate Cancer Patients

For effective clinical management of patients being treated with 223Ra, there is a need for radiographic response biomarkers to minimize disease progression and to stratify patients for subsequent treatment options. The objective of this study was to evaluate an automated bone scan index (aBSI) as a quantitative assessment of bone scans for radiographic response in patients with metastatic castration-resistant prostate cancer (mCRPC). Methods: In a multicenter retrospective study, bone scans from patients with mCRPC treated with monthly injections of 223Ra were collected from 7 hospitals in Sweden. Patients with available bone scans before treatment with 223Ra and at treatment discontinuation were eligible for the study. The aBSI was generated at baseline and at treatment discontinuation. The Spearman rank correlation was used to correlate aBSI with the baseline covariates: alkaline phosphatase (ALP) and prostate-specific antigen (PSA). The Cox proportional-hazards model and Kaplan–Meier curve were used to evaluate the association of covariates at baseline and their change at treatment discontinuation with overall survival (OS). The concordance index (C-index) was used to evaluate the discriminating strength of covariates in predicting OS. Results: Bone scan images at baseline were available from 156 patients, and 67 patients had both a baseline and a treatment discontinuation bone scan (median, 5 doses; interquartile range, 3–6 doses). Baseline aBSI (median, 4.5; interquartile range, 2.4–6.5) was moderately correlated with ALP (r = 0.60, P < 0.0001) and with PSA (r = 0.38, P = 0.003). Among baseline covariates, aBSI (P = 0.01) and ALP (P = 0.001) were significantly associated with OS, whereas PSA values were not (P = 0.059). After treatment discontinuation, 36% (24/67), 80% (54/67), and 13% (9/67) of patients demonstrated a decline in aBSI, ALP, and PSA, respectively. As a continuous variable, the relative change in aBSI after treatment, compared with baseline, was significantly associated with OS (P < 0.0001), with a C-index of 0.67. Median OS in patients with both aBSI and ALP decline (median, 134 wk) was significantly longer than in patients with ALP decline only (median, 77 wk; P = 0.029). Conclusion: Both aBSI at baseline and its change at treatment discontinuation were significant parameters associated with OS. The study warrants prospective validation of aBSI as a quantitative imaging response biomarker to predict OS in patients with mCRPC treated with 223Ra.




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Subtle Variations in Dietary-Fiber Fine Structure Differentially Influence the Composition and Metabolic Function of Gut Microbiota

ABSTRACT

The chemical structures of soluble fiber carbohydrates vary from source to source due to numerous possible linkage configurations among monomers. However, it has not been elucidated whether subtle structural variations might impact soluble fiber fermentation by colonic microbiota. In this study, we tested the hypothesis that subtle structural variations in a soluble polysaccharide govern the community structure and metabolic output of fermenting microbiota. We performed in vitro fecal fermentation studies using arabinoxylans (AXs) from different classes of wheat (hard red spring [AXHRS], hard red winter [AXHRW], and spring red winter [AXSRW]) with identical initial microbiota. Carbohydrate analyses revealed that AXSRW was characterized by a significantly shorter backbone and increased branching compared with those of the hard varieties. Amplicon sequencing demonstrated that fermentation of AXSRW resulted in a distinct community structure of significantly higher richness and evenness than those of hard-AX-fermenting cultures. AXSRW favored OTUs within Bacteroides, whereas AXHRW and AXHRS favored Prevotella. Accordingly, metabolic output varied between hard and soft varieties; higher propionate production was observed with AXSRW and higher butyrate and acetate with AXHRW and AXHRS. This study showed that subtle changes in the structure of a dietary fiber may strongly influence the composition and function of colonic microbiota, further suggesting that physiological functions of dietary fibers are highly structure dependent. Thus, studies focusing on interactions among dietary fiber, gut microbiota, and health outcomes should better characterize the structures of the carbohydrates employed.

IMPORTANCE Diet, especially with respect to consumption of dietary fibers, is well recognized as one of the most important factors shaping the colonic microbiota composition. Accordingly, many studies have been conducted to explore dietary fiber types that could predictably manipulate the colonic microbiota for improved health. However, the majority of these studies underappreciate the vastness of fiber structures in terms of their microbial utilization and omit detailed carbohydrate structural analysis. In some cases, this causes conflicting results to arise between studies using (theoretically) the same fibers. In this investigation, by performing in vitro fecal fermentation studies using bran arabinoxylans obtained from different classes of wheat, we showed that even subtle changes in the structure of a dietary fiber result in divergent microbial communities and metabolic outputs. This underscores the need for much higher structural resolution in studies investigating interactions of dietary fibers with gut microbiota, both in vitro and in vivo.




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An Extensive Meta-Metagenomic Search Identifies SARS-CoV-2-Homologous Sequences in Pangolin Lung Viromes

ABSTRACT

In numerous instances, tracking the biological significance of a nucleic acid sequence can be augmented through the identification of environmental niches in which the sequence of interest is present. Many metagenomic data sets are now available, with deep sequencing of samples from diverse biological niches. While any individual metagenomic data set can be readily queried using web-based tools, meta-searches through all such data sets are less accessible. In this brief communication, we demonstrate such a meta-metagenomic approach, examining close matches to the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in all high-throughput sequencing data sets in the NCBI Sequence Read Archive accessible with the "virome" keyword. In addition to the homology to bat coronaviruses observed in descriptions of the SARS-CoV-2 sequence (F. Wu, S. Zhao, B. Yu, Y. M. Chen, et al., Nature 579:265–269, 2020, https://doi.org/10.1038/s41586-020-2008-3; P. Zhou, X. L. Yang, X. G. Wang, B. Hu, et al., Nature 579:270–273, 2020, https://doi.org/10.1038/s41586-020-2012-7), we note a strong homology to numerous sequence reads in metavirome data sets generated from the lungs of deceased pangolins reported by Liu et al. (P. Liu, W. Chen, and J. P. Chen, Viruses 11:979, 2019, https://doi.org/10.3390/v11110979). While analysis of these reads indicates the presence of a similar viral sequence in pangolin lung, the similarity is not sufficient to either confirm or rule out a role for pangolins as an intermediate host in the recent emergence of SARS-CoV-2. In addition to the implications for SARS-CoV-2 emergence, this study illustrates the utility and limitations of meta-metagenomic search tools in effective and rapid characterization of potentially significant nucleic acid sequences.

