Machine Learning Approach to Metabolomic Data Predicts Type 2 Diabetes Mellitus Incidence.

Metabolomics, with its wealth of data, offers a valuable avenue for enhancing predictions and decision-making in diabetes. This observational study aimed to leverage machine learning (ML) algorithms to predict the 4-year risk of developing type 2 diabetes mellitus (T2DM) using targeted quantitative metabolomics data. A cohort of 279 cardiovascular risk patients who underwent coronary angiography and who were initially free of T2DM according to American Diabetes Association (ADA) criteria was analyzed at baseline, including anthropometric data and targeted metabolomics, using liquid chromatography (LC)-mass spectroscopy (MS) and flow injection analysis (FIA)-MS, respectively. All patients were followed for four years. During this time, 11.5% of the patients developed T2DM. After data preprocessing, 362 variables were used for ML, employing the Caret package in R. The dataset was divided into training and test sets (75:25 ratio) and we used an oversampling approach to address the classifier imbalance of T2DM incidence. After an additional recursive feature elimination step, identifying a set of 77 variables that were the most valuable for model generation, a Support Vector Machine (SVM) model with a linear kernel demonstrated the most promising predictive capabilities, exhibiting an F1 score of 50%, a specificity of 93%, and balanced and unbalanced accuracies of 72% and 88%, respectively. The top-ranked features were bile acids, ceramides, amino acids, and hexoses, whereas anthropometric features such as age, sex, waist circumference, or body mass index had no contribution. In conclusion, ML analysis of metabolomics data is a promising tool for identifying individuals at risk of developing T2DM and opens avenues for personalized and early intervention strategies.

Plasma apolipoprotein M predicts overall survival in metastatic breast cancer patients.

PURPOSE: Apolipoprotein M (APOM) is a plasma apolipoprotein closely involved with lipid metabolism and inflammation. In vitro studies suggest that APOM may also have a tumor-suppressive role in breast cancer. In the present study, we aimed to evaluate the impact of plasma APOM levels on the prognosis of breast cancer patients. METHODS: We measured APOM levels using an enzyme-linked immunosorbent assay in 75 patients with ER-positive/HER2-negative metastatic breast cancer. The endpoint was overall survival (OS) at 24 months. RESULTS: During the 24-month follow-up period, 34.7% of the patients died. Baseline APOM levels were significantly reduced in patients who deceased during follow-up compared to survivors (42.7 ± 14.5 µg/mL versus 52.2 ± 13.8 µg/mL; P = 0.003). Cox regression analysis showed a hazard ratio of 0.30 [95% confidence interval 0.15-0.61]; P < 0.001 per doubling of APOM levels. Correction for age, C-reactive protein, menopausal state, histology of the primary tumor, metastatic site, number of metastases, endocrine resistance, scheduled therapy line, and kind of scheduled therapy indicated that circulating APOM predicted OS independently of these parameters (HRper doubling = 0.23 [0.09-0.56; P = 0.001). CONCLUSIONS: Our study suggests that circulating APOM is significantly linked with reduced mortality in metastatic breast cancer patients.

Myricetin attenuates hypoxia-induced inflammation in human adipocytes.

BACKGROUND: Adipose tissue hypoxia plays a crucial role in the development of chronic low-grade systemic inflammation which has been associated with the pathogenesis of obesity-related diseases. Myricetin is a natural compound present in numerous plant-based foods with presumed anti-inflammatory and beneficial health effects. The impact of this flavonoid on hypoxia-induced expression of inflammatory adipokines and hypoxia-regulated pathways is unknown so far and has been addressed in the present study. METHODS: Differentiated human Simpson-Golabi-Behmel syndrome (SGBS) adipocytes were cultured with or without myricetin under normoxic and hypoxic conditions for varying time periods. The effect of hypoxia and myricetin on the expression of the investigated adipokines was measured by real-time RT-PCR. Western blot analysis was used for the detection of transcription factors involved in hypoxia-regulated pathways. RESULTS: Myricetin interfered in the hypoxia-induced regulation of adipokines and the underlying pathways, which are involved in transmitting the inflammatory response. It strongly repressed hypoxia-induced expression of apelin, leptin, chemerin, asprosin, and DPP-4 and HIF-1α accumulation in the nucleus was diminished. Furthermore, the activation of the key regulators in the inflammatory response NF-κB, Akt, and CREB was suppressed by myricetin under hypoxic conditions. Myricetin also decreased hypoxia-induced accumulation of the pro-tumorigenic transcription factors Snail and Slug in the nucleus. CONCLUSION: Taken together, our results indicated that myricetin regulated hypoxia-induced expression of adipokines and hypoxia-regulated pathways in human adipocytes. Our study therefore provided evidence of the anti-inflammatory effects of myricetin in hypoxia-treated human adipocytes.