IMPORTANCE Meta-metagenomic searches allow for high-speed, low-cost identification of potentially significant biological niches for sequences of interest.




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Sex Disparities in Cardiovascular Outcome Trials of Populations With Diabetes: A Systematic Review and Meta-analysis

BACKGROUND

Sex differences have been described in diabetes cardiovascular outcome trials (CVOTs).

PURPOSE

We systematically reviewed for baseline sex differences in cardiovascular (CV) risk factors and CV protection therapy in diabetes CVOTs.

DATA SOURCES

Randomized placebo-controlled trials examining the effect of diabetes medications on major adverse cardiovascular events in people ≥18 years of age with type 2 diabetes.

STUDY SELECTION

Included trials reported baseline sex-specific CV risks and use of CV protection therapy.

DATA EXTRACTION

Two reviewers independently abstracted study data.

DATA SYNTHESIS

We included five CVOTs with 46,606 participants. We summarized sex-specific data using mean differences (MDs) and relative risks (RRs) and pooled estimates using random effects meta-analysis. There were fewer women than men in included trials (28.5–35.8% women). Women more often had stroke (RR 1.28; 95% CI 1.09, 1.50), heart failure (RR 1.30; 95% CI 1.21,1.40), and chronic kidney disease (RR 1.33; 95% CI 1.17; 1.51). They less often used statins (RR 0.90; 95% CI 0.86, 0.93), aspirin (RR 0.82; 95% CI 0.71, 0.95), and β-blockers (RR 0.93; 95% CI 0.88, 0.97) and had a higher systolic blood pressure (MD 1.66 mmHg; 95% CI 0.90, 2.41), LDL cholesterol (MD 0.34 mmol/L; 95% CI 0.29, 0.39), and hemoglobin A1c (MD 0.11%; 95% CI 0.09, 0.14 [1.2 mmol/mol; 1.0, 1.5]) than men.

LIMITATIONS

We could not carry out subgroup analyses due to the small number of studies. Our study is not generalizable to low CV risk groups nor to patients in routine care.

CONCLUSIONS

There were baseline sex disparities in diabetes CVOTs. We suggest efforts to recruit women into trials and promote CV management across the sexes.




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Effects of Continuous Glucose Monitoring on Metrics of Glycemic Control in Diabetes: A Systematic Review With Meta-analysis of Randomized Controlled Trials

BACKGROUND

Continuous glucose monitoring (CGM) provides important information to aid in achieving glycemic targets in people with diabetes.

PURPOSE

We performed a meta-analysis of randomized controlled trials (RCTs) comparing CGM with usual care for parameters of glycemic control in both type 1 and type 2 diabetes.

DATA SOURCES

Many electronic databases were searched for articles published from inception until 30 June 2019.

STUDY SELECTION

We selected RCTs that assessed both changes in HbA1c and time in target range (TIR), together with time below range (TBR), time above range (TAR), and glucose variability expressed as coefficient of variation (CV).

DATA EXTRACTION

Data were extracted from each trial by two investigators.

DATA SYNTHESIS

All results were analyzed by a random effects model to calculate the weighted mean difference (WMD) with the 95% CI. We identified 15 RCTs, lasting 12–36 weeks and involving 2,461 patients. Compared with the usual care (overall data), CGM was associated with modest reduction in HbA1c (WMD –0.17%, 95% CI –0.29 to –0.06, I2 = 96.2%), increase in TIR (WMD 70.74 min, 95% CI 46.73–94.76, I2 = 66.3%), and lower TAR, TBR, and CV, with heterogeneity between studies. The increase in TIR was significant and robust independently of diabetes type, method of insulin delivery, and reason for CGM use. In preplanned subgroup analyses, real-time CGM led to the higher improvement in mean HbA1c (WMD –0.23%, 95% CI –0.36 to –0.10, P < 0.001), TIR (WMD 83.49 min, 95% CI 52.68–114.30, P < 0.001), and TAR, whereas both intermittently scanned CGM and sensor-augmented pump were associated with the greater decline in TBR.

LIMITATIONS

Heterogeneity was high for most of the study outcomes; all studies were sponsored by industry, had short duration, and used an open-label design.

CONCLUSIONS

CGM improves glycemic control by expanding TIR and decreasing TBR, TAR, and glucose variability in both type 1 and type 2 diabetes.




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Accuracy of the Ottawa score in risk stratification of recurrent venous thromboembolism in patients with cancer-associated venous thromboembolism: a systematic review and meta-analysis

In patients with cancer-associated venous thromboembolism, knowledge of the estimated rate of recurrent events is important for clinical decision-making regarding anticoagulant therapy. The Ottawa score is a clinical prediction rule designed for this purpose, stratifying patients according to their risk of recurrent venous thromboembolism during the first six months of anticoagulation. We conducted a systematic review and meta-analysis of studies validating either the Ottawa score in its original or modified versions. Two investigators independently reviewed the relevant articles published from 1st June 2012 to 15th December 2018 and indexed in MEDLINE and EMBASE. Nine eligible studies were identified; these included a total of 14,963 patients. The original score classified 49.3% of the patients as high-risk, with a sensitivity of 0.7 [95% confidence interval (CI): 0.6-0.8], a 6-month pooled rate of recurrent venous thromboembolism of 18.6% (95%CI: 13.9-23.9). In the low-risk group, the recurrence rate was 7.4% (95%CI: 3.4-12.5). The modified score classified 19.8% of the patients as low-risk, with a sensitivity of 0.9 (95%CI: 0.4-1.0) and a 6-month pooled rate of recurrent venous thromboembolism of 2.2% (95%CI: 1.6-2.9). In the high-risk group, recurrence rate was 10.2% (95%CI: 6.4-14.6). Limitations of our analysis included type and dosing of anticoagulant therapy. We conclude that new therapeutic strategies are needed in patients at high risk for recurrent cancer-associated venous thromboembolism. Low-risk patients, as per the modified score, could be good candidates for oral anticoagulation. (This systematic review was registered with the International Prospective Registry of Systematic Reviews as: PROSPERO CRD42018099506).




meta

The Use of Mendelian Randomization to Determine the Role of Metabolic Traits on Urinary Albumin-to-Creatinine Ratio




meta

Pervasive Small RNAs in Cardiometabolic Research: Great Potential Accompanied by Biological and Technical Barriers