Volatilomic Signatures of AGS and SNU-1 Gastric Cancer Cell Lines.

In vitro studies can help reveal the biochemical pathways underlying the origin of volatile indicators of numerous diseases. The key objective of this study is to identify the potential biomarkers of gastric cancer. For this purpose, the volatilomic signatures of two human gastric cancer cell lines, AGS (human gastric adenocarcinoma) and SNU-1 (human gastric carcinoma), and one normal gastric mucosa cell line (GES-1) were investigated. More specifically, gas chromatography mass spectrometry has been applied to pinpoint changes in cell metabolism triggered by cancer. In total, ten volatiles were found to be metabolized, and thirty-five were produced by cells under study. The volatiles consumed were mainly six aldehydes and two heterocyclics, whereas the volatiles released embraced twelve ketones, eight alcohols, six hydrocarbons, three esters, three ethers, and three aromatic compounds. The SNU-1 cell line was found to have significantly altered metabolism in comparison to normal GES-1 cells. This was manifested by the decreased production of alcohols and ketones and the upregulated emission of esters. The AGS cells exhibited the increased production of methyl ketones containing an odd number of carbons, namely 2-tridecanone, 2-pentadecanone, and 2-heptadecanone. This study provides evidence that the cancer state modifies the volatilome of human cells.

Evaluation of the associations between circulating microRNAs and kidney function in coronary angiography patients.

Circulating microRNAs (miRNAs) have been linked to chronic kidney disease. Little is known about the association between circulating miRNAs and kidney function in patients at high cardiovascular risk. We therefore investigated the association between a panel of candidate miRNAs and kidney function, based on estimated glomerular filtration rate (eGFR), in two independent cohorts of patients undergoing coronary angiography. The present study totally included 438 patients undergoing coronary angiography, who were divided into a discovery cohort (n = 120) and a validation cohort (n = 318). A candidate miRNA panel comprising 50 renal miRNAs was selected from the literature, and expression levels of circulating miRNAs were determined by real-time PCR. Out of the initially tested candidate miRNAs, 38 miRNAs were sufficiently detectable in plasma. Their association with kidney function was evaluated in the discovery cohort. Associations of seven of these miRNAs with eGFR were significant after multiple testing correction via false discovery rate estimation. To verify obtained results, miRNAs with significant false discovery rates were further analyzed in the validation cohort. miR-106b-5p, miR-16-5p, miR-19b-3p, miR-20a-5p, miR-25-3p, and miR-451a proved to be significantly associated with eGFR also in the validation cohort (all P < 0.001). Association between the identified renal miRNAs and kidney function was confirmed by analysis of covariance adjusting for age, sex, type 2 diabetes, hypertension, and albumin-to-creatinine ratio. In conclusion, our study showed that miR-16-5p, miR-19b-3p, miR-20a-5p, miR-25-3p, miR-106b-5p, and miR-451a are significantly linked to kidney function in patients undergoing coronary angiography.

VicTORia: a randomised phase II study to compare vinorelbine in combination with the mTOR inhibitor everolimus versus vinorelbine monotherapy for second-line chemotherapy in advanced HER2-negative breast cancer.

PURPOSE: Improving the outcome of patients with HER2-negative metastatic breast cancer experiencing tumour progression following first-line chemotherapy remains an urgent medical need. The purpose of the VicTORia trial was to show superiority of everolimus in combination with vinorelbine versus vinorelbine monotherapy as second-line chemotherapy for patients with advanced HER2 negative breast cancer. METHODS: In this randomised phase II trial, 133 patients were recruited in 32 centres in Germany. Patients were randomised 1:1 to second-line chemotherapy either with vinorelbine plus everolimus (arm1) or vinorelbine alone (arm2). Primary endpoint was progression-free survival (PFS). Secondary endpoints were PFS rate at 6 months, overall survival (OS), overall response rate (ORR) and safety. Baseline PI3 K mutational status was determined in plasma samples. RESULTS: Median progression-free survival was not different between arms (arm1 vs. arm2: 4.01 months, 95% CI 2.40-6.09 vs. 4.08, 95% CI 2.80-5.33). PFS rate at 6 months (arm1 vs. arm2: 39.4%, 95% CI 27.6-50.9% vs. 36.6%, 95% CI 24.6-48.6%), median OS (arm1 vs. arm2: 16.3 months, 95% CI 11.4-19.0 vs. 13.8 months, 95% CI 10.2-19.1) and ORR were not different between arms. Most frequent grade 3/4 adverse events were neutropenia (50% vs. 40%), gastrointestinal toxicities (19.1% vs. 6.1%), and infections (19.1% vs. 7.7%). PI3 K mutational status was neither associated with PFS nor with OS. CONCLUSION: Although well tolerated, the efficacy of everolimus and vinorelbine combination therapy was not superior to vinorelbine monotherapy. There was no correlation between PI3 K mutational status and efficacy. EudracCT No 2011-001024-38, ClinicalTrials.gov No NCT01520103.

Real-time PCR based detection of the lactase non-persistence associated genetic variant LCT-13910C>T directly from whole blood.

Primary hypolactasia is the main cause of lactose intolerance in adults. It is strongly associated with the single genetic variant LCT-13910C>T, located upstream of the lactase encoding gene. Consequently, analysis of LCT-13910C>T has been recommended as a direct genetic test for the trait. The aim of our study was to develop a TaqMan probe based real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood, circumventing DNA isolation. The LCT-13910C>T variant was determined using the DirectBlood Genotyping PCR Kit (myPOLS Biotec, Konstanz, Germany) together with an in-house TaqMan primer-probe assay. Validity and specificity of the assay was evaluated using EDTA anti-coagulated whole blood samples and corresponding DNA samples. Results from real-time PCR were compared with results obtained by Sanger sequencing from 105 blinded whole blood samples. Validity and specificity of the assay using whole blood were comparable to those using purified genomic DNA as substrate in PCR. Genetic analysis of blood samples were in complete agreement with results obtained by Sanger sequencing. In conclusion, we present a reliable real-time PCR protocol for the detection of the LCT-13910C>T variant directly from whole blood further facilitating diagnosis of primary hypolactasia in symptomatic patients.

Occurrence of the JAK2 V617F mutation in patients with peripheral arterial disease.

The acquired JAK2 V617F mutation is common in patients with myeloproliferative neoplasms. We previously showed that JAK2 V617F is also found in coronary patients, most of them affected by coronary atherosclerosis. Peripheral arterial disease (PAD) is another important manifestation of atherosclerosis. However, prevalence of the JAK2 V617F mutation and its effect on clinical or hematologic characteristics is unknown in PAD patients. In the present study we determined the prevalence of JAK2 V617F in a cohort of 287 patients with sonographically proven PAD and compared mutation frequency with mutational status of 997 healthy people from the KORA F4 study. JAK2 V617F screening and quantification of allele burden in both cohorts was performed with same allele-specific quantitative real-time PCR method. From a total of 287 PAD patients, 9 individuals were tested positive for the JAK2 V617F mutation. One patient showed elevated hemoglobin values, indicating polycythemia vera. Observed JAK2 V617F frequency (3.1%) in PAD patients showed a 5-fold, highly significant increase compared with healthy people (P < 0.001). Furthermore, occurrence of the mutation in PAD patients was significantly decreased in patients using aspirin (P = 0.003). We conclude that the prevalence of JAK2 V617F mutation is significantly increased in PAD patients compared to the general population. Future studies are warranted to confirm our observations and to define the underlying mechanisms behind our findings.

Evaluation of the prevalence and prospective clinical impact of the JAK2 V617F mutation in coronary patients.