Advances in small RNA sequencing have revealed the enormous diversity of small noncoding RNA (sRNA) classes in mammalian cells. At this point, most investigators in diabetes are aware of the success of microRNA (miRNA) research and appreciate the importance of posttranscriptional gene regulation in glycemic control. Nevertheless, miRNAs are just one of multiple classes of sRNAs and likely represent only a minor fraction of sRNA sequences in a given cell. Despite the widespread appreciation of sRNAs, very little research into non-miRNA sRNA function has been completed, likely due to some major barriers that present unique challenges for study. To emphasize the importance of sRNA research in cardiometabolic diseases, we highlight the success of miRNAs and competitive endogenous RNAs in cholesterol and glucose metabolism. Moreover, we argue that sequencing studies have demonstrated that miRNAs are just the tip of the iceberg for sRNAs. We are likely standing at the precipice of immense discovery for novel sRNA-mediated gene regulation in cardiometabolic diseases. To realize this potential, we must first address critical barriers with an open mind and refrain from viewing non-miRNA sRNA function through the lens of miRNAs, as they likely have their own set of distinct regulatory factors and functional mechanisms.




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Starvation causes female-to-male sex reversal through lipid metabolism in the teleost fish, medaka (Olyzias latipes) [RESEARCH ARTICLE]

Yuta Sakae, Akira Oikawa, Yuki Sugiura, Masatoshi Mita, Shuhei Nakamura, Toshiya Nishimura, Makoto Suematsu, and Minoru Tanaka

The teleost fish, medaka (Oryzias latipes), employs the XX/XY genetic sex determination system. We show here that the phenotypic sex of medaka is affected by changes in lipid metabolism. Medaka larvae subjected to 5 days of starvation underwent female-to-male sex reversal. Metabolomic and RT-qPCR analyses indicated that pantothenate metabolism was suppressed by starvation. Consistently, inhibiting the pantothenate metabolic pathway caused sex reversal. The final metabolite in this pathway is coenzyme A, an essential factor for lipogenesis. Inhibiting fatty acid synthesis, the first step of lipogenesis, also caused sex reversal. The expression of dmrt1, a critical gene for male development, was suppressed by starvation, and a dmrt1 (13) mutant did not show sex reversal under starvation. Collectively, these results indicate that fatty acid synthesis is involved in female-to-male sex reversal through ectopic expression of male gene dmrt1 under starvation.




meta

Metabolic Disorders with Kidney Transplant

Metabolic disorders are highly prevalent in kidney transplant candidates and recipients and can adversely affect post-transplant graft outcomes. Management of diabetes, hyperparathyroidism, and obesity presents distinct opportunities to optimize patients both before and after transplant as well as the ability to track objective data over time to assess a patient’s ability to partner effectively with the health care team and adhere to complex treatment regimens. Optimization of these particular disorders can most dramatically decrease the risk of surgical and cardiovascular complications post-transplant. Approximately 60% of nondiabetic patients experience hyperglycemia in the immediate post-transplant phase. Multiple risk factors have been identified related to development of new onset diabetes after transplant, and it is estimated that upward of 7%–30% of patients will develop new onset diabetes within the first year post-transplant. There are a number of medications studied in the kidney transplant population for diabetes management, and recent data and the risks and benefits of each regimen should be optimized. Secondary hyperparathyroidism occurs in most patients with CKD and can persist after kidney transplant in up to 66% of patients, despite an initial decrease in parathyroid hormone levels. Parathyroidectomy and medical management are the options for treatment of secondary hyperparathyroidism, but there is no randomized, controlled trial providing clear recommendations for optimal management, and patient-specific factors should be considered. Obesity is the most common metabolic disorder affecting the transplant population in both the pre- and post-transplant phases of care. Not only does obesity have associations and interactions with comorbid illnesses, such as diabetes, dyslipidemia, and cardiovascular disease, all of which increase morbidity and mortality post-transplant, but it also is intimately inter-related with access to transplantation for patients with kidney failure. We review these metabolic disorders and their management, including data in patients with kidney transplants.




meta

RIPK3 Orchestrates Fatty Acid Metabolism in Tumor-Associated Macrophages and Hepatocarcinogenesis

Metabolic reprogramming is critical for the polarization and function of tumor-associated macrophages (TAM) and hepatocarcinogenesis, but how this reprogramming occurs is unknown. Here, we showed that receptor-interacting protein kinase 3 (RIPK3), a central factor in necroptosis, is downregulated in hepatocellular carcinoma (HCC)–associated macrophages, which correlated with tumorigenesis and enhanced the accumulation and polarization of M2 TAMs. Mechanistically, RIPK3 deficiency in TAMs reduced reactive oxygen species and significantly inhibited caspase1-mediated cleavage of PPAR. These effects enabled PPAR activation and facilitated fatty acid metabolism, including fatty acid oxidation (FAO), and induced M2 polarization in the tumor microenvironment. RIPK3 upregulation or FAO blockade reversed the immunosuppressive activity of TAMs and dampened HCC tumorigenesis. Our findings provide molecular basis for the regulation of RIPK3-mediated, lipid metabolic reprogramming of TAMs, thus highlighting a potential strategy for targeting the immunometabolism of HCC.




meta

Single-Cell Immune Competency Signatures Associate with Survival in Phase II GVAX and CRS-207 Randomized Studies in Patients with Metastatic Pancreatic Cancer

The identification of biomarkers for patient stratification is fundamental to precision medicine efforts in oncology. Here, we identified two baseline, circulating immune cell subsets associated with overall survival in patients with metastatic pancreatic cancer who were enrolled in two phase II randomized studies of GVAX pancreas and CRS-207 immunotherapy. Single-cell mass cytometry was used to simultaneously measure 38 cell surface or intracellular markers in peripheral blood mononuclear cells obtained from a phase IIa patient subcohort (N = 38). CITRUS, an algorithm for identification of stratifying subpopulations in multidimensional cytometry datasets, was used to identify single-cell signatures associated with clinical outcome. Patients with a higher abundance of CD8+CD45ROCCR7CD57+ cells and a lower abundance of CD14+CD33+CD85j+ cells had improved overall survival [median overall survival, range (days) 271, 43–1,247] compared with patients with a lower abundance of CD8+CD45ROCCR7CD57+ cells and higher abundance of CD14+CD33+CD85j+ cells (77, 24–1,247 days; P = 0.0442). The results from this prospective–retrospective biomarker analysis were validated by flow cytometry in 200 patients with pancreatic cancer enrolled in a phase IIb study (P = 0.0047). The identified immune correlates provide potential prognostic or predictive signatures that could be employed for patient stratification.