The JAK2 V617F mutation is not only found in the majority of patients with myeloproliferative neoplasms (MPN), including essential thrombocythemia (ET), but also has been reported in individuals without overt MPN. A close relation of the JAK2 V617F mutation to atherothrombotic events has been described, at least in patients with MPN. The prevalence of the JAK2 V617F mutation and its clinical impact in coronary patients is unknown. To address this issue, DNA samples from 1,589 subjects undergoing coronary angiography with up to 11 years of follow up were genotyped using allele-specific real-time PCR assays. Prevalence of the JAK2 V617F mutation was 1.32% (n = 21) in coronary patients. Two JAK2 V617F positive patients showed baseline platelet counts indicative for ET and a third patient developed ET during follow up, finally resulting in a percentage of 0.188% of ET cases. This corresponds to an up to fivefold accumulation of ET cases in coronary patients compared with the general population. Our study showed no impact of the JAK2 V617F mutation on future atherothrombotic events or overall survival (HR = 1.04 [0.33-3.27]; P = 0.949 and HR = 0.35 [0.05-2.46]; P = 0.288, respectively). Therefore, our data suggest that JAK2 V617F positive coronary patients are not at increased risk for future atherothrombotic complications. Routine mutation screening in coronary patients is, therefore, not warranted. However, number of ET cases appears to be accumulated in coronary patients. For this reason, we recommend JAK2 V617F testing only in coronary patients showing abnormal blood cell counts for further clarification.

Ceramides improve cardiovascular risk prediction beyond low-density lipoprotein cholesterol.

Aims: Low-density lipoprotein cholesterol (LDL-C) is the best documented cardiovascular risk predictor and at the same time serves as a target for lipid-lowering therapy. However, the power of LDL-C to predict risk is biased by advanced age, comorbidities, and medical treatment, all known to impact cholesterol levels. Consequently, such biased patient cohorts often feature a U-shaped or inverse association between LDL-C and cardiovascular or overall mortality. It is not clear whether these constraints for risk prediction may likewise apply to other lipid risk markers in particular to ceramides and phosphatidylcholines. Methods and results: In this observational cohort study, we recorded cardiovascular mortality in 1195 patients over a period of up to 16 years, comprising a total of 12 262 patient-years. The median age of patients at baseline was 67 years. All participants were either consecutively referred to elective coronary angiography or diagnosed with peripheral artery disease, indicating a high cardiovascular risk. At baseline, 51% of the patients were under statin therapy. We found a U-shaped association between LDL-C and cardiovascular mortality with a trough level of around 150 mg/dL of LDL-C. Cox regression analyses revealed that LDL-C and other cholesterol species failed to predict cardiovascular risk. In contrast, no U-shaped but linear association was found for ceramide- and phosphatidylcholine-containing markers and these markers were able to significantly predict the cardiovascular risk even after multivariate adjustment. Conclusion: We thus suggest that ceramides- and phosphatidylcholine-based predictors rather than LDL-C may be used for a more accurate cardiovascular risk prediction in high-risk patients.

The value of earlier-in-life systolic and diastolic blood pressure for cardiovascular risk prediction.

Blood pressure (BP) varies over a lifetime. This cardiovascular observation study (OS) compared the predictive value of earlier- and later-in-life blood pressure (BP) in 1,497 cardiovascular disease patients utilizing readings taken during a health survey (HS) and 15 years later from the same subjects at the baseline of this OS. Prediction of the cardiovascular risk during the OS follow-up (21 years) was significantly more effective if the earlier BP readings at HS were used instead of recent OS readings (NRI = 0.30, p < 0.001). For HS readings, each 10 mm Hg increase of systolic and diastolic BP was associated with a 17% and 20% higher risk, respectively. At OS, systolic BP lost significance and diastolic BP reversed its association. Noteworthy, different BP categorizations (European vs. US guidelines) yielded similar results. This study highlights the poor predictive power of BP readings in elderly cardiovascular disease patients but emphasizes the significant prognostic value of earlier-in-life BP.

Circulating Glypican-4 Is a Predictor of 24-Month Overall Survival in Metastatic Breast Cancer.

INTRODUCTION: Endocrine treatment combined with CDK4/6 inhibitors is the preferred treatment strategy in patients presenting with ER-positive/HER2-negative breast cancer, but the clinical course remains highly variable among individual patients. There is an unmet need for prognostic or predictive biomarkers in this important group of patients. Recently, we have identified circulating glypican-4 (GPC4) as a new biomarker of inferior outcomes in patients with metastatic colorectal cancer. The impact of plasma GPC4 levels on the survival of breast cancer patients is unknown and has been addressed in the present study. METHODS: Our study included 47 patients with ER-positive/HER2-negative metastatic breast cancer prior to treatment with CDK4/6 inhibitors combined with endocrine therapy. The endpoint was overall survival (OS) at 24 months. GPC4 levels were measured in plasma using an enzyme-linked immunosorbent assay. RESULTS: Increased circulating GPC4 levels were significantly linked to advanced age, postmenopausal state, visceral metastases, and invasive lobular carcinoma. During the 2-year observational follow-up period, 25.5% of patients died. The area under the receiver operating characteristic curve (ROC-AUC) analysis revealed an AUC of 0.713 [0.555-0.871]; p = 0.029 for OS; and an optimal cutoff value of GPC4 for predicting OS of 4.77 ng/mL. No patient showing GPC4 values below this cutoff died during the observational period. Cox regression analysis showed a hazard ratio of 2.14 [95% confidence interval: 1.24-3.67]; p = 0.006 for one standard deviation change of plasma GPC4. CONCLUSIONS: Our study suggests circulating GPC4 as a significant predictor of poor survival in metastatic breast cancer patients.

Coronary Event Risk Test (CERT) as a Risk Predictor for the 10-Year Clinical Outcome of Patients with Peripheral Artery Disease.

(1) Background: Ceramides are a new kind of lipid biomarker and have already been demonstrated to be valuable risk predictors in coronary patients. Patients with peripheral artery disease (PAD) are a population with a worse prognosis and higher mortality risk compared to coronary artery disease (CAD) patients. However, the value of ceramides for risk prediction in PAD patients is still vague, as addressed in the present study. (2)Methods: This observational study included 379 PAD patients. The primary endpoint was all-cause mortality at 10 years of follow-up. A set of ceramides was measured by LC-MS/MS and combined according to the Coronary Event Risk Test (CERT) score, which categorizes patients into one of four risk groups (low risk, moderate risk, high risk, very high risk). (3) Results: Kaplan-Meier survival curves revealed that the overall survival of patients decreased with the increasing risk predicted by the four CERT categories, advancing from low risk to very high risk. Cox regression analysis demonstrated that each one-category increase resulted in a 35% rise in overall mortality risk (HR = 1.35 [1.16-1.58]). Multivariable adjustment, including, among others, age, LDL-cholesterol, type 2 diabetes, and statin treatment before the baseline, did not abrogate this significant association (HR = 1.22 [1.04-1.43]). Moreover, we found that the beneficial effect of statin treatment is significantly stronger in patients with a higher risk, according to CERT. (4) Conclusions: We conclude that the ceramide-based risk score CERT is a strong predictor of the 10-year mortality risk in patients with PAD.

Circulating glypican-4 is a new predictor of all-cause mortality in patients with heart failure.

BACKGROUND: Heart failure confers a high burden of morbidity and mortality. However, risk prediction in heart failure patients still is limited. Blood-based biomarkers hold promise to improve clinical risk assessment. Recently we have identified circulating glypican-4 (GPC4) as a significant predictor of mortality in coronary angiography patients and patients with peripheral artery disease. The impact of serum GPC4 on mortality in patients with heart failure is unknown and is addressed in this prospective cohort study. METHODS: We prospectively recorded all-cause mortality in 288 patients with heart failure. GPC4 levels were measured using an enzyme-linked immunosorbent assay at baseline. RESULTS: During the 24-month follow-up period, 28.1% (n = 81) of the patients died. Serum GPC4 significantly predicted all-cause mortality (hazard ratio (HR) per doublingof GPC4 = 3.57 [2.31-5.53]; P < 0.001). Subgroup analysis showed that GPC4 was significantly associated with all-cause mortality in patients with reduced ejection fraction (HR per doubling = 3.25 [1.75-6.04]; P < 0.001) as well as in those with preserved ejection fraction (HR per doubling = 3.07 [1.22-7.70]; P = 0.017). The association between serum GPC4 and all-cause mortality remained significant in multivariable Cox regression analysis correcting for traditional risk factors (P = 0.035). Results from C-statistics indicated an additional prognostic value of GPC4 relative to NT-proBNP for the prediction of two-year all-cause mortality (P = 0.030). CONCLUSION: Circulating GPC4 independently predicts all-cause mortality in patients with heart failure.