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The Role of Fnr Paralogs in Controlling Anaerobic Metabolism in the Diazotroph Paenibacillus polymyxa WLY78 [Environmental Microbiology]

Fnr is a transcriptional regulator that controls the expression of a variety of genes in response to oxygen limitation in bacteria. Genome sequencing revealed four genes (fnr1, fnr3, fnr5, and fnr7) coding for Fnr proteins in Paenibacillus polymyxa WLY78. Fnr1 and Fnr3 showed more similarity to each other than to Fnr5 and Fnr7. Also, Fnr1 and Fnr3 exhibited high similarity with Bacillus cereus Fnr and Bacillus subtilis Fnr in sequence and structures. Both the aerobically purified His-tagged Fnr1 and His-tagged Fnr3 in Escherichia coli could bind to the specific DNA promoter. Deletion analysis showed that the four fnr genes, especially fnr1 and fnr3, have significant impacts on growth and nitrogenase activity. Single deletion of fnr1 or fnr3 led to a 50% reduction in nitrogenase activity, and double deletion of fnr1 and fnr3 resulted to a 90% reduction in activity. Genome-wide transcription analysis showed that Fnr1 and Fnr3 indirectly activated expression of nif (nitrogen fixation) genes and Fe transport genes under anaerobic conditions. Fnr1 and Fnr3 inhibited expression of the genes involved in the aerobic respiratory chain and activated expression of genes responsible for anaerobic electron acceptor genes.

IMPORTANCE The members of the nitrogen-fixing Paenibacillus spp. have great potential to be used as a bacterial fertilizer in agriculture. However, the functions of the fnr gene(s) in nitrogen fixation and other metabolisms in Paenibacillus spp. are not known. Here, we found that in P. polymyxa WLY78, Fnr1 and Fnr3 were responsible for regulation of numerous genes in response to changes in oxygen levels, but Fnr5 and Fnr7 exhibited little effect. Fnr1 and Fnr3 indirectly or directly regulated many types of important metabolism, such as nitrogen fixation, Fe uptake, respiration, and electron transport. This study not only reveals the function of the fnr genes of P. polymyxa WLY78 in nitrogen fixation and other metabolisms but also will provide insight into the evolution and regulatory mechanisms of fnr in Paenibacillus.




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Two Functional Fatty Acyl Coenzyme A Ligases Affect Free Fatty Acid Metabolism To Block Biosynthesis of an Antifungal Antibiotic in Lysobacter enzymogenes [Environmental Microbiology]

In Lysobacter enzymogenes OH11, RpfB1 and RpfB2 were predicted to encode acyl coenzyme A (CoA) ligases. RpfB1 is located in the Rpf gene cluster. Interestingly, we found an RpfB1 homolog (RpfB2) outside this canonical gene cluster, and nothing is known about its functionality or mechanism. Here, we report that rpfB1 and rpfB2 can functionally replace EcFadD in the Escherichia coli fadD mutant JW1794. RpfB activates long-chain fatty acids (n-C16:0 and n-C18:0) for the corresponding fatty acyl-CoA ligase (FCL) activity in vitro, and Glu-361 plays critical roles in the catalytic mechanism of RpfB1 and RpfB2. Deletion of rpfB1 and rpfB2 resulted in significantly increased heat-stable antifungal factor (HSAF) production, and overexpression of rpfB1 or rpfB2 completely suppressed HSAF production. Deletion of rpfB1 and rpfB2 resulted in increased L. enzymogenes diffusible signaling factor 3 (LeDSF3) synthesis in L. enzymogenes. Overall, our results showed that changes in intracellular free fatty acid levels significantly altered HSAF production. Our report shows that intracellular free fatty acids are required for HSAF production and that RpfB affects HSAF production via FCL activity. The global transcriptional regulator Clp directly regulated the expression of rpfB1 and rpfB2. In conclusion, these findings reveal new roles of RpfB in antibiotic biosynthesis in L. enzymogenes.

IMPORTANCE Understanding the biosynthetic and regulatory mechanisms of heat-stable antifungal factor (HSAF) could improve the yield in Lysobacter enzymogenes. Here, we report that RpfB1 and RpfB2 encode acyl coenzyme A (CoA) ligases. Our research shows that RpfB1 and RpfB2 affect free fatty acid metabolism via fatty acyl-CoA ligase (FCL) activity to reduce the substrate for HSAF synthesis and, thereby, block HSAF production in L. enzymogenes. Furthermore, these findings reveal new roles for the fatty acyl-CoA ligases RpfB1 and RpfB2 in antibiotic biosynthesis in L. enzymogenes. Importantly, the novelty of this work is the finding that RpfB2 lies outside the Rpf gene cluster and plays a key role in HSAF production, which has not been reported in other diffusible signaling factor (DSF)/Rpf-producing bacteria.




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Metabolic Acidosis and Hypoglycemia in a Child with Leigh-Like Phenotype




meta

Overdiagnosis of lung cancer with low-dose computed tomography screening: meta-analysis of the randomised clinical trials

In low-dose computed tomography (LDCT) screening for lung cancer, all three main conditions for overdiagnosis in cancer screening are present: 1) a reservoir of slowly or nongrowing lung cancer exists; 2) LDCT is a high-resolution imaging technology with the potential to identify this reservoir; and 3) eligible screening participants have a high risk of dying from causes other than lung cancer. The degree of overdiagnosis in cancer screening is most validly estimated in high-quality randomised controlled trials (RCTs), with enough follow-up time after the end of screening to avoid lead-time bias and without contamination of the control group.

Nine RCTs investigating LDCT screening were identified. Two RCTs were excluded because lung cancer incidence after the end of screening was not published. Two other RCTs using active comparators were also excluded. Therefore, five RCTs were included: two trials were at low risk of bias, two of some concern and one at high risk of bias. In a meta-analysis of the two low risk of bias RCTs including 8156 healthy current or former smokers, 49% of the screen-detected cancers were overdiagnosed. There is uncertainty about this substantial degree of overdiagnosis due to unexplained heterogeneity and low precision of the summed estimate across the two trials.