Cell-free circulating RAS mutation concentrations significantly impact the survival of metastatic colorectal cancer patients.

PURPOSE: RAS mutations are predictors of an adverse outcome in EGFR-targeted therapies and have been proposed as prognostic biomarkers of survival in metastatic colorectal cancer (mCRC). The analysis of circulating tumor DNA from plasma samples, known as liquid biopsies, has indicated that the RAS mutation status may change over time, potentially affecting patients' prognosis. To further evaluate the clinical validity of RAS mutation retesting using liquid biopsies, we prospectively investigated the impact of the circulating quantitative RAS mutation status on the course of mCRC. METHODS: The present study included 81 consecutively recruited patients with mCRC. We used targeted next-generation sequencing of circulating cell-free DNA to determine and quantify plasma RAS mutation status. RESULTS: Patients with a RAS mutation detected by liquid biopsy (37%; n = 30) were at increased risk of death during the follow-up period compared to RAS wild-type patients. Patients with evidence of a RAS mutation in the primary tumor but a putative RAS mutation loss in plasma (28%; n = 11) showed a prolonged survival compared to patients with a preserved RAS mutation status. Also, circulating RAS mutation concentrations significantly affected the outcome: The mortality risk of patients with a high RAS mutation concentration increased fivefold compared to subjects with a putative RAS mutation loss or low RAS mutation concentration. CONCLUSION: Our results emphasize the clinical value of circulating RAS mutations in managing mCRC.

The optimal healthy ranges of thyroid function defined by the risk of cardiovascular disease and mortality: systematic review and individual participant data meta-analysis.

BACKGROUND: Reference intervals of thyroid-stimulating hormone (TSH) and free thyroxine (FT4) are statistically defined by the 2·5-97·5th percentiles, without accounting for potential risk of clinical outcomes. We aimed to define the optimal healthy ranges of TSH and FT4 based on the risk of cardiovascular disease and mortality. METHODS: This systematic review and individual participant data (IPD) meta-analysis identified eligible prospective cohorts through the Thyroid Studies Collaboration, supplemented with a systematic search via Embase, MEDLINE (Ovid), Web of science, the Cochrane Central Register of Controlled Trials, and Google Scholar from Jan 1, 2011, to Feb 12, 2017 with an updated search to Oct 13, 2022 (cohorts found in the second search were not included in the IPD). We included cohorts that collected TSH or FT4, and cardiovascular outcomes or mortality for adults (aged ≥18 years). We excluded cohorts that included solely pregnant women, individuals with overt thyroid diseases, and individuals with cardiovascular disease. We contacted the study investigators of eligible cohorts to provide IPD on demographics, TSH, FT4, thyroid peroxidase antibodies, history of cardiovascular disease and risk factors, medication use, cardiovascular disease events, cardiovascular disease mortality, and all-cause mortality. The primary outcome was a composite outcome including cardiovascular disease events (coronary heart disease, stroke, and heart failure) and all-cause mortality. Secondary outcomes were the separate assessment of cardiovascular disease events, all-cause mortality, and cardiovascular disease mortality. We performed one-step (cohort-stratified Cox models) and two-step (random-effects models) meta-analyses adjusting for age, sex, smoking, systolic blood pressure, diabetes, and total cholesterol. The study was registered with PROSPERO, CRD42017057576. FINDINGS: We identified 3935 studies, of which 53 cohorts fulfilled the inclusion criteria and 26 cohorts agreed to participate. We included IPD on 134 346 participants with a median age of 59 years (range 18-106) at baseline. There was a J-shaped association of FT4 with the composite outcome and secondary outcomes, with the 20th (median 13·5 pmol/L [IQR 11·2-13·9]) to 40th percentiles (median 14·8 pmol/L [12·3-15·0]) conveying the lowest risk. Compared with the 20-40th percentiles, the age-adjusted and sex-adjusted hazard ratio (HR) for FT4 in the 80-100th percentiles was 1·20 (95% CI 1·11-1·31) for the composite outcome, 1·34 (1·20-1·49) for all-cause mortality, 1·57 (1·31-1·89) for cardiovascular disease mortality, and 1·22 (1·11-1·33) for cardiovascular disease events. In individuals aged 70 years and older, the 10-year absolute risk of composite outcome increased over 5% for women with FT4 greater than the 85th percentile (median 17·6 pmol/L [IQR 15·0-18·3]), and men with FT4 greater than the 75th percentile (16·7 pmol/L [14·0-17·4]). Non-linear associations were identified for TSH, with the 60th (median 1·90 mIU/L [IQR 1·68-2·25]) to 80th percentiles (2·90 mIU/L [2·41-3·32]) associated with the lowest risk of cardiovascular disease and mortality. Compared with the 60-80th percentiles, the age-adjusted and sex-adjusted HR of TSH in the 0-20th percentiles was 1·07 (95% CI 1·02-1·12) for the composite outcome, 1·09 (1·05-1·14) for all-cause mortality, and 1·07 (0·99-1·16) for cardiovascular disease mortality. INTERPRETATION: There was a J-shaped association of FT4 with cardiovascular disease and mortality. Low concentrations of TSH were associated with a higher risk of all-cause mortality and cardiovascular disease mortality. The 20-40th percentiles of FT4 and the 60-80th percentiles of TSH could represent the optimal healthy ranges of thyroid function based on the risk of cardiovascular disease and mortality, with more than 5% increase of 10-year composite risk identified for FT4 greater than the 85th percentile in women and men older than 70 years. We propose a feasible approach to establish the optimal healthy ranges of thyroid function, allowing for better identification of individuals with a higher risk of thyroid-related outcomes. FUNDING: None.