Key points

  • Nine randomised controlled trials (RCTs) on low-dose computed tomography screening were identified; five were included for meta-analysis but only two of those were at low risk of bias.

  • In a meta-analysis of recent low risk of bias RCTs including 8156 healthy current or former smokers from developed countries, we found that 49% of the screen-detected cancers may be overdiagnosed.

  • There is uncertainty about the degree of overdiagnosis in lung cancer screening due to unexplained heterogeneity and low precision of the point estimate.

  • If only high-quality RCTs are included in the meta-analysis, the degree of overdiagnosis is substantial.

  • Educational aims

  • To appreciate that low-dose computed tomography screening for lung cancer meets all three main conditions for overdiagnosis in cancer screening: a reservoir of indolent cancers exists in the population; the screening test is able to "tap" this reservoir by detecting biologically indolent cancers as well as biologically important cancers; and the population being screened is characterised by a relatively high competing risk of death from other causes

  • To learn about biases that might affect the estimates of overdiagnosis in randomised controlled trials in cancer screening




    meta

    Surfactant Expression Defines an Inflamed Subtype of Lung Adenocarcinoma Brain Metastases that Correlates with Prolonged Survival

    Purpose:

    To provide a better understanding of the interplay between the immune system and brain metastases to advance therapeutic options for this life-threatening disease.

    Experimental Design:

    Tumor-infiltrating lymphocytes (TIL) were quantified by semiautomated whole-slide analysis in brain metastases from 81 lung adenocarcinomas. Multi-color staining enabled phenotyping of TILs (CD3, CD8, and FOXP3) on a single-cell resolution. Molecular determinants of the extent of TILs in brain metastases were analyzed by transcriptomics in a subset of 63 patients. Findings in lung adenocarcinoma brain metastases were related to published multi-omic primary lung adenocarcinoma The Cancer Genome Atlas data (n = 230) and single-cell RNA-sequencing (scRNA-seq) data (n = 52,698).

    Results:

    TIL numbers within tumor islands was an independent prognostic marker in patients with lung adenocarcinoma brain metastases. Comparative transcriptomics revealed that expression of three surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) was closely associated with TIL numbers. Their expression was not only prognostic in brain metastasis but also in primary lung adenocarcinoma. Correlation with scRNA-seq data revealed that brain metastases with high expression of surfactant genes might originate from tumor cells resembling alveolar type 2 cells. Methylome-based estimation of immune cell fractions in primary lung adenocarcinoma confirmed a positive association between lymphocyte infiltration and surfactant expression. Tumors with a high surfactant expression displayed a transcriptomic profile of an inflammatory microenvironment.

    Conclusions:

    The expression of surfactant metabolism-related genes (SFTPA1, SFTPB, and NAPSA) defines an inflamed subtype of lung adenocarcinoma brain metastases characterized by high abundance of TILs in close vicinity to tumor cells, a prolonged survival, and a tumor microenvironment which might be more accessible to immunotherapeutic approaches.




    meta

    Proteomic Analysis of CSF from Patients with Leptomeningeal Melanoma Metastases Identifies Signatures Associated with Disease Progression and Therapeutic Resistance

    Purpose:

    The development of leptomeningeal melanoma metastases (LMM) is a rare and devastating complication of the late-stage disease, for which no effective treatments exist. Here, we performed a multi-omics analysis of the cerebrospinal fluid (CSF) from patients with LMM to determine how the leptomeningeal microenvironment shapes the biology and therapeutic responses of melanoma cells.

    Experimental Design:

    A total of 45 serial CSF samples were collected from 16 patients, 8 of these with confirmed LMM. Of those with LMM, 7 had poor survival (<4 months) and one was an extraordinary responder (still alive with survival >35 months). CSF samples were analyzed by mass spectrometry and incubated with melanoma cells that were subjected to RNA sequencing (RNA-seq) analysis. Functional assays were performed to validate the pathways identified.

    Results:

    Mass spectrometry analyses showed the CSF of most patients with LMM to be enriched for pathways involved in innate immunity, protease-mediated damage, and IGF-related signaling. All of these were anticorrelated in the extraordinary responder. RNA-seq analysis showed CSF to induce PI3K/AKT, integrin, B-cell activation, S-phase entry, TNFR2, TGFβ, and oxidative stress responses in the melanoma cells. ELISA assays confirmed that TGFβ expression increased in the CSF of patients progressing with LMM. CSF from poorly responding patients conferred tolerance to BRAF inhibitor therapy in apoptosis assays.

    Conclusions:

    These analyses identified proteomic/transcriptional signatures in the CSF of patients who succumbed to LMM. We further showed that the CSF from patients with LMM has the potential to modulate BRAF inhibitor responses and may contribute to drug resistance.

    See related commentary by Glitza Oliva and Tawbi, p. 2083




    meta

    Plasma Thymidine Kinase Activity as a Biomarker in Patients with Luminal Metastatic Breast Cancer Treated with Palbociclib within the TREnd Trial

    Purpose:

    Thymidine kinase 1 (TK1) is downstream to the CDK4/6 pathway, and TK activity (TKa) measured in blood is a dynamic marker of outcome in patients with advanced breast cancer (ABC). This study explores TK1 as a biomarker of palbociclib response, both in vitro and in patients with ABC.

    Experimental Design:

    Modulation of TK1 levels and activity by palbociclib were studied in seven estrogen receptor–positive breast cancer cell lines: sensitive (PDS) and with palbociclib acquired resistance (PDR). TKa was assayed in plasma obtained at baseline (T0), after one cycle (T1), and at disease progression on palbociclib (T2) in patients enrolled in the "To Reverse ENDocrine Resistance" (TREnd) trial (n = 46).

    Results:

    Among E2F-dependent genes, TK1 was significantly downregulated after short-term palbociclib. Early TKa reduction by palbociclib occurred in PDS but not in PDR cells. In patients, median TKa (mTKa) at T0 was 75 DiviTum units per liter (Du/L), with baseline TKa not proving prognostic. At T1, mTKa decreased to 35 Du/L, with a minority of patients (n = 8) showing an increase—correlating with a worse outcome than those with decreased/stable TKa (n = 33; mPFS 3.0 vs 9.0 months; P = 0.002). At T2, mTKa was 251 Du/L; patients with TKa above the median had worse outcomes on post-study treatment compared with those with lower TKa (2.9 vs 8.7 months; P = 0.05).

    Conclusions:

    TK is a dynamic marker of resistance to palbociclib which may lead to early identification of patients in whom treatment escalation may be feasible. In addition, TKa may stratify prognosis in patients with acquired resistance to palbociclib.




    meta

    TBCRC 032 IB/II Multicenter Study: Molecular Insights to AR Antagonist and PI3K Inhibitor Efficacy in Patients with AR+ Metastatic Triple-Negative Breast Cancer

    Purpose:

    Preclinical data demonstrating androgen receptor (AR)–positive (AR+) triple-negative breast cancer (TNBC) cells are sensitive to AR antagonists, and PI3K inhibition catalyzed an investigator-initiated, multi-institutional phase Ib/II study TBCRC032. The trial investigated the safety and efficacy of the AR-antagonist enzalutamide alone or in combination with the PI3K inhibitor taselisib in patients with metastatic AR+ (≥10%) breast cancer.

    Patients and Methods:

    Phase Ib patients [estrogen receptor positive (ER+) or TNBC] with AR+ breast cancer received 160 mg enzalutamide in combination with taselisib to determine dose-limiting toxicities and the maximum tolerated dose (MTD). Phase II TNBC patients were randomized to receive either enzalutamide alone or in combination with 4 mg taselisib until disease progression. Primary endpoint was clinical benefit rate (CBR) at 16 weeks.

    Results:

    The combination was tolerated, and the MTD was not reached. The adverse events were hyperglycemia and skin rash. Overall, CBR for evaluable patients receiving the combination was 35.7%, and median progression-free survival (PFS) was 3.4 months. Luminal AR (LAR) TNBC subtype patients trended toward better response compared with non-LAR (75.0% vs. 12.5%, P = 0.06), and increased PFS (4.6 vs. 2.0 months, P = 0.082). Genomic analyses revealed subtype-specific treatment response, and novel FGFR2 fusions and AR splice variants.

    Conclusions:

    The combination of enzalutamide and taselisib increased CBR in TNBC patients with AR+ tumors. Correlative analyses suggest AR protein expression alone is insufficient for identifying patients with AR-dependent tumors and knowledge of tumor LAR subtype and AR splice variants may identify patients more or less likely to benefit from AR antagonists.




    meta

    Prospective Evaluation of Bone Metabolic Markers as Surrogate Markers of Response to Radium-223 Therapy in Metastatic Castration-resistant Prostate Cancer

    Purpose:

    Radium-223 is approved for metastatic castration-resistant prostate cancer (mCRPC) based on improved overall survival, and delay in skeletal related events. However, it is not associated with PSA or radiographic response, which poses a challenge in real-time assessment of its efficacy. Surrogate markers of treatment outcomes may facilitate tailoring treatment duration with radium-223, by limiting the duration of therapy with radium-223 in these patients. Here, we sought to investigate the utility of bone metabolic markers (BMMs) as surrogate markers of response to radium-223 in mCRPC.

    Patients and Methods:

    A prospective phase II trial of radium-223 plus enzalutamide (RE) versus enzalutamide alone was designed to assess surrogacy of BMMs with respect to response to radium-223. Enzalutamide was used as a comparator in lieu of placebo due to the progressive disease. Co-primary endpoints were relative change in serum BMM N-telopeptide (NTP) levels from baseline to 6 months between the two arms and safety and feasibility of the combination.

    Results:

    Thirty-nine men were randomized to RE (n = 27) or enzalutamide (n = 12). Combination was safe and feasible. Primary endpoint was met. A statistically significant relative change to NTP ratios between arms (0.64, 95% confidence interval, 0.51–0.81; P = 0.00048) favored RE versus enzalutamide. Overall, BMMs decreased with the RE therapy compared with enzalutamide. Improved PSA response rate in RE versus enzalutamide (P = 0.024), correlated with decline in BMMs.

    Conclusions:

    BMMs declined significantly with combination therapy, and were associated with improved outcomes. Upon external validation, BMMs may emerge as surrogate markers to monitor treatment with radium-223 in real-time.




    meta

    Targeting PD-1 or PD-L1 in Metastatic Kidney Cancer: Combination Therapy in the First-Line Setting

    Recent FDA approvals of regimens targeting programmed death 1 (PD-1) in combination with anti-CTLA-4 or with VEGF tyrosine kinase inhibitors are reshaping front-line therapy for metastatic kidney cancer. In parallel, therapeutics specific for programmed death ligand 1 (PD-L1), one of the two major ligands for PD-1, are under continued investigation. Surprisingly, not all PD-1 and PD-L1 agents lead to similar clinical outcomes, potentially due to biological differences in the cellular expression and regulation of these targets. Here, we review current clinical data on combination immune checkpoint inhibitor therapy in metastatic kidney cancer and discuss the relevant biology of PD-1 and PD-L1. The design of future rational combination therapy trials in metastatic renal cell carcinoma will rely upon an understanding of this biology, along with an evolving understanding of immune cell populations and their functional states in the tumor microenvironment.




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    An Individual Participant Data Population Pharmacokinetic Meta-analysis of Drug-Drug Interactions between Lumefantrine and Commonly Used Antiretroviral Treatment [Clinical Therapeutics]

    Treating malaria in HIV-coinfected individuals should consider potential drug-drug interactions. Artemether-lumefantrine is the most widely recommended treatment for uncomplicated malaria globally. Lumefantrine is metabolized by CYP3A4, an enzyme that commonly used antiretrovirals often induce or inhibit. A population pharmacokinetic meta-analysis was conducted using individual participant data from 10 studies with 6,100 lumefantrine concentrations from 793 nonpregnant adult participants (41% HIV-malaria-coinfected, 36% malaria-infected, 20% HIV-infected, and 3% healthy volunteers). Lumefantrine exposure increased 3.4-fold with coadministration of lopinavir-ritonavir-based antiretroviral therapy (ART), while it decreased by 47% with efavirenz-based ART and by 59% in the patients with rifampin-based antituberculosis treatment. Nevirapine- or dolutegravir-based ART and malaria or HIV infection were not associated with significant effects. Monte Carlo simulations showed that those on concomitant efavirenz or rifampin have 49% and 80% probability of day 7 concentrations <200 ng/ml, respectively, a threshold associated with an increased risk of treatment failure. The risk of achieving subtherapeutic concentrations increases with larger body weight. An extended 5-day and 6-day artemether-lumefantrine regimen is predicted to overcome these drug-drug interactions with efavirenz and rifampin, respectively.