A common variant of the MACC1 gene is significantly associated with overall survival in colorectal cancer patients.

BACKGROUND: The newly discovered metastasis-associated in colon cancer-1 (MACC1) gene is a key regulator of the HGF/MET pathway. Deregulation of HGF/MET signaling is reported as a prognostic marker for tumorigenesis, early stage invasion, and metastasis. High expression levels of MACC1 have been associated with colon cancer metastasis and reduced survival. Potential links between the genetic diversity of the MACC1 locus and overall survival are unknown. We therefore investigated the association between MACC1 tagging single nucleotide polymorphisms (SNPs) and overall survival in a large cohort of colorectal cancer patients. METHODS: The study included 318 subjects with histopathologically proven colorectal cancer at the Academic Teaching Hospital Feldkirch, Austria. Survival data were provided by the federal agency for statistics in Austria. Genomic DNA was isolated from formalin-fixed paraffin-embedded specimens; six tagging SNPs (rs1990172, rs3114446, rs10275612, rs3095007, rs3095009, and rs7780032), capturing most of the common variants of the MACC1 locus, were genotyped by SNaPshot assays. RESULTS: Over a mean follow up period of 5.3 (± 1.0) years, 94 deaths were recorded. Carriers of the G-allele of SNP rs1990172 showed a significantly decreased overall survival (additive HR = 1.38 [1.05-1.82]; p = 0.023). Multivariate analysis adjusted for age and UICC tumor stage confirmed this result (HR = 1.49 [1.12-1.98]; p = 0.007). Other investigated genetic variants of the MACC1 gene were not significantly associated with overall survival (p-values > 0.05). CONCLUSIONS: For the first time, our study investigated the influence of MACC1 tagging polymorphisms on overall survival suggesting SNP rs1990172 as a predictor for reduced overall survival in colorectal cancer patients. Further studies will be required to validate our findings.

Elevated levels of apolipoprotein D predict poor outcome in patients with suspected or established coronary artery disease.

BACKGROUND AND AIMS: Apolipoprotein D (apoD) is a lipocalin exerting neuroprotective effects. However, the relevance of apoD in respect to cardiovascular risk is largely unexplored. Therefore, this study aimed to evaluate the ability of apoD to predict future all-cause mortality, cardiovascular mortality, and cardiovascular events. METHODS: Serum apoD levels were measured in a cohort of 531 Caucasian individuals who underwent coronary angiography (356 males, 175 females; mean age 65 ± 10 years). Fatal and non-fatal events were recorded over a median follow-up period of 5.8 years. RESULTS: ApoD concentrations at baseline correlated significantly with age, presence of the metabolic syndrome, body mass index, lipoprotein levels, fasting glucose, and estimated glomerular filtration rate. Kaplan-Meier curve analyses by gender-stratified quartiles of apoD revealed that the cumulative incidence rates of mortality and cardiovascular events become higher with increasing apoD levels. The adjusted hazard ratios for participants in the highest quartile of apoD compared to those in the lowest quartile were 4.00 (95% confidence interval [CI] 1.49-10.74) for overall mortality, 5.47 (95% CI 1.20-25.00) for cardiovascular mortality, and 2.52 (95% CI 1.28-5.00) for cardiovascular events. CONCLUSIONS: High circulating levels of apoD are an indicator of poor prognosis in patients with suspected or established coronary artery disease.

Clonal Evolution in Patients with Hormone Receptor Positive, HER-2 Negative Breast Cancer Treated with Chemotherapy or CDK4/6 Inhibitors.

INTRODUCTION: Somatic evolution of the cancer genome resulting in genetically different subclones is thought to be involved in the development of treatment resistance but might also offer new therapeutic opportunities in metastatic breast cancer. No data are available if clonal evolution differs in patients treated with chemotherapy (chemo) or CDK4/6 inhibitors given with endocrine treatment (CE treatment). METHODS: We performed a prospective analysis of circulating tumor DNA (ctDNA) by targeted next-generation sequencing in 46 patients before the beginning of a systemic first-line (n = 37) or second-line (n = 9) treatment. Ct DNA was analyzed again upon disease progression. RESULTS: New mutations in ctDNA of patients with progressive disease were detected in 1/11 patients who started chemo, in 4/9 patients treated with chemo followed by CE maintenance treatment, and in 9/26 patients receiving CE therapy. The number of acquired new mutations did not differ significantly between the three therapy cohorts (all p values >0.05). However, in patients classified as secondary resistant (n = 37), occurrence of new mutations significantly differed between patients who started chemo (0/9) compared to patients treated with chemo followed by CE (4/11; p = 0.041) and patients receiving CE therapy (8/19; p = 0.024), respectively. CONCLUSION: Clonal evolution might differ significantly between metastatic breast cancer patients with hormone receptor positive and HER-2 negative disease treated with chemo or CDK4/6 inhibitors. These results should be confirmed in larger patient cohorts.

Serum glypican-4 is a marker of future vascular risk and mortality in coronary angiography patients.

BACKGROUND AND AIMS: Glypican-4 (GPC4) is a cell surface protein, but can be released into circulation under various clinical conditions. The association of circulating GPC4 with the risk of future cardiovascular events or death is unclear. In the present study, we aimed to investigate the association between serum GPC4 and major adverse cardiovascular events (MACE), vascular mortality, and all-cause mortality in a prospective cohort study. METHODS: Our study included 760 patients undergoing coronary angiography. During a mean follow up period of 6.3 years, the incidence of MACE, vascular mortality, and all-cause mortality was recorded. Serum GPC4 levels were determined using an enzyme-linked immunosorbent assay. RESULTS: Serum GPC4 was highly significantly associated with increased age, body mass index, brain natriuretic peptide, and oxidized low density lipoprotein, as well as with decreased estimated glomerular filtration rate. During the follow-up period, 145 patients died, including 67 vascular deaths. MACE occurred in 137 patients. Serum GPC4 was significantly associated with MACE, vascular mortality, and all-cause mortality independently of traditional cardiovascular risk factors, with adjusted hazard ratios (HR) and 95% confidence intervals for one standard deviation change of serum GPC4 of 1.32 [1.10-1.58], 1.38 [1.06-1.78], and 1.53 [1.29-1.82], respectively. The best cut-off value for serum GPC4 for predicting MACE, vascular mortality, and all-cause mortality was 7.24 ng/ml for all three endpoints. Respective adjusted HRs were 1.61 [1.07-2.43], 2.85 [1.62-5.01], and 2.92 [2.00-4.27]. CONCLUSIONS: Our study indicates that elevated serum GPC4 levels are significantly associated with an increased risk of MACE, vascular mortality, and all-cause mortality.