    meta

    Distinct Mechanisms of Dissemination of NDM-1 Metallo-{beta}-Lactamase in Acinetobacter Species in Argentina [Epidemiology and Surveillance]

    A 4-year surveillance of carbapenem-resistant Acinetobacter spp. isolates in Argentina identified 40 strains carrying blaNDM-1. Genome sequencing revealed that most were Acinetobacter baumannii, whereas seven represented other Acinetobacter spp. The A. baumannii genomes were closely related, suggesting recent spread. blaNDM-1 was located in the chromosome of A. baumannii strains and on a plasmid in non-A. baumannii strains. A resistance gene island carrying blaPER-7 and other resistance determinants was found on a plasmid in some A. baumannii strains.




    meta

    Accumulation of Major Linezolid Metabolites in Patients with Renal Impairment [Pharmacology]

    In patients with renal impairment (n = 22 of 39), the median serum concentrations of linezolid, PNU-142300, and PNU-142586 were 1.6-, 3.3-, 2.8-fold higher, respectively, than in patients without renal impairment. Metabolite concentrations in paired samples were poorly correlated with linezolid concentrations (r2 = 0.26 for PNU-142300 and 0.06 for PNU-142586). Linezolid and its metabolites share potential toxicophores that deserve characterization to mitigate higher myelosuppression risk in patients with renal impairment.




    meta

    Somatic Copy-Number Alterations Contribute to Brain Metastasis [Metastasis]

    In lung cancer, brain metastasis was associated with somatic amplification of MYC, YAP1, or MMP13.




    meta

    Epigenetic Therapy Can Suppress Premetastatic Changes in the Lung [Metastasis]

    Low-dose adjuvant epigenetic therapy (AET) reduced metastasis and promoted survival in mouse models.




    meta

    Non-Stem Cells Seed Colorectal Cancer Metastases and Gain Stem Traits [Metastasis]

    LGR5 cells seed colorectal cancer metastases and produce stemlike LGR5+ outgrowth-promoting cells.




    meta

    Comparative single-cell RNA sequencing (scRNA-seq) reveals liver metastasis-specific targets in a patient with small intestinal neuroendocrine cancer [RESEARCH REPORT]

    Genomic analysis of a patient's tumor is the cornerstone of precision oncology, but it does not address whether metastases should be treated differently. Here we tested whether comparative single-cell RNA sequencing (scRNA-seq) of a primary small intestinal neuroendocrine tumor to a matched liver metastasis could guide the treatment of a patient's metastatic disease. Following surgery, the patient was put on maintenance treatment with a somatostatin analog. However, the scRNA-seq analysis revealed that the neuroendocrine epithelial cells in the liver metastasis were less differentiated and expressed relatively little SSTR2, the predominant somatostatin receptor. There were also differences in the tumor microenvironments. RNA expression of vascular endothelial growth factors was higher in the primary tumor cells, reflected by an increased number of endothelial cells. Interestingly, vascular expression of the major VEGF receptors was considerably higher in the liver metastasis, indicating that the metastatic vasculature may be primed for expansion and susceptible to treatment with angiogenesis inhibitors. The patient eventually progressed on Sandostatin, and although consideration was given to adding an angiogenesis inhibitor to her regimen, her disease progression involved non-liver metastases that had not been characterized. Although in this specific case comparative scRNA-seq did not alter treatment, its potential to help guide therapy of metastatic disease was clearly demonstrated.




    meta

    BRAF V600E-mutated metastatic pediatric Wilms tumor with complete response to targeted RAF/MEK inhibition [RESEARCH REPORT]

    Wilms tumor (WT) is the most common renal malignancy of childhood and accounts for 6% of all childhood malignancies. With current therapies, the 5-yr overall survival (OS) for children with unilateral favorable histology WT is greater than 85%. The prognosis is worse, however, for the roughly 15% of patients who relapse, with only 50%–80% OS reported in those with recurrence. Herein, we describe the extended and detailed clinical course of a rare case of a child with recurrent, pulmonary metastatic, favorable histology WT harboring a BRAF V600E mutation. The BRAF V600E mutation, commonly found in melanoma and other cancers, and previously undescribed in WT, has recently been reported by our group in a subset of epithelial-predominant WT. This patient, who was included in that series, presented with unilateral, stage 1, favorable histology WT and was treated with standard chemotherapy. Following the completion of therapy, the patient relapsed with pulmonary metastatic disease, that then again recurred despite an initial response to salvage chemotherapy and radiation. Next-generation sequencing (NGS) on the metastatic pulmonary nodule revealed a BRAF V600E mutation. After weighing the therapeutic options, a novel approach with dual BRAF/MEK inhibitor combination therapy was initiated. Complete radiographic response was observed following 4 months of therapy with dabrafenib and trametinib. At 12 months following the start of BRAF/MEK combination treatment, the patient continues with a complete response and has experienced minimal treatment-related side effects. This represents the first case, to our knowledge, of effective treatment with BRAF/MEK molecularly targeted therapy in a pediatric Wilms tumor patient.




    meta

    Objectively-Measured Light-Intensity Physical Activity and Risk of Cancer Mortality: A Meta-analysis of Prospective Cohort Studies

    Background:

    The impact of light-intensity physical activity (LPA) in preventing cancer mortality has been questioned. To address this concern, the present meta-analysis aimed to quantify the association between objectively-measured LPA and risk of cancer mortality.

    Methods:

    We conducted a systematic literature search in PubMed and Scopus to January 2020. Prospective cohort studies reporting the association between objectively-measured LPA using activity monitors (e.g., accelerometers) and risk of cancer mortality in the general population were included. The summary hazard ratios (HR) per 30 min/day of LPA and 95% confidence intervals (CI) were obtained using a random-effects model. Dose–response analysis was used to plot their relationship.

    Results:

    Five prospective cohort studies were included, in which the definition of LPA based on accelerometer readings was mainly set within 100 to 2,100 counts/min. The summary HR for cancer mortality per 30 min/day of LPA was 0.86 (95% CI, 0.79–0.95; I2 < 1%), and the association between LPA and risk reduction in cancer mortality was linearly shaped (Pnonlinearity = 0.72). LPA exhibited a comparable magnitude of risk reduction in cancer mortality of moderate-to-vigorous physical activity regardless of equal time-length (0.87 per 30 min/day vs. 0.94 per 30 min/day, Pinteraction = 0.46) or equal amount (0.74 vs. 0.94 per 150 metabolic equivalents-min/day, Pinteraction = 0.11). Furthermore, replacing sedentary time by LPA of 30 min/day decreased the risk of cancer mortality by 9%.

    Conclusions:

    Objectively-measured LPA conferred benefits in decreasing the risk of cancer mortality.

    Impact:

    LPA should be considered in physical activity guidelines to decrease the risk of cancer mortality.




    meta

    Red and Processed Meat, Poultry, Fish, and Egg Intakes and Cause-Specific and All-Cause Mortality among Men with Nonmetastatic Prostate Cancer in a U.S. Cohort

    Background:

    Research on the relationship of meat, fish, and egg consumption and mortality among prostate cancer survivors is limited.

    Methods:

    In the Cancer Prevention Study-II Nutrition Cohort, men diagnosed with nonmetastatic prostate cancer between baseline in 1992/1993 and 2015 were followed for mortality until 2016. Analyses of pre- and postdiagnosis intakes of red and processed meat, poultry, fish, and eggs included 9,286 and 4,882 survivors, respectively. Multivariable-adjusted RRs and 95% confidence intervals (CI) were estimated using Cox proportional hazards models.

    Results:

    A total of 4,682 and 2,768 deaths occurred during follow-up in pre- and postdiagnosis analyses, respectively. Both pre- and postdiagnosis intakes of total red and processed meat were positively associated with all-cause mortality (quartile 4 vs. 1: RR = 1.13; 95% CI, 1.03–1.25; Ptrend = 0.02; RR = 1.22; 95% CI, 1.07–1.39; Ptrend = 0.03, respectively), and both pre- and postdiagnosis poultry intakes were inversely associated with all-cause mortality (quartile 4 vs. 1 RR = 0.90; 95% CI, 0.82–0.98; Ptrend = 0.04; RR = 0.84; 95% CI, 0.75–0.95; Ptrend = 0.01, respectively). No associations were seen for prostate cancer–specific mortality, except that higher postdiagnosis unprocessed red meat intake was associated with lower risk.

    Conclusions:

    Higher red and processed meat, and lower poultry, intakes either before or after prostate cancer diagnosis were associated with higher risk of all-cause mortality.

    Impact:

    Our findings provide additional evidence that prostate cancer survivors should follow the nutrition guidelines limiting red and processed meat consumption to improve overall survival. Additional research on the relationship of specific meat types and mortality is needed.




    meta

    Prospective Association of Energy Balance Scores Based on Metabolic Biomarkers with Colorectal Cancer Risk

    Background:

    Energy balance–related factors, such as body mass index (BMI), diet, and physical activity, may influence colorectal cancer etiology through interconnected metabolic pathways, but their combined influence is less clear.

    Methods:

    We used reduced rank regression to derive three energy balance scores that associate lifestyle factors with combinations of prediagnostic, circulating levels of high-sensitivity C-reactive protein (hsCRP), C-peptide, and hemoglobin A1c (HbA1c) among 2,498 participants in the Cancer Prevention Study-II Nutrition Cohort. Among 114,989 participants, we verified 2,228 colorectal cancer cases. We assessed associations of each score with colorectal cancer incidence and by tumor molecular phenotypes using Cox proportional hazards regression.

    Results:

    The derived scores comprised BMI, physical activity, screen time, and 14 food groups, and explained 5.1% to 10.5% of the variation in biomarkers. The HR and 95% confidence interval (CI) for quartile 4 versus 1 of the HbA1c+C peptide–based score and colorectal cancer was 1.30 (1.15–1.47), the hsCRP-based score was 1.35 (1.19–1.53), and the hsCRP, C-peptide, and HbA1c-based score was 1.35 (1.19–1.52). The latter score was associated with non-CIMP tumors (HRQ4vsQ1: 1.59; 95% CI: 1.17–2.16), but not CIMP-positive tumors (Pheterogeneity = 0.04).

    Conclusions:

    These results further support hypotheses that systemic biomarkers of metabolic health—inflammation and abnormal glucose homeostasis—mediate part of the relationship between several energy balance–related modifiable factors and colorectal cancer risk.

    Impact:

    Results support cancer prevention guidelines for maintaining a healthful body weight, consuming a healthful diet, and being physically active. More research is needed on these clusters of exposures with molecular phenotypes of tumors.




    meta

    Projected Reductions in Absolute Cancer-Related Deaths from Diagnosing Cancers Before Metastasis, 2006-2015

    Background:

    New technologies are being developed for early detection of multiple types of cancer simultaneously. To quantify the potential benefit, we estimated reductions in absolute cancer–related deaths that could occur if cancers diagnosed after metastasis (stage IV) were instead diagnosed at earlier stages.

    Methods:

    We obtained stage-specific incidence and survival data from the Surveillance, Epidemiology, and End Results Program for 17 cancer types for all persons diagnosed ages 50 to 79 years in 18 geographic regions between 2006 and 2015. For a hypothetical cohort of 100,000 persons, we estimated cancer-related deaths under assumptions that cancers diagnosed at stage IV were diagnosed at earlier stages.

    Results:

    Stage IV cancers represented 18% of all estimated diagnoses but 48% of all estimated cancer-related deaths within 5 years. Assuming all stage IV cancers were diagnosed at stage III, 51 fewer cancer-related deaths would be expected per 100,000, a reduction of 15% of all cancer-related deaths. Assuming one third of metastatic cancers were diagnosed at stage III, one third diagnosed at stage II, and one third diagnosed at stage I, 81 fewer cancer-related deaths would be expected per 100,000, a reduction of 24% of all cancer-related deaths, corresponding to a reduction in all-cause mortality comparable in magnitude to eliminating deaths due to cerebrovascular disease.

    Conclusions:

    Detection of multiple cancer types earlier than stage IV could reduce at least 15% of cancer-related deaths within 5 years, affecting not only cancer-specific but all-cause mortality.

    Impact:

    Detecting cancer before stage IV, including modest shifts to stage III, could offer substantial population benefit.